NEW MUTANT PTE ENZYMES

Description

[0001] The present application describes mutated phosphotriesterase enzymes having improved stability and activity, as well as their use in particular for degrading organophosphorus compounds.

[0002] Organophosphate (OP) insecticides have become the most widely used insecticides today. OPs are used in agriculture, in the home, in gardening, and in the veterinary sector.

[0003] Because these compounds inhibit certain esterase enzymes, exposure to OPs can lead to serious toxicity in several ways.

[0004] Irreversible inhibition of acetylcholinesterase (a key enzyme in the mammalian nervous system) by OPs causes severe damage in all vertebrates. Loss of enzyme function leads to the accumulation of acetylcholine in various body compartments, causing muscle contraction, paralysis, and respiratory distress. Increased pulmonary secretions combined with respiratory failure are the usual causes of death from organophosphate poisoning.

[0005] Some OPs were also developed during World War II: the discovery of OPs with increased toxicity and / or better stability led to the development of chemical weapons such as sarin, soman, tabun, or VX gas. Moreover, OP insecticides, being easily accessible and no less toxic compared to chemical weapons OPs, pose a significant risk to the population.

[0006] Closer to home, the current terrorist threat has led authorities to consider scenarios of chemical attacks using organophosphorus substances such as sarin gas, and in the face of these growing threats, the development of antidotes has never been more urgent.

[0007] OPs are readily absorbed by the body through inhalation, ingestion, or skin penetration due to the hydrophobic nature of these molecules. The incidence of poisoning depends on the absorption rate of the compound. Symptoms of acute OP poisoning occur during or after exposure, within minutes or hours, depending on the type of exposure.

[0008] Inhalation exposure results in the most rapid onset of poisoning symptoms, followed by the gastrointestinal route and finally, poisoning by skin contact.

[0009] Protective clothing and masks do not always provide sufficient protection against OPs. In patients poisoned by OPs via skin, clothing, or hair, decontamination should be performed with medical soap or textile detergents. Treatment of highly contaminated individuals is carried out by administering atropine or diazepam, which antagonize the effects of excessive acetylcholine concentration on target organs with muscarinic receptors.

[0010] Pralidoxime, an acetylcholinesterase reactivator, attenuates both the nicotinic and muscarinic effects of OP poisoning when administered less than 48 hours after poisoning.

[0011] Although progress in prophylaxis has been made with the 10 techniques mentioned above, existing protections and treatments for these types of poisoning remain unsatisfactory.

[0012] The first OP hydrolases were identified in several bacteria in the early 1990s (Cheng et al., 1993, Appl. Environ. Microbiol., 59: 3138-3140, Raveh et al., 1993, Biochem Pharmacol., 45: 2465-2474). These enzymes are capable of catalyzing the hydrolysis of phosphotriester bonds in OPs. Unfortunately, due to their low stoichiometric binding capacity to OPs, a large amount of enzymes is required to cure poisoned individuals. This makes the use of these enzymes disproportionate and expensive.

[0013] Other microbial enzymes generally called phosphotriesterases (PTEs) show a preference for organophosphorus compounds with PO or PS bonds. These enzymes are members of a superfamily, the aminohydrolases, which are enzymes that catalyze the hydrolysis of a wide range of compounds with different chemical properties (phosphoesters, esters, amides, etc.). Their coding genes,opd (organophosphate degradation), have been isolated from soil bacteria such as Pseudomonas diminuta, also called Brevundimonas diminuta (Munnecke et al., 1976, Appl. Environ. Microbiol., 32: 7-13), Flavobacterium sp. (Sethunathan et al., 1973, Can J Microbiol, 19: 873-875) and Agrobacterium radiobacter (Horne et al., 2003, FEMS Microbiol Lett, 222: 1-8), and opd-like genes have also been identified in archaea (Merone et al., 2005, Extremophiles, 9: 297-305). The catalytic properties of hyperthermophilic PTEs are being extensively studied because of their ability to hydrolyze pesticides and various nerve agents (Jackson et al., 2005, Biochem Biophys Acta, 1752: 56-64 / Jackson et al., 2008, J Mol Biol, 375: 1189-1196 / Wong et al., 2007, Biochemistry, 46: 13352-13369 / Elias et al., 2008, J Mol Biol, 379: 1017-1028 / Pompea et al., 2009, Extremophiles, 13: 461-470).

[0014] Hyperthermophilic PTEs have the advantage of being very stable and inexpensive to produce due to their ability to resist organic solvents or detergents at moderate temperatures. Thus, hyperthermophilic PTEs are promising for the development of bioscrubbers for neurotoxic agents.

[0015] The invention is set forth in the attached set of claims.

[0016] One of the aims of the present application is to describe PTE enzymes having improved stability.

[0017] Another aim of the present application is to describe the improvement, in addition to the stability, of the enzymatic activity of such a PTE enzyme.

[0018] In its most general aspect, the present application describes a mutated PTE enzyme derived from parathion hydrolase of sequence SEQ ID NO: 1, which mutated PTE enzyme has at least 90% identity with SEQ ID NO: 1 and comprises at least the following 7 mutations with respect to the sequence SEQ ID NO: 1: Substitution of threonine T by proline P, at position 13, Substitution of isoleucine I by valine V, at position 14, Substitution of alanine A by serine S, at position 60, Substitution of serine S by arginine R, at position 79, Substitution of tyrosine Y by histidine H, at position 124, Substitution of isoleucine I by valine V, at position 218, Substitution of glutamine Q by arginine R, at position 258.

[0019] The inventors have described that specific mutations in the sequence of parathion hydrolase of sequence SEQ ID NO: 1 allow the production of an enzyme having retained a catalytic activity substantially identical to that of wild-type parathion hydrolase, but having improved stability. The so-called "wild-type" parathion hydrolase is the enzyme of sequence SEQ ID NO: 1.

[0020] At least 90% identity means the ranges of values ​​of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and 100% identity.

[0021] By "enzyme with improved stability" is meant an enzyme whose melting temperature T m is increased compared to that of the unmutated parathion hydrolase.

[0022] To give an idea, the increase in stability can be translated into an increase in the melting temperature of at least 3% and in particular of at least 3% to 20%.

[0023] The expression "substantially identical catalytic activity" corresponds to a variation in the K cat / K m ratio of the mutated enzyme of less than a factor of 10 compared to the K cat / K m ratio of the non-mutated enzyme.

[0024] For example, K cat / K m ratios of 6.10 5< M -1< .s -1< and 3.10 5< M -1< .s -1< are considered close enough (factor 2) to consider that enzymes which have such K cat / K m ratios have substantially identical catalytic activity.

[0025] By "position" is meant the place of an amino acid in the polypeptide sequence, established from the N-terminal end of the enzyme of sequence SEQ ID NO: 1.

[0026] All 7 mutations cited above, at positions 13, 14, 60, 79, 124, 218 and 258 relative to the sequence SEQ ID NO: 1 will be designated subsequently as constituting the mutations of group A.

[0027] Thus, the expression “group A mutations” can be replaced by the following list: "Substitution of threonine T by proline P, at position 13, Substitution of isoleucine I by valine V, at position 14, Substitution of alanine A by serine S, at position 60, Substitution of serine S by arginine R, at position 79, Substitution of tyrosine Y by histidine H, at position 124, Substitution of isoleucine I by valine V, at position 218, Substitution of glutamine Q by arginine R, at position 258."

[0028] The percentage of identity with a given sequence refers to the percentage of amino acids that are identical to those in a reference sequence and are found in the same positions. This percentage of identity is established by a bioinformatics alignment such as BlastP.

[0029] By "mutation" is meant a point mutation, namely the presence of an amino acid at a given position which is different from that of a reference sequence, the sequence SEQ ID NO: 1 being chosen as the reference sequence.

[0030] Substitution refers to the replacement of an amino acid in a given sequence with a different amino acid.

[0031] In a particular embodiment, the present application describes a mutated PTE enzyme of sequence SEQ ID NO: 2, derived from parathion hydrolase having the sequence SEQ ID NO: 1, which mutated PTE enzyme has at least 90% identity with SEQ ID NO: 1 and comprises at least the following 7 mutations with respect to the sequence SEQ ID NO: 1: Substitution of threonine T by proline P, at position 13, Substitution of isoleucine I by valine V, at position 14, Substitution of alanine A by serine S, at position 60, Substitution of serine S by arginine R, at position 79, Substitution of tyrosine Y by histidine H, at position 124, Substitution of isoleucine I by valine V, at position 218, Substitution of glutamine Q by arginine R, at position 258, and mutated enzyme having a percentage identity of at least 90% with the above sequence SEQ ID NO: 2, subject to the presence of the above 7 mutations in the said sequence of the mutated enzyme.

[0032] According to another particular embodiment, the application describes a mutated PTE enzyme, comprising group A of mutations as defined above, in which at least 2 additional amino acids chosen from the amino acids occupying the following positions are mutated: Amino acid at position 45, Amino acid at position 48, Amino acid at position 74, Amino acid at position 100, Amino acid at position 141, Amino acid at position 153, Amino acid at position 177, Amino acid at position 201, Amino acid at position 222, Amino acid at position 225, Amino acid at position 235, Amino acid at position 238, Amino acid at position 239, Amino acid at position 240, Amino acid at position 242, Amino acid at position 271, Amino acid at position 276, Amino acid at position 277, Amino acid at position 287, Amino acid at position 310, the positions being defined relative to the sequence SED ID NO: 1.

[0033] Such an enzyme can be obtained from the sequence SEQ ID NO: 1 which, in addition to the mutations of group A, comprises 2 additional mutations at the positions defined above. For example, in addition to the 7 mutations of group A mentioned, 2 mutations can be made: one at position 141 and the other at position 277.

[0034] An "additional mutation" or "additional substitution" means any mutation or substitution occurring in addition to those in group A.

[0035] According to another particular embodiment, the application describes a mutated PTE enzyme, comprising the mutations of group A, in which the additional mutations at the positions defined above are chosen from the following list: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by valine V or methionine M, At position 74: substitution of isoleucine I by cysteine ​​C or alanine A, At position 100: substitution of phenylalanine F by glutamic acid E or valine V, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G or glutamine Q, At position 225: substitution of histidine H by tyrosine Y, At position 235: substitution of serine S by methionine M, At position 238: substitution of alanine A by valine V or serine S, At position 239: substitution of leucine L by tryptophan W,At position 240: substitution of leucine L by methionine M, At position 242: substitution of isoleucine I by asparagine N, At position 271: substitution of leucine L by threonine T, At position 276: substitution of serine S by leucine L, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S, At position 310: substitution of proline P by serine S.

[0036] For example, such a mutated enzyme can be obtained from the enzyme of sequence SEQ ID NO: 1 which, in addition to the 7 mutations of group A, comprises 2 additional ones, such as for example a substitution of alanine A at position 48 by valine V or methionine M and the substitution of proline P at position 310 by serine S. Such a mutated enzyme then comprises 9 mutations compared to the enzyme of sequence SEQ ID NO: 1, at positions 13, 14, 48, 60, 79, 124, 218, 258 and 310.

[0037] According to another particular embodiment, the application describes a mutated PTE enzyme comprising only group A of mutations, of sequence SEQ ID NO: 2, in which: The amino acid at position 45 is lysine K, The amino acid at position 48 is alanine A, The amino acid at position 74 is isoleucine I, The amino acid at position 100 is phenylalanine F, The amino acid at position 141 is threonine T, The amino acid at position 153 is lysine K, The amino acid at position 177 is glycine G, The amino acid at position 201 is aspartic acid D, The amino acid at position 222 is histidine H, The amino acid at position 225 is histidine H, The amino acid at position 235 is serine S, The amino acid at position 238 is alanine A, The amino acid at position 239 is leucine L, The acid amino acid at position 240 is leucine L, amino acid at position 242 is isoleucine I, amino acid at position 271 is leucine L, amino acid at position 276 is serine S, amino acid at position 277 is tyrosine Y, amino acid at position 287 is arginine R, amino acid at position 310 is proline P.

[0038] Thus, the mutated enzyme obtained comprises only the 7 mutations of group A, compared to the sequence SEQ ID NO: 1, and therefore has the sequence SEQ ID NO: 2.

[0039] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 2 additional mutations, at positions 225 and 271.

[0040] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 3, comprising the mutations of group A and in which the at least 2 additional mutations above are the following: At position 225: substitution of histidine H by tyrosine Y, At position 271: substitution of leucine L by threonine T.

[0041] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 2 additional mutations above, its sequence being the sequence SEQ ID NO: 3.

[0042] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 8 additional mutations, in position 74, 100, 222, 225, 238, 240, 242 and 276.

[0043] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 4, comprising the mutations of group A and in which the at least 8 additional mutations above are as follows: At position 74: substitution of isoleucine I by cysteine, At position 100: substitution of phenylalanine F by valine V, At position 222: substitution of histidine H by glutamine Q, At position 225: substitution of histidine H by tyrosine Y, At position 238: substitution of alanine A by valine V, At position 240: substitution of leucine L by methionine M, At position 242: substitution of isoleucine I by asparagine N, At position 276: substitution of serine S by leucine L.

[0044] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 8 additional mutations above, its sequence being the sequence SEQ ID NO: 4.

[0045] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 9 additional mutations, in position 45, 48, 100, 141, 153, 222, 242, 287 and 310.

[0046] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 5, comprising the mutations of group A and in which the at least 9 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by valine V, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 222: substitution of histidine H by glycine G, At position 242: substitution of isoleucine I by asparagine N, At position 287: substitution of arginine R by serine S, At position 310: substitution of proline P by serine S.

[0047] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 9 additional mutations above, its sequence being the sequence SEQ ID NO: 5.

[0048] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 11 additional mutations, at positions 45, 48, 100, 141, 153, 177, 201, 222, 242, 277 and 287.

[0049] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 6, comprising the mutations of group A and in which the at least 11 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by valine V, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S.

[0050] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 11 additional mutations above, its sequence being the sequence SEQ ID NO: 6.

[0051] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 12 additional mutations, at positions 45, 48, 74, 100, 141, 153, 177, 201, 222, 242, 277 and 287.

[0052] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 7, comprising the mutations of group A and in which the at least 12 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by valine V, At position 74: substitution of isoleucine I by alanine A, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S.

[0053] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 12 additional mutations above, its sequence being the sequence SEQ ID NO: 7.

[0054] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 13 additional mutations, at positions 45, 48, 100, 141, 153, 177, 201, 222, 238, 239, 242, 277 and 287.

[0055] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 8, comprising the mutations of group A and in which the at least 13 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by methionine M, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 238: substitution of alanine A by serine S, At position 239: substitution of leucine L by tryptophan W, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S.

[0056] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 13 additional mutations above, its sequence being the sequence SEQ ID NO: 8.

[0057] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 14 additional mutations, in position 45, 48, 74, 100, 141, 153, 177, 201, 222, 238, 239, 242, 277 and 287.

[0058] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 9, comprising the mutations of group A and in which the at least 14 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by methionine M, At position 74: substitution of isoleucine I by alanine A, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 238: substitution of alanine A by serine S, At position 239: substitution of leucine L by tryptophan W, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S.

[0059] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 14 additional mutations above, its sequence being the sequence SEQ ID NO: 9.

[0060] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 14 additional mutations, in position 45, 48, 100, 141, 153, 177, 201, 222, 235, 238, 239, 242, 277 and 287.

[0061] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 10, comprising the mutations of group A and in which the at least 14 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by methionine M, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 235: substitution of serine S by methionine M, At position 238: substitution of alanine A by serine S, At position 239: substitution of leucine L by tryptophan W, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W, At position 287: substitution of arginine R by serine S.

[0062] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 14 additional mutations above, its sequence being the sequence SEQ ID NO: 10.

[0063] According to another particular embodiment, the application describes a mutated PTE enzyme comprising the mutations of group A and comprising at least 15 additional mutations, at positions 45, 48, 74, 100, 141, 153, 177, 201, 222, 235, 238, 239, 242, 277 and 287.

[0064] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 11, comprising the mutations of group A and in which the at least 15 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by methionine M, At position 74: substitution of isoleucine I by alanine A, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 235: substitution of serine S by methionine M, At position 238: substitution of alanine A by serine S, At position 239: substitution of leucine L by tryptophan W, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W,At position 287: substitution of arginine R by serine S.,

[0065] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 15 additional mutations above, its sequence being the sequence SEQ ID NO: 11.

[0066] According to another particular embodiment, the application describes a mutated PTE enzyme of sequence SEQ ID NO: 12, comprising the mutations of group A and in which the at least 15 additional mutations above are as follows: At position 45: substitution of lysine K by alanine A, At position 48: substitution of alanine A by methionine M, At position 74: substitution of isoleucine I by cysteine ​​C, At position 100: substitution of phenylalanine F by glutamic acid E, At position 141: substitution of threonine T by asparagine N, At position 153: substitution of lysine K by arginine R, At position 177: substitution of glycine G by aspartic acid D, At position 201: substitution of aspartic acid D by glycine G, At position 222: substitution of histidine H by glycine G, At position 235: substitution of serine S by methionine M, At position 238: substitution of alanine A by serine S, At position 239: substitution of leucine L by tryptophan W, At position 242: substitution of isoleucine I by asparagine N, At position 277: substitution of tyrosine Y by tryptophan W,At position 287: substitution of arginine R by serine S.,

[0067] Thus, the obtained mutated enzyme comprises the 7 mutations of group A and at least the 15 additional mutations above, its sequence being the sequence SEQ ID NO: 12.

[0068] According to another particular embodiment, the application describes a mutated PTE enzyme according to group A of mutations, of sequence SEQ ID NO: 2, in which: The amino acid at position 45 is different from alanine A, The amino acid at position 48 is different from valine V and methionine M, The amino acid at position 74 is different from cysteine ​​C and alanine A, The amino acid at position 100 is different from glutamic acid E and valine V, The amino acid at position 141 is different from asparagine N, The amino acid at position 153 is different from arginine R, The amino acid at position 177 is different from aspartic acid D, The amino acid at position 201 is different from glycine G, The amino acid at position 222 is different from glycine G and glutamine Q, The amino acid at position 225 is different from tyrosine Y, The amino acid at position 235 is different from methionine M, The amino acid at position 238 is different from valine V and serine S, The amino acid at position 239 is different from tryptophan W, The amino acid at position 240 is different from methionine M,Amino acid at position 242 is different from asparagine N, Amino acid at position 271 is different from threonine T, Amino acid at position 276 is different from leucine L, Amino acid at position 277 is different from tryptophan W, Amino acid at position 287 is different from serine S, Amino acid at position 310 is different from serine S. ,

[0069] According to a particularly preferred embodiment, the application describes a PTE enzyme of sequences SEQ ID NO: 3, of sequence SEQ ID NO: 4 or of sequence SEQ ID NO: 5.

[0070] One aspect of the present application describes the use of at least 7 mutations, in particular 7 mutations, to increase the stability of a phosphotriesterase (PTE) enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by substituting in the sequence SEQ ID NO: 1: the amino acid in position 13 by proline P, the amino acid in position 14 by valine V, the amino acid in position 60 by serine S, the amino acid in position 79 by arginine R, the amino acid in position 124 by histidine H, the amino acid in position 218 by valine V, the amino acid in position 258 by arginine R, in order to obtain a mutated PTE enzyme, in particular of sequence SEQ ID NO: 2, which has improved stability compared to the stability of the enzyme of sequence SEQ ID NO: 1.

[0071] As seen previously, the 7 mutations at positions 13, 14, 60, 79, 124, 218, 258 constitute the group A mutations.

[0072] According to another particular embodiment, the application describes the use of at least the 7 mutations of group A, combined with additional mutations to increase the stability and the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by making in the sequence SEQ ID NO: 1 at least two additional substitutions chosen from the substitutions of: the amino acid in position 45 by alanine A, the amino acid in position 48 by valine V or methionine M, the amino acid in position 74 by cysteine ​​C or alanine A, the amino acid in position 100 by glutamic acid E or valine V, the amino acid in position 141 by asparagine N, the amino acid in position 153 by arginine R, the amino acid in position 177 by aspartic acid D, the amino acid in position 201 by glycine G, the amino acid in position 222 by glycine G or glutamine Q, the amino acid in position 225 by tyrosine Y, the amino acid in position 235 by methionine M, the amino acid in position 238 by valine V or serine S, amino acid at position 239 by tryptophan W, amino acid at position 240 by methionine M, amino acid at position 242 by asparagine N, amino acid at position 271 by threonine T, amino acid at position 276 by leucine L, amino acid at position 277 by tryptophan W,the amino acid at position 287 by serine S, the amino acid at position 310 by serine S. , in order to obtain a mutated PTE enzyme which has improved stability and improved catalytic activity for hydrolysis of organophosphorus compounds, compared to the stability and catalytic activity of the enzyme of sequence SED ID NO: 1.

[0073] By "increase in catalytic activity" or "enhanced catalytic activity" is meant an increase greater than a factor of 10 in the K cat / K m ratio of the mutated enzyme compared to that of the wild-type parathion hydrolase, of sequence SEQ ID NO: 1.

[0074] For example, a mutated enzyme having a K cat / K m = 6.10 6< M -1< .s -1< will be considered to have improved catalytic activity compared to an enzyme having a K cat / K m = 5.10 5< M -1< .s -1< .

[0075] According to another particular embodiment, the PTE enzyme is mutated by the use of at least the 7 mutations of group A, combined with additional mutations to increase the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by carrying out in the sequence SEQ ID NO: 1 at least 2 additional substitutions in order to obtain a mutated enzyme of sequence chosen from the following: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0076] In a particularly preferred embodiment, the PTE enzyme is mutated by using at least the 7 mutations of group A, combined with additional mutations to increase the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by making in the sequence SEQ ID NO: 1 at least 2 additional substitutions in order to obtain a mutated enzyme of sequence chosen from the following: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5.

[0077] Thus, the obtained mutated PTE enzyme has improved stability and catalytic activity for hydrolysis of organophosphorus compounds, compared to the stability and catalytic activity of the enzyme of sequence SED ID NO: 1.

[0078] A particular embodiment relates to the use of at least the 7 mutations of group A, in combination with 8 additional mutations to increase the stability and the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, said 8 additional mutations consisting of the substitution in the sequence SEQ ID NO: 1 of: the amino acid at position 74 by cysteine ​​C, the amino acid at position 100 by valine V, the amino acid at position 222 by glutamine Q, the amino acid at position 225 by tyrosine Y, the amino acid at position 238 by valine V, the amino acid at position 240 by methionine M, the amino acid at position 242 by asparagine N, the amino acid at position 276 by leucine L, in order to obtain a mutated PTE enzyme of sequence SEQ ID NO: 4 which has improved stability and improved catalytic activity for hydrolysis of organophosphorus compounds compared to the stability and catalytic activity of the enzyme of sequence SEQ ID NO: 1.

[0079] In one aspect of the application, the OP compounds hydrolyzed by the mutated PTE enzymes of the present application may be organophosphorus insecticides and organophosphorus chemical weapons.

[0080] In a particular aspect of the present application, the PTE enzyme hydrolyzed organophosphorus insecticides of the present application may be one of the following:

[0081] In a particular aspect of the present application, the chemical weapons organophosphates hydrolyzed by the PTE enzymes of the present application may be one of the following:

[0082] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 3, said mutated PTE enzyme of sequence SEQ ID NO: 3 being capable of hydrolyzing the following organophosphorus insecticides:

[0083] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 3, said mutated PTE enzyme of sequence SEQ ID NO: 3 being capable of hydrolyzing the following chemical weapons organophosphorus compounds:

[0084] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 4, said mutated PTE enzyme of sequence SEQ ID NO: 4 being capable of hydrolyzing the following organophosphorus insecticides:

[0085] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 4, said mutated PTE enzyme of sequence SEQ ID NO: 4 being capable of hydrolyzing the following chemical weapons organophosphorus compounds:

[0086] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 5, said mutated PTE enzyme of sequence SEQ ID NO: 5 being capable of hydrolyzing the following organophosphorus insecticides:

[0087] In a particularly preferred aspect, the application describes the mutated PTE enzyme of sequence SEQ ID NO: 5, said mutated PTE enzyme of sequence SEQ ID NO: 5 being capable of hydrolyzing the following chemical weapons organophosphorus compounds:

[0088] Another aspect of the present application describes the use of at least one mutated PTE enzyme as defined above, having a phosphotriesterase (PTE) catalytic activity capable of hydrolyzing organophosphorus compounds: for the decontamination of soils polluted by organophosphorus compounds, or for the decontamination of a surface, skin, mucous membranes or hair contaminated with organophosphorus compounds, or for the prevention or treatment of internal or external poisoning by ingestion or inhalation of an organophosphorus compound, or for the control of pollution of water polluted by organophosphorus compounds, or for the destruction of stocks of nerve agents, or for the decontamination of textiles and filters, or for the decontamination of paints, said at least one mutated PTE enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combination with each other.

[0089] A particularly preferred embodiment of the present application describes the use of at least one mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, having a phosphotriesterase (PTE) catalytic activity capable of hydrolyzing organophosphorus compounds: for the decontamination of soils polluted by organophosphorus compounds, or for the decontamination of a surface, skin, mucous membranes or hair contaminated with organophosphorus compounds, or for the prevention or treatment of internal or external poisoning by ingestion or inhalation of an organophosphorus compound, or for the control of pollution of water polluted by organophosphorus compounds, or for the destruction of stocks of neurotoxic agents, or for the decontamination of textiles and filters, or for the decontamination of paints.

[0090] Another aspect of the present application describes a kit for the decontamination of surfaces, skin or mucous membranes, hair, paints, fabrics or filters contaminated with organophosphorus compounds, said kit comprising at least one mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds and said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combinations with each other.

[0091] A particularly preferred embodiment of the present application describes a kit for the decontamination of surfaces, skin or mucous membranes, hair, paints, fabrics or filters contaminated with organophosphorus compounds, said kit comprising at least one mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 having an activity of catalysis of organophosphorus compounds, alone or in combinations with each other.

[0092] Another aspect of the present application describes a phytosanitary composition comprising as active ingredient at least one mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds, said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combinations with each other.

[0093] A particularly preferred embodiment of the present application describes a phytosanitary composition comprising as active ingredient at least one mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, having an activity of catalysis of organophosphorus compounds, alone or in combinations with each other.

[0094] Another aspect of the present application describes a pharmaceutical composition comprising as active ingredient at least one mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds, said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combination with each other; in combination with a pharmaceutically acceptable excipient.

[0095] A particularly preferred embodiment of the present application describes a pharmaceutical composition comprising as active ingredient at least one mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, having an activity of catalysis of organophosphorus compounds, alone or in combinations with each other; in combination with a pharmaceutically acceptable excipient.

[0096] Another aspect of the present application describes a mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds, said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combinations with each other, for its use in the treatment or prevention of poisoning by contact, inhalation or ingestion of organophosphorus compounds.

[0097] A particularly preferred embodiment of the present application describes a mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, having an activity of catalysis of organophosphorus compounds, alone or in combinations with each other, for its use in the treatment or prevention of poisoning by contact, inhalation or ingestion of organophosphorus compounds. Another aspect of the present application describes a method for treating poisoning by contact, inhalation or ingestion of organophosphorus compounds comprising the administration of at least one mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds, said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combination with each other. A particularly preferred embodiment of the present application describes a method for treating poisoning by contact, inhalation or ingestion of organophosphorus compounds comprising the administration of at least one mutated PTE enzyme of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, having an activity of catalysis of organophosphorus compounds,alone or in combination with each other Another aspect of the present application describes a method for preventing poisoning by contact, inhalation or ingestion of organophosphorus compounds comprising the administration of at least one mutated PTE enzyme as defined above, having an activity of catalysis of organophosphorus compounds, said at least one mutated enzyme being preferentially chosen from the mutated enzymes of sequence SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, alone or in combination with each other A particularly preferred embodiment of the present application describes a method for preventing poisoning by contact, inhalation or ingestion of organophosphorus compounds comprising the administration of at least one enzyme Mutated PTEs of sequence SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5,having an activity of catalysis of organophosphorus compounds, alone or in combination with each other, Legend of the figures :

[0098] There Figure 1 represents the evolution of circular dichroism (CD) as a function of temperature for the enzyme of sequence SEQ ID NO: 13. The Figure 2 represents the evolution of circular dichroism (CD) as a function of temperature for the enzyme of sequence SEQ ID NO: 4. Materials & methods 1. Production of enzymes

[0099] The genes encoding each enzyme were optimized for expression within E. coli and synthesized by GeneScript and then inserted into the plasmid pET22b using the restriction enzymes NdeI and NotI.

[0100] The production of the protein was carried out within E. coliBL21 (DE 3 )-pGro7 / GroEL in 2 liters of ZYP medium (Tryptone 10 g / L, Yeast extract 5 g / L, (NH 4 ) 2 SO 4 66 g / L, KH 2 PO 4 136 g / L, Na 2 HPO 4 142 g / L, Glycerol 250 g (w / v), Glucose 25 g, α-lactose 100 g, 100 µg / ml ampicillin and 34 µg / ml chloramphenicol) inoculated in pre-culture overnight with a ratio of 1 / 100.

[0101] Growth takes place at 37°C until reaching an OD 600nm = 0.8. Induction is carried out by adding 0.2% L-arabinose to the ZYP medium for each PTE as well as 0.2mM CoCl 2 for PTEs of sequence SEQ ID NO: 3 and SEQ ID NO: 4 and 0.1mM ZnCl 2 for PTE of sequence SEQ ID NO: 5 and a temperature change of 16°C for 20 hours.

[0102] The cells are then harvested by centrifugation (6420g, 30 min, 4°C), then resuspended in the lysis buffer (Tris 50 mM pH 8, NaCl 300 mM, DNAsel 10 µg / mL, lysozyme 0.25 mg / mL, PMSF 0.1 mM) for 4 hours at room temperature and finally stored at -80°C overnight.

[0103] Cells are sonicated (3 steps of 30 seconds) for mechanical lysis (Amplitude 45, time 00:30, pulse on 00:01, pulse off 00:01). Cell debris is finally removed by centrifugation (11000 rpm, 20 min, 4°C). Before proceeding to the purification step, filtration at 0.8 µm is required.

[0104] Purification is done by Strep-Tag affinity chromatography (StrepTrap ™ < HP 5ml). Washing and equilibration of the column is done with PTE buffer (50 mM Tris, 300 mM NaCl pH 8) while sample elution is done with 50 mM Tris, 300 mM NaCl, 2.5 mM desthiobiotin, pH 8 buffer for a flow rate of 2ml / min. 3. Stability measurement Determination of the melting temperature

[0105] Circular dichroism spectra were obtained using a Jasco J-815 CD spectrometer with a Pelletier-type temperature control system (Jasco MPTC-4905) in a 1 mm thick Starna ® quartz cell and using Spectra Manager software. Experiments were performed in 50 mM Tris buffer at pH 8. Since protein concentrations were in the range of 0.1 - 0.2 mg / mL, denaturation was performed at 222 nm with a temperature increase from 25 to 85°C (at 5°C / min). Data were analyzed with GraphPad Prism 6, using the Boltzmann sigmoid equation. The results can be seen in the figures 1 And 2 as well as in Table 1 which compares the melting temperature for the enzyme of sequence SEQ ID NO: 4 to that of the enzyme of sequence SEQ ID NO: 13 (influence of the presence of ancestral mutations on the stability of the enzyme). Table 1 : Melting temperature (Tm) as a function of the presence or absence of ancestral mutations. The presence of ancestral mutations (SEQ ID NO: 4) causes an increase in the melting temperature of the enzyme, and therefore its stability compared to the enzyme lacking said ancestral mutations (SEQ ID NO: 13) PTE Tm (°C) SEQ ID NO: 13 52,01 SEQ ID NO: 4 55,64 4. Activity measurement Determination of the activity on ethyl-paraoxon of formula:

[0106]

[0107] The data were analyzed with GraphPad Prism 6, using one phase decay modeling. The results are expressed in Table 2 which compares the Kcat / Km ratio values ​​for the enzymes of sequence SEQ ID NO: 4 and SEQ ID NO: 13 with respect to ethyl-paraoxon (influence of the presence of ancestral mutations on the catalytic activity of the enzyme with respect to this substrate). PTE buffer: 50 mM Tris, 300 mM NaCl, 100 µM CoCl 2, pH 8.

[0108] Table 2: Catalytic activity towards ethyl-paraoxon of the enzyme of sequence SEQ ID NO: 13 and SEQ ID NO: 4. PTE k cat / k M (M -1< .s -1< ) SEQ ID NO: 13 6.10 5< SEQ ID NO: 4 3.10 5< Determination of the activity on the DEVX of formula:

[0109]

[0110] The data were analyzed with GraphPad Prism 6, using Michaelis-Menten equation modeling. The results are expressed in Table 3 which compares the Kcat, Km and Kcat / Km ratio values ​​for enzymes with sequence SEQ ID NO: 4 and SEQ ID NO: 13 with respect to DEVX (influence of the presence of ancestral mutations on the catalytic activity of the enzyme with respect to this substrate). Buffer: NaCl 300mM, TRIS 50mM, CoCl 2 100µM, DTNB 4mM, pH8.

[0111] Table 3 : Catalytic activity towards DEVX of the enzyme of sequence SEQ ID NO: 13 and SEQ ID NO: 4. PTE k cat (s -1< ) k M (M) k cat / k M (M -1< .s -1< ) SEQ ID NO: 13 3,356 0,001767 ≈ 1,9.10 3< SEQ ID NO: 4 3.10 5< 0,001317 ≈ 1,3.10 3< 5. Measurement of the activity of mutated PTEs on organophosphorus insecticides

[0112] The catalytic parameters of the mutated PTEs of the application, including mutated PTEs of sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, are measured at 25 °C in triplicate in 96-well plates with a reaction volume of 200 µL and recorded by a microplate reader (Synergy HT, BioTek, USA) in a 6.2 mm cell using Gen5.1 software.

[0113] Kinetic assays were performed at organophosphorus insecticide concentrations ranging from 0.05 to 2 mM. The hydrolysis efficiency of organophosphorus insecticides by the mutated PTEs on demand was determined by measuring absorbance or fluorescence for 10 min using a microplate reader. The catalytic efficiency kcat / KM was then determined. Kinetic assays were performed in activity buffer (50 mM HEPES or Tris pH 8.0, 150 mM NaCl). Catalytic parameters were obtained by fitting the data to the Michaelis-Menten (MM) equation.

[0114] The organophosphate insecticides used to measure the activity of the mutated PTEs in demand are: 6. Measurement of the activity of mutated PTEs of the demand on organophosphorus chemical weapons a) Degradation kinetics and NMR analysis:

[0115] The degradation of chemical weapon organophosphates (CWNAs), including Soman and VX, by the mutated PTE enzymes of the application, including mutated PTEs of sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, is monitored over time by 31P NMR.

[0116] Up to 10 µl of CWNA is placed in an NMR tube and 0.6 to 1.0 mL of the requested mutated PTE enzyme, including mutated PTEs of sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, or water (control) is added. Signal recording is performed at room temperature (approximately 23°C) and signals are measured every 10 minutes for 1 hour.

[0117] The degradation efficiency of CWNAs is determined by measuring the integral of the products as the fraction of the sum of all 31P integrals. The obtained values ​​are reported as the standard deviation of the mean of two degradation measurements performed separately. b) Decontamination of surfaces:

[0118] The effectiveness of decontamination of surfaces contaminated with CWNA, including Soman and VX, by the mutated PTEs of the application, including mutated PTEs of sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, is evaluated over time

[0119] The material panels (5x5 cm) are impregnated with CWNA to achieve a contamination of 10 g / m2. Then, the panels are immersed in a solution containing the PTEs of the application, in particular mutated PTEs of sequences SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, for a period ranging from 15 minutes to 1 hour.

[0120] The panels are then washed with water and carefully dried with a wipe without rubbing. Any residual CWNA agent on the panel is then extracted with an appropriate solvent and analyzed and quantified by gas chromatography-mass spectrometry (GC-MS).

[0121] The chemical weapon organophosphates used to measure the activity of the mutated PTEs on demand and their ability to decontaminate impregnated surfaces are:

Claims

1. Mutated PTE enzyme derived from parathion hydrolase having sequence SEQ ID NO: 1, and containing the following mutations with respect to sequence SEQ ID NO: 1: - Substitution of threonine T by proline P, in position 13, - Substitution of isoleucine I by valine V, in position 14, - Substitution of alanine A by serine S, in position 60, - Substitution of serine S by arginine R, in position 79, - Substitution of tyrosine Y by histidine H, in position 124, - Substitution of isoleucine I by valine V, in position 218, - Substitution of histidine H by tyrosine Y, in position 225, - Substitution of glutamine Q by arginine R, in position 258, - Substitution of leucine L by threonine T, in position 271, said mutated PTE enzyme having sequence SEQ ID NO: 3, or containing the following mutations with respect to sequence SEQ ID NO: 1: - Substitution of threonine T by proline P, in position 13, - Substitution of isoleucine I by valine V, in position 14, - Substitution of alanine A by serine S, in position 60, - Substitution of isoleucine I by cysteine C in position 74 - Substitution of serine S by arginine R, in position 79, - Substitution of phenylalanine F by valine V, in position 100, - Substitution of tyrosine Y by histidine H, in position 124, - Substitution of isoleucine I by valine V, in position 218, - Substitution of histidine H by glutamine Q, in position 222, - Substitution of histidine H by tyrosine Y, in position 225, - Substitution of alanine A by valine V, in position 238, - Substitution of leucine L by methionine M, in position 240, - Substitution of isoleucine I by asparagine N, in position 242, - Substitution of glutamine Q by arginine R, in position 258, - Substitution of serine S by leucine L, in position 276, said mutated PTE enzyme having sequence SEQ ID NO: 4.

2. Process for obtaining a mutated PTE enzyme comprising the introduction of 9 mutations to increase the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by substituting in sequence SEQ ID NO: 1: - the amino acid in position 13 by proline P, - the amino acid in position 14 by valine V, - the amino acid in position 60 by serine S, - the amino acid in position 79 by arginine R, - the amino acid in position 124 by histidine H, - the amino acid in position 218 by valine V, - the amino acid in position 258 by arginine R, - the amino acid in position 225 by tyrosine Y, - the amino acid in position 271 by threonine T, to obtain a mutated PTE enzyme of sequence SEQ ID NO: 3, or comprising the introduction of 15 mutations to increase the phosphotriesterase (PTE) catalytic activity of the enzyme of sequence SEQ ID NO: 1 capable of hydrolyzing organophosphorus compounds, by substituting in sequence SEQ ID NO: 1 : - the amino acid in position 13 by proline P, - the amino acid in position 14 by valine V, - the amino acid in position 60 by serine S, - the amino acid in position 74 by cysteine, - the amino acid in position 79 by arginine R, - the amino acid in position 100 by valine V, - the amino acid in position 124 by histidine H, - the amino acid in position 218 by valine V, - the amino acid in position 222 by glutamine Q, - the amino acid in position 225 by tyrosine Y, - the amino acid in position 238 by valine V, - the amino acid in position 240 by methionine M, - the amino acid in position 242 by asparagine N, - the amino acid in position 258 by arginine R, - the amino acid in position 276 by leucine L, to obtain a mutated PTE enzyme of sequence SEQ ID NO: 4, said sequence SEQ ID NO: 3 and SEQ ID NO: 4 have an improved hydrolysis catalytic activity of organophosphorus compounds, compared to the catalytic activity of the enzyme of sequence SEQ ID NO: 1.

3. Use of at least one mutated PTE enzyme according to claim 1, and having a phosphotriesterase (PTE) catalytic activity capable of hydrolyzing organophosphorus compounds : - for the decontamination of soils polluted with organophosphorus compounds, or - for the decontamination of a surface, skin, mucous membranes or hair contaminated with organophosphorus compounds, or for the prevention or the treatment of an internal or of an external poisoning by ingestion or inhalation of an organophosphorus compound, or - for the control of pollution of water polluted with organophosphorus compounds, or - for the destruction of stocks of neurotoxic agents, said at least one mutated PTE enzyme being chosen among mutated enzyme of sequence SEQ ID NO: 3 or SEQ ID NO: 4, alone or in combination thereof.

4. Kit for the decontamination of surfaces, skin or mucous membranes contaminated with organophosphorus compounds, said kit comprising at least one mutated PTE enzyme according to claim 1, having a catalysis activity of organophosphorus compounds and said at least one mutated enzyme being chosen among the mutated enzymes of sequence SEQ ID NO: 3 or SEQ ID NO: 4, alone or in combination thereof.

5. Phytosanitary composition comprising as active ingredient at least one mutated PTE enzyme according to claim1, and having a catalysis activity of organophosphorus compounds, said at least one mutated enzyme being chosen among the mutated enzymes of sequence SEQ ID NO: 3 or SEQ ID NO: 4, alone or in combination thereof.

6. Pharmaceutical composition comprising as active ingredient at least one mutated PTE enzyme according to claim1, and having a catalysis activity of organophosphorus compounds, said at least one mutated enzyme being chosen among the mutated enzymes of sequence SEQ ID NO: 3 or SEQ ID NO: 4, alone or in combination thereof, in combination with a pharmaceutically acceptable excipient.

7. Mutated PTE enzyme according to claim1, having a catalysis activity of organophosphorus compounds, said at least one mutated enzyme being chosen among the mutated enzymes of sequence SEQ ID NO: 3 or SEQ ID NO: 4, alone or in combination thereof, for its use in the treatment or the prevention of poisonings by contact, inhalation or ingestion of organophosphorus compounds.