Patent Grant
7888103
This invention relates to method for detoxification of yperite 2,2′-dichlorodiethylsulfide—by using haloalkane dehalogenases (Enzyme Commission number EC 3.8.1.5) as a primary, chemically active component of decontamination compositions. Decontamination compositions are designated for detoxification of yperite (2,2-dichlorodiethylsulfide) on the surface of military hardware, transportation, industrial and agricultural hardware, technical devices and constructional objects (hereafter instrumentation), human or animal skin and elements of environment (water, soil, sediments and air), that are contaminated by this highly toxic blistering substance.
At the present time, decontamination compositions are being used in the armed forces, civil defense troops, fire services and rescue forces, that exhibit high unit consumption and undesirable aggressiveness on material, because their chemically active components are stechiometric agents, that are gradually consumed during their reaction with yperite. Their application on instrumentation leads to depreciation of decontaminated material or surfaces by corrosion and if these compositions get into soil or water, it endangers environment.
There have been described enzymes in the literature that exhibit activity against highly toxic organophosphorous (neural) substances, called organophosphate hydrolases, OPA anhydrases or DFPases. As the only example of biological detoxification of blistering yperite (2,2′-dichlorodiethylsulfide), the use of bacteria species Rhodococcus rhodochrous IGTS8 (ATCC 53968) was mentioned in the art so far, which has the ability to utilize a chemical analog of yperite 2-chloroethyl-ethylsulfide as the only source of carbon for its growth [Kilbane, J. J., and Jackowski, K. (1996) J. Chem. Tech. Biotechnol. 65, 370-374]. Detoxification activity of bacteria species Rhodococcus rhodochrous IGTS8 (ATCC 53968) is based on splitting the S—C bond in the molecule. The application of the enzyme splitting C—S bond in a non-toxic product of hydrolysis, thiodiglycol, has been published [Harvey, S., DeFrank, J. J., Valdes, J. J., Kamely, D, and Chakrabarty, A. M., (1990) Proceedings: Biotechnology-Biodegradation Workshop Symposium by US Army Research Office, 47-58; Kilbane, J. J., (1990) Resources Conserv. and Recycl. 3, 69-79].
Haloalkane dehalogenases are enzymes that are able to remove halogen from halogenated aliphatic compound by a hydrolytic replacement, forming the corresponding alcohols [Janssen, D. B. Pries, F., and Van der Ploeg, J. R. (1994) Annual Review of Microbiology 48, 163-191]. Hydrolytic dehalogenation proceeds by formal nucleophilic substitution of the halogen atom by hydroxyl ion. Structurally, haloalkane dehalogenases belong to the α/β-hydrolase fold superfamily [Ollis, D. L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S. M., Harel, M., Remington, S. J., Silman, I., Schrag, J., Sussman, J. L., Verschueren, K. H. G., and Goldman, A. (1992) Protein Engineering 5, 197-211]. Haloalkane dehalogenases contain a nucleophile elbow [Damborsky, J. (1998) Pure and Applied Chemistry 70, 1375-1383] that belongs to the most conserved structural features of the α/β-hydrolase fold. Another highly conserved region in haloalkane dehalogenases is the central β-sheet that is flanked on both sides by α-helixes that form hydrophobic core of the main domain. The main domain carries the catalytic triad: aspartic acid-histidine-aspartic acid/glutamic acid (Asp-His-Asp/Glu). The second domain is solely consisting of α-helixes and is placed like a cap on top of the main domain. The interface between the main and the cap domain forms the active site of the enzyme. Whereas there is significant similarity between main domains, the sequence and structure of the cap domain varies significantly in different haloalkane dehalogenases. It is supposed, that character and structure of the cap domain essentially determine the substrate specificity [Pries, F., Van den Wijngaard, A. J., Bos, R., Pentenga, M., and Janssen, D. B. (1994) Journal of Biological Chemistry 269, 17490-17494; Kmunicek, J., Luengo, S., Gago, F., Ortiz, A. R., Wade, R. C., and Damborsky, J. (2001) Biochemistry 40, 8905-8917].
In the drawing:
The disadvantages of the above mentioned existing decontamination compositions containing stechiometric agents, are to the great extent overcome by preparations in accordance with the present invention comprising a catalyst of hydrolytic detoxification of yperite (2,2′-dichlorodiethylsulfide), which is enzyme or mixture of enzymes haloalkane dehalogenases. The basic and active component of the compositions is at least one wild type or modified enzyme of the haloalkane dehalogenase family (EC 3.8.1.5). The present invention provides a method of detoxication of yperite by the use of haloalkane dehalogenases which comprises hydrolytic dehalogenation of yperite, wherein yperite is treated with the detoxification composition containing one or more wild type or modified haloalkane dehalogenases and is thereby converted to the non-toxic product thiodiglycol.
Haloalkane dehalogenase is expressed in the natural producer or in a heterologous host organism, e.g. in bacteria Escherichia coli, or in yeast Pichia pastoris. The enzyme used can be comprised in non-living or living cells, in the form of crude extract or purified protein. As an enzyme for the dehalogenase composition at least one haloalkane dehalogenase selected from the family of enzymes EC 3.8.1.5 is used, e.g. haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064, DmbA from Mycobacterium bovis 5033/66 or LinB from Sphingomonas paucimobilis UT26.
Haloalkane dehalogenases constitute an important group of enzymes that are able to cleave the halogen-carbon bond in halogenated aliphatic compounds. They exhibit a broad substrate specificity including haloalkanes, halocycloalkanes, haloalkenes, haloethers and haloalcohols. The mechanism of dehalogenation is based on the nucleophilic attack to the carbon atom to which the halogen is bound and proceeds to cleavage of halogen ion and formation of alkyl-enzyme intermediate. The intermediate is subsequently hydrolyzed, yielding the corresponding alcohol, halogen ion and proton. The enzyme haloalkane dehalogenase transforms yperite into non-toxic bis(2-hydroxyethyl)sulfide by hydrolytic dehalogenation.
In the decontamination compositions haloalkane dehalogenase can be in crude extract or purified, immobilized on a carrier material, free in aqueous solution, in a monophasic organic or aqueous solution or in organic/aqueous biphasic systems. Enzymes can be immobilized by absorption on the inorganic or organic carrier material (such as: Celite, activated charcoal, aluminium oxide, cellulose, synthetic resins or Sephadex which is based on synthetically derived polysaccharide (dextran) or covalent attachment onto the surface of organic material (such as: cellulose, dextran, starch, chitin, agarose) or inorganic material (such as: porous glass), or synthetic polymeric carrier material (such as: VA-Epoxy Biosynt, Eupergit): The enzyme may be immobilized also by cross-linking (linkage to each other) or entrapping enzyme into a solid matrix or a compartment confined by a membrane.
The enzyme haloalkane dehalogenase may be dissolved, crystalline, lyophilized or precipitated. The liquid medium is an organic solvent, a mono-phasic aqueous solution of organic solvent or bi-phasic system consisting of organic solvent and water. The enzyme can be confined to a restricted area, where it remains catalytically active—entrapped into a solid matrix or into compartments restricted by a membrane. Enzymes may be entrapped into a biological matrix, e.g., agar gel, alginate gel, κ-carragenan. The enzyme can be entrapped also to inorganic stable matrices, e.g., silica gel. A tight network that is able to carry isolated enzyme can be obtained by polymerization of synthetic monomers, e.g., polyacrylamide, in the presence of the enzyme. Depending on the immobilization technique, the properties of the enzyme such as catalytic rate, stability and binding affinity may be significantly altered. The hydrolytic detoxification of yperite catalysed by the enzyme can be performed in the temperature range of 10-70° C. with reaction optimum of about 40° C.
Additional components are aqueous buffer systems (e.g., phosphate buffer, tris-sulfate buffer, glycine buffer, acetate buffer or citrate buffer) which stabilize the neutral pH being close to optimum interval of 7.0-8.5. The pH activity profile is broader and allows pH interval from 4 to 12 while maintaining a reasonable activity. Other additional components are surfactants or organic solvents that facilitate dissolving of yperite in aqueous solvents. Addition of water-miscible organic solvents, e.g., methanol, tert-butanol, acetone, dioxane, acetonitrile, dimethyl formamide, dimethyl sulfoxide, tetrahydrofuran, 3-methyl-3-pentanol and pyridine, can be used at concentration up to 70% of the total volume depending on the enzyme stability.
Decontamination compositions based on haloalkane dehalogenases can consist of two macroscopic phases, namely the aqueous phase containing the dissolved enzyme and a second phase consisting of organic solvents, partially water soluble or insoluble in water, e.g., ethyl acetate, diethyl ether, methyl tert-butyl ether, cyclohexanol, n-propylacetate, ethyl chloroacetate, bis(2-chloroethyl)ether, isopropyl acetate, butyl acetate, isobutyl acetate, hexanol, isoamyl acetate, n-amyl acetate, toluene, octanol, isoheptane, n-butyl ether, cyclohexane, 2-methylpentane, n-hexane, methylcyclohexane and n-octane. Organic phase enhances solubility of yperite in the decontamination composition which penetrates into water phase. The reaction takes place in aqueous phase, where the enzyme is in natural environment and is not in direct contact with organic solvent, where the most of dissolved yperite is located. The transfer of reactant and product between the two phases, reactant to the enzyme, product from the enzyme, can be increased by enlarging the surface between the two phases (producing a fine dispersion) or by stirring. The bulk water can be replaced by addition of water immiscible organic solvent. The enzyme is then suspended in a monophasic organic solvent. The optimum catalytic activity of the enzyme in organic solvent can be obtained by adjustment and maintenance of the water content. This can be conventionally obtained by a pair of salt/hydrate, e.g., CaCl2.H2O/2 H2O, Nal anh./2 H2O, Na2HPO4 anh./2 H2O, NaOAc anh./3 H2O, NaBr anh./2 H2O, Na4P2O7 anh./7 H2O, Na2HPO4.2 H2O/7 H2O, Na2SO4 anh./10 H2O, Na2HPO4.7 H2O/12 H2O, that are added to the solvent and function as a water content buffer. The enzyme solubility in lipophilic organic solvents can be modified by covalent attachment of the amphiphatic polymer polyethylene glycol to the surface of enzyme. Linkage of the polymer chain onto the enzyme surface is achieved by reaction of ε-amino groups of lysine residues with “linker”, e.g., cyanuric chloride. Protein stabilizers such as polyalcohols, e.g., sugar alcohols or glycerol, inactivated proteins, e.g., bovine serum albumin, or polymers, which show a certain structural resemblance with water, e.g., polyethylene glycol, polyvinyl alcohol, can be added to the reaction medium to enhance the enzyme stability.
Haloalkane dehalogenases used according to this invention can be further produced by means of rational design based on structural analysis, e.g., protein crystallography, nuclear magnetic resonance and circular dichroism spectroscopy, and biochemical characterization, e.g., steady-state kinetics, transient-state kinetics, stability and thermo stability assays, spectroscopic analyses and the like, followed by computer modelling, e.g., sequence comparisons, phylogenetic analysis, homology modelling, molecular docking, molecular mechanics, molecular dynamics, quantum mechanics and multivariate statistics, and DNA mutagenesis, e.g., cassette mutagenesis, site-directed mutagenesis, chemical mutagenesis, error-prone PCR, site saturation mutagenesis, ensemble mutagenesis, recursive ensemble mutagenesis, scanning saturation mutagenesis, mutator strains, etc. The procedure includes altering at least one amino acid residue of the haloalkane dehalogenase with another amino acid residue or recombining two or more members of the haloalkane dehalogenases to obtain a modified enzyme with improved efficacy. Modified nucleic acids can be introduced into a cell, in which they can be expressed to provide an altered haloalkane dehalogenase.
Advantages of decontamination compositions with haloalkane dehalogenases are that
The enzyme is prepared by homologous expression in native producer or by heterologous expression in a host organism, e.g., bacterium Escherichia coli or yeast Pichia pastoris. According to the invention, the enzyme present in living or non-living cells is used in the form of crude extract or purified protein.
To overproduce wild type of enzyme haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26 (Sequence 1 and 2) the corresponding gene was cloned in the pPICZαA expression vector. Cloned plasmids were then transferred into Pichia pastoris GS115. Pichia pastoris GS115 was then cultured at 28° C. in growth medium (1 weight % of yeast extract, 2 weight % of peptone, 4×10−5 weight % of Biotine and 1 weight % of casamino acid in 100 mM potassium phosphate buffer, pH 6.5). The induction of the enzyme synthesis was initiated by addition of 0.7 volume % of methanol when the culture reached an optical density of 2 at 600 nm. After induction the culture was incubated at 28° C. for 10 h and then harvested. Ammonium sulfate was added to supernatant to a final concentration of 75% of saturation. Solution was stirred 30 min until the added ammonium sulfate was dissolved. The supernatant was centrifugated 15 min at 11 000 g. Pellet was than re-suspended in 20 mM potassium phosphate buffer, pH 7.5 with content of 0.5 M sodium chloride and 10 mM imidazole. The haloalkane dehalogenase was then purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). The His-tagged haloalkane dehalogenase was bound to the resin placed in the equilibrating buffer, which contained 20 mM potassium phosphate buffer pH 7.5; 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed off by buffer containing 60 mM imidazole. Then the His-tagged enzyme LinB was eluted by buffer containing 160 mM imidazole. The active fractions were dialyzed overnight against 50 mM potassium phosphate buffer, pH=7.5. The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol to enhance long-lasting enzyme stability.
Hydrolytic dehalogenation catalyzed by haloalkane dehalogenase (wild type or modified) converts the toxic yperite into non-toxic bis(2-hydroxyethyl)sulfide. The hydrolytic dehalogenation of yperite was catalyzed by haloalkane dehalogenase at 37° C. in 50 mM phosphate buffer (pH 7.5; adjusted by addition of 1M NaOH solution). The yperite was added into reaction buffer to its final concentration of 94.3 mg.l−1 of yperite in buffer. Reaction was initiated by addition of solution of enzyme LinB (50 mM potassium phosphate buffer pH 7.5; 1×10−5 to 1×10−4 mol.l−1 of haloalkane dehalogenase LinB, 10 vol % of glycerol and 1 mmol.l−1 2-mercaptoethanol). Operation of haloalkane dehalogenase leads to rapid and complete decontamination of the yperite. The kinetics of reaction is shown in
Sequence 1 and 2. Sequence of the gene linB and haloalkane dehalogenase LinB isolated from bacterium Sphingomonas paucimobilis UT26.
To overproduce enzyme haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 (Sequence 3 and 4), the corresponding gene was cloned in the pAQN vector containing tac promotor (Ptac) under the control of laclq. Escherichia coli BL21 containing pAQN plasmid was cultured in 250 ml Luria broth at 37° C. The induction of enzyme synthesis was initiated by addition of isopropyl-β-D-thiogalactopyranoside to a final concentration of 0.5 mM when the culture reached an optical density of 0.6 at 600 nm. After induction, the culture was incubated at 30° C. for 4 h and then harvested. The cells were disrupted by sonication using a Soniprep 150 (Sanyo, UK). The supernatant was used after centrifugation at 100 000 g for 1 h. The haloalkane dehalogenase was purified on a Ni-NTA Sepharose column HR 16/10 (Qiagen, Germany). His-tagged haloalkane dehalogenase was bound onto the resin placed in the equilibrating buffer, which contained 20 mM potassium phosphate buffer pH 7.5, 0.5 M sodium chloride and 10 mM imidazole. Unbound and weakly bound proteins were washed off by buffer containing 60 mM imidazole. The His-tagged haloalkane dehalogenase was then eluted by buffer containing 160 mM imidazole. The active fractions were dialysed overnight against 50 mM potassium phosphate buffer, pH 7.5. The enzyme was stored at 4° C. in 50 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 1 mM 2-mercaptoethanol enhancing long-term enzyme stability.
Enzyme haloalkane dehalogenase DhaA or its variant is the part of decontamination composition that contains aforementioned enzyme at concentration 1×10−6 to 1×10−4 mol.l−1, further 1 to 5 vol % of aliphatic hydrocarbon of general formula CnH2n+2 or cyclic aliphatic hydrocarbon of general formula CnH2n, where n is 6 to 12, further 5 to 20 vol % of aliphatic alcohol of general formula CnH2n+1OH, where n is 2 to 4, further 3 to 15 weight % of anion active tenside of general formula CnH2n+1OSO3Me, where n is 10 to 16 and Me stands for counter ion (Na+, K+ or monoethanol amonium), 1 to 10 weight % alkylbenzenesulfonate of general formula R(3)—(Ar)SO3−.Me+, where R(3) stands for alkyl with 11 to 13 atoms of carbon, and Me+ indicates sodium ion, further components of glycine buffer to adjust pH of aqueous solution in the range from 7 to 9, or else glycine of total concentration 0.1 mol.l−1, etc. Required pH 8.2 is reached by addition of 1M NaOH. The rest to make 100% is water. The catalytic power of haloalkane dehalogenase DhaA at decontamination of yperite is kcat/Km=5.7 s−1.mM−1.
Sequence 3 and 4. Sequence of the gene dhaA and haloalkane dehalogenase DhaA isolated from bacterium Rhodococcus rhodochrous NCIMB 13064.
To overproduce enzyme haloalkane dehalogenase DmbA from Mycobacterium bovis 5033/66 (Sequence 5 and 6), the same procedure as in example 1 is followed. Enzyme haloalkane dehalogenase DmbA or its variant is the part of decontamination composition that contains aforementioned enzyme at concentrations 1×10−6 to 1×10−4 mol.l−1, further 1 to 20 vol % of aliphatic alcohol of general formula CnH2n+1OH, where n is 2 to 4, further 3 to 15 weight % of anion active surfactant of general formula CnH2n+1OSO3Me, where n is 10 to 16 and Me stands for counter ion (Na+, K+ or monoethanol amonium), 1 to 10 weight % of ethoxy nonylphenol of general formula C9H19—Ar—O—(C2H4O)nH, where n is 9 to 10, further components of glycine buffer to adjust pH of aqueous solution in the range from 7 to 9, or else glycine of total concentration 0.1 mol.l−1, etc. Required pH is reached by addition of 1M NaOH. The rest to make 100% is water. The catalytic power of haloalkane dehalogenase DmbA at decontamination of yperite is kcat/Km=6.0 s−1.mM−1.
Sequence 5 and 6. Sequence of the gene dmbA and haloalkane dehalogenase DmbA isolated from bacterium Mycobacterium bovis 5033/66.
Enzyme haloalkane dehalogenase LinB or its variant is the part of decontamination composition that contains the enzyme at concentrations 1×10−6 to 1×10−4 mol.l−1, further 1 to 15 weight % of anion active surfactant of general formula CnH2n+1OSO3Me, where n is 10 to 16 and Me stands for counter ion (Na+, K+ or monoethanol ammonium), 1 to 10 weight % of ethoxylated nonylphenol of general formula C9H19—Ar—O—(C2H4O)nH, where n is 9 to 10, further components of phosphate buffer to adjust pH of aqueous solution in the range from 7 to 8.5; that is KH2PO4 a K2HPO4 in the required ratio and in a total concentration 50 mmol.l−1, etc. The rest to make 100% is water.
This invention is utilizable in industry to eliminate yperite from the surfaces of military hardware, transportation, industrial and agricultural hardware, technical devices and constructional objects, of the human or animal skin and elements of environment, that are contaminated by this highly toxic blistering substance. This technology is utilizable in armed forces and also in civil services, generally there, where the use of detoxication compositions to decontaminate blistering substances is necessary.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2005-352 | Jun 2005 | CZ | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CZ2006/000036 | 6/1/2006 | WO | 00 | 11/30/2007 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2006/128390 | 12/7/2006 | WO | A |
| Number | Date | Country |
|---|---|---|
| WO 0156380 | Aug 2001 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20080248557 A1 | Oct 2008 | US |
