ES2651192B2 - ALBUMS AND PEPTIDES ABLE TO HYDROLYZE ORGANOPHOSPHORATED COMPOUNDS AND CARBAMATES, AND USES OF THE SAME - Google Patents

Abstract

Albumin and peptides capable of hydrolyzing organophosphorus compounds and carbamates, and uses thereof. # The present invention discloses the amino terminal sequence of bird albumin bound to divalent cations such as copper or zinc, as well as any recombinant or fusion protein that comprise said sequence for use as a medicine, specifically for the treatment of poisonings produced by organophosphorus compounds. The present invention also describes compositions and methods of treatment for said type of poisonings. On the other hand, the present invention discloses the use of said sequences and peptides that comprise them for the decontamination and / or bioremediation of areas or surfaces contaminated by organophosphorus compounds and for the isolation of chiral compounds.

Description

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ALBUMS AND PEPTIDES ABLE TO HYDROLYZE ORGANIC PHOSPHORED COMPOUNDS AND CARBAMATES, AND USES OF THE SAME.

DESCRIPTION

TECHNICAL FIELD

The present invention relates to an isolated peptide that corresponds to the amino terminal region of the albumine of birds and which is capable of hydrolyzing organophosphorus compounds and carbamates. The invention also relates to nucleic acid constructs, fusion proteins, vectors, host cell and compositions comprising polynucleotides or peptides, as well as methods for producing and using the polypeptides.

STATE OF THE TECHNIQUE

Pesticides are products of chemical synthesis that include compounds of the organochlorine, organophosphorus and carbamate type, which caused strong problems of contamination to the environment and resistance of insects, in addition to harmful effects on non-white organisms. Organophosphorus compounds (OPs) are esters, amides or thiols derived from phosphoric or phosphorothioic acid and carbamates are esters of carbamic acid. 30% of organophosphorus compounds are racemic mixtures that develop potent stereoselective neurotoxicity in living beings. In this regard, the World Health Organization (WHO) has reported that human poisoning by organophosphorus compounds affects a high proportion of people in agriculture, and is also considered a public health and environmental health problem. Organophosphorus compounds are used for the preparation of lubricants, nerve agents used in warlike conflicts and especially insecticides and pesticides for the control of insects (vectors) in public health and in agricultural activities. Hundreds of tons of these compounds are synthesized annually, including malation, paration, chlorpyrifos, methamidophos, monocrotophos, trichloronate, methamidophos, acetate and diazinon, among others. Despite the development of bioinsecticides, the demand for organophosphorus compounds of industrial origin is required due to their high efficiency for the best control of insects, especially in tropical countries.

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Generally, in work environments where OPs are present, there are no safety systems, sensors, or degradation systems, biocatalysts, efficient for air detection and for the control of liquid waste of these compounds due to lack of Enzymatic systems that degrade them in situ, thus avoiding the intoxication and contamination to which they give rise. For these reasons, the control of OP emissions during their production, as well as the elimination of industrial waste and the stored quantities of these compounds by the chemical industry represent an occupational and environmental risk of ecosystem toxicity.

Another field in which there is a need for effective systems of elimination of organophosphorus compounds is worldwide in the destruction of the thousands of tons of chemical weapons arsenals (war gases). The chemical and incineration methods represent a high risk, so biotechnological approaches with biocatalysts in mediums with mild temperature and pH conditions, are a desirable alternative and of important potential application.

Currently, two types of pharmacological strategies are used in the medical clinic for the treatment of PO poisonings: a) acetylcholine antagonists (atropine) and, b) acetylcholinesterase acetylcholinesterase reactive nucleophilic agents (oximes). Despite these pharmacological alternatives in severe cases of intoxication, death occurs in short periods of time (24-72 hours) due to the high concentrations of these compounds in the body. For this reason, the search for biological alternatives that degrade or hydrolyze these compounds is an urgent need worldwide.

In this sense, there is a small number of proteins in the state of the art that hydrolyze OPs compounds called phosphotriesterases, among the most studied and used are paraoxonase-1 (PON1) from mammalian serum and phosphotriesterase from Psedomonas diminuta. Both metalloprotins depend on divalent cations for their activity, with the particularity that in the presence of copper-divated cation (Cu2 +) they lose their hydrolyzing capacity, that is, Cu2 + is an inhibitor of phosphotriesterase enzymes. Despite the efforts made, no satisfactory results have been obtained with respect to the treatment and degradation of OPs compounds by said enzymes. For example, the enzyme PON1 is a

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Very limited enzyme in terms of the number of OPs compounds it hydrolyzes, as well as its stereoselective enzymatic inactivity against chiral OPs. As for bacterial phosphotriesterases, although they hydrolyse OPs compounds at a faster rate than the PON1 enzyme, they are generally stereoselective in favor of less toxic isomers, and show immunological adverse effects during in vivo application in mammals including humans. On the other hand, it is also known that albumin has the ability to hydrolyze organophosphorus compounds. Site 411 (Tyr411) of chicken albumin has been identified as the site responsible for the hydrolysis of the organophosphorus compound O-hexyl, O-2,5 dichlorophenylphosphoramidate (HDCP) (Sogorb et al., Chem. Res. Toxicol. 1998 ; 11: 14411446). Likewise, other studies have suggested the interaction of organophosphorus compounds with the 411 site of mammalian albumin using organophosphorus compounds of toxicological interest such as diisopropylfluorophosphate (DFP) (Schuh, Arch. Toxicol. 1970; 26: 262-72) . Said hydrolyzing activity of organophosphorus compounds of chicken, rabbit, chicken and human albumines is very low, since said enzymes, in their original state, cannot be used in the fields of biomedicine and biotechnology for the degradation of organophosphorus compounds.

Therefore, there is a need in the state of the art for finding biological alternatives to those described above that are capable of effectively degrading OPs and carbamates compounds, without giving rise to immunological adverse effects.

DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to an isolated peptide, from here we will call it "peptide of the invention", which consists of an amino acid sequence having at least 95%, 96%, 97%, 98 %, 99% identity with the amino acid sequence of SEQ ID NO: 1, in a more preferred embodiment the peptide of the invention comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% identity with the amino acid sequence of SEQ ID NO NO: 2.

Said sequences SEQ ID NOs: 1 or 2 correspond to the N-terminal regions of the bird albumins, more preferably of the chicken or turkey albumines. Such isolated peptide sequences are demonstrated in the present invention that are

the specific regions within the peptides of the bird albumins, preferably of the chicken and turkey albumines, to which at least one metal cation cofactor is attached, allowing said proteins to exhibit organophosphatase activity, thus being able to hydrolyze 5 organophosphorus compounds, carbamic and / or carboxylic esters.

The Table shows a comparison of the amino acids present in the N-terminal zone of albumins of different species, specifically those amino acids to which the divalent metal cations are attached to enable the function of hydrolysis of organophosphorus and / or carbamic compounds by part of the bird albumines. Table 1 shows the amino acid sequence of the N-terminal region of albumin as they are in serum after post-translational modifications of the precursor proteins. As can be seen in this comparison of Table 1, the albumine of the human species, rabbit, bovine, pig, 15 and dog, does not have the glutamic (E) in position 3 and the N-terminal regions of the albumins of Cobra and lamprey do not present any homologla with the N-terminal region of turkey or chicken albumine (SEQ ID NO: 2).

Table 1. Comparison of the amino acids present in the N-terminal zone of 20 albumines of different species.

 Species

 Amino Acids SEQ ID NO:

 Chicken

 DAEHK 2

 Turkey

 DAEHK 2

 Human

 DA-HK 5

 Rabbit

 EA-HK 6

 Sheep

 DT-HK 7

 Bovine

 DT-HK 8

 Pork

 DT-YK 9

 Dog

 EA-YK 10

 Rat

 EA-HK 11

 Cobra

 TSSTG 12

 Lamprey

 EDESF 13

The N-terminal zone of chicken albumins (SEQ ID NO: 14) and turkey (SEQ ID NO: 15), specifically the region that goes from amino acid 1 to 40, is identical in both species, except that the amino acid located in position 24 of the N-terminal region of chicken albumin (SEQ ID NO: 14) is F, while in the same 5 region in turkey albumin (SEQ ID NO: 15) is V (See Table 2) , but said positions of these amino acids are outside the specific region of SEQ ID NO: 1 and more specifically SEQ ID NO: 2, to which the divalent metal cation is attached and which provides the specific ability to hydrolyze organophosphorus compounds and / or carbamics, so it does not affect the hydrolytic capacity of said compounds.

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Table 2 shows the N-terminal zone of albumin and albumin precursors of different species. Underlined is the region of interest to which the metal divalent cation is attached to provide the hydrolysis activity of organophosphorus compounds and / or carbamic esters described in the present invention.

Table 2. N-terminal zone of albumins and albumine precursors of different species.

 Species

 Region N-terminal SEQ ID NO:

 Seric chicken albumin precursor [Gallus gallus]

 MKWVTLISFIFLFSSATSRNLQRFARDAEHK S 14

 Turkey albumina [Meleagris gallopavo]

 MKWVTLISFIFLFSSATSRNLQRVARDAEHK SEIA 15

 Human albumin [Homo sapiens]

 MKWVTFISLLFLFSSAYSRGVFRRDAHKSEV 16

 Rabbit albumin precursor

 MKWVTFISLLFLFSSAYSRGVFRREAHKSEI AHR 17

 Albumina dog [Canis lupus familiaris]

 EAYKSEIAHR 18

 Rat albumin [Rattus norvegicus]

 MKWVTFLLLLFISGSAFSRGVFRREAHKSEI AHR 19

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As can be seen in Tables 1 and 2, the sequence (SEQ ID NO: 2) of the N-terminal region of the bird albines, more particularly of the turkey and chicken albines, is a different region from the one presented by the albumines of the rest of vertebrates. Said region of SEQ ID NO: 2 linked to divalent metal cations, preferably cations such as zinc (Zn2 +) and / or copper (Cu2 +), form a catallotic center in the protein sequence of bird albumins capable of effectively hydrolyzing and without adverse immunological effects, organosphosphorus compounds and / or carbamates, which are toxic compounds for both animals, including man, and the environment. In addition, given the properties and structure-activity relationship that organophosphorus compounds and / or carbamates have with other compounds of the esters or amides type, the sequence SEQ ID NO: 2, as well as any recombinant protein or bird albumin comprising said sequence SEQ ID NO: 2, can be used to make chemical modifications in candidate compounds for use as a medicine, for example, phosphoramidates derived from (-) - beta-D- (2T, 4R) - diolano-thymine (DOT) that have been proposed as antivirals, or drugs derived from clinical esters such as lactones.

The catalytic ability to degrade and / or hydrolyze organophosphorus compounds (organophosphatase activity) and / or carbamic esters of the sequence SEQ ID NO: 2, is given by the presence in said sequence SEQ ID NO: 2 of the amino acid acid glutamic (E) and the amino acid histidine (H), in positions 3 and 4, respectively. Therefore, the amino acids identified as Xaa at positions 1, 2 and 5 of SEQ ID NO: 1 could be any amino acid.

For the purposes of the present invention the term "organophosphatase activity" is defined herein as the activity directed to catalyze the hydrolysis of OPs compounds. The peptides and / or enzymes that exhibit this activity, act on OPs compounds such as malation, paration, chlorpyrifos, methamidophos, monocrotophos, trichloronate, acetafo, diaxinon, diazinon, paraoxin, among others, including esters of phosphonic and phosphonic acids and esters of carbamic acids and carboxylic esters. A list of oganophosphorus compounds and carbamates is shown in Table 3.

Table 3. List of organophosphorus pesticides and carbamates.

 ORPHANOPHOSPHORATED

 CARBAMATES

 Methamidophos

 Methoxy

 Acetate

 Carbaryl

 Diazinon

 Pirimicarb

 Chlorpyrifos

 Carbofuran

 Fenamiphos

 Oxamilo

 Profenofos

 Sphevalerate

 Metidation

 Carbaryl

 Dimethoate

 Phenypiroxate

 Etoprofos

 Methoxy

 Methyl azinphs

 Phenoxycarb

 Paraoxon

 Fosmet

 Malation

 Caduzafos

 Cryptoxyphos

 Dialifor

 Phonophos

 Fensulfotion

 Isophenphs

 Trichloronate

Organophosphorus compounds (OPs) are synthetic organophosphorus esters and related compounds such as phosphoramidates. They have the general formula 5 (RR'X) P = O or (RR'X) P = S, where R and R 'are short chain groups. For organophosphorus insecticides molecule X is a good aromatic or aliphatic leaving group, which is a requirement for irreversible inhibition of acetylcholinesterase. The peptides described in the present invention, which include chicken and turkey albumines comprising SEQ ID NO: 2 of the invention, hydrolyze the phosphoester bonds of the OPs compounds. These OPs can be oxon and / or tion type OPs such as HDCP and trichloronate, among others (see Table 3). In addition to the compounds described in Table 3, the peptides described in the present invention are capable of hydrolyzing war gas type compounds such as sarin, cyclosarin, tabun, soman, VX).

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For the purposes of the present invention, the terms "peptide", "polypeptide", or "peptide sequence" are used interchangeably to refer to the peptide or peptides of the invention. The term "isolated polypeptide" or "isolated peptide" as used herein, refers to a polypeptide that is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, more preferably at least 90% pure, and even more preferably at least 95%, 96%, 97%, 98%, 99% pure, as determined by any technique known to the person skilled in the art, such as , SDS-PAGE. By "substantially purified polypeptide" or "substantially pure peptide", we mean a peptide that has generally been separated from lipids, nucleic acids, other peptides, and other contaminating molecules with which it is associated in its native state. Preferably, the substantially purified peptide is at least 60% free, more preferably at least 75% free, more preferably at least 90%, more preferably at least 95%, more preferably at least 96% pure, more preferably at least 97% pure. , more preferably at least 98% pure, even more preferably at least 99%, more preferably at least 99.5% pure, and even more preferably 100% pure, free of other components with which they are naturally or natively associated. The polypeptides of the present invention are preferably in a substantially pure form. This can be achieved, for example, by the preparation of the polypeptide by means of well-known recombinant methods or by classical purification methods. In this document, the term "substantially pure polypeptide" is synonymous with the terms "isolated polypeptide" and "isolated polypeptide."

In the present invention the term "homology" refers to the concept of "sequence homology", referring to the degree of similarity between two amino acid or nucleotide sequences. In the present invention the term "orthologue" refers to two chains of amino acids or nucleotides, from two different organisms, which share a high degree of homology.

The term "identity", as used in this description, refers to the proportion of identical amino acids or nucleotides between two amino acid sequences or nucleotides that are compared. Preferably, "identity" means the ratio of nucleic acid or amino acid residues that are identical between two nucleic acid or amino acid sequences, with respect to the full length of the reference sequence. The percent identity

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existing between two amino acid sequences can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences. As an example, the degree of identity can be determined by the ClustalW method, the Wilbur-Lipman method, the GAG program, which includes GAP, BLAST or BLASTN, EMBOSS Needle and FASTA. In addition, the Smith Waterman algorithm can be used to determine the degree of identity between two sequences.

For sequence comparison, typically one of the sequences acts as a reference sequence with which the "problem" sequences are compared. When a sequence comparison algorithm is used to determine its identity, the reference sequence and the problem sequence (s) are entered into the program, and the parameters thereof are configured. You can use the program parameters that appear by default or be configured. Preferably said parameters will be those that appear by default. Thus, the sequence comparison algorithm calculates the percentage of identity between the problem sequence (s) and the reference sequence based on the program parameters. Two examples of algorithms that are useful for determining percent sequence identity are BLAST and BLAST 2.0, described in Altschul et al. (1997) Nucleic Acids Res 25 (17): 3389-3402 and Altschul et al. (1990) J. Mol Biol 215 (3) -403-410, respectively. Preferably, the degree of identity referred to in the present invention is calculated by BLAST. The software to carry out the BLAST analysis is publicly available in the National Center for Biotechnology Information (NCBI).

In a particular embodiment, the polypeptide or peptide sequence of the invention has a sequence identity with SEQ ID NO: 1 of at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% and more preferably at least 100%.

In another preferred embodiment, the polypeptide of the invention comprises at least one of the sequences selected from any of the following: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15 and / or any of its combinations. In another more preferred embodiment, the polypeptide of the invention consists of at least one sequence selected from any of the following:

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SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15 and / or any combination thereof.

As demonstrated in the examples included herein, the bird albumines, specifically the chicken albumins (SEQ ID NO: 3) and turkey (SEQ ID NO: 4), which comprises in their N-terminal region the SEQ ID NO: 2, act as biocatalysts for the degradation of organophosphorus compounds, carbamic esters and / or carboxylic esters, always in the presence of at least one metal cation.

In another preferred embodiment, the polypeptide of the invention is characterized in that it is covalently linked to at least one cofactor, said cofactor being preferably a metal cation with 2+ charge.

For the purposes of the present invention, the term "cofactor" refers to any chemical, non-protein, thermostable and low molecular weight component necessary for the action of a protein or enzyme, whether metal ions, organic molecules or coenzymes. Examples of cofactors are, but not limited to, divalent cations, preferably Zn2 +, Fe2 +, Cu2 +, K +, Mn2 + or Mg2 +, as! like other organic cofactors like vitamins, NAD (P) +, NAD (P) H, ATP, ADP or FAD.

In a more preferred embodiment of the present invention, the cofactors are preferably divalent cations and more preferably cations such as zinc (Zn2 +) or copper (Cu2 +), with copper, Cu2 + being preferred.

In another preferred embodiment, the polypeptide of the invention is characterized in that SEQ ID NO: 1, and more specifically, SEQ ID NO: 2, are the amino terminal sequences of bird albumin.

In another more preferred embodiment, the polypeptide of the invention is characterized in that the bird albumin is a serine albumin. In another preferred embodiment, the bird albumin is preferably selected from chicken albumin (SEQ ID NO: 3) or turkey (SEQ ID NO: 4).

For the purposes of the present invention, "albumin" generally refers to native or recombinant albumin of any species. Biochemical studies

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have revealed that albumins of animal origin have a structural homologla of 60%. More preferably, albumin is from birds, mammals and / or reptiles. More preferably, the albumine is of avian origin. Even more preferably, the albumine used is native or recombinant avian serine albumine, preferably chicken or turkey albumine.

In another aspect, the present invention relates to artificial or mutant variants of the peptide of the invention, which comprise a conservative substitution, deletion, and / or insertion of one or more amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 These variants refer to limited variations in the amino acid sequence, which allow the maintenance of the functionality of the peptide. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein. Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids ( phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).

Variations can be artificially generated variations, such as by mutagenesis or direct synthesis. These variations do not cause essential modifications in the characteristics or essential properties of the peptide. Therefore, peptides or polypeptides whose amino acid sequence is identical or homologous to the sequences described in the present invention are also included within the scope of the present invention. In addition, of the 20 standard amino acids, non-standard amino acids (such as A-hydroxyproline, 6 - [lambda] lysine / -methyl, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) can be substituted for amino acid residues of a wild type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can be substituted by amino acid residues. Additionally, if desired, unnatural amino acids or analogs of chemical amino acids can be introduced as a substitution or addition into the peptide of the present invention. Such amino acids include, but are not limited to, the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyl acid, 4-aminobutyl acid, 2-aminobutyl acid, 6-amino hexanoic acid,

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2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, p-alanine, amino fluoroamino acid such as p-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.

Also included within the scope of the invention are the peptides of the present invention that are differentially modified during or after the synthesis, for example, by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, lipid binding (for example fatty acids ), derivatization by known protective / blocking groups, proteolytic division, binding with an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and / or bioactivity of the peptide of the invention.

The peptides of the present invention can be produced in a variety of ways, including the production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated peptide of the present invention is produced by the cultivation of a cell capable of expressing the peptide under conditions effective to produce the peptide, and the recovery of the peptide. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that allow the production of proteins. An effective medium refers to any medium in which a cell is cultured to produce a peptide of the present invention. Such a medium generally comprises an aqueous medium having sources of nitrogen, phosphate and assimilable carbon, and salts, minerals, appropriate metals and other nutrients, such as vitamins. The cells of the present invention can be grown in conventional fermentation bioreactors, oscillating flasks, test tubes, microtiter plates, and petri dishes. The culture can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such cultivation conditions are within the skill of an expert in the trade.

Due to the degeneracy of the genetic code, in which several nucleotide triplets give rise to the same amino acid, there are several nucleotide sequences that give rise to the same amino acid sequence. Therefore, another aspect of the invention relates to an isolated polynucleotide or isolated nucleotide, now in

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hereinafter "polynucleotide of the invention", which encodes at least one polypeptide of the invention.

The terms "nucleotide sequence", "nucleotide sequence", "nucleic acid", "oligonucleotide" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length that may or may not be chemical or biochemically modified. They refer, therefore, to any polyiribonucleotide or polydeoxyribonucleotide, both single-stranded and double-stranded. The polynucleotide of the invention can therefore be DNA, RNA, or derivatives of both DNA and RNA, including cDNA. The polynucleotide of the invention can be obtained artificially by conventional donation and selection methods, or by sequencing. The polynucleotide, in addition to the coding sequence, can carry other elements, such as, but not limited to, introns, non-coding sequences at the 5 'or 3' ends, ribosome binding sites, or stabilizing sequences. These polynucleotides can additionally include coding sequences for additional amino acids that may be useful, for example, but not limited, to increase the stability of the peptide generated from it or allow a better purification thereof.

The term "isolated polynucleotide" or "isolated nucleotide" as used herein refers to a polynucleotide that is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, more preferably at least 90% pure, and even more preferably at least 95% pure, as determined by any technique known to the person skilled in the art, such as, SDS-PAGE. The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other foreign or unwanted nucleotides and in a form suitable for use within genetically created protein production systems. This can be achieved, for example, by preparing the polynucleotide by means of well-known recombinant methods or by classical purification methods. In this document, the term "substantially pure polynucleotide" is synonymous with the terms "isolated polynucleotide" and "isolated polynucleotide." The polynucleotides of the present invention are preferably in a substantially pure form.

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Nucleic acid sequences encoding the polypeptides of the invention can, for example, be designed based on the amino acid sequences provided in the present invention. Therefore, another object described in the present invention relates to an isolated nucleotide sequence encoding the polypeptide of the invention. In a more preferred embodiment the polynucleotide of the invention comprises at least one of the sequences selected from any of the following: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and / or any combination thereof. In another more preferred embodiment, the polypeptide of the invention consists of at least one of the sequences selected from any of the following: SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and / or any combination thereof.

Another aspect of the present invention relates to an isolated polynucleotide comprising a nucleotide sequence complementary to the polynucleotide of the invention.

The polynucleotide of the invention can be introduced into a genetic construct, for example, into a donation vector or expression vector, to allow its replication or expression. Preferably, said vector is an appropriate vector for the expression and purification of the polypeptide of the invention. Therefore, another aspect of the invention relates to a genetic construct comprising at least one of the polynucleotides of the invention, hereafter referred to as the "genetic construct of the invention", operatively linked to one or more control sequences that direct Polypeptide production in a host.

The term "genetic construction" as used herein refers to a nucleic acid molecule, both single-stranded and double-stranded, that is isolated from a gene of natural origin or that is modified to contain nucleic acid segments in a manner that would otherwise They will not exist in nature. The term "genetic construction" or "genetic construction" is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of a coding sequence of the present invention. Therefore, the genetic construction of the invention may further comprise one or more control or regulatory sequences of the gene expression, such as promoter sequences, llder sequences, transcription terminator sequences, polyadenylation sequences, signal sequences, etc. An isolated polynucleotide encoding a

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The polypeptide of the present invention can be manipulated in a variety of ways to provide expression of the polypeptide. Manipulation of the polynucleotide sequence before insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art. The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for the expression of a polynucleotide encoding a polypeptide of the present invention.

The term "control sequences" as defined herein includes all components that are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence can be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a llder, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and stop signals for translation and transcription. Control sequences may be provided with links in order to introduce specific restriction sites that facilitate the binding of control sequences with the coding region of the nucleotide sequence encoding a polypeptide. Appropriate control sequences for the expression of a polynucleotide in eukaryotic cells are known in the state of the art. As used herein, the term "promoter" refers to a nucleotide sequence, generally "upstream" or "upstream" of the transcription start point, which is capable of initiating transcription in a cell. This term includes, for example, but not limited to, constitutive promoters, cell-type specific promoters and inducible or repressible promoters. In general, control sequences depend on the origin of the host cell.

The term "operably linked" denotes a configuration in which a control sequence is placed in an appropriate position in relation to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

When used here the term "coding sequence" or "coding sequence" means a nucleotide sequence, which specifically and directly results in a

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specific amino acid sequence of its protein product. The limits of the coding sequence are generally determined by an open reading frame, which normally begins with the ATG initiation codon or alternative initiation codons such as GTG and TTG. The coding sequence can be a nucleotide sequence of DNA, cDNA, or recombinant.

The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Another object described in the present invention relates to a "fusion peptide" or "fusion polypeptide" comprising at least the polypeptide of the present invention.

In a preferred embodiment, the fusion peptide according to the present invention refers to any native or recombinant peptide, to which the peptide of the invention is covalently bound and which is capable of maintaining organophosphatase activity, as described in the present document

In a preferred embodiment, the genetic construction of the invention is an expression vector. An "expression vector" is a linear or circular DNA molecule that comprises at least one polynucleotide of the invention, and which is operably linked to additional nucleotides that are provided for expression. Said vector comprising the polynucleotide of the invention can be introduced into a host cell such that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector.

For the purposes of the present invention, the term "vector" or "expression vector" refers to a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention encoding the polypeptide of the present invention, inserted into any vector, and that is operatively linked to the additional nucleotides that propitiate its expression and that is capable of delivering the nucleic acid molecule into a host cell. Said vector may contain heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally adjacent to the nucleic acid molecules of the present invention and that are preferably derived from a different species from the

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species from which, the nucleic acid molecule (s) are derived. When creating the expression vector, the coding sequence is located in the vector such that it is operably linked to the control sequences suitable for its expression. Thus, the expression vectors referred to in the present invention comprise the polynucleotide of the invention, a promoter, and termination signals of transcription and translation. The various nucleic acids and control sequences described herein can be linked together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow insertion or replacement of the polypeptide of the invention at said sites.

The expression vector to which the invention relates can be any vector, RNA or DNA, either prokaryotic or eukaryotic, and is usually a virus or a plasmid. Preferably, the expression vector is also able to replicate within the host cell. Expression vectors of the present invention include any of the vectors that can function (ie, direct gene expression) in the recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, other animal cells, and vegetables The choice of the vector will normally depend on the compatibility of the vector with the host cell in which it is to be introduced. The expression vector may be a plasmid, a cosmid, a phage, a virus, an artificial bacterial chromosome (BAC), an artificial yeast chromosome (YAC), or the like. Vectors can be closed linear or circular plasmids. The vector may be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome (s) in which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon can be used. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, plant and mammalian cells.

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The vectors of the present invention preferably contain one or more selectable markers that allow easy selection of transformed, transfected, transduced cells, or the like. A selectable marker is a gene whose product provides biocidal or viral resistance, heavy metal resistance, prototroph to auxotrophs, and the like.

More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of the genetic product (s). An increase in the number of copies of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the genome of the host cell or including with the polynucleotide an amplifiable selectable marker gene, where the cells containing the amplified copies of the marker gene selectable, and therefore, additional copies of the polynucleotide, can be selected by culturing the cells in the presence of the appropriate selection agent.

The genetic construction of the invention, as mentioned above, can be introduced into a competent host cell to carry out the expression of the polypeptide of the invention. Therefore, another aspect of the invention relates to a "cell" or "host cell" comprising the polypeptide, the polynucleotide sequence or the genetic construct of the invention, "host cell of the invention" or "cell of the invention" .

The term "host cell" or "recombinant host cell", as used herein, includes any type of cell that is susceptible to transformation, transfection, transduction, and the like with the genetic construction of the invention.

More than one copy of a polynucleotide of the present invention can be inserted into the host cell to increase the production of the gene product. An increase in the number of copies of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the genome of the host cell or by including a selectable marker gene amplifiable with the polynucleotide where cells containing amplified copies of the gene selectable marker, and thereby additional copies of the polynucleotide, can be selected for culturing the cells in the presence of the appropriate selectable agent. The procedures used to link the elements described above to construct the vectors

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The recombinant expression agents of the present invention are well known to one skilled in the art (see, for example, Sambrook et al., 1989, supra).

The transformation of a nucleic acid molecule into a cell can be achieved by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and fusion of protoplasts. A recombinant cell can remain unicellular or can grow in a tissue, an organ or a multicellular organism. The transformed nucleic acid molecules of the present invention can remain extrachromosomal or can be integrated into one or more sites within a chromosome of the transformed (i.e., recombinant) cell such that its ability to be expressed is preserved.

The choice of a host cell largely depends on the gene that encodes the polypeptide and its source. The host cell can be a prokaryotic or a eukaryotic.

Suitable host cells for transformation include any non-human cell that can be transformed with a polynucleotide of the present invention. The host cells can be both untransformed cells and cells that are already transformed with at least one nucleic acid molecule (for example, nucleic acid molecules encoding one or more proteins of the present invention). The host cells of the present invention may be capable of endogenously producing (i.e., of course) the proteins of the present invention or may be capable of producing the aforementioned proteins after being transformed with at least one nucleic acid molecule of the present invention. The host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal cells (including yeast), parasites, arthropods, animals and plants. Preferred host cells include bacterial, mycobacterial, yeast, vegetable and animal cells, preferably non-human mammal. The most preferred host cells include Agrobacterium, Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (hamster kidney) cells, MDCK cells (normal dog kidney cell line for herpesvirus culture) canine), CRFK cells (normal cat kidney cell line for feline herpesvirus culture), CV-1 cells (cell line

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of African monkey kidney used, for example, to grow raccoon poxvirus), COS cells (eg, COS-7), and Vero cells. Particularly preferred host cells are E. coli, including K-12 derivatives of E. coli; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and G8 cells of non-tumorigenic mouse myoblast (for example, ATCC CRL 1246). Other suitable non-human mammalian host cells include other non-human kidney cell lines, other non-human fibroblast cell lines (eg, chicken or murine embryo fibroblast cell lines), non-human myeloma cell lines , Chinese hamster ovary cells, mouse NIH / 3T3 cells and / or LMTK cells.

Recombinant DNA technologies can be used to improve the expression of polynucleotide molecules transformed by manipulation, for example, the number of copies of the polynucleotide molecules within a host cell, the efficiency with which the polynucleotide molecules are transcribed, the efficiency with which the resulting transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, polynucleotide molecules operatively linked to high copy plasmids, integration of polynucleotide molecules into one or more chromosomes of the host cell, addition of vector stability sequences for plasmids, substitutions or modifications of the transcription control signals (for example, promoters, operators, enhancers), substitutions or modifications of the translation control signals (for example, ribosome binding sites, Shine-Dalgarno sequences), modification of the polynucleotide molecules of the present invention that correspond to the use of the host cell codon, and the deletion of sequences that destabilize transcripts. The activity of an expressed recombinant protein of the present invention can be enhanced by fragmentation, modification, or derivatization of the polynucleotide molecules encoding said protein.

In a preferred embodiment, the host cell of the invention therefore comprises at least one polynucleotide of the invention recombinantly introduced by the genetic construction of the invention. Such polynucleotides can encode the mature polypeptide or a pre-protein consisting of a signal peptide

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bound to the mature enzyme that will have to be further processed in order to produce the polypeptide of the invention.

The host cell of the invention expresses at least one of the peptides selected from any of the following SEQ ID NO: 1, SEQ ID NO: 2, as well as any peptide comprising said sequences in its N-terminal region such that said Peptides are functional as described herein, that is, they maintain their hydrolytic activity against organosphosphorus compounds and / or carbamates. Among these peptide sequences are the bird albumin, preferably chicken albumin (SEQ ID NO: 3) and turkey albumin (SEQ ID NO: 4). The term "functional" means that the expressed peptide (s) retains its hydrolyzing catalytic activity of phosphoric, carbamic and / or carboxylic esters. This activity can be measured by means of any suitable procedure known in the state of the art, preferably by means of any of the procedures described below in the examples that accompany this document.

The term "expression" includes any stage involved in the production of the peptide of the invention that includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

The expression of the invention peptide can be carried out in the host cell of the invention by any method known in the art, such as the transformation of a suitable host cell with at least one polynucleotide of the invention and / or with any combination of these, or with the genetic construction of the invention, and the culture of the transformed host cell under conditions that induce the expression of said polynucleotide in order to obtain the secreted peptide and / or any combination thereof.

The host cell can be cultured in a suitable nutrient medium, solid or liquid, for the production of the peptide of the invention, under conditions that allow it to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts using procedures well known in the art. If the peptides of the invention are secreted in the nutrient medium, they can be recovered directly from the medium.

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On the contrary, if they remain inside the host cell, they will be recovered by techniques known to those skilled in the art.

In turn, said peptides of the invention can be detected using methods known in the art specific for polypeptides. These detection procedures may include the use of specific antibodies, the formation of a product, or the disappearance of a substrate. In addition, said peptides can be recovered using methods known in the art. For example, from the nutrient medium by conventional procedures that include, but are not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

The peptides of the present invention can be purified by a variety of methods known in the art that include, but are not limited to, chromatography (eg, ion exchange, affinity, hydrophobic,

chromatofocalization, and size exclusion), electrophoretic procedures (for example, preparative isoelectric focusing), differential solubility (for example, precipitation in ammonium sulfate), SDS-PAGE, or extraction.

Thus, in another preferred embodiment, the host cell of the invention can express at least one of the peptides of the invention, or any combination thereof, for example, but not limited to, the peptides comprising the sequences SEQ ID NO: 1, 2, 3, 4, 14, 15, and / or any combination thereof.

Another object described in the present invention relates to a composition, hereafter referred to as "composition of the invention", which comprises polynucleotide sequences, or peptide sequences, or genetic construction, or fusion proteins, or vectors, or the cells, described throughout this document, together with at least one cofactor characterized in that it is a divalent cation.

In a preferred embodiment, the composition of the invention is characterized in that it comprises a peptide sequence selected from any of the following: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15, and / or any combination thereof; more preferably the sequences are SEQ ID NO: 3 and / or SEQ ID NO: 4.

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In another more preferred embodiment, the composition of the invention is characterized in that the divalent cations are selected from any of the following list: Zn2 +, Fe2 +, Cu2 +, Mn2 +, Mg2 + and / or any combination thereof, more preferably where divalent cation is Zn2 + or Cu2 +, preferably Cu2 +.

In a more preferred embodiment, the composition of the invention is a composition that exhibits hydrolyzing catalytic activity of organophosphorus compounds such as malation, paration, chlorpyrifos, methamidophos, monocrotophos, trichloronate, acetafo, diaxinon, diazinon, paraoxin, among others, including esters of phosphonic and phosphonic acids and esters of carbamic acids and carboxylic esters.

In a preferred embodiment, the composition of the invention is characterized in that it is a pharmaceutical or veterinary composition. Such compositions may further comprise physiologically acceptable carriers or excipients. An excipient can be any material that the animals, plants, plant or animal material, or environment (including soil and water samples) that are treated, can tolerate. Examples of such excipients include water, saline solution, Ringer's solution, dextrose solution, Hank's solution, and other physiologically balanced aqueous saline solutions. Non-aqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides can also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Excipients may also contain lower amounts of additives, such as substances that improve isotonicity and chemical stability. Examples of regulatory solutions include phosphate regulatory solution, bicarbonate regulatory solution and Tris regulatory solution, 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, controlled release polymeric vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

In another preferred embodiment, the pharmaceutical or veterinary compositions described in the present invention may also comprise another active ingredient. The term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological or other activity

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different effect on the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the elaboration of the drug and are present therein in a modified form intended to provide the specific activity or effect.

In a preferred embodiment, the active ingredients that can accompany the pharmaceutical or veterinary compositions described in the present invention are selected from any of the following: acetylcholine receptor blocking agents, preferably atropine (avoids the toxic effects of organophosphorus and carbamates ), oximes, preferably pralidoxime, or any substance or compound capable of reactivating cholinesterase inhibited by organophosphates, non-catallotic bioscavengers (protelnas or peptides that bind and capture organophosphorus compounds and / or carbamates); Any other compound capable of avoiding adverse effects of organophosphorus and / or carbamate intoxication, such as anxiolytics, anticonvulsants, sedatives, such as diazepan and / or calcium blockers.

In another preferred embodiment, the composition of the invention further comprises a pharmaceutically or veterinarily acceptable vehicle or excipient.

The term "vehicle" applied to the pharmaceutical and veterinary compositions described in the present invention refers to a diluent, adjuvant, excipient or carrier with which the peptide of the invention should be administered, the nucleotide sequence encoding it, the genetic construction , or the cell as described in the invention.The function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the composition.Therefore, the vehicle is a substance that is used in the composition to dilute any of the components thereof to a certain volume or weight; or even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the composition. When the presentation form is In this case, the pharmaceutically acceptable vehicle is the diluent, and the vehicle must be allowed and evaluated so that They do not cause damage to the organisms to which they are administered.

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An excipient can be any material that animals, including humans, can tolerate. Examples of such excipients include water, saline solution, Ringer's solution, dextrose solution, Hank's solution, and other physiologically balanced aqueous saline solutions. Non-aqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides can also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Excipients may also contain lower amounts of additives, such as substances that improve isotonicity and chemical stability. Examples of regulatory solutions include phosphate regulatory solution, bicarbonate regulatory solution and Tris regulatory solution, 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, controlled release polymeric vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

The composition of the invention can be found in a solid, liquid or suspended state, even inside nanoparticles, preferably in solution.

The composition of the present invention can be administered by any suitable route for a specific molecule, directly (for example, locally, such as by injection, subcutaneous injection or topical administration at the site of tissue) or by systemic route (for example, parenterally or orally). When the composition of the present invention is to be provided parenterally, such as by intravenous, subcutaneous, ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular administration. , intranasally or by aerosol administration, the composition preferably comprises part of a physiologically fluid or aqueous compatible suspension or solution. Therefore, the vehicle or excipient is physiologically acceptable so that when the desired agent is supplied to the subject, the solution does not otherwise adversely affect the electrolyte balance and / or the volume of the subject. The aqueous medium for the agent can therefore comprise normal physiological saline solution.

In another preferred embodiment, the composition of the invention is characterized in that it is a biocatalyst.

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For the purposes of the present invention the term "biocatalyst" refers to any protein or peptide that catalyzes a reaction, typically corresponds to the concept of enzyme. It implies that a chemical compound (substrate or substrates) in the presence of the biocatalyst undergoes a chemical transformation resulting in a product or products of the reaction, while spontaneously in its absence, the reaction either does not occur or occurs more slowly .

Another object of the present invention relates to a controlled release formulation that is capable of slowly releasing the composition of the present invention in an animal or plant material, or the environment (including soil and water samples). As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Appropriate controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Preferred controlled release formulations are biodegradable (i.e., bioerodible).

A preferred controlled release formulation is capable of releasing the composition of the present invention in soil or water that is in an atomized area with a phosphoric, carbamic and / or carboxylic ester type compound. The formulation is preferably released for a period of time ranging from about 1 to about 12 months. A preferred controlled release formulation of the present invention is capable of performing a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least less than 9 months, and even more preferably for at least about 12 months.

The concentration and / or effective amount of the polypeptide, nucleotide sequence, vector, or host cell of the present invention that will be necessary to hydrolyze an organophosphorus or carbamate type compound, such as phosphoric, carbamic and / or carboxylic ester, will depend on the nature of the sample that is decontaminated, the concentration of the ester in the sample, and the formulation of the composition. The concentration and / or effective amount of the polypeptide, sequence

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Nucieotldica, vector, or host cell within the composition can be easily determined experimentally, as will be understood by an expert.

Another object described in the present invention relates to a biosensor and / or bioremediator comprising the polypeptide, or the polynucleotide, or the construction of nucleic acid, or the vector, or the cell, or the composition as defined in the present invention. , wherein said biosensor and / or bioremediator is capable of hydrolyzing and / or degrading organophosphorus compounds and / or carbamates.

For the purposes of the present invention the term "biosensor" refers to an analytical device that is generally formed by a biologically active material, such as an enzyme and a transducer that converts a biochemical reaction into a quantifiable electronic signal that can be processed. , transmit, and measure A general review of biosensors that have been used for the detection of phosphoric, carbamic and / or carboxylic ester compounds is provided by Rekha et al. (Rekha, M. et al. Critical Reviews in Biotechnology. 2000; 20: 213-235) The objects described in the present invention can be adapted for use in such biosensors.

For the purposes of the present invention the term "bioremediation" refers to the process for the elimination and / or destruction of contaminating compounds through living organisms and / or substances and / or compounds synthesized by them.

Another object described in the present invention relates to the use of the polypeptide, or of the polynucleotide, or of a nucleic acid construct, or of a vector, or of a cell, or of a fusion polypeptide, or of a composition according to the present. invention for the preparation of a medicine. Alternatively, the present invention relates to the polypeptide, or to the polynucleotide, or to the construction of nucleic acid, or to the vector, or to the cell, or to the fusion polypeptide, or to the composition according to the present invention, for use as a medicament.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in animals, including humans. In the context of the present invention, the disease refers to poisonings produced by organophosphorus compounds, carbamic esters and / or carboxylic esters.

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In a preferred embodiment, the present invention describes the use of bird albumin, preferably chicken albumin (SEQ ID NO: 3) and / or turkey (SEQ ID NO: 4) together with a divalent cation, such as described herein, for the preparation of a medicament. Alternatively, the present invention relates to bird albumin, preferably chicken albumin (SEQ ID NO: 3) and / or turkey (SEQ ID NO: 4) together with a divalent cation for use as a medicine.

The compositions of the invention will contain a prophylactic or therapeutically effective amount as defined herein, to provide the desired effect.

As used herein, the term "therapeutically or prophylactically effective amount" refers to the amount of polypeptide (s), nucleotide sequence (s) encoding the peptide (s). of the invention, fusion polypeptide, vector, cells of the invention, or composition of the invention that is capable of producing the desired effect. In general, the therapeutically effective amount to be administered will depend among other factors, on the subject's own characteristics, the severity of the disease and / or infection, the form of administration, etc.

Another object described in the present invention relates to the use of the polypeptide, or the polynucleotide, or a nucleic acid construct, or a vector, or a cell, or a fusion polypeptide, or a composition according to the present. invention for the preparation of a medicament for the treatment of poisonings by organophosphorus compounds, carbamic esters and / or carboxylic esters. Alternatively, the present invention relates to the polypeptide, or to the polynucleotide, or to the construction of nucleic acid, or to the vector, or to the cell, or to the fusion polypeptide, or to the composition according to the present invention, for the treatment of compound poisoning. organophosphates, carbamic esters and / or carboxylic esters.

In a preferred embodiment, the present invention describes the use of bird albumin, preferably chicken albumin (SEQ ID NO: 3) and / or turkey (SEQ ID NO: 4) together with a divalent cation, such as It is described herein for the preparation of a medicament for the treatment of poisoning by organophosphorus compounds, carbamic esters and / or carboxylic esters.

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Alternatively, the present invention relates to bird albumin, preferably chicken albumin (SEQ ID NO: 3) and / or turkey (SEQ ID NO: 4) together with a divalent cation for use as a medicament for treatment. of poisoning caused by organophosphorus compounds, carbamic esters and / or carboxylic esters.

Another object described in the present invention relates to the use of the polypeptide, or of the polynucleotide, or of a nucleic acid construct, or of a vector, or of a cell, or of a fusion polypeptide, or of a composition, or of a biosensor according to the present invention, to hydrolyze organophosphorus molecules.

Another object described in the present invention relates to the use of the polypeptide, or of the polynucleotide, or of a nucleic acid construct, or of a vector, or of a cell, or of a fusion polypeptide, or of a composition, or of a biosensor according to the present invention as a useful biocatalyst in bioremediation processes. In a preferred embodiment, said use is preferably directed to catalyze hydrolysis reactions and / or degradation of organophosphorus compounds, carbamic esters and / or carboxylic esters.

Another object described in the present invention relates to the use of the polypeptide, or of the polynucleotide, or of a nucleic acid construct, or of a vector, or of a cell, or of a fusion polypeptide, or of a composition, or of a biosensor according to the present invention for the isolation of chiral compounds.

Another object described in the present invention relates to the use of bird albumin for the treatment of organophosphorus compound poisoning. Alternatively, it refers to bird albumin for use in the treatment of organophosphorus compound poisoning. In a preferred embodiment, albumin is selected from turkey albumin or chicken albumin.

Another object described in the present invention relates to the use of bird albumin to hydrolyze organophosphorus molecules. In a preferred embodiment, albumin is selected from turkey albumin or chicken albumin.

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Another object described in the present invention relates to the use of bird albumin for bioremediation processes. In a preferred embodiment, albumin is selected from turkey albumin or chicken albumin.

Another object described in the present invention relates to the use of bird albumin for the isolation of chiral compounds. In a preferred embodiment, albumin is selected from turkey albumin or chicken albumin.

Another object described in the present invention relates to the use of bird albumin as a biosensor. In a preferred embodiment, albumin is selected from turkey albumin or chicken albumin.

Another of the objects described in the present invention relates to methods for producing a polypeptide according to the present invention, which comprises (a) culturing a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive to the polypeptide production; (b) recover and / or collect said cells or take a sample of the cell culture medium; c) isolate and purify the polypeptide.

Preferably, the cell is any of the cells described herein, more preferably, the cell is from the genus Saccharomyces cerevisiae, and more preferably Pichia pastoris.

Although Escherichia coli (E. coli) was the first host used to express recombinant human proteins, it has drawbacks to make some post-translational modifications, such as glycosylation. Because of this, Saccharomyces cerevisiae (S. cerevisiae) yeast has been used. However, because it forms glycosylated derivative proteins different from human cells, it produces antigenic proteins. More recently, however, other unconventional yeasts are being used, such as Pichia pastoris (P. pastoris).

The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a host cell comprising a genetic construct comprising a nucleotide sequence encoding the polypeptide of the invention under conditions conducive to production of the

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polypeptide, and (b) recover and / or collect said cells or take a sample of the cell culture medium; c) isolate and purify the polypeptide.

In a preferred embodiment of the methods described herein, these are characterized in that in step b) a crude extract or homogenized clarified extract is prepared. In another more preferred embodiment of the method of the invention, it is optional to enrich the level of purity of the peptide in the homogenized extract or in the crude extract.

In the production methods of the present invention, the cells are grown in a nutrient medium suitable for the production of the polypeptide using methods well known in the art. For example, the Pichia pastoris cell can be grown by agitation flask culture, small-scale or large-scale fermentation (including continuous, batch, batch feed, or solid state fermentation) in laboratory or industrial fermenters made in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated.

The culture is grown in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (for example, in catalogs of the American Type Culture Collection).

If the polypeptide is secreted in the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

Polypeptides can be detected using methods known in the art that are specific for polypeptides. These detection methods may include the use of specific antibodies, formation of an enzymatic product, or disappearance of an enzyme substrate. For example, an enzymatic assay can be used to determine the activity of the polypeptide as described herein.

The resulting polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by

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conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, precipitation, use of specific antibodies, the formation of a product, or the disappearance of a substrate.

The polypeptides of the present invention can be purified by a variety of methods known in the art, including, but not limited to, chromatography (eg, ion exchange, affinity, hydrophobic interaction, gel filtration, HPLC), electrophoretic methods (preparatory isoelectric focusing, preparative electrophoresis in polyacrylamide-SDS gels), differential solubility (precipitation with ammonium sulfate), preparative ultracentrifugation in sucrose gradient. Once the desired degree of purity has been reached, which may require more than one chromatographic step, it is often necessary to concentrate the protein or eliminate salts and ions that may be harmful for later use. In that case, known techniques are used, such as lyophilization or ultrafiltration.

Another object described in the present invention relates to a method for isolating isomers from a raceminal mixture of organophosphorus compounds comprising the following steps: a) incubating a racemic mixture of organophosphorus compounds with at least one of the peptides, sequences nucleotide, construction of nucleic acid, vector, cell, fusion polypeptide, composition, or biosensor described herein, in the presence of at least one metal cation, as described herein; b) recover and / or collect the compounds obtained; and c) isolate, purify and identify the isomer.

Step a) can be carried out in reactors in which the racemic mixture of organophosphorus compounds is mixed in the presence of at least one metal cation, and the biocatalyst and / or composition of the invention. The detection of the isomers obtained is carried out by any methodology known by the average expert in the present technical field, such as, for example, without being limiting, solvent extraction techniques or chromatographic techniques.

Another object described in the present invention relates to a method of detoxification and treatment of a subject that has been exposed to contamination by organophosphorus molecules comprising administration to said subject of

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a therapeutically effective amount of the polypeptide, or of the polynucleotide, or of the construction of nucleic acid, or of the vector, or of the cell, or of the fusion polypeptide, or of the composition, as described throughout the present invention.

For the purposes of the present invention the term "subject" refers to refers to all animals classified as mammals and includes but is not limited to farm and domestic animals, primates and humans, for example humans, nonhuman primates, cows, horses , pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a human being male or female of any age or race. Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (A) illustrates the effect of copper concentration on the hydrolysis levels of O-hexyl, O-2.5 dichlorophenylphosphoramidate (HDCP) in chicken serum and its comparison in the presence of (B) calcium, EDTA and Tris.

Figure 2 shows the hydrolysis levels of each of the two HDCP isomers due to their incubation with chicken serum (S) (10 pL) and their respective concentration of albumin (A) (216 pg) in the presence of copper or EDTA for 60 and 120 minutes under physiological conditions of pH and temperature.

Figure 3 shows the activating effect of copper (Cu2 +) on the hydrolysis of the R-HDCP isomer by the albumine of chicken serum and the hydrolysis of the two isomers of turkey serum albumine.

Figure 4 shows the hydrolysis capacity of different vertebrate albumine species (excluding birds) on the HDCP compound and the effect of different concentrations of copper (Cu2 +). Each point represents the mean ± SD of three experiments performed with 216 pg of sheep albumine (OSA), dog (DSA), pig (PSA) or lamprey (LSA) incubated with 400 pM racemic HDCP (200 pM of each isomer) and different concentrations of copper for 60 min at 37 ° C and pH 7.4. Figure 5 shows the effect of different cations on the hydrolysis of both HDCP isomers of turkey serum albumin.

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Figure 6 shows the effect of copper on the hydrolysis of both trichloronate isomers by turkey serum albumin.

Figure 7 illustrates the effect of pH on the hydrolysis of both HDCP isomers in the presence of EDTA (A) and Cu2 + (B) by chicken serum albumin.

Figure 8 shows the effect of pre-incubation time with Zn2 + and Cu2 + on the hydrolysis of HDCP isomers of chicken and turkey serum albumines.

Figure 9 illustrates the effect of Ni2 + pre-incubation on the hydrolysis of HDCP isomers of chicken and turkey albumines in the presence of copper (100 pM) and incubated at 37 ° C, pH 7.4 for 60 minutes.

EXAMPLES

Next, the invention will be illustrated by tests carried out by the inventors, which show the effectiveness and usefulness of the product and methods of the invention. These examples are included for illustrative purposes only and are not to be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Materials and methods.

The organophosphorus compounds used in the examples described below are HDCP and trichloronate, in racemic form, using the S and R isomers of both compounds. The range of concentrations used for them has been from 100 pM to 400 pM, with concentrations of 400 pM being preferred, equivalent to 200 pM for each isomer.

The enzymes used have been chicken or turkey albumine, present in chicken serum (CSA) (10 pL) or turkey (TSA) (10 pL) respectively, as well as commercial albumines (Sigma Aldrich) from different species, such as fish, preferably lamprey (LSA), mammals, such as sheep (OSA), dog (DSA) and pig (PSA), and birds such as chicken or turkey, at a concentration of 216 pg dissolved in 100 pL of Tris buffer 10 mM, pH 7.4. On the other hand, the volume of 10 mM Tris buffer used in the reaction was that necessary to complete 1 mL of enzymatic reaction for all cases.

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The divalent metal cofactors used have been, Cu2 +, Zn2 + or Ca2 +, among others, in the form of metal salts such as, for example, copper sulfate (1-500 qM), calcium sulfate (2.5 mM) or zinc sulfate (1 -500 qM). Additionally, 5 mM EDTA chelator has been used. With the purpose of obtaining in the final reaction volume of 1 mL at a range of concentrations ranging from 1 nM to 2.5 mM, volumes from 0.2 qL to 250 qL of 10 mM solutions of the metal salts mentioned above were used, and 250 qL of 20 mM EDTA, to obtain concentrations of 5 mM in each case.

The hydrolysis reaction of the organophosphorus compounds HDCP and trichloronate analyzed was stopped with a specific solution based on the subsequent analysis that is carried out for the determination of the concentration of the compounds released in said hydrolysis reaction, 1,2 dichlorophenol (DCP ) or trichlorophenol (TCP) respectively.

For the analysis of the concentration of the DCP or TCP compounds released by colorimetric methods, the reaction is stopped with the addition of 750 qL of a solution comprising 2% SDS / 0.25 mg of aminoantipyrine / mL in 50 mM Tris buffer / EDTA 1 mM pH 8. Subsequently, 375 qL of 0.4% potassium ferricyanide used as a chelator and for color formation were added. Said color formation was analyzed by a UV / VIS spectrophotometer measuring the absorbance at a wavelength of 510 nm. The absorbance values of each organophosphorus compound analyzed were corrected using the absorbance values measured for spontaneous hydrolysis controls (in the absence of serum or protein) and for the serum colorimetric blank. The amount of DCP or TCP respectively released, was determined by comparison with their respective calibration curve. The results are expressed in qmoles of DCP / minute / mL of serum or qg of albumin, or TCP / minute / mL of serum or qg of albumin.

For the chromatographic analysis of the S and / or R isomers of each organosphosphorus compound, and of the DCP or TCP compounds obtained after their hydrolysis reaction, the reaction is stopped with the addition of 40 qL of a 0.2 solution M of HCl, which in addition to stopping the reaction, provides an optimal acidic medium for the extraction of the R and S isomers of the organophosphorus compounds HDCP and trichloronate. Subsequently, 2 mL of 1,2-dichloroethane or heptane are added to quantify the residual amount of each isomer of each compound

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Tested organophosphate, HDCP or trichloronate, respectively. Said mixture is kept under stirring for 45 minutes and centrifuged in the cold at 1000 x g for 15 minutes. From the lower layer (organic phase - 1,2-dichloroethane), it was removed with a 1 mL micro-syringe and 25 pL of each sample was deposited in the autosampler vials of the high resolution liquid chromatography system with Ev / VIS detector and columns chiral, OA-4100, (HPLC Technology Ltd) and CHIRALCEL-OD (Daicel Chem. Ind., LTD) .ico HPLC. The chromatographic conditions used have been:

a) Mobile phase: hexane / 1,2-dichloroethane / ethanol 92: 5: 3 (v / v / v) at a flow of 1.5 mL / min in the case of HDCP and heptane 1 mL / min in the case of trichloronate .

b) Detection: UV / VIS detector at X 230 nm.

c) Column: Techocel OA-4100 chiral, 25 cm x 4.6 mm for the case of HDCP and Daicel OD 25 cm x 4.6 mm for the case of trichloronate.

Under these conditions, the elution volumes were: 1 = 9-10 min and 2 = 10.5-11.5 minutes for the R-HDCP and S-HDCP isomers and 7-8.2 and 8.3-9 min for the R-trichloronate isomers and S-trichloronate. The quantification of the chromatographic peaks was performed with the corresponding area value of each stereoisomer. The results are expressed in pM concentration of residual R or S isomers for each time established in the different tests.

All tests were performed at a temperature of 37 ° C for different times depending on the test.

Example 1. Hydrolysis of organophosphorus compounds by treatment with albumines present in chicken serum (CSA) or turkey (TSA) in the presence of divalent cations (Cu2 + and Zn2 +).

To demonstrate the hydrolytic capacity on organophosphorus compounds (HDCP or trichloronate) of bird albines, preferably of chicken and / or turkey albumin, racemic mixtures of said organophosphorus compounds, HDCP or trichloronate were contacted with chicken serum (CSA ) or turkey (TSA), comprising their corresponding albumines, in the presence or not, of divalent metal cations.

Briefly, a solution containing 216 pg of turkey serum albumin (TSA) or chicken (CSA) dissolved in 100 pL of 10 mM Tris is deposited in a test tube,

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to which 100 pL of a standard 4 mM solution of the organophosphorus compound HDCP or trichloronate are added, and 250 pL of a metal salt, in this particular case copper sulphate of a 10 mM standard solution is used. Next, 550 pL of 10 mM Tris, pH 7.4, are added to adjust the volume of the reaction to 1 mL in the test tube. The reaction is incubated at 37 ° C for a time ranging from 60 to 120 minutes. After this time, the reaction is stopped and the products obtained from the hydrolysis reaction, as well as the residual R and S isomers are analyzed by colorimetric and / or chromatographic methods, as previously described in the materials and methods section.

For the analysis of the hydrolysis product formed (DCP or TCP) using colorimetric techniques as previously described, the reaction is stopped with the addition of 750 pL of 2% SDS / 0.25 mg aminoantipyrine / mL of 50 mM Tris / 1 mM EDTA pH 8 and 375 pL of 0.4% potassium ferricyanide. The mixture is allowed to stand for approximately 15 minutes and subsequently the color intensity (absorbance) is quantified by 510 nm spectrophotometer in the UV / VIS spectrophotometer. The absorbance values of the experimental groups were corrected as mentioned above. The amount of isomeric residuals of both organophosphorus compounds was determined with a calibration curve of concentrations 0, 100, 200 and 300 pM for each isomer of the organophosphorus compound tested.

As shown in Figure 1A, for the particular case of HDCP, chicken serum (CSA) is capable of hydrolyzing the organophosphorus HDCP compound giving rise to DCP compound, in the presence of concentrations of 10 to 300 pM Cu2 +, about 20 times more with respect to the hydrolysis of this same compound when it comes into contact with chicken serum (CSA) in the presence of calcium (2.5 mM), EDTA (5 mM) or Tris under physiological conditions of pH and temperature during 60 or 120 minutes of incubation (Figure 1B).

For the analysis by chromatographic methods of the isomers and of the hydrolysis product obtained after the hydrolysis reaction of the HDCP, the protocol described above in the Materials and Methods section is followed. Briefly, the reaction is stopped with the addition of 40 pL of a 0.2 M solution of HCl (provides an optimal acidic medium for the extraction of the isomers of the organophosphorus compounds) and 2 mL of 1,2-dichloromethane are added for that matter. of HDCP or

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heptane in the case of trichloronate. The mixture is then stirred for 45 minutes and centrifuged at 1000 x g for 15 minutes in a refrigerated centrifuge at 4 ° C. From the organic phase (1,2-dichloroethane) 1 mL is removed and 20 pL are deposited in the HPLC autosampler vials that have the conditions described above and the concentration of the R or S isomers is analyzed, as! as of the product resulting from the hydrolysis, DCP, in the specific case of the HDCP compound.

As seen in Figures 2 and 3, the hydrolysis of each of the R and S isomers of the HDCP compound in the presence of CSA (10 pL) or chicken albumin (210 pg), is dependent on Cu2 + (100 pM of copper sulphate), since in the presence of 5 mM EDTA the hydrolysis of HDCP is inhibited in the analyzed times, 60 and 120 minutes (Figure 2). On the other hand, Figure 2 shows that chicken albumin in the presence of copper is capable of hydrolyzing the R-HDCP isomer to a greater extent than the S-HDCP, demonstrating its stereoselectivity for the R isomer, relative to S.

To demonstrate whether turkey serum or turkey albumin is in turn stereoselective (a) for the R isomer with respect to the S of the HDCP compound, as with serum and chicken albumin, the hydrolytic capacity was compared of chicken serum (CSA) versus turkey serum (TSA) in a hydrolysis reaction for HDCP in the presence of copper. As shown in Figure 3, the hydrolytic capacity of turkey serum (TSA) was greater, about double, than the hydrolytic capacity of albumin present in chicken serum (CSA), since turkey serum albumin (TSA) hydrolyzed the two isomers of HDCP at the same rate, that is, it is not stereoselective (Figure 3), while chicken, as we have said before, if it is stereoselective for the R-HDCP isomer (Figure 3).

To demonstrate that the hydrolytic capacity of organophosphorus compounds by chicken and turkey albumines, in the presence of metal cations, resides in the N-terminal region, specifically in those albumines that comprise SEQ ID NO: 1, and more specifically, SEQ ID NO: 2, different albumins of different species were tested in the presence of copper as a metal cation, to show that those albumines that do not have the SEQ ID NO: 1 sequences in their N-terminal region, or more specifically SEQ ID NO: 2, do not show hydrolytic activity of said organophosphorus compounds. The albumins tested were the mammalian albumines present in the sheep serum

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(OSA), dog (DSA) and pig (PSA), or fish albumines such as lamprey (LSA). These albumines were obtained commercially from Sigma Aldrich (Spain).

As shown in Figure 4, the albumines present in the OSA, DSA, PSA or LSA sera have a low capacity for hydrolysis of the HDCP compound. On the other hand, as shown in Figure 3, the capacity of HDCP hydrolysis by albumin present in both turkey serum (TSA) and chicken serum (CSA) is very high, specifically for the case of turkey albumin that has a high hydrolytic activity for both isomers of the HDCP compound, while the albumin present in chicken serum (CSA), shows a high stereoselective hydrolytic capacity for the R-HDCP isomer.

Additionally, an experiment was also carried out to demonstrate which were the divalent cations that most activated the hydrolytic capacity against organophosphorus compounds of the albumin tested. For this, turkey albumine was incubated together with HDCP and in the presence of different divalent cations (zinc, iron, calcium, manganese, magnesium, nickel, cobalt and copper), at the concentration of 100 pM for each of them and analyzed the concentration of each isomer of the HDCP. As shown in Figure 5, copper and zinc were the only two metal cations that activated the hydrolysis of HDCP by turkey albumin. Specifically, the greater hydrolytic capacity of albumin compared to HDCP was carried out in the presence of copper.

In the same way that the hydrolytic capacity of turkey and chicken albumines for the organophosphorus compound HDCP was analyzed, this capacity was tested for the commercial organophosphorus trichloronate insecticide. The results show that turkey albumin has catallotic activity for trichloronate that is slightly stereoselective in favor of the S-trichloronate isomer (in its "uncle" form) (Figure 6), unlike the hydrolytic capacity for HDCP that does not I present this stereoselectivity (Figure 3).

Subsequently, it was analyzed if the pH could influence the hydrolytic capacity of organophosphorus compounds by poultry albines, specifically chicken and / or turkey albumin, in the presence of metal cations. For this, the albumin present in the chicken serum was incubated in the presence of 100 pM of Zn2 + or Cu2 + at different pHs within the range of pH 4 to pH 10. As observed in the

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Figure 7, the catalytic capacity of the HDCP compound by said albumine was reduced when the pH of the medium was greater than 7 and was completely inhibited at pH greater than 9. On the other hand, it was also analyzed whether the catalytic activity of the albumine of chicken or turkey, in the presence of divalent cations is affected when these proteins were pre-incubated at different times (0, 15 and 30 minutes) in the presence of metal cations (100 pM of Zn2 + or Cu2 +). The results shown in Figure 8 demonstrate that the pre-incubation of the bird albumin in the presence of Zn2 + or Cu2 + and subsequent incubation with Cu2 +, makes that particularly for the case of preincubation with Zn2 +, the chicken albumin loses its stereoselective hydrolysis capacity on the R-HDCP isomer, while turkey albumin decreases its hydrolytic capacity for both isomers of the HDCP compound. In contrast, preincubation with Ni2 +, under the same conditions of concentrations and incubation times as for Zn2 +, was not able to inhibit or affect the HDCP hydrolysis characteristic of the two albumin in the presence of copper (Figure 9).

In subsequent studies, turkey or chicken albumines were incubated in the presence of copper and HDCP and compounds that react with specific amino acids. Such compounds have been, N, N'-dicyclohexylcarbodiimide that reacts with the aspartic and glutamic amino acids, diisopropyl phosphorus phosphate and paraoxon that react with serine and tyrosine, N-ethylmaleimide (N-NEM), p-nitrophenol and 5,5'-dithiobis- 2- nitrobenzoic acid (DTNB) that react with the -SH groups of cystel, and others. The presence of these compounds does not alter the property of said proteins to hydrolyze HDCP in the presence of copper, confirming that these amino acids are not involved at the site where this catalysis occurs. This test demonstrates that the catalytic property of organophosphorus compounds by chicken or turkey albumines involves their N-terminal region.

In conclusion, the particular catalytic activity of breaking organophosphorus ester compounds (phosphotriesterase activity) of chicken and turkey albumines is due to the high affinity that copper and zinc cations have in their N-terminal DAEHK sequence (SEQ ID NO: 2).

Claims (25)

5 10 fifteen twenty 25 30 1. Composition comprising a peptide sequence comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1, or a sequence nucleotide coding for said peptide sequence that has at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1, and at least one cofactor characterized in that it is a divalent cation. 2. Composition according to revindication 1 characterized in that it comprises a peptide sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 15 and / or any of their combinations; or a nucleotide sequence selected from SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and / or any combination thereof. 3. Composition according to claim 2 characterized in that the peptide sequence is SEQ ID NO: 3, SEQ ID NO: 4 and / or any combination thereof. 4. Composition according to any of claims 1 to 3 characterized in that the divalent cation is selected from any of the following list: Zn2 +, Fe2 +, Cu2 +, Mn2 +, Mg2 + and / or any combination thereof. 5. Composition according to any of claims 1 to 4 characterized in that the divalent cation is Zn2 + or Cu2 +, preferably Cu2 +. 6. Composition according to any one of claims 1 to 5 characterized in that it comprises SEQ ID NO: 3 and / or SEQ ID NO: 4, and Cu2 +. 7. Composition according to any one of claims 1 to 6 characterized in that it is a pharmaceutical or veterinary composition. 8. Composition according to any one of claims 1 to 7, characterized in that it additionally comprises at least one active ingredient selected from any of the following: cholinergic receptor blocker, preferably atropine, calcium blocker, acetylcholinesterase blocker, butyrylcholinesterase blocker, anxiolytic , anticonvulsant, sedative, oximes, preferably pralidoxime, cholinesterase activators and / or any combination thereof. 5 10 fifteen twenty 25 30 35 9. Composition according to any one of claims 1 to 8 characterized in that it further comprises a vehicle or excipient, pharmaceutically or veterinarily acceptable. 10. Composition according to any one of claims 1 to 9 characterized in that it is in a liquid or solid state. 11. Composition according to any of claims 1 to 10 characterized in that it is a controlled release composition. 12. Composition according to any of claims 1 to 11 characterized in that it is a biocatalyst. 13. Biosensor and / or bioremediator comprising the composition according to any of claims 1 to 12. 14. Use of the composition according to any of claims 1 to 12 for the preparation of a medicament. 15. Use of the composition according to any one of claims 1 to 12 for the preparation of a medicament for the treatment of poisonings by organophosphorus compounds, carbamic esters and / or carboxylic esters. 16. Use the composition according to any one of claims 1 to 6, or of a biosensor and / or bioreader according to revindication 13, as a biocatalyst in bioremediation processes. 17. Use according to revindication 16 characterized in that it catalyzes hydrolysis reactions of organophosphorus compounds, carbamic esters and / or carboxylic esters. 18. Isolated peptide sequence comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO: 1. 19. Isolated peptide sequence according to revindication 18 characterized in that it comprises a peptide sequence of SEQ ID NO: 2. 20. Isolated nucleotide sequence encoding a polypeptide according to any one of claims 18 to 19. 21. Isolated nucleotide sequence complementary to the nucleotide sequence according to claim 20. 22. Genetic construction comprising the peptide sequence according to any of claims 18 to 19 or the nucleotide sequence according to any of claims 20 to 21. 23. Recombinant expression vector comprising the nucleotide sequence according to any one of claims 20 to 21. 10 24. Host cell or microorganism comprising a recombinant vector according to claim 23. 25. Method for the synthesis of a peptide sequence according to any of claims 18 to 19, comprising: a) Cultivating a cell comprising a peptide sequence according to any one of claims 18 to 19, or a nucleotide sequence according to any of claims 20 to 21, under conditions that allow its growth, and synthesis of said peptide sequence, b) Recover and / or collect said cells or take a sample of the culture medium thereof, and c) Isolate and purify the peptide sequence.