Decontamination Composition and Application Thereof

Abstract

A decontamination composition, containing chloroperoxidase. The decontamination composition can rapidly degrade chemical warfare agents, namely mustard gas (HD), Lewisite (L) and VX, and the decontamination products of mustard gas and Lewisite are non-toxic, thereby effectively preventing the harm of the chemical warfare agents to the environment and organism, and reducing the degree of harm of the products to soil, vegetation and organism compared with conventional decontaminant agents.

Claims

1. A decontamination composition, which comprises a chloroperoxidase; preferably, the chloroperoxidase is CPO (EC 1.11.1.10).

2. The decontamination composition according to claim 1, which further comprises one or more selected from the group consisting of hydrogen peroxide, halide ion, buffer solution and cosolvent; preferably, the decontamination composition further comprises an ingredient or combination selected from the group consisting of: hydrogen peroxide; halide ion; buffer solution; cosolvent; hydrogen peroxide and halide ion; hydrogen peroxide and buffer solution; hydrogen peroxide and cosolvent; halide ion and buffer solution; halide ion and cosolvent; buffer solution and cosolvent; hydrogen peroxide, halide ion, and buffer solution; hydrogen peroxide, halide ion, and cosolvent; hydrogen peroxide, buffer solution, and cosolvent; halide ion, buffer solution, and cosolvent; and hydrogen peroxide, halide ion, buffer solution and cosolvent.

3. The decontamination composition according to claim 1 or 2, characterized by one or more of the following: (1) the halide ion is selected from the group consisting of fluoride ion, chloride ion and bromide ion; preferably chloride ion; (2) the buffer solution is selected from the group consisting of phosphate buffer solution, borate buffer solution and citrate buffer solution; preferably phosphate buffer solution, such as KH.sub.2PO.sub.4 buffer solution; (3) the cosolvent is selected from alcohols, such as tert-butyl alcohol.

4. The decontamination composition according to any one of claims 1 to 3, characterized by one or more of the following: (1) the chloroperoxidase has a concentration of nanomolar level to millimolar level; for example, 1 nM to 100 mM, preferably 20 nM to 10 mM; (2) the hydrogen peroxide has a concentration of 0 to 50 mM; (3) the halide ion has a concentration of 0 to 0.5M; (4) the buffer solution has a pH of 2.0 to 5.0; (5) the cosolvent has a concentration of 1 to 10% (v/v).

5. A decontamination preparation, which comprises the decontamination composition according to any one of claims 1 to 4; preferably, the hydrogen peroxide and other ingredients in the decontamination composition are located in the same preparation unit; preferably, the hydrogen peroxide and other ingredients in the decontamination composition are located in different preparation units; preferably, the decontamination preparation is a decontamination solution.

6. A protective equipment, which comprises the decontamination composition according to any one of claims 1 to 4 or the decontamination preparation according to claim 5.

7. Use of the decontamination composition according to any one of claims 1 to 4, the decontamination preparation according to claim 5 or the protective equipment according to claim 6 in the decontamination of a chemical poison; preferably, the chemical poison is a nerve agent or a blister agent; preferably, the nerve agent is an organophosphorus agent, for example selected from the group consisting of sarin, tabun, soman and VX; preferably, the blister agent is mustard gas, Lewisite, nitrogen mustard or a combination thereof, for example, a combination of Lewisite and mustard gas.

8. A method for decontaminating a chemical poison, which comprises the following steps: (1) rapidly mixing the ingredients of the decontamination composition according to any one of claims 1 to 4, or the decontamination preparation according to claim 5, to obtain a mixture system; (2) pouring or spraying or contacting the mixture system to an area, such as water, soil or biological surface, where the chemical poison leaks; preferably, the chemical poison is a nerve agent or a blister agent; preferably, the nerve agent is an organophosphorus agent, for example, selected from the group consisting of sarin, tabun, soman, and VX; preferably, the blister agent is mustard gas, Lewisite, nitrogen mustard, or a combination thereof, for example, a combination of Lewisite and mustard gas.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present application. The illustrative examples of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the attached drawings:

[0045] FIG. 1: (a) the photos under light microscope, which show the comparison of effect on the survival of zebrafish embryos, of the group using the decontamination formulation with the group using only hydrogen peroxide, of the control group of mustard gas without being decontaminated, and of the groups using traditional decontaminants; (b) survival rate of embryos.

[0046] FIG. 2: (a) the decontamination effect on the chemical poisons of mustard gas, Lewisite and VX, of the group using the decontamination formulation, of the group using only hydrogen peroxide, and of the chemical poison control group: (b) in the case of Lewisite, the response of zebrafish larvae to the decontamination formulation; (c) in the case of Agent Yellow, the response of zebrafish larvae to the decontamination formulation (the group of the decontamination formulation of the present invention, and the chemical poison control group).

[0047] FIG. 3: (a) Michaelis-Menten analysis of enzymatic oxidation of mustard gas; (b) kinetics of mustard gas degradation and mustard sulfoxide formation; (c) mass spectrum and NMR spectrum of enzymatic degradation products of mustard gas; (d) proposed degradation mechanism of mustard gas.

[0048] FIG. 4: the optimal condition exploration diagram of each formula of the decontamination formulation of the present invention.

SPECIFIC MODELS FOR CARRYING OUT THE PRESENT INVENTION

[0049] The technical solutions in the examples of the present invention will be clearly and completely described below with reference to the accompanying drawings in the examples of the present invention. Obviously, the described examples are only some of the examples of the present invention, rather than all the examples. The following description of at least one exemplary example is merely illustrative in nature and is in no way intended to limit the present invention, its application or uses. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0050] Chloroperoxidase (CPO, EC 1.11.1.10) is a multifunctional glycoprotein secreted by the marine fungus Caldariomyces fumago, which is one of the most versatile catalysts in heme enzymes. However, the broad substrate selectivity of CPO results in its poor decomposition efficiency for HD.

[0051] Zebrafish (Danio rerio) embryos are often used as vertebrate models for environmental and animal physiological monitoring because the models have advantages in terms of chemical permeability, short disease incubation period, sensitivity to chemical treatments, and ease of observation and operation. In addition, the operating conditions of CPO do not conflict with the developmental conditions of zebrafish, which allowed us to establish a new environmental model combining the enzyme and zebrafish embryos to detect decontamination efficiency.

Example 1

[0052] All procedures were performed in a well-ventilated fume hood at 255 C., freshly prepared H.sub.2O.sub.2 stock solution was used in each experiment. Zebrafish embryos were collected from a spawning apparatus, rinsed with embryo medium E3, and divided in a 24-well plate with 10 embryos per well. A pipette was used to carefully remove the culture medium from the wells of the culture plate, then 1 ml of test solution was added to the culture well within 4 hours after fertilization, the culture plate was placed flatwise and incubated at 271 C., and the embryonic death rate were recorded every day, lasting for 3 days. The data were subsequently expressed as mean #standard deviation of the results of 3 independent parallel experiments. CPO reaction conditions were optimized by using blank embryo culture medium E3 as negative control, and specific solutions of 0.1-5% (vol %) TBA, 1-100 mM KH.sub.2PO.sub.4, 0.1-50 mM KCl, and 0.05-100 M HD (pH 3.0-7.0) were added to the embryo culture medium. By establishing a system for degrading 0.1 mM HD in 5 min with 20 nM CPO and 0.2 mM H.sub.2O.sub.2 at pH 4.5, the toxicity of HD and its degradation products was evaluated.

[0053] As shown in FIG. 1, all the embryos treated with 0.1 mM HD died within 24 h, while the survival rates of the CPO-treated embryos and the negative control embryos were 878% and 936%, respectively. The results were expressed as mean+standard deviation, n=3. In the last two cases, the embryos and larvae showed no malformation or morphological change. However, the embryos exposed to 0.5% sodium hypochlorite were immediately damaged.

Example 2

[0054] The HD stock solution (0.1 mM), which was obtained by dissolving pure HD in TBA, was added to the buffer solution comprising 0.1 M KH.sub.2PO.sub.4, 0.5 M KCl, 0.022 M CPO and 5 mM H.sub.2O.sub.2 with pH of 2.75 to obtain HD stock solution having a final concentration of 1 mM. In another experiment, H.sub.2O.sub.2 was reacted with HD in the absence of CPO to confirm the key role of the enzyme in this process. The control samples were prepared separately by decomposing HD in buffers without CPO or H.sub.2O.sub.2. After reacting for 1 minute, the organic layer was quenched with an equal volume of CH.sub.2Cl.sub.2 and separated, then dried over anhydrous Na.sub.2SO.sub.4, subsequently transferred to a gas chromatography vial and stored at 20 C., and finally analyzed by gas chromatography-mass spectrometry. Similar methods were used to study the degradation of 1 mM L and VX under the conditions of CPO concentration of 0.220 UM and reaction time of 1 min and 5 min respectively. It could be seen from FIG. 2a that complete decomposition of HD and LVX was achieved quickly by CPO-catalyzed oxidation with the consumption of enzyme at nanomolar level and H.sub.2O.sub.2 concentration of 5 mM, revealing CPO is capable of promoting the degradation of various CWAs simultaneously. When the concentration of H.sub.2O.sub.2 was 1 mM, HD and L were completely consumed within Imin, 1 mM HD was completely consumed within 40 s, and 1 mM VX was completely consumed within 5 min. Under the conditions that the concentration of H.sub.2O.sub.2 was 5 mM and CPO was absent, the residual amounts of HD, L, and VX obtained were 83.855.01%, 78.3611.56%, and 35.720.77% after 1 h, Imin, and 5 min of reaction, respectively. Therefore, except for that the degrading rate of VX reached 60% within 5 minutes, the non-catalytic reaction between the chemical poisons and H.sub.2O.sub.2 were relatively slow.

Example 3

[0055] Zebrafish larvae three days after fertilization were used to evaluate the degradation efficiency for L and Agent Yellow (HD+L) by CPO. Zebrafish larvae were divided and added into 24-well plates at 6 larvae per well. 0.1 mM L solution, CPO and H.sub.2O.sub.2 were added to the modified embryonic E3 medium, with the final concentration of 20 nM for L and 0.2 mM for CPO and H.sub.2O.sub.2 respectively, and the reaction lasted for 5 minutes. Additional experiments were performed with samples comprising only 0.1 mM L and 0.1 mM L+0.2 mM H.sub.2O.sub.2 in embryonic E3 medium. In addition, 0.5% sodium hypochlorite was tested as an oxidizing agent. The degradation of a mixture of 0.1 mM HD and 0.1 mM L was studied in 20 nM CPO and 0.5 mM H.sub.2O.sub.2, respectively. In the modified embryonic E3 medium comprising TBA (0.1%) and KH.sub.2PO.sub.4 (1-10 mM), the optimal reaction conditions for HD were determined by adjusting pH to 4.5 with HCl. In the CPO system, L could be effectively degraded, while the oxidation speed without CPO was not fast enough and toxic light absorption products could be produced, as shown in FIGS. 2b to 2c. In the presence of 0.5% sodium hypochlorite, zebrafish larvae were immediately damaged, died within a few minutes, and decomposed after 5 minutes. In 1 mM L, cessation of heartbeat was observed within 20 minutes, and the body of dead larvae was twisted and had a corrosive appearance. The larvae treated with 0.1 mM L and 0.2 mM H.sub.2O.sub.2 died within 20 minutes and had dark skin. Similar results were observed for the degradation of Agent Yellow at 0.1 mM HD+0.1 mM L. That was to say, CPO could effectively degrade HD and L, so it could be used as a real chemical weapons decontamination agent for the degradation of Agent Yellow.

Example 4

[0056] The optimal conditions were set as: 0.1 M KH.sub.2PO.sub.4, 0.5 M KCl, pH 2.75, 5% TBA, 5 mM H.sub.2O.sub.2, 20 nM CPO, temperature of 252 C., and total volume of 7 ml. The degradation rate of HD catalyzed by CPO was monitored under optimal reaction conditions. Every 5 seconds or 10 seconds, 1 ml of the reaction mixture was taken out and immediately mixed with 1 ml of CH.sub.2Cl.sub.2 under stirring with a magnetic stirrer for 1 minute to obtain a sample for later detection. Michaelis-Menten analysis was used to determine the affinity of CPO for HD, and GraphPad Prism 5 was used for graphing analysis. As shown in FIG. 3, the K.sub.m, V.sub.max, and k.sub.cat values were 0.17 mM, 0.06 mM s.sup.1 (R.sup.2=0.935), 2717 M.sup.1 s.sup.1, respectively. For extremely-efficient enzymes, the reaction rates exceeded 10.sup.7 M.sup.1 s.sup.1, while for ultra-efficient enzymes, the k.sub.cat/K.sub.m ratios were 108 to 1010 M.sup.1 s.sup.1. The corresponding value of CPO was 1.5810.sup.7 M.sup.1 s.sup.1, indicating that CPO had the ability to effectively promote the HD oxidation.

[0057] Various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety.