Method For The Detection And/or Detoxification Of Compounds

  • Published: Nov 27, 2008
  • Earliest Priority: May 18 2007
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Title: METHOD FOR THE DETECTION AND/OR DETOXIFICATION OF

COMPOUNDS

The present disclosure relates to methods of attaching hydrolytic enzymes to paper and paper products, to the paper and paper products prepared using this method as well as various uses of these products, in particular for detection and/or detoxification of toxic substances. BACKGROUND

Organophosphates (OPs) are toxic compounds that can severely damage the nervous system. They work by inhibiting acetylcholinesterase, which is an enzyme important for transmitting nerve signals (Costa, L.G. (2006) Clin. Chim. Acta. 366:1-13). OPs are found in pesticides and chemical warfare agents. Of pesticides used worldwide, 38% contain OPs, resulting in 3 million deaths per year (Singh, B. K. and Walker, A. (2006) FEMS Microbiol. Rev. 30:428-471). An assay that is easily administered in order to test for the presence of OP pollutants will help to prevent OP poisoning.

Construction of a non-thermal stable organophosphorous enzyme from Flavobacterium fused with a Clostridium celluovorans Cellulose binding domain at the N-terminus has been previously described (Richins, R.D., Mulchandani, A. and Chen, W. 2000. Biotechnol Bioeng.69:591-596.).

In 2005, a thermophilic phosphotriesterase (PTE) termed &øPox was identified in Sulfolobus solfataricus (Merone et a (2005) Extremophiles. 9:297-305). This organism is native to hot springs, and thrives at 800C (She et al. (2001) Proc. Natl. Acad. Sci. USA. 98:7835-7840).

SUMMARY OF THE DISCLOSURE

A bioactive paper that can be used as a biosensor for organophosphorus compounds has been developed. The bioactive paper was prepared by addition of a cellulose binding domain (CBD) to the C-terminus of a thermostable hydrolytic PTE enzyme (from Sulfolobus solfataricus P2, SSO2522 or SsoPox) or by attaching or linking this enzyme directly to paper. In both cases, the enzyme retained activity when bound to paper. Studies based on detection of organophosphorus pesticide, paraoxon, have been conducted. Hydrolytic cleavage of paraoxon resulted in the production of the yellow colored p-nitrophenolate. This system can therefore be a qualitative or quantitative biosensor and/or detoxifier/remover of organophosphorous compounds and/or N-acyl homoserine lactones.

Accordingly, the present disclosure includes a method of producing a bioactive paper for detecting, removing and/or detoxifying one or more compounds comprising at least one hydrolyzable bond, comprising contacting the paper with a thermostable hydrolytic enzyme under conditions for the attachment of the enzyme to the paper, wherein said enzyme hydrolyzes the one or more compounds. Also included in the present disclosure is a bioactive paper having a thermostable hydrolytic enzyme attached thereto. In an embodiment of the disclosure the one or more compounds comprising at least one hydrolyzable bond are selected from an organophosphorus compound, and an N-acyl homoserine lactone and hydrolysis of the one or more compounds by the thermostable hydrolytic enzyme results in the removal and/or detoxification of the one or more compounds. In a further embodiment of the present disclosure, hydrolysis of the one or more compounds by the thermostable hydrolytic enzyme results a detectable change on the paper. In another embodiment the detectable change is quantifiable.

The present disclosure also includes a method of detecting one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a solution comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and

(b) observing a detectable change on the paper due to the hydrolysis of the one or more compounds by the enzyme, wherein the detection of a change on the paper indicates the presence of the one or more of the compounds in the solution. The present disclosure also includes a method of removing or detoxifying one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a sample comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and (b) optionally, observing a detectable change on the paper due to the hydrolysis of the one of more compound by the enzyme, wherein, the contacting of the sample with the paper, results in hydrolysis of the one or more compounds and the hydrolysis of the one or more compounds removes or detoxifies the one or more compounds.

In an embodiment of the disclosure, the one or more compounds comprising at least one hydrolyzable bond are selected from an organophosphorus compound, and an

N-acyl homoserine lactone.

In an embodiment of the disclosure, the thermostable hydrolytic enzyme is modified to include a Cellulose Binding Domain (CBD) and the enzyme is attached to the paper via the CBD. In a further embodiment of the disclosure, the thermostable hydrolytic enzyme is a phosphotriesterase (PTE).

The present disclosure also includes uses of bioactive paper having a thermostable hydrolytic enzyme attached thereto to detect, detoxify and/or remove one or more compounds having at least one hydrolysable bond, for example, a compound selected from an organophosphorus compound and an N-acyl homoserine lactone in, for example, environmental or food-based samples. For example, the bioactive paper of the present disclosure can be used to make paper-based filters or paper-based facemasks to detect, detoxify and/or remove pesticides minimizing exposure of individuals to these toxic chemicals. Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will now be described in relation to the drawings in which:

Figure 1 shows SDS-PAGE analysis of native SsoPox purification.

Figure 2 shows SEQ ID NO: 1 Figure 3 shows a photograph of native SsoPox chemically immobilized on Whatman

3mm paper and treated with paraoxon. Also shown are control experiments as described in the Examples. Figure 4 shows a photograph of the immobilization of SsoPox-CBD on Whatman 3mm paper and treated with paraoxon. Also shown are control experiments as described in the Examples.

DETAILED DESCRIPTION OF THE DISCLOSURE DEFINITIONS

The term "paper" and "paper products" as used herein refers to a commodity of thin material produced by the amalgamation of fibers, typically vegetable fibers composed of cellulose, which are subsequently held together by hydrogen bonding.

While the fibers used are usually natural in origin, a wide variety of synthetic fibers, such as polypropylene and polyethylene, may be incorporated into paper as a way of imparting desirable physical properties. The most common source of natural cellulose fibers is wood pulp from pulpwood trees. Other vegetable fiber materials, including those of cotton, hemp, linen and rice, may also be used. Cellulose is available in many forms including, cotton wool, filters, beads, powders, fibers, hydrogels, membranes and sheets of defined porosity.

The term "attached" means covalently bound or otherwise linked (e.g. through hydrophobic, electrostatic, hydrogen-bonding, bioaffinity, covalent interactions or combinations thereof) to the surface of the paper.

As used herein, the term "thermostable hydrolytic enzyme" is meant to include any enzyme that acts on a substrate to catalyze the hydrolysis of a bond and the activity of which is not significantly reduced at elevated temperatures, for example at temperatures in the range of about 6O0C to about 100 0C, suitably about 80 0C. In an embodiment of the present disclosure, the hydrolytic enzyme is a phosphodiesterase

(PTE). In a particular embodiment of the disclosure, the thermostable hydrolytic enzyme is a thermostable PTE from Sulfolobus solfataricus.

The term "detoxify" as used herein means to render a compound or compounds non-toxic to humans or animals by chemically degrading the compound. The chemical degradation occurs by hydrolyzing the compound.

The term "toxic" as used herein means that exposure of a human or animal to a compound, by any means (e.g. ingestion, inhalation and/or external contact), has a negative effect on the health and well-being of the human or animal.

The terms "a" and "an" as used herein can mean one or more than one.

In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives. Finally, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

METHODS OF THE DISCLOSURE

Phosphotriesterases (PTEs) from several organisms have been shown to degrade organophosphates (OPs) into less toxic products (Dumas et al. (1989) J. Biol. Chem. 264: 19659-19665; Vilanova, M.A. and Sogorb, E. (1999) Crit. Rev. Tox. 29:21-57). For example, the hydrolysis of the organophosphate compound paraoxon by PTE is shown in Scheme 1. One of the by-products from the degradation (hydrolysis) of paraoxon is p- nitrophenol. This compound absorbs light at a wavelength of approximately 405 nm, thus it is visible as a bright yellow colour. When PTE is in the presence of paraoxon, p- nitrophenol will be formed, and a bright yellow colour will appear. This reaction is utilized herein in a colorimetric assay for detecting OPs. PTEs are also known to degrade N-acyl homoserine lactone compounds.

Scheme 1

Paraoxon diethyl phosphate p-nitrophenol

The phosphotriesterase enzyme SsoPox is a thermostable enzyme, that remains stable even under other harsh conditions. This characteristic renders SsoPox an attractive candidate for chemical immobilization to paper. Accordingly, in one embodiment of the disclosure, native SsoPox was chemically immobilized on paper. In another embodiment of the disclosure, SsoPox-CBD fusion protein was also successfully immobilized on paper. Both methods of immobilization yielded paper that can detect OPs.

In an embodiment of the application, the SsoPox-CBD fusion protein comprised Sso?ox and a family Ha cellulose binding domain (CBD) from Cellulomonas fimi. This CBD can bind to Avicel cellulose irreversibly (Ong et al. (1993) Biotechnol. Bioeng. 42:410-419).

Thermostable hydrolytic enzymes have also been found to degrade N-acyl homoserine lactones by hydrolysis of the lactone ring as shown in Scheme 2 (Afriat L, Roodveldt C, Manco G, Tawfik DS. 2006. Biochemistry 45: 13677-13686). N-acyl homoserine lactones are bacterial quorum sensing molecules that regulate a number of processes, and enzymes that degrade these signals may be useful to control bacterial production of virulence factors, biofilm formation and biofouling (Gonzalez JE, Keshavan ND. 2006. Microbiol MoI Biol Rev. 70:859-875). For example, it has been shown that transgenic plants engineered to express a bacterial lactonase are more resistant to damage by the plant pathogen, Erwinia caratovora (Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH. (2001) Nature 411 :813-817).

Scheme 2

Paper functionalized with a thermostable hydrolytic enzyme such as SsoFox is therefore useful as a bioactive filter to remove N-acyl homoserine lactones in an aqueous environment thereby reducing bacterial colonization and biofilm formation in pipes, bioreactors etc.

Accordingly, the present disclosure includes a method of producing a bioactive paper for detecting, removing and/or detoxifying one or more compounds comprising at least one hydrolyzable bond, the method comprising contacting the paper with a thermostable hydrolytic enzyme under conditions for the attachment of the enzyme to the paper, wherein said enzyme hydrolyzes the one or more compounds. Also included in the present disclosure is a bioactive paper having a thermostable hydrolytic enzyme attached thereto. In an embodiment of the disclosure the one or more compounds comprising at least one hydrolysable bond are selected from an organophosphorus compound and an N-acyl homoserine lactone and hydrolysis of the one or more compounds by the thermostable hydrolytic enzyme results in the detoxification of the one or more compounds. In a further embodiment of the present disclosure, hydrolysis of the one or more compounds by the thermostable hydrolytic enzyme results a detectable change on the paper. In another embodiment the detectable change is quantifiable. The present disclosure also includes a method of detecting one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a solution comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and (b) observing a detectable change on the paper due to the hydrolysis of the one or more compounds by the enzyme, wherein the detection of a change on the paper indicates the presence of the one or more of the compounds in the solution.

The present disclosure also includes a method of removing or detoxifying one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a sample comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and

(b) optionally, observing a detectable change on the paper due to the hydrolysis of the one of more compound by the enzyme, wherein, the contacting of the sample with the paper, results in hydrolysis of the one or more compounds and the hydrolysis of the one or more compounds removes or detoxifies the one or more compounds.

In an embodiment of the disclosure, the thermostable hydrolytic enzyme is modified to include a Cellulose Binding Domain (CBD) and the enzyme is attached to the paper via the CBD. The CBD is any such entity known in the art. There are well over 100 different CBDs known in the art (Shoseyov et al. inNovations: Newsletter for

Novagen Inc. No. 7, August 1997) that are classified into at least 11 families. In an o

embodiment of the present disclosure the CBD is from Cellulomonas fimi or an analog or fragment thereof, said analog or fragment retaining the cellulose binding portion of the native protein. The fusion of the CBD to the thermostable hydrolytic enzyme may be performed using standard recombinant DNA technology (see e.g. Sambrook et al. (2001), Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press). The CBD may be fused to the C-terminus, the N-terminus or intermediately within the thermostable hydrolytic enzyme. In an embodiment of the disclosure the CDB is fused to the C- terminus of the thermostable hydrolytic enzyme.

In another embodiment of the disclosure, the thermostable hydrolytic enzyme is attached to the paper by means of a covalent bond or is otherwise linked (e.g. through electrostatic, hydrogen-bonding, bioaffinity, covalent interactions or combinations thereof) to the surface of the paper. In an embodiment of the disclosure, the enzyme is attached to the paper by a covalent linkage. For example, the paper is first treated with periodate or another suitable oxidizing agent which oxidizes the glucosyl units in the paper to aldehyde units which react with amine groups in the enzyme to form linkages via Schiff bases.

In further embodiments of the disclosure, the thermostable hydrolytic enzyme may be attached to the paper through entrapment inside or on the surface of a sol-gel material (for example a silica or titania based sol-gel material). In another embodiment the thermostable hydrolytic enzyme may be attached to the paper through entrapment inside or on the surface of a microgel, for example microgel-based inks that have been specifically developed for paper- supported biosensing applications (Su, S. et al. Biomacromolecules, 2008, 9:935-941). In another embodiment, special derivatized paper may be used to allow the enzyme to be attached to the surface of the paper. In still another embodiment, the cellulose paper is impregnated with alumina nanofibrils which then bind to the thermostable hydrolytic enzyme.

In a further embodiment of the disclosure, the thermostable hydrolytic enzyme is a phosphotriesterase (PTE). In a particular embodiment of the disclosure, the thermostable hydrolytic enzyme is a thermostable PTE from Sulfolobus solfataricus. In an embodiment of the disclosure, the one or more compounds comprising at least one hydrolyzable bond are any compound that is able to be hydrolyzed by the thermostable hydrolytic enzyme. In a further embodiment the one or more compounds are selected from an organophosphorus compound and an N-acyl homoserine lactone. The organophosphorus is any organophosphorus compound that is hydrolyzed by the enzyme. In specific embodiments of the disclosure, the organophosphorus compound is selected from paraoxon, methylparaoxon, parathion, methyl parathion, Dursban, coumaphos and diazinon (Merone, L., Mandrich, L., Rossi, M. and Manco, G. (2005) Extremophiles. 9: 297-305). In an embodiment of the disclosure, the organophosphorus compound is paraoxon.

In a further embodiment of the disclosure, the hydrolysis of the one or more compounds comprising at least one hydrolyzable bond produces a detectable change on the paper. For example, hydrolysis of paraoxon and similar compounds results in the generation of />-nitrophenol which is a yellow compound, the formation of which, on the paper, is detectable by visualization. In an embodiment of the disclosure, the hydrolysis of the compound may be detected by observing a color change on the paper (colormetric assay). The color change may be quantified, for example, using a digital camera with a macrofocus lens and using standard image analysis software or using an image scanner. In further embodiments of the disclosure, the compound is modified to include a moiety that is detectable upon cleavage by the enzyme. A detectable change in the enzyme and/or compound includes, for example, changes in the color, fluorescence, ultraviolet or infrared properties of the enzyme and/or compound.

In an embodiment of the present disclosure, the contacting of the paper with a thermostable hydrolytic enzyme under conditions for the attachment of the enzyme to the paper is done using a micropipette. Alternatively, the colloidal support particles are formulated as an ink and are deposited on the paper using any printing technique known in the art. For a review of the printing techniques that may be applied to bioactive paper and that are known in the art see Aikio, S. et al., Bioactive paper and fibre products: Patent and literary survey, VTT Working Papers, Julkaisija - Utgivare Publisher, (2006) ISBN 951-38-6603-3. When the enzyme does not contain a CBD, the paper is suitably pretreated with sodium periodate prior to contacting with the enzyme. The periodate oxidizes the glucosyl units in the paper producing free aldehdye groups which form Schiff bases with the amine groups in the enzyme. In embodiments of the disclosure, the assay for detecting the one or more compounds may be performed, for example, in a manner analogous to paper chromatography. The enzyme is attached to a particular area of a paper strip (the detectable area) and then the solution suspected of containing the compound(s) to be detected is put on the paper strip and the strip placed in a developing buffer. Once the developing buffer has traveled up the paper strip carrying the solution to be tested, a color or fluorescence change should be detected in the detection area for a positive result. Another method for performing the assay to detect the one or more compounds with paper, is to deposit the enzyme on the paper and then do an incubation experiment (i.e. immerse the paper in the solution comprising, or suspected of comprising, one or more of the compounds to get a signal in the detection area.) To detect the interaction between the enzyme and the compound to be detected, an observable change in the "detection" area on the paper occurs as a result of the interaction between the enzyme and the compound. The "detection area" refers to that area on the paper or paper product where the enzyme has been deposited. Such detection methods are known to those skilled in the art and may include, for example, a detectable change in the color, fluorescence, ultraviolet or infrared properties of the enzyme and/or compound.

In further embodiments of the disclosure, the detectable change on the paper due to the hydrolysis of the compound by the enzyme is compared to a control to assist in the quantification of the compound and/or activity of the enzyme.

The present disclosure also includes uses of the bioactive paper having a thermostable hydrolytic enzyme attached thereto, to detect, detoxify and/or remove one or more compounds comprising at least one hydrolyzable bond, such as organophosphorus compounds in, for example, environmental or food-based samples. For example, the bioactive paper of the present disclosure can be used to make paper- based filters or paper-based facemasks to detect and/or remove pesticides minimizing exposure of individuals to these toxic chemicals.

The present disclosure further includes uses of the bioactive paper having a thermostable hydrolitic enzyme attached thereto, to hydrolyse, and thereby remove, N- acyl homoserine lactone in environmental or food-based samples. The removal of N- acyl homoserine lactone is useful to disrupt a variety of bacterial processes that depend on quorum sensing eg. production of virulence factors, biofilm formation, food spoilage and biofouling. For example, bioactive papers as described above may be used for packaging foods, such as fruit, vegetables, meat, bread, or other items such as fresh flowers, or as paper liners for cosmetics.

The following non-limiting examples are illustrative of the present disclosure: EXAMPLES

Example 1 : EXPRESSION AND PURIFICATION OF NATIVE SroPox

The gene SSO2522 was PCR amplified from the genomic DNA of Sulfolobus solfataricus P2 and subcloned into the expression vector pT7-7. Two plasmid constructs were made: one that encodes the native hydrolase enzyme and the other that encodes the hydrolase with an in-frame translational fusion at the C-terminus to the Cellulose

Binding Domain (CBD) Cex (Ong, E., Gilkes, N.R., Miller, R.C., Warren, R.A.J, and

Kilburn, D.G. (1993) Biotechnol. Bioeng. 42:410-419.) from Cellulomonas fimi. The two plasmids were transformed separately into E. coli Rosetta 2 and E. coli BL21(DE3) cells, respectively. The protein was expressed and purified by anion and hydrophobic column chromatography (Figure 1).

Example 2: EXPRESSION AND PURIFICATION OF PTE

A PTE gene optimized for expression enhancement (Figure 2) [SEQ ID NO: 1] in plasmid pT7-7 was subcloned into the broad host range expression vector, pVLT31 (de Lorenzo, V., Eltis, L., Kessler, B. & Timmis, K. N. (1993) Gene 123, 17-24.) using Xbal and HindIII restriction sites

Over-expression of PTE in Pseudomonas putida KT2442 A starter culture is prepared by inoculating a single colony from a streak plate into 50 mL of LB broth supplemented with 15 μg/mL of tetracycline. This culture is grown overnight (-16 hours) at 37°C, shaking at 210 rpm. The next day, 25 mL of the starter culture is inoculated into 1 L of LB broth containing 15 μg/mL of tetracycline. The culture is grown until early log phase (optical density at 600 nm between 0.4 and 0.6). IPTG and CoCl2 are added to a final concentration of 0.5 mM and 0.2 mM, respectively, to induce overexpression of PTE. The culture is incubated overnight (-24 hours) at 37°C, shaking at 210 rpm. Subsequently, cells are harvested by at 3000 x g for 10 minutes at 4°C. The resulting cell pellet is washed with 25 mM Tris-HCl pH 8.0.

PTE Purification

The cell pellet is resuspended in Buffer A (20 mM Hepes pH 8.0, 0.2 mM CoCl2). For 1 g of cells, 3 mL of Buffer A is added. The cell suspension is treated with lysozyme (0.2 mg/mL) and DNase (0.1 mg/mL) at room temperature for 20 minutes, and then frozen at -2O0C overnight. Next day, the cell suspension is thawed out, kept on ice, and sonicated for six 1 -minute cycles using a Heat Systems sonicator - ultrasonic processor with a macro tip at power 5. Cell debris is removed by centrifugation at 58,545 X g for 20 minutes at 6°C. The supernatant is heat treated at 50°C for 15 minutes. Precipitate is removed by centrifugation at 75,600 X g for 20 minutes at 100C. The heat treatment procedure is repeated for the supernatant at 600C and 700C. The heat treated lysate is loaded into a column (2 x 13 cm) containing Source 15Q resin (Amersham Pharmacia Inc.) that has been equilibrated with Buffer A + 1 M NaCl. PTE is eluted at around 15 mSi. Fractions with paraoxonase activity are pooled.

Example 3: DETECTION OF PARAOXON BY CHEMICALLY-IMMOBILIZED NATIVE SsoPox

The hydrolase SsoVox (without CBD fusion) was also expressed in E. coli. The enzyme was purified by heat treatment and chromatography. This enzyme was immobilized on paper (Whatman 3mm) treated with periodate (Figure 3). The periodate oxidizes the glucosyl units in paper producing free aldehyde groups that can then form Schiff bases with the amine groups on the enzyme. Paper strips treated with 0.0 IM NaIO4 and 0.1 M Na2CO3/NaHCO3 buffer (pH 9) are shown in Figure 3 A. Paper strips treated with water and 0.1 M Na2CO3/NaHCO3 buffer (pH 9) are shown in Figure 3B. Various amounts of hydrolase and control enzyme were spotted on the treated and untreated paper and incubated for 2h in covered Petri dishes to keep them moist. They were then washed in 1OmL buffer (2OmM Hepes + 0.2mM CoCl2, pH 8.0) and blocked with 1OmL of 5% bovine serum albumin. After which the paper strips were dipped in 5mM paraoxon and incubated at 60°Cfor Ih. Paper containing immobilized SsoVox turned yellow while no color change developed in controls.

Example 4: IMMOBILIZATION VIA CELLULOSE BINDING DOMAIN

Soluble cell extract of recombinant E. coli expressing the hydrolase-Cex fusion protein showed activity towards paraoxon (Figure 4). Strips of Whatman 3mm paper were wetted with 250μL water and then 250μL E. coli cell lysate (A) or water was applied (B). The paper strips were incubated in Petri dishes (to keep moist) at room temperature for 4h; the strips were then washed three times with 1OmM potassium phosphate buffer (pH 7.0) and once with water. The strips were dipped in 5mM paraoxon and incubated at 6O0C for an hour. Colour change was detected for paper strips treated with the enzyme after 1 hour, which demonstrates that paper -immobilized &oPox-CBD can serve as OP sensor (See Figure 3).

While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

WE CLAIM:

1. A method of producing a bioactive paper for detecting, removing and/or detoxifying one or more compounds comprising at least one hydrolysable bond, the method comprising contacting the paper with a thermostable hydrolytic enzyme under conditions for the attachment of the enzyme to the paper, wherein the enzyme hydrolyzes the one or more compounds

2. The method according to claim 1, wherein the thermostable hydrolytic enzyme is modified to include a Cellulose Binding Domain (CBD) and the enzyme is attached to the paper via the CBD.

3. The method according to claim 2, wherein the CBD is from Cellulomonas fimi or an analog or fragment thereof, said analog or fragment retaining the cellulose binding portion of the native protein.

4. The method according to claim 3, wherein the CBD is fused to the C-terminus of the thermostable hydrolytic enzyme.

5. The method according to claim 1, wherein the enzyme is attached to the paper via hydrophobic, covalent, electrostatic, hydrogen-bond or bioaffinity linkages or via combinations thereof.

6. The method according to any one of claims 1-5, wherein the thermostable hydrolytic enzyme is a phosphotriesterase (PTE).

7. The method according to claim 6, wherein the thermostable PTE is from Sulfolobus solfataricus.

8. The method according to any one of claims 1-7, wherein the one or more compounds comprising at least one hydrolysable bond are selected from an organophosphorus compound and an N-acyl homoserine lactone.

9. The method according to claim 8, wherein the organophosphorous compound is selected from paraoxon, methylparaoxon, parathion, methyl parathion, Dursban, coumaphos and diazinon.

10. The method according to claim 9, wherein the organophosphorus compound is paraoxon.

11. A bioactive paper having a thermostable hydrolytic enzyme attached thereto prepared according the method according to any one of claims 1-10.

12. A method of detecting one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a solution comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and

(b) observing a detectable change on the paper due to the hydrolysis of the one or more compounds by the enzyme, wherein the detection of a change on the paper indicates the presence of the one or more of the compounds in the solution.

13. The method according to claim 12, wherein the one or more compounds comprising at least one hydrolysable bond are selected from an organophosphorus compound and an N-acyl homoserine lactone.

14. The method according to claim 13, wherein the organophosphorus compound is selected from paraoxon, methylparaoxon, parathion, methyl parathion, Dursban, coumaphos and diazinon.

15. The method according to claim 14, wherein the organophosphorus compound is paraoxon.

16. The method according to any one of claims 12-15, wherein the thermostable hydrolytic enzyme is modified to include a Cellulose Binding Domain (CBD) and the enzyme is attached to the paper via the CBD.

17. The method according to claim 16, wherein the CBD is from Cellulomonas fimi or an analog or fragment thereof, said analog or fragment retaining the cellulose binding portion of the native protein.

18. The method according to claim 17, wherein the CBD is fused to the C-terminus of the thermostable hydrolytic enzyme.

19. The method according to any one of claims 12-15, wherein the enzyme is attached to the paper via hydrophobic, covalent, electrostatic, hydrogen-bond or bioaffϊnity linkages or via combinations thereof.

20. The method according to any one of claims 12-19, wherein the thermostable hydrolytic enzyme is a phosphodiesterase (PTE).

21. The method according to claim 20, wherein the thermostable PTE is from Sulfolobus solfataricus.

22. The method according to any one of claims 12-21, wherein the detectable change is selected from changes in the color, fluorescence, ultraviolet and infrared properties.

23. The method according claim 22, wherein the detectable change is a change in color.

24. The method according claim 23, wherein the color change is quantified using a digital camera with a macrofocus lens and using standard image analysis software or using an image scanner.

25. The method according to any one of claims 12-24, wherein the detectable change on the paper due to the hydrolysis of the compound by the enzyme is compared to a control.

26. A method of removing or detoxifying one or more compounds comprising at least one hydrolyzable bond, the method comprising:

(a) contacting paper with a thermostable hydrolytic enzyme attached thereto with a sample comprising, or suspected of comprising, one or more of the compounds, said compounds being hydrolyzed by the enzyme; and (b) optionally, observing a detectable change on the paper due to the hydrolysis of the one of more compound by the enzyme, wherein, the contacting of the sample with the paper, results in hydrolysis of the one or more compounds and the hydrolysis of the one or more compounds removes or detoxifies the one or more compounds.

27. The method according to claim 26, wherein the one or more compounds comprising at least one hydrolysable bond are selected from an organophosphorus compound and an N-acyl homoserine lactone.

28. The method according to claim 27, wherein the organophosphorus compound is selected from paraoxon, methylparaoxon, parathion, methyl parathion, Dursban, coumaphos and diazinon.

29. The method according to claim 28, wherein the organophosphorus compound is paraoxon.

30. The method according to any one of claims 26-29, wherein the thermostable hydrolytic enzyme is modified to include a Cellulose Binding Domain (CBD) and the enzyme is attached to the paper via the CBD.

31. The method according to claim 30, wherein the CBD is from Cellulomonas fimi or an analog or fragment thereof, said analog or fragment retaining the cellulose binding portion of the native protein. o

32. The method according to claim 31, wherein the CBD is fused to the C-terminus of the thermostable hydrolytic enzyme.

33. The method according to any one of claims 26-29, wherein the enzyme is attached to the paper via hydrophobic, covalent, electrostatic, hydrogen-bond or bioaffinity linkages or via combinations thereof.

34. The method according to any one of claims 26-33, wherein the thermostable hydrolytic enzyme is a phosphotriesterase (PTE).

35. The method according to claim 34, wherein the thermostable PTE is from Sulfolobus solfataricus.

36. A use of the bioactive paper according to claim 1 1 , to detect, remove and/or detoxify toxic compounds comprising at least one hydrolyzable bond, wherein the hydrolysis of the compounds results in detection, removal and/or detoxification of the compound.

37. A use of the bioactive paper according to claim 11 to prepare paper-based filters or paper-based facemasks to detect, remove and/or detoxify compounds comprising at least one hydrolyzable bond, wherein the hydrolysis of the compounds results in detection, removal and/or detoxification of the compound.

38. The use according to claim 36 or 37 wherein the one or more compounds comprising at least one hydrolysable bond are selected from an organophosphorus compound and an N-acyl homoserine lactone.

39. A use of the bioactive paper according to claim 11 to reduce bacterial colonization or biofilm formation.

40. A use of the bioactive paper according to claim 11 in vegetable and fruit, meat, bread packaging.


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