ANTI-FOULING PAINTS AND COATINGS

Abstract

Disclosed herein are a materials such as a coating, an elastomer, an adhesive, a sealant, a textile finish, a wax, and a filler for such a material, wherein the material includes an proteinaceous molecule such as a peptide and/an enzyme that confer a metal binding, an anti-fouling and/or an antibiotic property to the material. In particular, disclosed herein are marine coatings such as a marine paint that comprise an anti-fouling peptide sequence that reversibly binds a metal cation that is toxic to a fouling organism. Also disclosed herein are methods of reducing fouling on a surface by treating the surface with a metal binding peptide.

Claims

1. (canceled)

2. A method of imparting marine anti-fouling properties on a surface of an object, comprising providing an object having a surface thereof in contact with an aqueous marine environment during use of the object, wherein the aqueous marine environment comprises at least one metal ion that chemically impedes growth of at least one marine-based surface-fouling organism when brought into contact therewith at a sufficient concentration thereof; prior to the object being subject to an instance of said use, treating the surface of the object with a coating composition comprising a metal binding peptide to which said at least one metal ion of the aqueous marine environment binds when contacted therewith; and subjecting the object to an instance of said use after performing said treating to thereby cause a quantity of said at least one metal ion from within the aqueous marine environment to bind to the coating composition comprising the metal binding peptide for creating an anti-fouling coating on the surface of the object, wherein the quantity of said at least one metal ion bound to the coating composition comprising the metal binding peptide is at least 60% greater than a quantity of said at least one metal ion bound from within the aqueous marine environment to the coating composition when not comprising the metal binding peptide.

3. The method of claim 2 wherein the metal binding peptide is His6 peptide.

4. The method of claim 2 wherein: the at least one metal ion is at least one of copper ion and nickel ion; and the aqueous marine environment is ocean water comprising at least one of copper ion and nickel ion whereby the anti-fouling coating comprises at least one of copper ion and nickel ion.

5. The method of claim 4 wherein the metal binding peptide is His6 peptide.

6. The method of claim 2 wherein the metal binding peptide comprises a His-tag sequence.

7. The method of claim 6 wherein the metal binding peptide is His6 peptide.

8. The method of claim 6 wherein the coating composition comprises a paint and an aqueous solution of the His6 peptide mixed at a ratio of 1 about part of the aqueous solution of the His6 to 7 about parts of the paint.

9. The method of claim 8 wherein: the at least one metal ion is at least one of copper ion and nickel ion; and the aqueous marine environment is ocean water comprising at least one of copper ion and nickel ion whereby the anti-fouling coating comprises at least one of copper ion and nickel ion.

10. The method of claim 9 wherein the metal binding peptide is His6 peptide.

11. An anti-fouling bottom surface of an aquatic vessel made by a process comprising: providing an aquatic vessel having at least a portion of a bottom surface thereof in continuous contact with an aqueous marine environment during use of the vessel, wherein the aqueous marine environment comprises at least one metal ion that chemically impedes growth of at least one marine-based surface-fouling organism when brought into contact therewith at a sufficient concentration thereof, wherein the at least a portion of the bottom surface is exposed to the aqueous marine environment when submersed therein; and prior to the object being subject to an instance of said use, treating the at least a portion of the bottom surface with a coating composition comprising a metal binding peptide to which said at least one metal ion binds when contacted therewith to cause a quantity of said at least one metal ion from within the aqueous marine environment to bind to the coating composition comprising the metal binding peptide when the at least a portion of the bottom surface is submersed within the aqueous marine environment after performing said treating to thereby cause for creating an anti-fouling coating on the at least a portion of the bottom surface, wherein the quantity of said at least one metal ion bound to the coating composition comprising the metal binding peptide is at least 60% greater than a quantity of said at least one metal ion bound from within the aqueous marine environment to the coating composition when not comprising the metal binding peptide.

12. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 11 wherein the metal binding peptide is His6 peptide.

13. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 11 wherein: the at least one metal ion is at least one of copper ion and nickel ion; and the aqueous marine environment is ocean water comprising at least one of copper ion and nickel ion whereby the anti-fouling coating comprises at least one of copper ion and nickel ion.

14. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 13 wherein the metal binding peptide is His6 peptide.

15. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 11 wherein the metal binding peptide comprises a His-tag sequence.

16. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 15 wherein the metal binding peptide is His6 peptide.

17. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 15 wherein the coating composition comprises a paint and an aqueous solution of the His6 peptide mixed at a ratio of 1 about part of the aqueous solution of the His6 to 7 about parts of the paint.

18. The anti-fouling bottom surface of the aquatic vessel made by a process of claim 17 wherein: the at least one metal ion is at least one of copper ion and nickel ion; and the aqueous marine environment is ocean water comprising at least one of copper ion and nickel ion whereby the anti-fouling coating comprises at least one of copper ion and nickel ion.

Description

SPECIFIC EXAMPLES

[0283] The general effectiveness of various embodiments is demonstrated in the following Examples. Some methods for preparing compositions are illustrated. Starting materials are made according to procedures known in the art or as illustrated herein. The following Examples are provided so that the embodiments might be more fully understood. These Examples are illustrative only and should not be construed as limiting in any way, as other material formulations such as a polymeric material, a surface treatment (e.g., a different paint formulation), and/or a filler, comprising different biomolecular compositions (e.g., a different purified or partly purified enzyme, a different cell-based particulate material comprising an enzyme, a peptide, a polypeptide) may be prepared.

[0284] Example 1This Example describes a screening method for identifying an anti-biological activity of a peptide library.

[0285] The screening method for a coating generally includes: creating a synthetic peptide combinatorial library using known methods and materials; testing a battery of biological entities that are known to, or suspected of, infesting a material formulation (e.g., a building material, an object) having a biological-infestation susceptible surface with aliquots of the synthetic peptide library, wherein each aliquot comprises an equimolar mixture of peptides in which at least one of the terminal amino acid residues (e.g., C-terminal residues) are defined and which residues are in common for each peptide in the mixture; admixing said aliquots with a coating typically used on such building material and coating a surface with the admixture; allowing an appropriate period of time for growth of the biological entity under suitable culture conditions; comparing the growth of the treated biological entity(s) with untreated control biological entity(s); identifying which of the aliquots demonstrate an anti-biological activity [e.g., reduced the growth of the biological entity(s)]; and, optionally, assessing the relative growth inhibitory activity of each aliquot compared to that of other aliquots (e.g., comparing IC.sub.50 data). Of course, such a screening method may be adapted for other material formulations than a coating, may be used assay for anti-biological activity in (e.g., on the surface of, within) a material formulation, and may be used to assay other proteinaceous molecules than those of a peptide library.

[0286] In the above-referenced U.S. Pat. Nos. 6,020,312; 5,885,782; and 5,602,097 an iterative process was used to identify active peptide sequences with broad spectrum anti-biological (e.g., antifungal) activity. A representative method employs a hexapeptide library with the first two amino acids in each peptide chain individually and specifically defined and with the last four amino acids consisting of equimolar mixtures of 20 amino acids. Four hundred (400) (20.sup.2) different peptide mixtures each comprising 130,321 (19.sup.4)(cysteine was eliminated) individual hexamers were evaluated. In such a peptide mixture, the final concentration for each peptide was 9.38 ng/ml, in a mixture comprised of 1.5 mg (peptide mix)/ml solution. This mixture profile assumed that an average peptide has a molecular weight of 785. This concentration was sufficient to permit testing for anti-biological activity. Both D- and L-amino acid comprising peptides may be constructed and tested to identify peptide compositions that can inhibit or kill biological entity(s) that can grow on the surfaces of inanimate objects. Peptide compositions comprising substantially homogeneous peptide compositions, as well as mixtures of peptides derived from amino acids that are between 3 to 25 residues in length (a length readily accomplished using standard peptide synthesis procedures), especially six residues in length, are disclosed in U.S. Pat. Nos. 6,020,312; 5,885,782; and 5,602,097. An example of an anti-biological peptide that inhibits or kills one or more fungus that infests and grows on the surfaces of inanimate objects comprises a hexapeptide having the amino acid sequence Phe Arg Leu Lys Phe His (SEQ ID No. 41).

[0287] Another example of a method for selecting antibiotic compositions includes first creating a synthetic peptide combinatorial library as described herein. Next, as further described in detail herein, a step of contacting a battery of fungal cells with aliquots of the synthetic peptide combinatorial library, each of which aliquots represents an equimolar mixture of peptides in which at least the two C-terminal amino acid residues are defined and which residues are in common for each peptide in said mixture may be accomplished. After allowing an appropriate period for growth, a next step may be accomplished in which the growth of the battery of fungal cells as compared to untreated control cells may be measured. Lastly, a determination may be made of which of the aliquots reduces (e.g., reduces to the relatively greatest extent) the growth of fungal cells in a coating overall in the battery of fungal cells. Of course, the same method may be carried out in which each of the aliquots represents an equimolar mixture of peptides in which at least three, four, five or more C-terminal amino acid residues are defined (depending upon the overall length of the ultimate peptide in the SPCL). Typically, such increasingly defined aliquots may be sequentially tested to select the succeeding candidate peptides for testing. Thus, an additional step in the method entails utilizing the determination of which of the aliquots reduces the growth of fungal cells in a coating overall in said battery of fungal cells to select which aliquots to next test of a synthetic peptide combinatorial library where at least one additional C-terminal amino acid residue may be defined.

[0288] Example 2This Example describes methods of identifying an anti-biological biomolecular composition (e.g., a proteinaceous molecule).

[0289] The testing methods described in U.S. Pat. Nos. 6,020,312; 5,885,782; and 5,602,097 may be employed to screen one or more peptidic agent(s) (e.g., a peptide library) for anti-biological activity against a wide variety of genera and species. The methods may be modified to screen against organisms that are known to, or suspected of, infesting, for example, construction materials or other vulnerable materials and surfaces. In some embodiments, examples of cells used for screening the peptide library include members of the fungal genera Stachybotrys (especially Stachybotrys chartarum), Aspergillus species (sp.), Penicillium sp., Fusarium sp., Alternaria dianthicola, Aureobasidium pullulans (aka Pullularia pullulans), Phoma pigmentivora and Cladosporium sp. Cell culture conditions may also be modified appropriately to provide favorable growth and proliferation conditions, using techniques of the art. The above-mentioned methods may be used to identify one or more peptidic agent(s) [e.g., a peptide(s), group(s) of peptide(s)] demonstrating broad-spectrum anti-biological activity. Similar methods may be used to identify one or more peptidic agent(s) [e.g., a peptide(s), group(s) of peptide(s)] that target specific genera or species. For example, certain of the peptides of particular usefulness in the coatings, as disclosed in U.S. Pat. Nos. 6,020,312; 5,602,097; and 8,885,782, exhibit variable abilities to inhibit fungal growth as adjudged by the minimal inhibitory concentrations (MIC mg/ml) and/or the concentrations to inhibit growth of fifty percent of a population of fungal spores (IC.sub.50 mg/ml). MICs may range depending upon peptide additive and target organism from about 3 to about 300 mg/ml, while IC.sub.50's may range depending upon peptide additive and target organisms from about 2 to about 100 mg/ml. Target organisms susceptible to these amounts include Fusarium oxysporum, Fusariam Sambucinum, Rhizoctonia Solani, Ceratocystis Fagacearum, Pphiostoma ulmi, Pythium ultimum, Magaporthe Aspergillus nidulans, Aspergillus fumigatus, and Aspergillus Parasiticus. Alternatively, any other suitable peptide/polypeptide/protein screening method could be used instead to identify anti-biological peptide candidates for testing as active anti-biological agents in a material formulation (e.g., a paint, a coating).

[0290] The mode of action of anti-biological peptides, polypeptides and/or proteins, by which they exert their anti-biological effect(s) (e.g., inhibitory effect, bioicidal activity), can be varied. For example, certain peptides may operate to destabilize fungal cell membranes, while the modes of action of others could include disruptions of macromolecular synthesis or metabolism. While the modes of action of some antifungal peptides have been determined (see, e.g., Fiedler, H. P., et al. 1982. J Chem. Technol. Biotechnol. 32:271-280; Isono, K. and S. Suzuki. 1979. Heterocycles 13:333-351), mechanisms which explain their modes of action and specificity have typically not yet been determined. Initial studies to elucidate antifungal mode of action of peptides involves a physical examination of mycelia and cells to determine if the peptides can perturb membrane functions responsible for osmotic balance, as has been observed for other peptides (Zasloff, M. 1987. Proc. Natl. Acad. Sci. USA 84:5449-5453). Disruption of appressorium formation may also be the mechanism by which some peptides inhibit fungal growth (see e.g., published U.S. patent application Ser. No. 10/601,207, expressly incorporated herein by reference in its entirety). For the purposes of preparing and using anti-biological peptides, polypeptides and/or proteins as active anti-biological agents in a material formulation (e.g., a paint, a coating), the mechanism by which the anti-biological effect may be exerted on a biological entity (e.g., one or more cells) may, in some embodiments, may not be understood.

[0291] Example 3This Example describes coating formulations comprising a metal binding and/or an anti-biological biomolecular composition.

[0292] A paint or coating composition may comprise an anti-biological biomolecular composition (i.e., one or more peptides, polypeptides or proteins) such as, for example, as described herein (e.g., a metal binding proteinaceous composition, an anti-fouling proteinaceous composition). For example, an anti-biological proteinaceous molecule may be used as a partial or complete substitute (replacement) for another biocide and/or biostatic typically used in a biological entity prone composition. It is contemplated that about 0.0001% to about 100%, including all intermediate ranges and combinations thereof, of a conventional anti-biological agent component in a coating formulation may be substituted by an anti-biological biomolecular composition. For example, the concentration of anti-biological proteinaceous molecule may exceed 100%, by weight or volume, of the non-proteinaceous anti-biological component (e.g., a bioicide, a bioistatic) being replaced. In another example a conventional non-proteinaceous anti-biological component may be replaced with an anti-biological proteinaceous molecule equivalent to 0.001% to 500% (by weight, or by volume), including all intermediate ranges and combinations thereof, of the substituted anti-biological component. For example, to produce a coating with similar biological resistance properties as a non-substituted formulation, it may require that 20% (e.g., 0.2 kg) of a chemical bioicide may be replaced by 10% (e.g., 0.1 kg) of an anti-biological proteinaceous molecule. In another exemplary formulation, to produce a coating with similar biological resistance as a non-substituted formulation, it may require replacing 70% of a chemical bioicide (e.g., 0.7 kg) with the equivalent of 127% (e.g., 1.27 kg) of anti-biological proteinaceous molecule. The various assays described herein, or as would be known in the art in light of the present disclosure, may be used to determine the biological resistance properties of a material formulation (e.g., a coating, a film) produced by direct addition of an anti-biological biomolecular composition and/or substitution of some or all of a non-biomolecular or chemical anti-biological component by an anti-biological biomolecular composition. Such additives may be directly admixed with the coating, applied as a primer coating, applied as an overcoat, or any combination of these application techniques.

[0293] It is contemplated that any previously described formulation of a coating composition may be modified to incorporate a biomolecular composition. Examples of described coating compositions include over 200 industrial water-borne coating formulations (e.g., air dry coatings, air dry or force air dry coatings, anti-skid of non-slip coatings, bake dry coatings, clear coatings, coil coatings, concrete coatings, dipping enamels, lacquers, primers, protective coatings, spray enamels, traffic and airfield coatings) described in Industrial water-based paint formulations, 1988, over 550 architectural water-borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, interior paints, interior enamels, interior coatings, exterior/interior paints, exterior/interior enamels, exterior/interior primers, exterior/interior stains), described in Water-based trade paint formulations, 1988, the over 400 solvent borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, exterior sealers, exterior fillers, exterior primers, interior paints, interior enamels, interior coatings, interior primers, exterior/interior paints, exterior/interior enamels, exterior/interior coatings, exterior/interior varnishes) described in Solvent-based paint formulations, 1977; and the over 1500 prepaint specialties and/or surface tolerant coatings (e.g., fillers, sealers, rust preventives, galvanizers, caulks, grouts, glazes, phosphatizers, corrosion inhibitors, neutralizers, graffiti removers, floor surfacers) described in Prepaint Specialties and Surface Tolerant Coatings, by Ernest W. Flick, Noyes Publications, 1991.

[0294] From these representative formulations, it will be readily appreciated that a wide variety of coating compositions (e.g., paints) may be improved by addition of a biomolecular composition. Some of these include industrial water-borne coating formulations (e.g., air dry coatings, air dry or force air dry coatings, anti-skid of non-slip coatings, bake dry coatings, clear coatings, coil coatings, concrete coatings, dipping enamels, lacquers, primers, protective coatings, spray enamels, traffic and airfield coatings); architectural water-borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, interior paints, interior enamels, interior coatings, exterior/interior paints, exterior/interior enamels, exterior/interior primers, and exterior/interior stains); solvent borne coating formulations (e.g., exterior paints, exterior enamels, exterior coatings, exterior sealers, exterior fillers, exterior primers, interior paints, interior enamels, interior coatings, interior primers, exterior/interior paints, exterior/interior enamels, exterior/interior coatings, and exterior/interior varnishes); and prepaint specialties and/or surface tolerant coatings (e.g., fillers, sealers, rust preventives, galvanizers, caulks, grouts, glazes, phosphatizers, corrosion inhibitors, neutralizers, graffiti removers and floor surfacers).

[0295] For example, an anti-biological and/or metal binding coating (e.g, a paint) comprising a biomolecular composition may then be tested and used as described elsewhere herein, or the product may be employed for any other suitable purpose as would be recognized in the art in light of this disclosure. For instance, the physical properties (e.g., purity, density, solubility, volume solids and/or specific gravity, rheology, viscometry, and particle size) of the resulting anti-biological liquid paint or other coating product, can be assessed using standard techniques that are known in the art and/or as described in PAINT AND COATING TESTING MANUAL, 14.sup.th ed. of the Gardner-Sward Handbook, J. V. Koleske, Editor (1995), American Society for Testing and Materials (ASTM), Ann Arbor, Mich., and applicable published ASTM test methods. Alternatively, any other suitable testing method of the art in light of the present disclosures may be employed for assessing physical properties of the coating mixture comprising an above-described biomolecular composition.

[0296] Example 4This Example describes latex paints with an anti-biological peptidic agent.

[0297] Both the interior latex (Olympic Premium, flat, ultra white, 72001) and acrylic paints (Sherwin Williams DTM, primer/finish, white, B66W1; 136-1500) appeared to be toxic to both Fusarium and Aspergillus. Therefore, eight individual wells (48-well microtito plate) of each paint type were extracted on a daily basis with 1 ml of phosphate buffer for 5 days (1-4 & 6) and then allowed the plates were allowed to dry before running the assay. Each well comprised 16 ul of respective paint.

[0298] Extract testing: The extract from two wells each of the two paints for each day was evaluated for toxicity by mixing the extract 1:1 with 2 medium and inoculating with spores (10.sup.4) of Aspergillus or Fusarium. The extracts had no affect on growth of either test fungus.

[0299] Well testing: The extracted and non-extracted wells for each of the paints were tested with a range of inoculum levels in growth medium using the two different fungi. For Fusarium the range was 10.sup.1-10.sup.4 and for Aspergillus 10.sup.2-10.sup.5. Well Testing of Acrylic Paint Plates: Both Fusarium and Aspergillus grew in all extracted wells at all inoculum levels. Only Aspergillus grew in non-extracted wells at the 10.sup.5 level and not at lower levels indicative of an inherent biocidal capability. Well Testing of Latex Paint Plates: Fusarium grew in the extracted wells only at the 10.sup.4 inoculum level but not at 10.sup.1-10.sup.3. Aspergillus grew in all extracted wells showing an inoculum level effect. No growth was observed for either Fusarium or Aspergillus in non-extracted wells.

[0300] Conclusion: Extraction of the toxic factor(s) found in both paints was possible. However, it appeared that it may be less extractable from the latex paint.

[0301] Evaluation of peptidic agent activity in presence of acrylic and latex paints: It was established that it was possible to extract both acrylic and latex paints dried in a 48-well format to make them non-toxic to the test microorganismsFusarium and Aspergillus. Using that information an assay was designed to determine the effect the paint has on peptidic agent activity against two test organisms.

[0302] Assay design: Coat 48-well plastic plates with 161 of acrylic or latex paint. Dry for two days under hood. Extract designated wells with 1-ml phosphate buffer changing the buffer on a daily basis for 7 days. Control wells were not extracted to confirm paint toxicity. Add 201 of peptidic agent series in duplicate to designated dry paint coated wells. Peptide, SEQ ID No. 41, series were added in a two-fold dilution series to wells and allowed to dry. The concentration of peptide added ranged from 200 g/201 to 1.5 g/201. Inoculated paint-coated plates as follows: Extracted control wells received 1801 of medium+201 of spore suspension (10.sup.4 spores/20 l of medium). Inoculum was either Fusarium or Aspergillus in each case. Non-extracted control wells received 180 l of medium+20 l of spore suspension (10.sup.4 spores/20 l of medium). Extract wells with dried peptide series received 180 l of medium+20 l of spore suspension (10.sup.4 spores/20 l of medium). In duplicate. Extract wells that did not have dried peptide series received 160 l of medium+20 l of spore suspension (10.sup.4/20 l of medium)+20 l peptide series as above. In duplicate. Plates were observed for growth over a 5-day period.

[0303] Growth and peptide controls: Used sterile non-paint coated 48 well plastic plates

[0304] Growth control wells for each test fungus received 180 l of medium+20 l of spore suspension (10.sup.4 spores/20 l of medium). Peptide activity controls received 160 l of medium+20 l of spore suspension (10.sup.4 spores/20 l of medium)+20 l peptide series as above. Peptide series were added in a two-fold dilution series to wells and range from 200 g/20 l to 1.5 g/201. Therefore, the range of peptide tested was 200 g/200 l or 1.0 g/1 (1000 g/ml) to 0.0075 g/1 (7.5 g/ml). Uninoculated medium served as blank for absorbance readings taken at 24, 48, 72, 96 and 120h.

[0305] Results: Unextracted wells comprising either latex or acrylic paint inhibited growth of both Fusarium and Aspergillus. Extracted wells comprising either latex or acrylic paint allowed growth of both Fusarium and Aspergillus. The calculated MIC for Fusarium in peptide activity control assays was 15.62 g/ml. For Aspergillus the calculated MIC was 61.4 g/ml. For extracted acrylic-coated plates the following results were obtained. Controls as stated in above. For Fusarium with dried peptide, inhibition was seen at 1000 and 500 g/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000, 500 and 250 g/ml after 4 days, and 1000 and 500 g/ml after 5 days. For Aspergillus with dried peptide, inhibition was seen at 1000 g/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 and 500 g/ml after 5 days.

[0306] For extracted latex-coated plates the following results were obtained. Controls as stated above.

[0307] For Fusarium with dried peptide, inhibition was seen at 1000 g/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 g/ml after 5 days. For Aspergillus with dried peptide, inhibition was seen at 1000 g/ml after 5 days. Spores exposed to liquid peptide added to dry paint wells were inhibited at 1000 g/ml after 5 days.

[0308] Example 5This Example describes the combined use of an anti-biological biomolecular composition and another anti-biological agent.

[0309] A material formulation (e.g, a paint composition) comprising one or more conventional anti-biological substance may be modified by addition of one or more of the anti-biological proteinaceous molecules(s) (e.g., an anti-fouling peptide) described herein. For example, the anti-biological proteinaceous molecule may comprise one or more pure anti-biological peptides of defined sequence, and/or it may include a peptide library aliquot comprising a mixture of peptides in which at least two (e.g., three, four) of the N-terminal amino acid residues are defined (as in SEQ ID Nos. 1-24). In another example, if the proteinaceous molecule comprises a mixture of metal binding peptides, at least one may have anti-biological activity.

[0310] Combining a non-proteinaceous anti-biological agent with one or more anti-biological proteinaceous molecule may provide anti-biological activity above that seen with either the proteinaceous molecule) or the non-proteinaceous molecule alone. The expected additive inhibitory activity of the combination may be calculated by summing the inhibition levels of each component alone. The combination may be assayed on the test organism to derive an observed additive inhibition. If the observed additive inhibition is greater than that of the expected additive inhibition, synergy is exhibited. More specifically, a synergistic combination of an anti-biological proteinaceous molecule (e.g., an aliquot of a peptide library comprising at least one anti-biological peptide), occurs when two or more anti-biological (e.g., cell growth-inhibitory) substances distinct from the proteinaceous molecule are observed to be more inhibitory to the growth of an evaluated organism than the sum of the inhibitory activities of the individual components alone.

[0311] An example of an assay method for determining additive or synergistic combinations comprises first creating a synthetic peptide combinatorial library. Each aliquot of the library represents an equimolar mixture of peptides in which at least the two C-terminal amino acid residues are defined. Using the testing methods described in one or more of U.S. Pat. Nos. 6,020,312, 5,885,782, and 5,602,097 it is possible to determine for each such aliquot of the synthetic peptide combinatorial library, a calculated concentration at which it may inhibit a target organism in a coating. Next, the aliquot of the synthetic peptide combinatorial library may be mixed with at least one non-peptide anti-biological compound to create a test mixture. As with the peptide component of the mixture, the baseline ability of the non-peptide anti-biological substance to inhibit the assay's organism may be determined initially. Next, the assay's organism may be contacted with the mixture being assayed, and the inhibition of growth of the organism may be measured as compared to at least one untreated control. More controls may be used, such as a control for each individual component of the mixture. Similarly, where there are more than two components being assayed, the number of controls to be used may be increased in a manner of the art of growth inhibition testing. From the separate assay results for the proteinaceous and non-proteinaceous molecules the expected additive anti-biological effect (e.g., inhibition of growth) may be determined using standard techniques. For example, after the growth inhibition assays are complete for the combination of proteinaceous and non-proteinaceous molecules, the actual or observed effect on the inhibition of growth may be determined. The expected additive effect and the observed effect are then compared to determine whether a synergistic inhibition of growth of the organism has occurred. The methods used to detect synergy may utilize non-peptide antimicrobial agents in combination with the inhibitory proteinaceous molecule described herein.

[0312] As described herein, an anti-biological proteinaceous molecule may be used in combination with one or more existing anti-biological agents (e.g., a biocide, a biostatic) identified herein or in the art. It is expected that some such combinations of the anti-biological proteinaceous molecule with another anti-biological agent may provide, for example, a broader range of activity against various organisms, a synergistic anti-biological (e.g, a preservative) effect, and/or a longer duration of effect.

[0313] Another example combination includes an anti-biological proteinaceous molecule and a preservative and/or antimicrobial agent that acts against non-fungal organisms (e.g., a bactericide, an algacide), as it is contemplated that many fungal prone material formulations and/or surfaces coated with a surface treatment are also susceptible to damage by a variety of organisms. Examples of these preservatives and antimicrobial agents are described herein.

[0314] Example 6This Example describes the combined use of an anti-biological biomolecular composition and/or an OP degrading enzyme.

[0315] A multifunctional material formulation (e.g., surface treatment) may combine an anti-biological property with the ability to degrade an organophosphorus compound. An enzyme that functions to degrade an organophosphorus compound is contemplated as having an anti-biological (e.g., anti-fouling) activity. Examples of such a composition may be in the form of a coating, a paint, a non-film forming coating, an elastomer, an adhesive, an sealant, a material applied to a textile, a polymeric material, or a wax, and may be modified by addition of one or more anti-biological biomolecular composition(s) (e.g., a metal binding peptide having an anti-fouling activity) selected as described herein and an organophosphorus compound detoxifying agent such as an OP degrading enzyme or cellular material comprising such activity.

[0316] Example 7This Example describes adhesives, sealants, and elastomers comprising a metal binding and/or an anti-biological biomolecular composition.

[0317] The metal binding and/or anti-biological biomolecular composition(s) (e.g., a metal binding peptide having an anti-fouling activity) described herein are expected to be additionally useful for coating or mixing into adhesive(s) and sealant(s) such as a grout and/or a caulk, such as those that are in frequent contact with, or constantly exposed to biological entity (e.g., fungal) nutrients and/or moisture. Examples of adhesives and sealants (e.g., caulks, acrylics, elastomers, phenolic resin, epoxy, polyurethane, anaerobic and structural acrylic, high-temperature polymers, water-based industrial type adhesives, water-based paper and packaging adhesives, water-based coatings, hot melt adhesives, hot melt coatings for paper and plastic, epoxy adhesives, plastisol compounds, construction adhesives, flocking adhesives, industrial adhesives, general purpose adhesives, pressure sensitive adhesives, sealants, mastics, urethanes) for various surfaces (e.g., metal, plastic, textile, paper), and techniques of preparation and assays for properties, have been described in Skeist, I., ed., Handbook of Adhesives, 3rd Ed., Van Nostrand Reinhold, New York, 1990; Satriana, M. J. Hot Melt Adhesives: Manufacture and Applications, Noyes Data Corporation, New Jersey, 1974; Petrie, E. M., Handbook of Adhesives and Sealants, McGraw-Hill, New York, 2000; Hartshorn, S. R., ed., Structural Adhesives-Chemistry and Technology. Plenum Press, New York, 1986; Flick, E. W., Adhesive and Sealant Compound Formulations, 2nd Ed., Noyes Publications, New Jersey, 1984; Flick, E., Handbook of Raw Adhesives 2nd Ed., Noyes Publications, New Jersey, 1989; Flick, E., Handbook of Raw Adhesives, Noyes Publications, New Jersey, 1982; Dunning, H. R., Pressure Sensitive Adhesives-Formulations and Technology, 2nd Ed., Noyes Data Corporation, New Jersey, 1977; and Flick, E. W., Construction and Structural Adhesives and Sealants, Noyes Publications, New Jersey, 1988. For example, an adhesive, sealant or elastomer composition comprising one or more conventional anti-biological substance(s) may be modified by addition of and/or substitution by one or more of the anti-biological proteinaceous molecule(s) described in herein. Examples of adhesive include a thermoplastic adhesive, a thermoset adhesive, an elastomeric adhesive, an alloy adhesive, a non-polymeric adhesive, or a combination thereof. Examples of an adhesive includes a cellulosic adhesive, a cyanoacrylate adhesive, a dextrin adhesive, an ethylene-vinyl acetate copolymer adhesive, a melamine formaldehyde adhesive, a natural rubber adhesive, a neoprene/phenolic adhesive, a neoprene rubber adhesive, a nitrile rubber adhesive, a nitrile/phenolic adhesive, a phenolic adhesive, a phenol/resorcinol formaldehyde adhesive, a phenoxy adhesive, a polyamide adhesive, a polybenzimidazole adhesive, a polyethylene adhesive, a polyester adhesive, a polyimide adhesive, a polyisobutylene adhesive, a polysulfide adhesive, a polyurethane adhesive, a polyvinyl acetal adhesive, a polyvinyl acetal/phenolic adhesive, a polyvinyl acetate adhesive, a polyvinyl alcohol adhesive, a reclaimed rubber adhesive, a resorcinol adhesive, a silicone adhesive, a styrenic TPE adhesive, a styrene butadiene adhesive, a vinyl phenolic adhesive, a vinyl vinylidene adhesive, an acrylic acid diester adhesive, an epoxy adhesive, an epoxy/phenolic adhesive, an epoxy/polysulfide adhesive, a urea formaldehyde adhesive, a urea formaldehyde/melamine formaldehyde adhesive, a urea formaldehyde/phenol resorcinol adhesive, or a combination thereof. Examples of a thermosetting adhesive comprise an acrylic adhesive, an acrylic acid diester adhesive, a cyanoacrylate adhesive, a cyanate ester adhesive, an epoxy adhesive, a melamine formaldehyde adhesive, a phenolic adhesive, a polybenzimidazole adhesive, a polyester adhesive, a polyimide adhesive, a polyurethane adhesive, a resorcinol adhesive, a urea formaldehyde adhesive, or a combination thereof. Examples of a thermoplastic adhesive comprise an acrylic adhesive, an ethylene-vinyl acetate copolymer adhesive, a carbohydrate adhesive (e.g., a dextrin adhesive, a starch adhesive), a cellulosic adhesive (e.g., a cellulose acetate adhesive, cellulose acetate butyrate adhesive, cellulose nitrate adhesive), a polyethylene adhesive, a phenoxy adhesive, a polyamide adhesive, a polyvinyl acetal adhesive, a polyvinyl acetate adhesive, a polyvinyl alcohol adhesive, a protein adhesive (e.g., an animal adhesive, a soybean adhesive, a blood adhesive, a fish adhesive, a casein adhesive), a vinyl vinylidene adhesive, or a combination thereof. Examples of an elastomeric adhesive comprise a butyl rubber adhesive, a natural rubber adhesive, a neoprene rubber adhesive, a nitrile rubber adhesive, a polyisobutylene adhesive, a polysulfide adhesive, a reclaimed rubber adhesive, a silicone adhesive, a styrenic TPE adhesive, a styrene butadiene adhesive, or a combination thereof. Examples of an alloy adhesive comprise an epoxy/polyamide adhesive, an epoxy/phenolic adhesive, an epoxy/polysulfide adhesive, a neoprene/phenolic adhesive, a nitrile/phenolic adhesive, a phenol/resorcinol formaldehyde adhesive, a polyvinyl acetal/phenolic adhesive, a vinyl/phenolic adhesive, a urea formaldehyde/phenol resorcinol adhesive, a urea formaldehyde/melamine formaldehyde adhesive, or a combination thereof. Examples of a non-polymeric adhesive include a mucilage adhesive.

[0318] It is contemplated that a biomolecular composition may also be incorporated into an elastomer. An elastomer may comprise a polymer that can undergo large, but reversible, deformations upon a relatively low physical stress. It is contemplated that an elastomer composition may incorporate a biomolecular composition, such as by preparation with the biomolecular composition and/or direct addition such as by a multi-pack composition. Elastomers (e.g., tire rubbers, polyurethane elastomers, polymers ending in an anionic diene, segmented polyerethane-urea copolymers, diene triblock polymers with styrene-alpha-methylstyrene copolymer end blocks, poly(p-methylstyrene-b-p-methylstyrene), polydimethylsiloxane-vinyl monomer block polymers, chemically modified natural rubber, polymers from hydrogenated polydienes, polyacrylic elastomers, polybutadienes, trans-polyisoprene, polyisobutene, cis-1,4-polybutadiene, polyolefin thermoplastic elastomers, block polymers, polyester thermoplastic elastomer, thermoplastic polyurethane elastomers) and techniques of elastomer synthesis and elastomer property analysis have been described, for example, in Walker, B. M., ed., Handbook of Thermoplastic Elastomers, Van Nostrand Reinhold Co., New York, 1979; Holden, G., ed., et. al., Thermoplastic Elastomers, 2n.sup.d Ed., Hanser Publishers, Verlag, 1996. An example of an elastomer includes a thermoplastic elastomer, a melt processable rubber (NPR), a synthetic rubber (SR), a natural rubber (NR), a non-polymeric elastomer, or a combination thereof. In some embodiments, the elastomer comprises a thermoplastic elastomer, a melt processable rubber, a synthetic rubber, a natural rubber, a propylene oxide elastomer, an ethylene-isoprene elastomer, an ethylene-vinyl acetate elastomer, a non-polymeric elastomer, or a combination thereof. In other embodiments, the composition comprises an adhesive, a sealant, or a combination thereof.

[0319] Example 8This Example describes a textile finish comprising a metal binding and/or an anti-biological biomolecular composition.

[0320] A metal binding and/or an anti-biological biomolecular composition (e.g., a metal binding peptide having an anti-fouling activity) may also be incorporated into a material applied to a textile, such as, for example, a textile finish. Textile finishes (e.g., soil-resistant finishes, stain-resistant finishes) and related materials for application to a textile are described, for example, in Johnson, K., ANTISTATIC COMPOSITIONS FOR TEXTILES AND PLASTICS, Noyes Data Corporation, New Jersey, 1976; Rouette, H. K., ENCYCLOPEDIA OF TEXTILE FINISHING, Springer, Verlag, 2001; TEXTILE FINISHING CHEMICALS: AN INDUSTRIAL GUIDE, by Ernest W. Flick, Noyes Publications, 1990; and HANDBOOK OF FIBER FINISH TECHNOLOGY, by Philip E. Slade, Marcel Dekker, 1998. One type of water repellent and/or oil repellent textile finish comprises Scotchguard (3M Corporate Headquarters, Maplewood, Minn., U.S.A.). For example, a textile finish comprising one or more conventional anti-biological substance(s) may be modified by addition of and/or substitution by one or more of the anti-biological proteinaceous molecule(s) described in herein.

[0321] Example 9This Example describes a polymeric material comprising a metal binding and/or an anti-biological biomolecular composition.

[0322] It is contemplated that a biomolecular composition (e.g., an enzyme, a proteinaceous sequence, a metal binding peptide, an anti-biological peptide) may also be incorporated into a polymeric material such as a plastic (e.g., a thermoplastic, a thermoset). A polymeric material may comprise a plurality of polymers (polymer blends), an ionomer, a thermoplastic polymer, a thermoset polymer, or an elastomer. A thermoplastic comprises a thermoplastic polymer, while a thermoset plastic comprises a thermosetting polymer. A thermoplastic polymeric material may, for example, comprise a biodegradable polymer, a cellulosic polymer, a fluoropolymer, a polyether, a polyamide, a polyacrylonitrile, a polyamide-imide, a polyarylate, a polybenzimidazole, a polybutylene, a polycarbonate, a thermoplastic polyester, a polyetherimide, a polyethylene, a polyimide, a polyketone, an acrylic, a polymethylpentene, a polyphenylene oxide, a polyarylene sulphide, a polypropylene, a polyurethane, a polystyrene, a polysulfone resin, a polyterpene, a polyvinyl acetal, a polyvinyl acetate, a thermoplastic vinyl ester, a polyvinyl ether, a polyvinyl carbazole, a polyvinyl chloride, a polyvinylidene chloride, a polyimidazopyrrolone, a polyacrolein, a polyvinylpyridine, a polyvinylamide, a polyurea, a polyquinoxaline, or a combination thereof. A thermoplastic polymer may comprise an environmentally degradable polymers (e.g., a biodegradable polymer), a natural polymer, a photodegradable polymer, a synthetic biodegradable polymer (e.g., a poly(alkylene oxalate)s, a polyamino acid, a pseudo-polyamino acid, a polyanhydride, a polycaprolactone, a polycyanoacrylate, a polydioxanone, a polyglycolide, poly(hexamethylene-co-trans-1,4-cyclohexane dimethylene oxalate), a polyhydroxybutyrate, a polyhydroxyvalerate, a polylactide, a poly(ortho ester), a poly (p-dioxanone), a polyphosphazene, a poly(propylene fumarate), a polyvinyl alcohol), a biological degradable polymer (e.g., a collagen, a fibrinogen/fibrin, a gelatin, a polysaccharide), a cellulosic polymer (e.g., cellulose acetate, a cellulose acetate butyrate, a cellulose acetate propionate, a cellulose methylcellulose, a methylcellulose, a cellulosehydroxyethyl, an ethylcellulose, a hydroxypropylcellulose), a fluoropolymer, an ethylene chlorotrifluoroethylene, an ethylene tetrafluoroethylene, a fluoridated ethylene propylene, a polyvinylidene fluoride, a polychlorotrifluoroethylene, a polytetrafluoroethylene, a polyvinyl fluoride), a polyoxymethylene, a polyamide, an aromatic polyamide, a polyacrylonitrile, a polyamide-imide, a polyarylate, a polybenzimidazole, a polybutylene, a polycarbonate, a polyester (e.g., a liquid crystal polyester polycarbonate, a polybutylene terephthalate, a polycyclohexylenedimethylene terephthalate, a poly(ethylene terephthalate)), a polyetherimide, polyethylene (e.g., a very low-density polyethylene, a low-density polyethylene, a linear low-density polyethylene, a medium-density polyethylene, a high-density polyethylene, an ultrahigh molecular weight polyethylene, a chlorinated polyethylene, a chlorosulfonated polyethylene, a phosphorylated polyethylene, an ethylene-acrylic acid copolymer, an ethylene-methyl acrylate copolymer, an ethylene-ethyl acrylate copolymer, an ethylene-n-butyl acrylate copolymer, an ethylene-vinyl acetate copolymer, an ethylene-vinyl alcohol copolymer), a polyimide, a polyketone, a poly(methylmethacrylate), a polymethylpentene, a polyphenylene oxide, a polyphenol sulfide, a polyphthalamide, a polypropylene, a polyurethane, a polystyrene (e.g., styrene-acrylonitrile copolymer, a styrene-butadiene copolymer, an acrylonitrile butadiene styrene terpolymer, an acrylonitrile-chlorinated polyethylene-styrene terpolymer, an acrylic styrene acrylonitrile terpolymer), a polysulfone resin (e.g., a polysulfone, a polyaryl sulfone, a polyether sulfone), a polyvinyl chloride (e.g., a chlorinated polyvinyl chloride), a polyvinylidene chloride, or a combination thereof. A thermoset polymeric material may comprise, for example, an alkyd resin, an allyl resin, an amino resin, a bismaleimide resin, a cyanate ester resin, an epoxy resin, a furane resin, a phenolic resin, a thermosetting polyester resin, a polyimide resin, a polyurethane resin, a silicone resin, a vinyl ester resin, a casein, or a combination thereof. Polymeric materials often comprise an additive, such as a filler, a plasticizer, a lubricant, a flame retarder, a colorant, a blowing agent, an anti-aging additive, a cross-linking agent, etc. or a combination thereof. Polymeric materials and methods of preparation of preparing a polymeric material and assays for a polymeric material's properties have been described, for example, Handbook of Plastics, Elastomers, & Composites Fourth Edition (Harper, C. A. Ed.) McGraw-Hill Companies, Inc, New York, 2002; and Tadmor, Z. and Costas, G. G. Principles of Polymer Processing Second Edition, John Wiley & Sons, Inc. Hoboken, N.J., 2006.

[0323] Example 10This Example demonstrates the ability of a lysozyme to survive the incorporation process into a coating, demonstrates lysozyme hydrolytic activity in a coating environment, and demonstrates the ability of lysozyme to survive in can conditions for 48 hours. A Sherwin-Williams Acrylic Latex paint was used.

[0324] Materials, reagents and equipment used are shown in the tables below.

TABLE-US-00004 TABLE 4 Materials and Reagents 0.1M potassium phosphate buffer, pH 6.4 Micrococcus lysodeikticus (Worthington Biochemicals, #8736) Sherwin-Williams Acrylic Latex paint Lysozyme (chicken egg white) (Sigma Product #L 6876, CAS 12650-88-3) 15 mL plastic test tubes

TABLE-US-00005 TABLE 5 Equipment Paint spreader (1-8 mil) Polypropylene blocks Lightnin Labmaster Mixer Rotator shaker Pipettes and Pipetteman Klett-Sumerson Colorimeter (Filter D35: 540 nm)

[0325] The reagents prepared included a Micrococcus cell suspension comprising 9 mg M. lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution. The paint formulations used are shown in the table below.

TABLE-US-00006 TABLE 6 Paint Preparation Sherwin-Williams Acrylic Latex Control (no additive) Sherwin-Williams Acrylic Latex with 1 mg/mL lysozyme

[0326] The paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time for the Sherwin-Williams was 72 hrs. To demonstrate in can durability, the Sherwin-Williams Acrylic Latex comprising lysozyme wet paint was sealed and shelf stored at ambient temperature. After 48 hrs in can, films were drawn onto polypropylene surfaces with a thickness of 8 mils and were allowed to cure 72 hrs prior to assay. Coupons were generated as free films from the polypropylene surface. Films were generated in three sizes: 2 cm.sup.2: 1 cm by 2 cm; 4 cm.sup.2: 1 cm by 4 cm; or 6 cm.sup.2: 1 cm by 6 cm.

[0327] For qualitative assessment, individual films were placed into labeled 15 mL tubes. Films of each size (2, 4 and 6 cm.sup.2) were evaluated in triplicate. In addition to a control paint with no additive, two other controls were utilized, a positive control and a negative control. The positive control comprised: lysozyme in buffer added to each of three 15 mL tubes in concentrations approximating the amount of lysozyme in the films (i.e., 40 g, 80 g, and 120 g). Each amount was assayed in triplicate. The negative control comprised: 5 mL of 0.36 mg/mL M. lysodeikticus cell suspension pipetted into a single 15 mL tube. 5 mL 0.36 mg/mL Micrococcus lysodeikticus cell suspension was added to all reaction tubes to begin the reaction. The tubes were placed on a rocker at ambient conditions for approximately 22 hours. Where possible, the films were removed from the suspension and determine opacity using the Klett-Summerson Colorimeter (turbidity unit: Klett Unit or KU).

[0328] Particulate matter in the samples interfered with quantitation; photographs of each set of 2 cm.sup.2 paint films and controls following 22 hour contact to M. lysodeikticus cell suspension were taken, and observations recorded in the Tables below.

TABLE-US-00007 TABLE 7 Qualitative Observations (visual assessments) Lysozyme Film Size Sample.sup.1 (g) (cm.sup.2) Clarity Suspension/Solution Controls M. lysodeikticus Translucent Lysozyme 40 Transparent.sup.2 80 Transparent 120 Transparent Control Films S-W 2, 4, 6 Translucent Films Comprising Lysozyme S-W 2, 4, 6 Transparent .sup.1Each evaluation was performed in triplicate .sup.2Thinned in opacity, with some suspended particulate matter

[0329] The strips comprising lysozyme of all three sizes of coupons cleared the M. lysodeikticus suspension, indicating that the lysozyme maintains activity in the coating environment. Cleared suspensions (lysozyme comprising coupons and controls) comprised large particles which interfere with the quantitation of the cleared suspensions. The particulate matter was less detectable in the 2 cm.sup.2 set comprising lysozyme, so this size coupon was used for the quantitative demonstrations.

TABLE-US-00008 TABLE 8 Quantiative Assessment of Lysozyme In-Film Activity (2 cm.sup.2 film, 4 hr time point, 3 independent assays, each performed in triplicate.) Replicate 1 Replicate 2 Replicate 3 In can Cell Cell Cell Formulation (hrs) KU lysis KU lysis KU lysis Suspension Controls M. lysodeikticus 81.5 0.0% 101 0% Lysozyme 17 27 S-W Acrylic Latex Control Films 75 18% 74 19% 71 22% 79 13% 82 10% 76 17% 83 9% 81 11% 73 20% Films Comprising Lysozyme 8 91% 20 78% 11 88% 13 86% 11 88% 15 84% 13 86% 5 95% 0 100% Control Films 48 hrs 82 10% 65 29% 68 25% Films Comprising 48 hrs 36 61% 26 72% 37 59% Lysozyme KU = Klett Units, measure of turbidity at 540 nm.

[0330] A lysozyme in Sherwin-Williams Acrylic Latex was able to lyse about 88% of the M. lysodeikticus culture over 4 hours, relative to the control which exhibited about a 15% drop in opacity. After in-can shelving for 48 hrs (i.e., the lysozyme was mixed into the Sherwin-Williams Acrylic Latex, capped and shelved for 48 hrs prior to drawing down the films), the lysozyme remained active, lysing about 64% of the M. lysodeikticus culture relative to the about 21% lysis exhibited by the control panels.

[0331] Example 11This Example demonstrates the retention of lysozyme vs. loss due to leaching in a paint film in a saturated condition at 1, 2 and 24 hours after submersion.

[0332] Materials, reagents and equipment used are shown in the Tables below.

TABLE-US-00009 TABLE 9 Materials and Reagents 0.1M potassium phosphate buffer, pH 6.4 Micrococcus lysodeikticus (Worthington Biochemicals, #8736) Lysozyme (chicken egg white) (Sigma Product #L 6876, CAS 12650-88-3) Sherwin-Williams Acrylic Latex paint 15 mL plastic test tubes

TABLE-US-00010 TABLE 10 Equipment Paint spreader (1-8 mil) Polypropylene blocks Lightnin Labmaster Mixer Rotator shaker Pipetter and tips Klett-Sumerson Colorimeter (Filter D35: 540 nm)

[0333] The reagents prepared included a Micrococcus cell suspension comprising 9 mg M. lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution.

[0334] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex Control (no additive), and a Sherwin-Williams Acrylic Latex comprising 1 mg/mL lysozyme. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 120 hrs. The Sherwin-Williams Acrylic Latex comprising a lysozyme wet paint was sealed and shelf stored at ambient temperature. After 48 hrs in can storage, films were drawn onto polypropylene surfaces with a thickness of 8 mils and were allowed to cure 72 hrs prior to assay. Materials for assay were generated from the polypropylene surface as a 2 cm (12 cm) free film.

[0335] The assay procedure included placing individual films into labeled 15 mL tubes. 24 hours prior to addition of Micrococcus lysodeikticus cell suspension, 5 mL KPO.sub.4 buffer was added to the 24-hour control and coupon comprising a lysozyme tube, as well as one tube comprising 41 lysozyme solution (positive control) and one tube comprising 5 mL of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for 24 hrs.

[0336] 2 hours prior to addition of M. lysodeikticus, 5 mL potassium phosphate buffer was added to the 2-hour control and lysozyme tubes each comprising a coupon, as well as one tube comprising 41 g lysozyme solution (positive control) and one tube comprising 5 mL of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for 2 hrs.

[0337] 1 hour prior to addition of M. lysodeikticus cell suspension, 5 mL potassium phosphate buffer was added to 1-hour control and coupon comprising a lysozyme tubes, as well as one tube comprising 41 g lysozyme solution (positive control) and one tube comprising 5 mL of the M. lysodeikticus cell suspension (negative control). These tubes were placed on the shaker for one hour.

[0338] The paint coupons were then transferred from each tube to a second reaction tube. 5 mL of the M. lysodeikticus cell suspension was added to both film and KPO.sub.4 buffer incubation buffer. The tubes were placed on the rotating shaker horizontally and shaken for approximately 4 hours, at which time each tube was measured in a Klett-Summerson Photoelectric Colorimeter to determine opacity.

TABLE-US-00011 TABLE 11 Assessment of lysis and enzyme leaching (free film) after 1, 2 and 24 hr, relative to the internal control (i.e., the no lysozyme films). Replicate 1 Replicate 2 Replicate 3 Average Cell Cell Cell Cell Formula- Time lysis lysis lysis Lysis tion (hrs) KU (dKU) KU (dKU) KU (dKU) KU (dKU) KPO.sub.4 Buffer Control 1 hr 110 0% 90 0% 104 0% 101 0% Lysozyme 1 hr 62 39% 42 59% 52 49% 52 49% Control 2 hr 92 0% 102 0% 106 0% 100 0% Lysozyme 2 hr 74 26% 65 35% 65 35% 68 32% Control 24 hr 95 0% 95 0% 92 0% 94 0% Lysozyme 24 hr 80 15% 62 34% 55 41% 66 30% Film Control 1 hr 64 0% 54 0% 38 0% 52 0% Lysozyme 1 hr 3 94% 40 23% 4 92% 16 81% Control 2 hr 63 0% 73 0% 72 0% 69 0% Lysozyme 2 hr 10 86% 23 67% 45 35% 26 54% Control 24 hr 65 0% 65 0% 68 0% 66 0% Lysozyme 24 hr 30 55% 52 21% 52 21% 45 32% KU = Klett Unit, measure of turbidity at 540 nm

[0339] At the three time points assayed, lysozyme leached out of films that comprised a lysozyme. The ability of the films comprising a lysozyme to lyse M. lysodeikticus was inversely related to the time the coupon was submerged. Over the first 2 hrs the films lost approximately 21%3% of the lytic activity per hour. This loss decreased substantially over the following 22 hrs, with the loss slowing to approximately 3% per hour. After 24 hours of liquid submersion, approximately one-third of the activity of a coupon comprising a lysozyme was retained. Though reduction of activity due to leaching may continue, activity may also be permanently retained in the films. The total percentage lysis by coupon and buffer pairs decreased with increasing leaching time.

[0340] Example 12This Example demonstrates the surface efficacy of paint films comprising a lysozyme in actively lyse M. lysodeikticus in a minimally hydrated environment.

[0341] Materials, reagents and equipment used are shown in the tables below.

TABLE-US-00012 TABLE 12 Materials and Reagents 0.1M potassium phosphate buffer, pH 6.4 Micrococcus lysodeikticus (Worthington Biochemicals, #8736) Lysozyme (chicken egg white) (Sigma Product #L 6876, CAS 12650-88-3) Sherwin-Williams Acrylic Latex paint 15 mL plastic test tubes

TABLE-US-00013 TABLE 13 Equipment Paint spreader (1-8 mil) Polypropylene blocks Lightnin Labmaster Mixer Rotator shaker Pipetter and tips Klett-Sumerson Colorimeter (Filter D35: 540 nm)

[0342] The reagents prepared included a Micrococcus cell suspension comprising 9 mg Micrococcus lysodeikticus in 25 mL sodium phosphate buffer, and a lysozyme solution comprising a 5 mg/mL stock solution.

[0343] The paint formulations prepared for the assay included a Sherwin-Williams Acrylic Latex Control (no additive), and a Sherwin-Williams Acrylic Latex with 1 mg/mL lysozyme. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 72 hrs. Assay materials were generated from the polypropylene surface as a 2 cm (12 cm) free film.

[0344] The assay procedure included placing individual coupons into separate Petri dishes. Each set of control coupons and coupons comprising a lysozyme was assayed in triplicate. Two controls were set up for this experiment: a M. lysodeikticus suspension control comprising 90 L 20 mg/mL M. lysodeikticus cell suspension that was pipetted into a petri dish; and a 1 mg/mL lysozyme control comprising 40.64 L 1 mg/mL lysozyme solution (an amount approximately equal to the amount of lysozyme in the 2 cm.sup.2 coupon comprising a lysozyme) that was pipetted into a petri dish. M. lysodeikticus cell suspension was distributed onto the surface of each individual coupon in a minimal volume (90 Petri dishes were kept on a flat surface. After 4 hours, KPO.sub.4 buffer was added to all samples to recover the unlysed portion of the M. lysodeikticus cell suspension. The suspension was removed from each dish with a pipette and placed into individual test tubes. Each suspension was read in the Klett-Summerson Photoelectric Colorimeter, using potassium phosphate buffer as a control.

TABLE-US-00014 TABLE 14 Surface Efficacy of Films comprising lysozyme in a low hydration environment. Replicate 1 Replicate 2 Replicate 3 Average Cell Cell Cell Cell Formulation KU lysis KU lysis KU lysis KU Lysis Suspension/ Solution Controls M. lysodeikticus 80 Lysozyme 10 S-W Acrylic Latex Control Films 75 6% 70 13% 78 3% 74 7% Lysozyme Films 35 56% 19 76% 31 61% 28 65% KU = Klett units, measure of turbidity at 540 nm.

[0345] The paint comprising a lysozyme contacted with 0.18 mg of a M. lysodeikticus suspension for 4 hours lysed 65%10% of the Micrococcus cells, compared to only 7%5% of cells lysed by the paint controls. This demonstrated that lysozyme can function in the low water (i.e., a minimally hydrated) environment of a coating. It is contemplated that a biological assay including a spray application of an assay organism would also demonstrate biostatic and/or biocidal activity.

[0346] Example 13This Example demonstrates the ability of a chymotrypsin to survive the incorporation process into a coating and demonstrates chymotrypsin activity in a coating environment.

[0347] A chymotrypsin free film assay was used for determining the activity of chymotrypsin, as measured by ester hydrolysis (esterase) activity of a p-nitrophenyl acetate substrate, in free-films using a plate reader. A functioning vent hood was used for the assay when appropriate for material handling. A Sherwin-Williams Acrylic Latex paint was used. Equipment and reagents that were used are shown in the tables below.

TABLE-US-00015 TABLE 15 Equipment Plate Reader 2 ml microtubes

TABLE-US-00016 TABLE 16 Reagents -Chymotrypsin from bovine pancreas, Type II (Sigma Cat# C4129) 4-Nitrophenyl acetate, MW 181.15 (Sigma Cat# N8130) Trizma base (Sigma Cat# T1503)

[0348] Sample preparation included: 14.5 mM p-nitrophenyl acetate (66 mg/25 ml) in isopropyl alcohol, and 200 mM TRIS; pH 7.1 (adjust to pH 7.1 with HCl).

[0349] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising 200 mg/mL -Chymotrypsin. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 days. Materials for assay were generated from the polypropylene surface as 1 cm.sup.2, 2 cm.sup.2 and 3 cm.sup.2 free films.

[0350] The plate reader assay comprised: cutting free films into appropriate size pieces; adding 600 L ddH.sub.2O into a 2 ml microtube; then adding 750 L 200 mM TRIS to each microtube; adding 150 L of 14.5 mM p-nitrophenyl acetate to each tube; and taking the 0 time sample, then adding the free film to the tube (control sample is free film with no chymotrypsin).

[0351] The analysis included: taking out 100 l and reading the absorbance at 405 nm, at the appropriate time points; and determining the initial rate slope by plotting absorbance vs. time to calculate chymotrypsin activity.

TABLE-US-00017 TABLE 17A Absorbance at 405 nm Chymotrypsin in Sherwin-Williams Acrylic Latex Time Blank 3 cm X 1 cm Control 0 0.0480 0.0429 0.0446 0.0480 0.0429 0.0446 15 0.0482 0.0489 0.0479 0.0518 0.0541 0.0541 30 0.0571 0.0558 0.0555 0.0596 0.0612 0.0609 45 0.0608 0.0617 0.0617 0.0679 0.0709 0.0690 60 0.0683 0.0690 0.0679 0.0773 0.0826 0.0781 Slope 0.0004 0.0004 0.0004 0.0005 0.0006 0.0005

TABLE-US-00018 TABLE 17B Absorbance at 405 nm Chymotrypsin in Sherwin-Williams Acrylic Latex Time 3 cm X 1 cm Enzyme 2 cm X 1 cm Enzyme 0 0.0480 0.0429 0.0446 0.0480 0.0429 0.0446 15 0.2364 0.2356 0.2347 0.1690 0.1801 0.1749 30 0.4504 0.4375 0.4208 0.3040 0.3149 0.3172 45 0.6395 0.6267 0.6441 0.4348 0.4579 0.4474 60 0.8358 0.7957 0.7970 0.5682 0.5942 0.5930 Slope 0.0132 0.0126 0.0128 0.0087 0.0092 0.0091

TABLE-US-00019 TABLE 17C Absorbance at 405 nm Chymotrypsin in Sherwin-Williams Acrylic Latex Time 1 cm 1 cm Enzyme 0 0.0480 0.0429 0.0446 15 0.1156 0.1155 0.1164 30 0.1886 0.1932 0.1872 45 0.2688 0.2745 0.2684 60 0.3427 0.3479 0.3578 Slope 0.0050 0.0051 0.0052

TABLE-US-00020 TABLE 18A Absorbance Averages Chymotrypsin in Sherwin-Williams Acrylic Latex Absorbance Average Chymo- Chymo- Chymo- Control trypsin trypsin trypsin Time Blank 3 cm.sup.2 3 cm.sup.2 2 cm.sup.2 1 cm.sup.2 0 0.0452 0.0452 0.0452 0.0452 0.0452 15 0.0483 0.0533 0.2356 0.1747 0.1158 30 0.0561 0.0606 0.4362 0.3120 0.1897 45 0.0614 0.0693 0.6368 0.4467 0.2706 60 0.0684 0.0793 0.8095 0.5851 0.3495

TABLE-US-00021 TABLE 18B Absorbance Averages Standard Deviations Chymotrypsin in Sherwin-Williams Acrylic Latex AbsorbanceStandard Deviation Chymo- Chymo- Chymo- Control trypsin trypsin trypsin Time Blank 3 cm.sup.2 3 cm.sup.2 2 cm.sup.2 1 cm.sup.2 0 0.0026 0.0026 0.0026 0.0026 0.0026 15 0.0005 0.0013 0.0009 0.0056 0.0005 30 0.0009 0.0009 0.0148 0.0071 0.0031 45 0.0005 0.0015 0.0090 0.0116 0.0034 60 0.0006 0.0029 0.0228 0.0147 0.0077

TABLE-US-00022 TABLE 19 Absorbance vs. Time Slope Slope U U U Sample (A/min) (umol/min) Average Deviation Blank 0.0004 0.0776 0.09 0.01 0.0004 0.0949 0.0004 0.0881 Control 0.0005 0.1090 0.12 0.02 3 cm.sup.2 0.0006 0.1404 0.0005 0.1195 Chymotrypsin 0.0132 2.8876 2.82 0.06 3 cm.sup.2 0.0126 2.7679 0.0128 2.7935 Chymotrypsin 0.0087 1.9062 1.97 0.06 2 cm.sup.2 0.0092 2.0145 0.0091 1.9983 Chymotrypsin 0.0050 1.0837 1.11 0.03 1 cm.sup.2 0.0051 1.1222 0.0052 1.1359

[0352] A chymotrypsin in Sherwin-Williams Acrylic Latex was able to hydrolyze the model substrate at rate 20 faster than the control. The test coupons demonstrate a dose response which corresponds to a hydrolytic capacity of 0.86 umol/min/cm.sup.2, as formulated in this demonstration.

[0353] Quality control included reading and become familiar with the operating instructions for equipment used in the analysis. Operating instructions and preventive maintenance records were placed near the relevant equipment, and kept in a labeled central binder in the work area. Working solutions which are out of date or prepared incorrectly were disposed of and not used.

[0354] Safety procedures and precautions included wearing a full length laboratory coat; and not eating, drinking, smoking, use of tobacco products or application of cosmetics near the procedure. Consumables and disposable items that come in contact with or are used in conjunction with samples disposal were in the proper hazard containers. This includes, but is not limited to, pipette tips, bench-top absorbent paper, diapers, kimwipes, test tubes, etc. Biohazard containers were considered full when their contents reach three-quarters of the way to the top of the bag or box. Bench-top biohazard bags were placed into a large biohazard burn box when full. Biohazard containers were not filled to overflowing. Biohazard bags were disposed of by closing with autoclave tape, and autoclaving immediately. Spills and spatters were immediately cleaned from durable surfaces by applying 70% ethanol (for bacteriological spills) to the spill, followed by wiping or blotting. All equipment used in sample analyses were wiped down on a daily basis or whenever tests were performed. Absorbent pads were placed under samples when useful. Hands were washed with antibacterial soap before exiting the room, when a test was finished, and before the end of the day. The Material Safety Data Sheet (MSDS) applicable to each chemical was read. MSDS documents have been prominently posted in the laboratory. During a fire alarm during laboratory operations, evacuation procedures were followed. Nitrile protective gloves were worn whenever handling organophosphates. All organophosphate waste was disposed of properly.

[0355] Example 14This Example demonstrates the ability of a cellulase to survive the incorporation process into a coating and demonstrates cellulase activity in a coating environment. A Glidden Latex paint was used. A plate reader was used to assay a free-film comprising a cellulase for the enzyme's activity.

[0356] Equipment and reagents that were used are shown in the table below.

TABLE-US-00023 TABLE 20 Equipment and Reagents Equipment Plate Reader Reagents Sodium Acetate (Sigma Cat# S8625) 4-Nitrophenyl -D-cellobioside (Sigma Cat# N5759) Cellulase (TCI Cat# C0057) Sodium Hydroxide

[0357] Sample preparation included: 14.5 mM 4-Nitrophenyl -D-cellobioside in ddH.sub.2O; 50 mM sodium acetate buffer; pH 5.0 (adjust to pH 5.0 with HCl); and 2 N NaOH in ddH.sub.2O.

[0358] The plate reader assay comprised: placing free films into 2 ml microtubes; add 1.2 ml 50 mM sodium acetate buffer, 0.15 ml 14.5 mM 4-Nitrophenyl -D-cellobioside and 0.15 ml ddH.sub.2O, in the 2 ml microtube; placing tubes on rocker; taking out 100 l from the tubes into a 96-well plate at desired time points; adding 200 l of 2 N NaOH and reading the absorbance at 405 nm; and determining the initial rate slope by plotting absorbance vs. time to calculate cellulase activity.

[0359] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex control (no additive), and a Sherwin-Williams Acrylic Latex comprising 100 g/gal, 200 g/gal and 300 g/gal cellulase. Each paint was mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 8 mil. Cure time was 24 hrs. Materials for assay were generated from the polypropylene surface as a 3 cm.sup.2 free film.

TABLE-US-00024 TABLE 21A Glidden Latex Cellulase Free Films - Dose Response - pNP Absorbance at 405 nm Time (min) Blank Control 100 g/gal 0 0.0600 0.0600 0.0600 0.0600 0.0600 0.0600 0.0600 30 0.0496 0.0588 0.0488 0.0476 0.0744 0.0753 0.0716 60 0.0496 0.0605 0.0505 0.0532 0.0975 0.1158 0.1007 120 0.0507 0.0519 0.0522 0.0514 0.1691 0.1823 0.1672 180 0.0550 0.0643 0.0583 0.0511 0.2351 0.2312 0.2073 240 0.0512 0.0614 0.0518 0.0548 0.2876 0.2919 0.2720 300 0.0491 0.0574 0.0601 0.0575 0.3187 0.3123 0.3083 360 0.0528 0.0680 0.0540 0.0655 0.3322 0.3215 0.3309 Slope 0.0001 0.0001 0.0000 0.0000 0.0009 0.0011 0.0009 (A/ min)

TABLE-US-00025 TABLE 21B Glidden Latex Cellulase Free Films - Dose Response - pNP Absorbance at 405 nm Time (min) 200 g/gal 300 g/gal 0 0.0600 0.0600 0.0600 0.0600 0.0600 0.0600 30 0.0986 0.0866 0.0927 0.1207 0.1170 0.1146 60 0.1387 0.1341 0.1432 0.1637 0.1711 0.1670 120 0.2285 0.2219 0.2364 0.2864 0.2685 0.2965 180 0.2891 0.2740 0.3071 0.3304 0.3262 0.3833 240 0.3174 0.3281 0.3270 0.3543 0.3638 0.4118 300 0.3449 0.3467 0.3511 0.3759 0.3891 0.4051 360 0.3714 0.3588 0.3632 0.3808 0.3964 0.3651 Slope (A/min) 0.0014 0.0014 0.0015 0.0019 0.0017 0.0020

TABLE-US-00026 TABLE 22A Glidden Latex Cellulase Free FilmsDose ResponsepNP Absorbance at 405 nm Averages Average Time 100 200 300 (min) Blank Control g/gal g/gal g/gal 0 0.0600 0.0600 0.0600 0.0600 0.0600 30 0.0496 0.0517 0.0738 0.0926 0.1189 60 0.0496 0.0547 0.1047 0.1387 0.1674 120 0.0507 0.0518 0.1729 0.2289 0.2775 180 0.0550 0.0579 0.2245 0.2901 0.3283 240 0.0512 0.0560 0.2838 0.3242 0.3591 300 0.0491 0.0583 0.3131 0.3476 0.3825 360 0.0528 0.0625 0.3282 0.3645 0.3886

TABLE-US-00027 TABLE 22B Glidden Latex Cellulase Free FilmsDose ResponsepNP Absorbance at 405 nm Averages' Deviations Deviation Time 100 200 300 (min) Control g/gal g/gal g/gal 0 0.0000 0.0000 0.0000 0.0000 30 0.0061 0.0019 0.0060 0.0026 60 0.0052 0.0098 0.0046 0.0052 120 0.0004 0.0082 0.0073 0.0127 180 0.0066 0.0151 0.0166 0.0030 240 0.0049 0.0105 0.0059 0.0067 300 0.0015 0.0052 0.0032 0.0093 360 0.0075 0.0058 0.0064 0.0110

[0360] A cellulase in a Glidden Latex was able to hydrolyze the model substrate at a rate approximately 100 faster than the control. Quality control and safety procedures were as described in Example 13.

[0361] Example 15This Example demonstrates preparation of technical papers coated with a latex coating comprising an antimicrobial enzyme additive, an antimicrobial peptide additive, or a combination thereof. The additives may be embedded in the coating.

[0362] The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat (Reactive Surfaces, Ltd.; also described in U.S. patent application Ser. Nos. 10/884,355; 11/368,086; and Ser. No. 11/865,514, each incorporated by reference). Materials that were used are shown in the tables below.

TABLE-US-00028 TABLE 23 Materials 30 mM Potassium Phosphate Buffer, was prepared by weighing out 416 mg of potassium phosphate into 2 50 mL conical tubes, and adding 50 mL of water to each tube. Micrococcus lysodeikticus (Worthington Biochemicals, #8736), was prepared by weighing out 18 mg of Micrococcus into a single 50 mL conical tube, adding KP0.sub.4 buffer to 50 mLs, and mixing by inversion. Lysozyme from chicken egg white (Sigma Product #L 6876; CAS no. 12650-88-3), was prepared by weighing out 1 g, 0.5 g and 0.1 g lysozyme into 3 2 mL eppendorf tubes. Dilute Acetic Acid Solution was prepared by measuring 1 mL of glacial acetic acid into 11 mLs of water into a 15 mL conical tube, and adding 50 l of the dilute acetic acid to 1 mL of water. ProteCoat was used at 125 mg ProteCoat per g coating, dispensed as 250 mg ProteCoat, and resuspended in 2 mL dilute acetic acid solution as appropriate. 5 15 mL conical tubes, glass stir rod P1000 and P200 Pipetteman and Tips 5 15 mL conical tubes

[0363] Paint formulations comprising enzyme were prepared as follows: 1 g lysozyme per 100 g coating; 0.5 g lysozyme per 100 g coating; 0.1 9 lysozyme per 100 g coating; and a negative control (no additive). Paint formulations comprising a peptide additive were prepared as follows: 125 mg ProteCoat per 1 g coating; 250 mg ProteCoat per 1 g coating; 375 mg ProteCoat per 1 g coating; or a negative control (no additive). Paint formulations comprising peptide and lysozyme were prepared as follows: 375 mg ProteCoat per 1 g lysozyme (1 g) coating; 250 mg ProteCoat per 1 g lysozyme (0.5 g) coating; 375 mg ProteCoat per 1 g lysozyme (0.1 g) coating, and a negative Control (no additive). All paint formulations were mixed well. The paper was cut into quarters, coatings drawn onto paper surfaces with a spreader, and wet weight determined. The coated paper was dried at about 37.8 C. for approximately 10 min, and dry weight determined.

[0364] A single coating material and one paper stock was evaluated. The paper comprised celluosic fibers typically used in technical paper applications, and had an acrylic latex coating added to the fibers.

TABLE-US-00029 TABLE 24 Coating dry components added to paper Ingredient % Dry Weight Kaolin Clay About 0.000000001% to about 90% (filler/pigment) Titanium Dioxide About 0.000000001% to about 90% (pigment) Calcium Carbonate About 0.000000001% to about 90% (filler/pigment) Acrylic Latex About 0.000000001% to about 80% (Binder)

[0365] To prepare the antimicrobial paper (AM-Paper), the antimicrobial additives were formulated for each coating on percentage dry weight to standardize the coating for comparison. The antimicrobial additives are listed in the table below.

TABLE-US-00030 TABLE 25 Formulation details for antimicrobial papers Anti- Desig- Additive Final Dry Additive microbial nation Formulation Weight (gsm) (%) Control 17.6 None 21 None Enzymatic A Powder 21.9 0.2% B Powder 19.4 1% C Powder 23.2 2% D Suspension 23 0.2% E Suspension 23 1% F Suspension 20.7 2% ProteCoat G Suspension 18.6 1% H Powder 23.9 2.5% I Suspension 20.6 0.5% J Powder 20.9 1.25% K Powder 20.9 0.25% L Powder 20.7 0.75% Enzyme + ProteCoat Powder 22.5 2% + 0.5% Powder 21.9 1% + 0.25%

[0366] The antimicrobial additives were weighed out, added to pre-weighed coating suspensions and mixed by hand for 10 to 20 minutes. After mixing, the coating was applied by draw down, in which approximately 3-5 mLs of coating was applied along one 8.5 edge of an 8.511 pre-weighed paper, and then spread evenly over the surface of the paper with a calibrated rod by drawing the rod down the full length of the paper. The coated paper was then placed into a 100 C. oven for 10 to 15 minutes to dry. After drying, the coated paper was weighed to determine the amount of coating on each sheet.

[0367] To conduct an assay to qualitatively assess antimicrobial activity, a paper strip of approximately 1 cm5 cm was cut from the control and each antimicrobial paper. 5 mL of the M lysodeikticus suspension was poured into each of 415 mL conical tubes. The prepared strip was dropped into the suspension, and mixed occasionally by inversion. Clearing changes were observed.

[0368] Example 16This Example demonstrates and provides a standard spectrophotometric assay procedure for lysozyme activity in a plate reader.

[0369] Equipment and reagents that were used are shown in the table below.

TABLE-US-00031 TABLE 26 Equipment and Reagents Equipment Thermo Multiskan Ascent Plate Reader 96-well assay plates Multi-channels and single-channel pipettes and tips Reagents Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI): [Sigma, cat # T3253, Molecular Formula: NH.sub.2C(CH.sub.2OH).sub.3HCI, Molecular Weight: 157.60, CAS Number 1185-53-1, pKa (25 C.) 8.1] Micrococcus Iysodeikticus cell (Worthington Biochemicals, cat #8736) Lysozyme: chicken egg white, Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C. Standard: 25 I of a 500,000 units (10 mg)/ mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 350 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C.

[0370] Micrococcus lysodeikticus cell suspension was made by adding 9 mg Micrococcus lysodeikticus to 25 mL 10 mM Tris-HCl, pH 8.0 and mixing well. Lysozyme solution was prepared by adding 10 mg lysozyme in 1 mL 10 mM Tris-HCl, pH 8.0, and mixing well. Reaction buffer was 10 mM Tris-HCl, pH 8.0, with an alternative reaction buffer being 0.1 M KPO.sub.4 pH 6.4.

[0371] A standard curve of the M. lysodeikticus was prepared. The lysozyme stock solution was diluted with the reaction buffer to create the following series: 10 mg/mL (undiluted); 5.0 mg/mL; 2.5 mg/mL; 1 mg/mL; 0.5 mg/mL; 0.1 mg/mL; 0.05 mg/mL; 0.01 mg/mL; 0.005 mg/mL; 0.001 mg/mL; 0.0005 mg/mL; 0.0001 mg/mL, and 0 mg/mL. The controls included 3 replicates of 194 L M. lysodeikticus cell suspension plus 6 L buffer; and 3 replicates of 200 L buffer.

[0372] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at 570 nm and determining the best fit to a standard curve. For a 200 L assay, 180 L M. lysodeikticus in reaction buffer was added to each well 1 to 12 of 3 rows. The reaction was started by adding 20 L of each lysozyme dilution to each well in the triplicate series. The plate was immediately placed into the reader, and the changes in absorbance at 570 nm (OD.sub.570) recorded. The number of reads may be 10-20 with second intervals. The plate reader's velocity table contained data for reaction rate in mOD/min. This assay can be scaled by increasing each suspension proportionately (e.g., a 2 mL reaction is used for material strip analysis).

[0373] Analysis of the data included calculating the initial velocities for the recorded slopes: [mOD.sub.540/min]/[slope standard curve (mOD/mg M. lysodeikticus]/[Iysozyme].

TABLE-US-00032 TABLE 27 Assay Standardization Coupon Size None Test Organism Micrococcus lysodeikticus Contamination level 2.5 10.sup.8 cells/mL Assay Time 4 hr

TABLE-US-00033 TABLE 28 Standardization of Assay [Lysozyme], (g/mL).sup.a OD.sub.570 % Lysis 0 0.3 0.00 0.78 0.26 13.33 1.56 0.07 76.67 3.13 0.02 93.33 6.25 0.005 98.33 12.5 0.005 98.33 25 0.011 96.33 50 0.065 78.33 .sup.ag/mL = ppm

[0374] The M. lysodeikticus assay as described can detect lytic activity down to the fractional to low ppm range. The rate of lysis, in suspension, is 32% (about 8.010.sup.7 cells) of the M. lysodeikticus suspension per g lysozyme.

[0375] Example 17This Example demonstrates a spectrophotometric assay for antimicrobial paper with a lytic additive. Lysozyme was used as the lytic additive.

[0376] Equipment and reagents that were used are shown in the table below.

TABLE-US-00034 TABLE 29 Equipment and Reagents Equipment Spectrophotometer (Thermo Multiskan Ascent Plate Reader) Cuvettes (96-well assay plates) Multi-channels and single-channel pipettes and tips Reagents Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI): [Sigma, cat # T3253, Molecular Formula: NH.sub.2C(CH.sub.2OH).sub.3HCI, Molecular Weight: 157.60, CAS Number 1185-53-1, pKa (25 C.) 8.1] Micrococcus Iysodeikticus cell (Worthington Biochemicals, cat #8736) Lysozyme: chicken egg white, Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C. Standard: 25 I of a 500,000 units (10 mg)/ mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 350 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C.

[0377] Micrococcus lysodeikticus cell suspension was made by adding 9 mg M. lysodeikticus to 25 mL 10 mM Tris-HCl, pH 8.0 and mixing well. Lysozyme solution was prepared by adding 10 mg lysozyme in 1 mL 10 mM Tris-HCl, pH 8.0, and mixing well. Reaction buffer was 10 mM Tris-HCl, pH 8.0, with an alternative reaction buffer being 0.1 M KP0.sub.4 pH 6.4. Antimicrobial paper coated with a coating comprising lysozyme and control paper was prepared in accordance with Example 15.

[0378] A standard curve of the M. lysodeikticus was prepared. The lysozyme stock solution was diluted with the reaction buffer to create the following series: 10 mg/mL (undiluted); 5.0 mg/mL; 2.5 mg/mL; 1 mg/mL; 0.5 mg/mL; 0.1 mg/mL; 0.05 mg/mL; 0.01 mg/mL; 0.005 mg/mL; 0.001 mg/mL; 0.0005 mg/mL; 0.0001 mg/mL and 0 mg/ml. The controls included 3 replicates of 194 L M. lysodeikticus cell suspension plus 6 L buffer; and 3 replicates of 200 L buffer. Pipet tips used fitted the pipette (e.g., multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc may alter volume. Quality control and safety procedures were as described in Example 13.

[0379] Antimicrobial paper was cut into appropriately sized strips from both the antimicrobial and control paper. For a 5 mL assay in a 15 mL tube, standard sizes included 510 mm, 520 mm, and 540 mm. These strips could be combined to provide a desired step series.

[0380] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at OD.sub.570 and determining the best fit to a standard curve. For a 5 mL assay, M. lysodeikticus was added in reaction buffer to an OD.sub.600 of 0.5. The reaction was started with the addition of the stripes. The tubes were immediately placed at 28 C. for a designated time (e.g., 4 hr and 24 hr). The absorbance at 570 nm was recorded.

[0381] Analysis of the data included calculating the initial velocities for the recorded slopes: [OD.sub.600 min]/[slope standard curve (OD/mg M. lysodeikticus]/[Iysozyme]

[0382] Example 18This Example demonstrates a biological assay for antimicrobial activity of paper strips comprising an antimicrobial enzyme additive against a microorganism.

[0383] The antimicrobial enzyme additive comprised lysozyme, the microorganism used was vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

TABLE-US-00035 TABLE 30 Equipment and Reagents Equipment: Petri Plates Reagents: Nutrient Yeast Extract (NBY) NBY Soft Agar Lysozyme: chicken egg white, Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C. Standard: 25 I of a 500,000 units (10 mg)/ mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 350 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C.

[0384] Micrococcus lysodeikticus cell suspension was made by adding 9 mg Micrococcus lysodeikticus to NBY and mixing well, with OD.sub.600 about 0.5. Antimicrobial paper coated with a latex coating comprising lysozyme and control paper was prepared in accordance with Example 15.

[0385] The assay includes cutting appropriated sized strips of both antimicrobial and control papers (e.g., a. 1010 mm, 2020 mm, 4040 mm, or 5050 mm). 100 L of the prepared M. lysodeikticus suspension was transferred to 15 mL tube containing 5 mL NBY Soft Agar, held molten at 55 C., and mixed well. Pipet tips used fitted the pipette (e.g., multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc may alter volume. The mixture was immediately poured over a prepared sterile agar plate, rotating the dish to completely cover the agar with the M. lysodeikticus overlay. The dish was covered and allowed to solidify on level surface. The prepared antimicrobial paper(s) were placed (face down) on the soft agar overlay. Coupon(s) up to 2020 mm were able to be paired with a control on a single petri dish. The dishes were left at 28 C. overnight, and visually evaluated for a zone of clearance around the antimicrobial coupon(s) relative to the control. Quality control and safety procedures were as described in Example 13.

[0386] Example 19This Example demonstrates a biological assay for the antimicrobial activity of a paper strip comprising ProteCoat against fungal spores.

[0387] The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

TABLE-US-00036 TABLE 31 Equipment and reagents Equipment: Petri Plates Incubator Autoclave Preval Sprayer Reagents: Nutrient Yeast Extract (NBY) NBY Soft Agar Micrococcus Iysodeikticus cell (Worthington Biochemicals, cat #8736) ProteCoat was used at 125 mg ProteCoat per g coating, dispensed as 250 mg ProteCoat, and resuspended in 2 mL dilute acetic acid solution as appropriate.

[0388] Fusarium oxysporium spores were prepared by maintaining cultures of Fusarium oxysporum f sp. lycoperici race 1 (RM-1)[FOLRM-1 on Potato Dextrose Agar (PDA) slants. Microconidia of the Fusarium oxysporum f sp. lycoperici, were obtained by isolating a small portion of an actively growing culture from a PDA plate and transferring to 50 ml a mineral salts medium FLC (Esposito and Fletcher, 1961). The culture was incubated with shaking (125 rpm) at 25 C. After 960 h the fungal slurry consisting of mycelia and microconidia were strained twice through eight layers of sterile cheese cloth to obtain a microconidial suspension. The microcondial suspension was then calibrated with a hemacytometer. All fungal inocula were tested for the absence of contaminating bacteria before their use in experiments. Antimicrobial paper coated with a latex coating comprising ProteCoat and control paper was prepared in accordance with Example 15.

[0389] The assay procedure included: cutting appropriated sized strips of both antimicrobial and control papers (e.g., 4040 mm or 5050 mm); centering the strips on a sterile Potato Dextrose Agar plate, treated side up; diluting spores to 210.sup.3 per mL Potato Dextrose broth; transferring to a calibrated preval sprayer (i.e., dispense 50 L per single pump action); dispersing spores in a hood onto the agar and paper surface with a single pump action (delivers approximately 100 spores to the area); covering and leaving at ambient conditions; and observing growth over several days, though time of assay will depend on organism. Pipet tips fitted the pipette (e.g., multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc may alter volume. Quality control and safety procedures were as described in Example 13.

[0390] Example 20This Example demonstrates a paper coating comprising an antimicrobial enzyme additive. The antimicrobial enzyme comprised a lysozyme.

[0391] Assay standardization and data are shown in the following tables.

TABLE-US-00037 TABLE 32 Assay Enzymatic Additive-Lysozyme Example Techniques Used Example 15 and 17 Coupon Size Variable, 200-600 mm.sup.2 Paper Age 3 months Test Organism Micrococcus lysodeikticus Contamination level 2.5 10.sup.8 cells/mL Assay Time 4 and 24 hrs

TABLE-US-00038 TABLE 33A Test Strips and Data Paper Paper coupon (mm Type mm) Area (mm.sup.2) [lysozyme], g 0 0 0.2% 5 40 200 8.76 1.0% 5 40 200 38.80 2.0% 5 40 200 92.80 2.0% 5 40 + 5 10 250 116.00 2.0% 5 40 + 5 20 300 139.20 2.0% 5 40 + 5 40 400 185.00 2.0% 5 40 + 5 40 + 5 10 450 208.80 2.0% 5 40 + 5 40 + 5 20 500 232.00 2.0% 5 40 + 5 40 + 5 40 600 278.40

TABLE-US-00039 TABLE 33B Antimicrobial Strips and Data Paper Paper coupon (mm 4 hrs 24 hrs Type mm) OD.sub.570 % Lysis OD.sub.570 % Lysis 0 0.305 0.00 0.27 0.00 0.2% 5 40 0.301 1.31 0.275 1.85 1.0% 5 40 0.277 9.18 0.2 25.93 2.0% 5 40 0.172 43.61 0.0015 99.44 2.0% 5 40 + 5 10 0.099 67.54 0.001 99.63 2.0% 5 40 + 5 20 0.136 55.41 0.0025 99.07 2.0% 5 40 + 5 40 0.017 94.43 0.005 99.81 2.0% 5 40 + 5 40 + 5 10 0.023 92.46 0.001 99.63 2.0% 5 40 + 5 40 + 5 20 0.024 92.13 0.001 99.63 2.0% 5 40 + 5 40 + 5 40 0.015 95.08 0.0015 99.44

[0392] The rate of lysis upon contact with a coupon cut from antimicrobial treated paper, is approximately 0.5% (1.3510.sup.7 cells) per g lysozyme. This corresponds to a reduction in activity, per g of lysozyme, of approximately 65% over that observed in suspension. Treated papers of identical size with antimicrobial loadings of 0.2%, 1.0% and 2.0%, demonstrated antimicrobial function. The antimicrobial concentration on a per unit of area for those loadings, is provided in the following table.

TABLE-US-00040 TABLE 34 Antimicrobial concentration per unit area Lysozyme Paper Coating (gsm) % lysozyme g/m.sup.2 g/m.sup.2 g/mm.sup.2 A 21.9 0.2% 0.0438 4.38 10.sup.8 0.0438 B 19.4 1.0% 0.194 1.94 10.sup.7 0.194 C 23.2 2.0% 0.464 4.64 10.sup.7 0.464

[0393] Example 21This Example qualitatively demonstrates an antimicrobial enzyme additive combined with an antimicrobial peptide additive to provide antimicrobial functionality to a paper coating formulation.

[0394] An adaptation of ASTM 02020-92 was used as the assay to demonstrate the growth of a microorganism in a petri dish was inhibited by contact with the treated paper. The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat Reactive Surfaces, Ltd.; also described in U.S. patent application Ser. Nos. 10/884,355; 11/368,086; and Ser. No. 11/865,514, each incorporated by reference).

[0395] The spectrophotometric lysozyme assay uses Micrococcus lysodeikticus bacterial cells as a substrate, and measures the change in the turbidity of the cell suspension as described in Example 11 and Example 12. The efficacy of an antimicrobial peptide (e.g., ProteCoat) may be monitored biologically. Though the contemplated mechanism of action for an antimicrobial or anti-fouling peptide is similar, i.e. disruption of the structural components of the microbial cell, the cell wall may remain relatively intact. As an antifungal or antimicrobial peptide's biocidal or biostatic activity inhibits the cell, the cell may not lyse for detection of a change in turbidity. Biological assay conditions are shown in the table below.

TABLE-US-00041 TABLE 35 Enzymatic Additive-Lysozyme (Qualitative) Example Techniques Used Example 13 Coupon Size 100 mm.sup.2 Paper Age 3 months Test Organism Micrococcus lysodeikticus Growth Conditions 28 C.

[0396] A zone of clearing was seen around the antimicrobial paper in contact with a petri dish covered by M. lysodeikticus, whereas the control paper had no such zone. The coupon of paper was about half the size of the smallest coupons in the quantitative M. lysodeikticus assay, yet growth inhibition was seen.

[0397] Assay conditions for Fusarium oxysporum is shown at the table below.

TABLE-US-00042 TABLE 36 Enzymatic Additive-ProteCoat (Qualitative) Example Techniques Used Example 14 Coupon Size 40 40 mm Paper Age 3 months Test Organism Fusarium oxysporum Contamination level 100 spore, aerosol delivery Growth Conditions Ambient

[0398] Overgrowth of both test and control ProteCoat paper by the fungus, Fusarium oxysporium, was observed. The developmental state of the mycelium on the antimicrobial paper was retarded over that seen in the control paper, indicative of biostatic, and possibly biocide activity.

[0399] Example 22This Example demonstrates synergism between an antimicrobial enzyme additive combined with an antimicrobial peptide additive in a coating applied to papers, and to demonstrate antimicrobial activity of a paper comprising the antimicrobial peptide.

[0400] The antimicrobial enzyme additive comprised lysozyme, and the antimicrobial peptide additive comprised ProteCoat (Reactive Surfaces, Ltd.; also described in U.S. patent application Ser. Nos. 10/884,355; 11/368,086; and Ser. No. 11/865,514, each incorporated by reference). Assay conditions are shown at the tables below.

TABLE-US-00043 TABLE 37 Enzymatic Additive-2% Lysozyme + 0.5% ProteCoat (Titration Assay) Example Techniques Used Example 19 Coupon Size Variable, 0-400 mm.sup.2 Paper Age 3 months Test Organism Micrococcus lysodeikticus Contamination level 2.5 10.sup.8 cells/mL Assay Time 3 and 20 hrs

TABLE-US-00044 TABLE 38A Activity in Treated Papers Area Lysozyme ProteCoat Paper Strips (mm mm) (mm.sup.2) mg g/mL mg g/mL 2% 0 0 Lysozyme 5 5 25 11.60 2.90 0.00 0.00 5 10 50 23.20 5.80 0.00 0.00 5 20 100 46.40 11.60 0.00 0.00 5 40 200 92.80 23.20 0.00 0.00 5 40 + 5 5 225 104.40 26.10 0.00 0.00 5 40 + 5 10 250 116.00 29.00 0.00 0.00 5 40 + 5 20 300 139.20 34.80 0.00 0.00 5 40 + 5 40 400 185.60 46.40 0.00 0.00 2% 0 Lysozyme + 5 5 25 11.60 2.90 2.90 0.73 0.5% 5 10 50 23.20 5.80 5.80 1.45 ProteCoat 5 20 100 46.40 11.60 11.60 2.90 5 40 200 92.80 23.20 23.20 5.80 5 40 + 5 5 225 104.40 26.10 26.10 6.53 5 40 + 5 10 250 116.00 29.00 29.00 7.25 5 40 + 5 20 300 139.20 34.80 34.80 8.70 5 40 + 5 40 400 185.60 46.40 46.40 11.60

TABLE-US-00045 TABLE 38B Activity in Treated Papers Strips Area 3 hrs 20 hrs Paper (mm mm) (mm.sup.2) OD.sub.600 % Lysis OD.sub.600 % Lysis 2% 0 0.266 0.00 0.258 0.00 Lysozyme 5 5 25 0.259 2.63 0.25 3.10 5 10 50 0.259 2.63 0.23 10.85 5 20 100 0.256 3.76 0.145 43.80 5 40 200 0.228 14.29 0.038 85.27 5 40 + 5 5 225 0.199 25.19 0.019 92.64 5 40 + 5 10 250 0.148 44.36 0.011 95.74 5 40 + 5 20 300 0.177 33.46 0.013 94.96 5 40 + 5 40 400 0.09 66.17 0.012 95.35 2% 0 0.266 0.00 0.258 0.00 Lysozyme + 5 5 25 0.255 4.14 0.23 10.85 0.5% 5 10 50 0.248 6.77 0.057 77.91 ProteCoat 5 20 100 0.237 10.90 0.016 93.80 5 40 200 0.195 26.69 0.012 95.35 5 40 + 5 5 225 0.199 25.19 0.012 95.35 5 40 + 5 10 250 0.15 43.61 0.012 95.35 5 40 + 5 20 300 0.124 53.38 0.01 96.12 5 40 + 5 40 400 0.031 88.35 0.012 95.35

[0401] The concentration of lysozyme in the papers corresponded to between 2 and 50 ppm, whereas ProteCoat was between 0.5 and 12 ppm. The comparison of lysis between the 2% lysozyme paper, and the combined paper which contained 2% lysozyme and 0.5% ProteCoat indicates synergism between the additives. For example, the 100 mm.sup.2 coupon size exhibited 44% lysis, whereas the combined paper exhibited 93%. This is an observed/expected (93/44+0) of 2.1, indicative of significant synergism. To further demonstrate this activity, the assay was repeated by titrating the 2% lysozyme paper with individual swaths of 2.5% ProteCoat paper. 510, 520, and 540 mm.sup.2 lysozyme paper strips with increasing amount of Protecoat paper were added to tubes in 4 ml total volume 2.510.sup.8 Micrococcus cells/ml. The assay conditions are shown at the tables below.

TABLE-US-00046 TABLE 39 Enzymatic Additive-2% Lysozyme & 2.5% ProteCoat (Titration) Example Techniques Used Example 19 Coupon Size Variable Lysozyme 0-200 mm.sup.2 ProteCoat 0-200 mm.sup.2 Paper Age 3 months Test Organism Micrococcus lysodeikticus Contamination level 2.5 10.sup.8 cells/mL Assay Time 4 and 22 hrs

TABLE-US-00047 TABLE 40 Activity of Protecoat paper with 50, 100 and 200 mm.sup.2 Lysozyme paper against Micrococcus lysodeikticus Square Square area Strips area (mm.sup.2) (mm.sup.2) [lysozyme] [Protecoat] Paper (mm mm) Lysozyme Protecoat (ug/ml) (ug/ml) Control 0 0 0 0 (0) 0 (0) 2% 5 10 50 0 23.2 (5.8) 0 (0) Lysozyme 2.5% 5 5 50 25 23.2 (5.8) 15 (3.75) Protecoat 5 10 50 50 23.2 (5.8) 30 (7.5) 5 20 50 100 23.2 (5.8) 60 (15) 5 40 50 200 23.2 (5.8) 120 (30) 5 40 2 50 400 23.2 (5.8) 240 (60) Control 0 0 0 0 (0) 0 (0) 2% 5 20 100 0 46.4 (11.6) 0 (0) Lysozyme 2.5% 5 5 100 25 46.4 (11.6) 15 (3.75) Protecoat 5 10 100 50 46.4 (11.6) 30 (7.5) 5 20 100 100 46.4 (11.6) 60 (15) 5 40 100 200 46.4 (11.6) 120 (30) 5 40 2 100 400 46.4 (11.6) 240 (60) 2% 5 40 200 0 92.8 (23.2) 0 (0) Lysozyme 2.5% 5 5 200 25 92.8 (23.2) 15 (3.75) Protecoat 5 10 200 50 92.8 (23.2) 30 (7.5) 5 20 200 100 92.8 (23.2) 60 (15) 5 40 200 200 92.8 (23.2) 120 (30) 5 40 2 200 400 92.8 (23.2) 240 (60)

[0402] An example of a calculation for the lysozyme content in 2% lysozyme paper was: 23.22% g/m.sup.2=0.464 g/m.sup.2=0.464 g/mm.sup.2. An example of a calculation for the Protecoat content in 2.5% Protecoat paper was: 23.92.5% g/m.sup.2=0.60 g/m.sup.2=0.60 g/mm.sup.2.

TABLE-US-00048 TABLE 41 Activity of Protecoat paper with 50, 100 and 200 mm.sup.2 Lysozyme paper against Micrococcus lysodeikticus Strips 4 hrs 23 hrs Paper (mm mm) OD.sub.600 % Lysis OD.sub.600 % Lysis Control 0 0.278 0 0.276 0 2% 5 10 0.269 3.24 0.206 25.36 Lysozyme 2.5% 5 5 0.264 5.04 0.235 14.86 Protecoat 5 10 0.268 3.60 0.213 22.83 5 20 0.269 3.24 0.197 28.62 5 40 0.266 4.32 0.172 37.68 5 40 2 0.24 13.67 0.027 90.22 Control 0 0.254 0 0.229 0 2% 5 20 0.224 11.81 0.026 88.65 Lysozyme 2.5% 5 5 0.22 13.39 0.023 89.96 Protecoat 5 10 0.204 19.69 0.013 94.32 5 20 0.212 16.54 0.019 91.70 5 40 0.178 29.92 0.014 93.89 5 40 2 0.194 23.62 0.027 88.21 2% 5 40 0.203 20.08 0.019 91.70 Lysozyme 2.5% 5 5 0.181 28.74 0.009 96.07 Protecoat 5 10 0.175 31.10 0.01 95.63 5 20 0.165 35.04 0.012 94.76 5 40 0.128 49.61 0.012 94.76 5 40 2 0.145 42.91 0.019 91.70

TABLE-US-00049 TABLE 42A % Lysis (relative to control without Protecoat added) at given time 4 hr Square Area 50 mm.sup.2 100 mm.sup.2 200 mm.sup.2 (mm.sup.2) of Lysozyme Lysozyme Lysozyme Protecoat paper paper paper paper 0 3.24 11.81 20.08 25 5.04 13.39 28.74 50 3.60 19.69 31.10 100 3.24 16.54 35.04 200 4.32 29.92 49.61 400 13.67 23.62 42.91

TABLE-US-00050 TABLE 42B % Lysis (relative to control without Protecoat added) at given time 22 hr Square Area 50 mm.sup.2 100 mm.sup.2 200 mm.sup.2 (mm.sup.2) of Lysozyme Lysozyme Lysozyme Protecoat paper paper paper paper 0 25.36 88.65 91.70 25 14.86 89.96 96.07 50 22.83 94.32 95.63 100 28.62 91.70 94.76 200 37.68 93.89 94.76 400 90.22 88.21 91.70

[0403] The assay was repeated by titrating the 2% lysozyme paper with individual swaths of 2.5% ProteCoat paper. Lysozyme in technical papers added to an assay at concentrations greater than 10 ppm exhibited antimicrobial activity in the M. lysodeikticus assay. Lysozyme at approximately 5 ppm in the assay did not exhibit significant antimicrobial activity over the course of the assay (20 hrs). The addition of ProteCoat papers, with between 3 and 60 ppm ProteCoat to the assay significantly enhanced the lytic activity of lysozyme, or possibly the reverse. This was also true with the 5 ppm lysozyme, in which the lytic activity was doubled by the addition of between 3 and 60 ppm ProteCoat to the assay. The peptide additive may be enhancing the activity of the enzyme, or the enzyme enhancing the activity of the peptide, or both, to produce these results.

[0404] Example 23This Example demonstrates a spectrophotometric assay for an antimicrobial coating with a lytic additive. The lytic additive comprised a lysozyme.

[0405] The antimicrobial coatings were created using acrylic latex, commercially available paints. Equipment and reagents that were used are shown in the table below.

TABLE-US-00051 TABLE 43 Equipment and Reagents Equipment Spectrophotometer (Thermo Multiskan Ascent Plate Reader) Cuvettes (96-well assay plates) Multi-channels and single-channel pipettes and tips Reagents Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCI): [Sigma, cat #T3253, Molecular Formula: NH.sub.2C(CH.sub.2OH).sub.3 .Math. HCI, Molecular Weight: 157.60, CAS Number 1185-53-1, pKa (25 C.) 8.1] Micrococcus lysodeikticus cell (Worthington Biochemicals, cat #8736) Lysozyme: chicken egg white {Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C.. Standard: 25 I of a 500,000 units (10 mg)/mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 350 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C.)

[0406] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg Micrococcus lysodeikticus to 1 mL 10 mM Tris pH 8.0 and mixing well. A lysozyme solution was prepared by adding 10 mg lysozyme in 1 mL ddH.sub.20, and mixing well.

[0407] The lysozyme stock solution was mixed into Sherwin Williams Acrylic (SW) or Glidden latex paint (1 part water:7 part paint). 4 mil, 6 mil, and 8 mil free films were created from Sherwin Williams paint comprising a lysozyme, a Glidden paint comprising a lysozyme, and controls for both. The plate controls included 3 replicates of 50 L M. lysodeikticus cell suspension plus 50 L buffer; and 3 replicates of 100 L buffer. Pipet tips used fitted the pipette (e.g., multichannel pipettes). The liquid level was correct in the tips, as air bubbles, etc may alter volume. Quality control and safety procedures were as described in Example 13.

[0408] The antimicrobial films were cut into appropriately sized strips from both the antimicrobial and control coating. For a 5 mL assay in a 15 mL tube, standard size was 11 cm.

[0409] Analysis of samples included determining activity by monitoring the clearing of the cell suspension at OD.sub.405 and determining the best fit to a standard curve. The reaction was started with the addition of 5 ml of the M. lysodeikticus stock. The tubes were immediately placed on a rocker for 3 hr; 100 l samples were taken at 3 hr, and the absorbance at 405 nm was recorded.

TABLE-US-00052 TABLE 44 Sample Lysis Averages and Deviations Avg. % Lysis at Standard Sample 3 hr Deviation SW Control 4 mils 11.1057 0.5752 6 mils 12.2932 0.3812 8 mils 12.2802 0.5752 SW Lysozyme 4 mils 65.0651 1.3638 6 mils 74.5744 3.8272 8 mils 84.2325 4.1432 Glidden Control 4 mils 4.8514 0.4912 6 mils 5.1005 0.0569 8 mils 5.1749 0.6266 Glidden 4 mils 18.3760 0.5846 Lysozyme 6 mils 23.1840 3.6201 8 mils 29.1666 1.9095

[0410] Analysis of the data included calculating the initial velocities for the recorded slopes: [OD.sub.405 min]/[slope standard curve (OD/mg M. lysodeikticus]/[Iysozyme].

[0411] Example 24This Example demonstrates a biological assay for antimicrobial activity of coatings comprising an antimicrobial enzyme additive against a microorganism.

[0412] The antimicrobial enzyme additive comprised lysozyme, the microorganism used comprised vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

TABLE-US-00053 TABLE 45 Equipment and Reagents Equipment: Petri Plates Reagents: Luria Broth Agar (LBA) Lysozyme: chicken egg white, Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C.. Standard: 25 I of a 500,000 units (10 mg)/mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 350 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C..

[0413] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg M. lysodeikticus to 10 mM Tris, pH 8.0, and mixing well. A lawn of M. lysodeikticus was generated by spreading 200 l of this suspension onto a LBA plate, using a glass spreading rod. An antimicrobial latex coating comprising lysozyme and a control film was prepared in accordance with Example 23.

[0414] The assay includes cutting appropriated sized strips of both antimicrobial and control latex films (e.g., a 11 cm). In triplicate the free films are carefully placed onto the surface of the petri dishes spaced out equally. This procedure was repeated for each of the paint film types/thicknesses.

[0415] The paint films comprising a lysozyme were active in lysing M. lysodeikticus, producing circular zones of clearing. The difference in Zone of Clearing Diameter between the different thicknesses of film was deemed negligible.

TABLE-US-00054 TABLE 46 Diameter (cm) of Zones of Clearing Sample 4 mils 6 mils 8 mils Glidden Lysozyme 2.8 2.8 2.8 2.8 2.9 2.8 2.7 2.9 2.9 Glidden Control 0 0 0 0 0 0 0 0 0 Sherwin Williams Lysozyme 2.1 1.9 2.2 2.1 1.9 1.9 2 2 1.8 Sherwin Williams Lysozyme 0 0 0 0 0 0 0 0 0

[0416] Example 25This Example demonstrates a qualitative biological assay for survivability of an antimicrobial latex coating comprising an antimicrobial enzyme additive against a microorganism.

[0417] The antimicrobial enzyme additive comprised lysozyme, the microorganism used comprised vegetative, gram-positive M. lysodeikticus. The assay was adapted from ASTM 02020-92, Method A, Standard Test for Mildew (Fungus) Resistance of Paper and Paperboard (Reapproved 2003). Equipment and reagents that were used are shown in the table below.

TABLE-US-00055 TABLE 47 Equipment and Reagents Equipment: Petri Plates Reagents: Luria Broth Agar (LBA) Lysozyme: chicken egg white, Sigma cat #L6876; 50,000 U/mg; CAS 12650-88-3; molecular weight: 14.3 kD; solubility (H.sub.2O) 10 mg/mL; stability-1 month at 2-8 C.. Standard: 25 I of a 500,000 units (10 mg)/mL (10 mM Tris-HCI) will typically lyse E. coli from >1 mL of culture media cell pellet resuspended in 35 l buffer (10 mM Tris HCI, pH 8.0, with 0.1M NaCI, 1 mM EDTA, and 5% [w/v] Triton X-100). Typical incubation conditions for lysis are 30 min at 37 C..

[0418] A Micrococcus lysodeikticus cell suspension was made by adding 1.5 mg M. lysodeikticus to 10 mM Tris, pH 8.0, and mixing well. A lawn of M. lysodeikticus was generated by spreading 200 l of this suspension onto a LBA plate, using a glass spreading rod.

[0419] The paint formulations that were prepared included a Sherwin-Williams Acrylic Latex or a Glidden Acrylic Latex as controls (no additive), and both a Sherwin-Williams Acrylic Latex or a Glidden Acrylic Latex comprising 10 mg/mL Lysozyme (ddH.sub.2O). Each paint was made by adding 1 part additive to 7 parts paint, and then mixed with a glass stirring rod and a paint mixer. Each film was immediately drawn onto polypropylene surfaces with a thickness of 4 mil, 6 mil, and 8 mil. Cure time was 24 days. Materials for assay were generated from the polypropylene surface as 1 cm.sup.2 free films.

[0420] The assay includes cutting appropriately sized strips of both antimicrobial and control latex films (e.g., a 11 cm). In triplicate the free films were carefully placed onto the surface of the petri dishes spaced out equally. This procedure was repeated for each of the paint film types/thicknesses.

[0421] After 24 hrs incubation, the diameter of the zones of clearing was measured for each film. Using sterile tweezer, the films were removed and transfer to a new LBA plate spread with M. lysodeikticus in the same orientation as the plates the films were removed from. Repeat the procedure of measuring the zones of clearing through transfer to a new plate every day for 5 days.

TABLE-US-00056 TABLE 48 Average Diameter (cm) of Zones of Clearing Stand- Stand- Stand- ard ard ard Devia- Devia- Devia- 4 mils tion 6 mils tion 8 mils tion Day 1 Glidden N/A N/A N/A N/A 0 0 Control Glidden 2.5667 0.0577 2.5333 0.0577 2.7000 0.0000 Lysozyme Day 2 Glidden N/A N/A N/A N/A 0 0 Control Glidden 2.0000 0.0000 2.0000 0.0000 2.2000 0.0000 Lysozyme Day 3 Glidden N/A N/A N/A N/A 0 0 Control Glidden 1.4667 0.0577 1.6667 0.0577 1.9000 0.0000 Lysozyme Day 4 Glidden N/A N/A N/A N/A 0 0 Control Glidden 1.4333 0.1155 1.5667 0.0577 1.8000 0.0000 Lysozyme Day 5 Glidden N/A N/A N/A N/A 0 0 Control Glidden 1.2667 0.0577 1.4500 0.0707 1.6333 0.0577 Lysozyme .sup.1N/A in this chart just means not available/ not applicable.

[0422] There were no 4 mil or 6 mil controls tested due to a limited LBA plate supply, though 8 mil control films were tested. The standard deviations for the 8 mil controls to 0, because all 3 controls produced a 0 cm zone of clearing in each case.

[0423] The paint films comprising lysozyme were active in lysing M. lysodeikticus, producing circular zones of clearing, for five cycles of contaminant control. The difference in Zone of Clearing Diameter between the different thicknesses of each film appeared negligible.

[0424] Example 26To provide a description that is both concise and clear, various examples of ranges have been identified herein. Any range cited herein includes any and all sub-ranges and specific values within the cited range, this example provides specific numeric values for use within any cited range that may be used for an integer, intermediate range(s), subrange(s), combinations of range(s) and individual value(s) within a cited range, including in the claims. Examples of specific values (e.g., %, kDa, C., m, kg/L, Ku) that can be within a cited range include 0.000001, 0.000002, 0.000003, 0.000004, 0.000005, 0.000006, 0.000007, 0.000008, 0.000009, 0.00001, 0.00002, 0.00003, 0.00004, 0.00005, 0.00006, 0.00007, 0.00008, 0.00009, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01, 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.10, 99.20, 99.30, 99.40, 99.50, 99.60, 99.70, 99.80, 99.90, 99.91, 99.92, 99.93, 99.94, 99.95, 99.96, 99.97, 99.98, 99.99, 99.999, 99.9999, 99.99999, 99.999999, 99.9999999, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 270, 275, 280, 290, 300, 310, 320, 325, 330, 340, 350, 360, 370, 375, 380, 390, 400, 410, 420, 425, 430, 440, 450, 460, 470, 475, 480, 490, 500, 510, 520, 525, 530, 540, 550, 560, 570, 575, 580, 590, 600, 610, 620, 625, 630, 640, 650, 660, 670, 675, 680, 690, 700, 710, 720, 725, 730, 740, 750, 760, 770, 775, 780, 790, 800, 810, 820, 825, 830, 840, 850, 860, 870, 875, 880, 890, 900, 910, 920, 925, 930, 940, 950, 960, 970, 975, 980, 990, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1225, 1250, 1275, 1300, 1325, 1350, 1375, 1400, 1425, 1450, 1475, 1500, 1525, 1550, 1575, 1600, 1625, 1650, 1675, 1700, 1725, 1750, 1775, 1800, 1825, 1850, 1875, 1900, 1925, 1950, 1975, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5250, 5500, 5750, 6000, 6250, 6500, 6750, 7000, 7250, 7500, 7750, 8000, 8250, 8500, 8750, 9000, 9250, 9500, 9750, 10,000, 25,000, 50,000, 75,000, 100,000, 250,000, 500,000, 1,000,000, or more. Additional examples of the use of this definition to specify sub-ranges are given herein. For example, a cited range of 25,000 to 100,000 would include specific values of 50,000 and/or 75,000, as well as sub-ranges such as 25,000 to 50,000, 25,000 to 75,000, 50,000 to 100,000, 50,000 to 75,000, and/or 75,000 to 100,000. In another example, the range 875 to 1200 would include values such as 910, 930, etc. as well as sub-ranges such as 940 to 950, 890 to 1150, etc.

[0425] In embodiments wherein a value or range is denoted in exponent form, both the integer and the exponent values are included. For example, a range of 1.010.sup.17 to 2.510.sup.7, would include a description for a sub-range such as 1.2410.sup.17 to 8.710.sup.11. However, general sub-ranges for each type of unit (e.g., %, kDa, C., m, kg/L, Ku) are contemplated, as the values typically found within a particular type of unit are of a sub-range of the integers described above. For example, integers typically found within a cited percentage range, as applicable, include 0.000001% to 100%. Examples of values that can be within a cited molecular mass range in kilo Daltons (kDa) as applicable for many coating components include 0.50 kDa to 110 kDa. Examples of values that can be within a cited temperature range in degrees Celsius ( C.) as may be applicable in the arts of a polymeric material, a surface treatment (e.g., a coating), and/or a filler include 10 C. to 500 C. Examples of values that can be within a thickness range in micrometers (m) as may be applicable to coating and/or film thickness upon a surface include 1 m to 2000 m. Examples of values that can be within a cited density range in kilograms per liter (kg/L) as may be applicable in the arts of a material formulation include 0.50 kg/L to 20 kDa. Examples of values that can be within a cited shear rate range in Krebs Units (Ku), as may be applicable in the arts of a material formulation, include 20 Ku to 300 Ku.

[0426] Example 27This Example is directed to the assay for active phosphoric triester hydrolase expression in cells.

[0427] Routine analysis of parathion hydrolysis in whole cells is accomplished by suspending cultures in 10 milli-Molar (mM) Tris hydrocholoride at pH 8.0 comprising 1.0 mM sodium EDTA (TE buffer). Cell-free extracts are assayed using sonicated extracts in 0.5 milliLiters (ml) of TE buffer. The suspended cells or cell extracts are incubated with 10 microLiters (l) of substrate, specifically 100 g of parathion in 10% methanol, and p-nitrophenol production is monitored at a wavelength of 400 nm. To induce the opd gene under lac control, 1.0 mol of isopropyl--D-thiogalactopyranoside (Sigma) per ml is added to the culture media.

[0428] Example 28This Example is directed to the preparation of an enzyme powder. In a typical preparation, a single colony of bacteria that expresses the opd gene is selected and cultured in a rich media. After growth to saturation, the cells are concentrated by centrifugation at 7000 rotations per minute (rpm) for 10 minutes for example. The cell pellet is then resuspended in a volatile organic solvent such as acetone one or two times to desiccate the cells and to remove a substantial portion of the water contained in the cell pellet. The pellet may then be ground or milled to a powder form. The powder may be frozen or stored at ambient conditions for future use, or may be added immediately to a surface coating formulation. Additionally, the powder may be freeze dried, combined with a cryoprotectant (e.g., cryopreservative), or a combination thereof.

[0429] Example 29This Example is directed to the formation of an OPH powder and latex coating. In an example of use of the powder prepared as described in Example 28, 3 mg of the milled powder was added to 3 ml of 50% glycerol. The suspension was then added to 100 ml of Olympic premium interior flat latex paint (Olympic, One PPG Place, Pittsburgh, Pa. 15272 USA). This paint with biomolecular composition was then used to demonstrate the activity of the paint biomolecular composition in hydrolysis of a pesticide or a nerve agent analog.

[0430] Example 30This Example demonstrates, in a first set of assays, a paint product as prepared in Example 29 was applied to a hard, metal surface. The surface used in the present Example was a non-galvanized steel surface that was cleaned through being degreased, and pretreated with a primer coat. A control surface was painted with the identical paint with no biomolecular composition. Paraoxon, an organophosphorus nerve gas analog was used as an indicator of enzyme activity. Paraoxon, which is colorless, is degraded to form p-nitrophenol, which is yellow in color, plus diethyl phosphate, thus giving a visual indication of enzyme activity. In multiple assays, the surface with control paint remained white, indicating no production of p-nitrophenol, and the surface painted with the paint and biomolecular composition turned yellow within minutes, indicating an active OPH enzyme in the paint. This demonstration has shown that the surface remains active for more than 65 days, which was the maximum duration of the protocol.

[0431] Example 31This Example demonstrates anti-fouling activity of a cell-based particulate material comprising an organophosphorus hydrolase in a marine coating that was superior to the activity of a different purified control enzyme (i.e., acetylasesubtilisin). A marine coating comprising an anti-biological peptide was also evaluated for anti-fouling activity.

[0432] To evaluate the anti-fouling properties of various enzymes and peptides, a marine coating (i.e., an emulsion blend of water dispersed resin system) had either: an cell-based particulate material comprising an organophosphorus hydrolase (EC 3.1.8.1) similar to that described for Example 28; an acetylasesubtilisin, which is a non-specific alkaline protease secreted by bacteria of the genus Bacillus (Novozymes); or a peptide preparation (ProteCoat, comprising SEQ ID no. 40); blended into the coating sample. The final concentration of a cell-based particulate material comprising an organophosphorus hydrolase or the peptide preparation in the coating samples was about 3% to about 5% weight of these anti-biological agents. The concentration of the acetylasesubtilisin in the coating sample was unknown. A Ross blender was used to disperse the cell-based particulate material comprising an organophosphorus hydrolase into the coating resin sample. A marine coating sample not comprising any enzymatic or peptidic anti-biological agent was used as a control. The pot life time was about 45 to about 60 min prior to application. Each coating sample was applied at room temperature to a different steel surface previously coated with an epoxy undercoat. The cure time for each coating to tack was 20 min, cure time to tack free was 2 hr, and cure time to usable service was between about 24h (fast) to 48h (normal). The dry film thickness was about 5 to about 6 mils for each coating sample.

[0433] The coated steel surfaces were statically and continuously submerged in Atlantic Ocean's waters, during summer, for about 1 month. Coating properties such as adhesion and hardness was not altered by incorporation of the anti-biological agents into the coating samples. No corrosion was detected in the steel surfaces for any coating sample.

[0434] All coating samples were fouled. The coating comprising an cell-based particulate material comprising an organophosphorus hydrolase, however, was easily cleaned from the surface relative to the other coating samples. Specifically, removal of fouling from the coating sample comprising the cell-based particulate material comprising an organophosphorus hydrolase did not require mechanical scrapping, unlike the other coating samples. The organophosphorus hydrolase bioadditive reduced and/or prevented adherence of the fouling organisms and fouling film. Barnacles that did attach were unable to glue themselves to the surface sufficiently to require mechanical removal methods. Some grasses may have attached to the surface of some coated surface when free of barnacles.

[0435] The marine coating used was a 2K system, that is, a 2-pack coating where separate coating components are admixed shortly before application. The addition of an additional enzyme or peptide to the coating effectively made the coating a 3K (i.e., 3 pack) system. It is contemplated that such a multi-pack system comprising a container with an anti-fouling bioadditive will retain greater bioactivity during pot-life storage, as the bioadditive is not contacted with other potentially damaging coating components prior to preparation for use. It is further contemplated that anti-fouling activity for a coating comprising an cell-based particulate material comprising an OP compound degrading enzyme, cell debris from a microorganism typically used to produce the OP compound degrading enzyme (e.g., Escherichia coli), and/or a purified OP compound degrading enzyme, may also possess such anti-biological and/or anti-fouling activity, possibly for an extended period of time (e.g., about 1, 2, 3, 4, or 5 years or more). It is also contemplated that combinations of such anti-biological enzyme and/or peptide with each other and/or another anti-biological agent (e.g., a preservative, a co-biocide, an anti-fouling agent, an anti-microbial agent) may produce additive and/or synergistic anti-biological (e.g., anti-fouling activity) in a coating.

[0436] Example 32This Example demonstrates immobilization of streptavidin in a coating. The streptavidin is for promoting the retention of a biotin labeled hexahistidine metal binding sequence in the coating.

[0437] Streptavidin was added to a Joncryl 74 coating, so that the streptavidin would function as an anchor for biotin labeled hexahistidine in the coating.

[0438] The equipment used included polypropylene sheet(s), a drawdown bar, and a balance. The reagents used included: streptavidin (Cat #PRO-283, Lot #1209STREP01 from ProSpec.com; ProSpec, Bonsal American, 8201 Arrowridge Blvd., Charlotte, N.C. 28273, USA), Joncryl 74 (48.5% Solids; BASF Corporation, 8310 16.sup.th Street; P.O. Box 902; Sturtevant, Wis., 53177, USA) and 25% aqueous glutaraldehyde. The Joncryl 74 coating was a soft film forming acrylic polymer emulsion, with approximate properties of: a 8.1 pH; a 700 cps viscosity; a 1.03 g/cm.sup.3 density at 25 C.; a 16 C. Tg; a 50 acid number (NV); a >200,000 molecular weight (Mw); a freeze/thaw stability property; a minimum film forming temperature of <5 C.; a 1.0 total wt. % VOC; grease (i.e., ink and overprint varnish formulations) resistance, water resistance, rub resistance, and a high slide angle coating property suitable for use on folding carton and multiwall bag applications.

[0439] Joncryl 74 neat coating samples were drawn down onto two separate polypropylene sheets (i.e., two 5 mil draws per sheet with an uncoated section of polypropylene between each draw). The films were allowed to cure overnight at room temperature.

[0440] Streptavidin was rehydrated using 2 mL sterile water to make a final concentration of 5 mg/mL. 10 mL Joncryl 74 (4.80 g solids) were blended with 1 mL (5 mg) of the streptavidin, and 120 L of 25% aqueous glutaraldehyde added for a final cross-linker concentration of 0.5%. The blended overcoating was allowed to react for 5 min. The overcoat material was drawn at 3 mil thickness in three separate (i.e., independent) locations across the 5 mil supporting undercoat on a polypropylene sheet as well as the uncoated section of the polypropylene sheet. For a control, 5 mL Joncryl 74 (2.4 g solids) was blended with 500 L (2.5 mg) streptavidin without glutaraldehyde. Three separate control resin films were similarly drawn at 3 mil thickness across the undercoat layer of another polypropylene sheet as well as the uncoated section of the polypropylene sheet. The overcoats were cured at room temperature overnight. The undercoat and overcoat coated sections, as well as the overcoat only sections, of the polypropylene sheets were sectioned for analysis. It is contemplated that the thinner film of the overcoat only sections may result in less noise and/or lower sensitivity in an absorbance dependant assay.

[0441] The resin system with the glutaraldehyde incorporated turned yellow within 5 min of blending. The coating was drawn over the control films quickly at 3 mils. No color changes were observed with the glutaraldehyde free streptavidin-Joncryl blend. It is contemplated that that the aldehydes likely reacted with the coating polymer amines creating chromatic aberrations. The aldehyde functional groups may have reacted with the polymer and the proteins to crosslink them to the coating material. There is no data to indicate that the color change was due to the aldehyde covalently linking with the protein.

[0442] Example 33This Example demonstrates Biotin labeled histidine (HH-Biotin, HH-Bio oligopeptide) incubation onto a coating comprising streptavidin (streptavidin coating).

[0443] Upper coatings of Joncryl 74 comprising immobilized streptavidin as previously described was used to bind Biotin labeled histidine (HH-Biotin, HH-Bio oligopeptide) tag.

[0444] The equipment used included a balance, a pipette, and a 40 C. shaker plate. The reagents used included a His6-Bio-0240 (Lot #10-9712-17687, 93.5%; structure His-His-His-His-His-His-OCO.sub.2-OCO.sub.2-(K-Ahx-biotin)-amide; 21st Century Biochemicals, 33 Locke Drive, Marlboro, Mass. 01752, U.S.A.), sterile water, scintillation vial, and wax paper. 36.5 mg HH bio oligopeptide was weighed into a scintillation vial and 3.65 mL sterile water added. The HH-Bio oligopeptide sample was placed on the 40 C. incubator shaker for 10 min to dissolve the sample. The HH-Bio oligopeptide sample was checked after incubation 250rpm for 10 min for residual crystals. If the HH-Bio oligopeptide sample was completely dissolved, the HH-Bio oligopeptide sample was removed from the incubator and set at room temperature until cool. If no crystals formed upon cooling, the HH-Biotin solution sample was ready for use.

[0445] The polypropylene sheet having the 3 mil Joncryl 74 films (3 samples total) comprising streptavidin was placed on a flat surface. 1 mL of the HH-Biotin solution was placed on the coating. A section of wax paper was placed on top of the HH-Biotin solution. The HH-Biotin solution was spread under the waxpaper to cover the area of the film that is to contact the biotin. The HH-Biotin solution was incubated for 2 hours at room temperature before removing the wax paper.

[0446] It is contemplated that the coating samples will undergo further assays to demonstrate streptavidin and/or labeled hexahistidine incorporation efficiency and/or metal binding (e.g., Ni.sup.+, Cu.sup.+, Ag.sup.+, Co.sup.+) binding properties. For example, elemental scanning electron microscopy may be conducted quantitate the amount of a metal and the metal's location in a film.

[0447] Example 34This Example demonstrates minimal inhibitory concentration (MIC) observed in a 96-well plate assay.

[0448] All metals and the Protecoat (i.e., comprising SEQ ID no. 40) tested for comparison were prepared as a 10 mg/ml stocks and the highest tested concentration for each is 5 mg/ml (5000 g/ml). After inoculating each of the test wells with 100 l of a 210.sup.4 cells/ml inoculum (final well volume=200 l), the plates are incubated at 37 C. for 24 hours and then are replica plated onto agar to observe the MIC. All tested against E. coli ATCC #8739. The MIC for each coating is shown in the Table below.

TABLE-US-00057 TABLE 49 MIC of Protecoat vs Metal Compounds Protecoat Standard MIC (g/m1) Protecoat in ddH2O 156 (#12166 C) Metals Cobalt(II) chloride hexahydrate 625 Copper(II) carbonate 5000 Copper(II) sulfate 625 Nickel sulfate 625 Silver nitrate 20 Copper Powder Greater than 5000

[0449] Example 35This Example describes reversible binding of biomolecular composition(s), ligand(s) for the biomolecular composition(s), and material formulation (e.g., coating) component(s) that may occur to create a rechargeable material.

[0450] For example, it is contemplated that a proteinaceous molecule comprising a binding sequence that may reversibly bind a component of the material formulation, based on the association equilibrium constant Ka and/or the dissociation equilibrium constant Kdiss (Kd), so that the proteinaceous molecule may be released from a material formulation by a mechanism such as elution. The content of the proteinaceous molecule in the material formulation may then be recharged by contacting a proteinaceous molecule to the material formulation so it is rebound as part of the material formulation. An example of such a rechargeable system would be a polypeptide comprising a streptavidin sequence and another bioactive proteinaceous sequence (e.g., a metal binding sequence, an anti-fouling sequence, an-anti-biological sequence, an enzyme sequence) and a material formulation comprising an affinity resin comprising biotin as a component, wherein the unbound biotin may be used to bind additional polypeptide molecule(s), which may comprise the same or different bioactive proteinaceous sequence(s), upon contact of the additional polypeptide molecule(s) with the material formulation after a period of time where some of the previously bound polypeptide molecules have been lost due to elution or other environmental conditions(s). For example, the bioactive proteinaceous sequence of such a polypeptide molecule may be binding a metal cation to also recharge the anti-fouling metal content of a material formulation such as a marine coating. Of course, other binding sequence(s) and compositions described herein (e.g., an immobilization agent) or as would be know in the art in light of the present disclosures may be used in a rechargeable material formulation/biomolecular composition system.

[0451] In some embodiments, it is contemplated that a material formulation comprising a binding sequence and/or an immobilized biomolecular composition may be reprogrammed to bind a like or different ligand for the binding sequence and/or a like or different biomolecular composition bound by the immobilization agent. Reprogramming refers to replacement of a bound (e.g., immobilized) material formulation component (e.g., a metal ion, a proteinaceous molecule) for a different bound component. For example, a material formulation may comprise a plurality of streptavadin molecules to bind a proteinaceous sequence comprising a biotin. As one or more proteinaceous molecule(s) become unbound from the streptavadin, an additional biotin linked proteinaceous molecule, which may comprise the same or different sequences, may bind the free streptavidin. Reprogramming may be conducted by contacting a material formulation with the additional material (e.g., a solution comprising a different biotin linked proteinaceous molecule) to become bound to the immobilization agent. In another example, a material formulation at least one unbound (free) biotin molecule may be reprogrammed by being contacted with streptavidin comprising proteinaceous molecule comprising a different sequence that a proteinaceous molecule that previously was bound to the free biotin molecule.

[0452] It is also contemplated that numerous proteinaceous molecules, such as those of peptide library(s), whether synthetically or recombinantly produced through genetic modification technique(s), may be screened for variations in binding association and/or dissociation equilibrium constant(s), using the assays and/or techniques described herein or as would be known in the art, to identify and/or optimize the coefficient(s) of binding for a particular application (e.g., binding for a particular metal ligand for use in an anti-fouling coating).

[0453] For example, it is contemplated that a metal binding sequence may be selected for having binding coefficient(s) sufficient to accumulate a metal found in a preparation (e.g., a solution) contacted with a material formulation to recharge the metal binding sequence. In another example, the metal binding sequence may be selected for a binding coefficient capable of accumulating a toxic metal at a concentration typically found in the environment the material formulation will be used in. In a specific example, a marine coating comprising a metal binding sequence may accumulate a metal ion from ocean water that contacts the marine coating. In another example, the binding coefficient of the metal binding sequence may be selected to be capable of such accumulation and/or modulating the net loss/rate of release of metal from a material formulation based on the expected concentration of metal ion available in the ocean water, the material formulation, and/or a material used to recharge (e.g., a solution/slurry of metal) the metal content of the material formulation. The table below gives exemplary concentrations of metals dissolved in ocean water.

TABLE-US-00058 TABLE 50 Metal Metal Concentrations In Ocean Water Metal Typical Concentration in Ocean Water* Alumina about 10 to about 20 nmol/L Antimony about 0.5 to about 2 nmol/L Arsenic about 15 to about 25 nmol/L Chromium about 1 to about 4 nmol/L Cobalt about 20 to about 40 pmol/L Copper about 1 to about 5 nmol/L Iron about 70 to about 760 pmol/L Manganese about 0.25 to about 1 nmol/L Mercury about 0.7 to about 1.1 pmol/L Silver about 7 to about 20 pmol/L Zinc about 0.5 to about 10 nmol/L *(see, for example, Handbook on the Toxicology of Metals pp. 263-278, 2007)

[0454] Example 36:This Example demonstrates the ability of a coating to bind metals by the incorporation of metal binding peptides.

[0455] The materials used are shown on the Table below.

TABLE-US-00059 TABLE 51 Materials Nickel sulfate (Sigma Product #656895) Copper sulfate (Sigma Product #451657) Cobalt chloride hexahydrate (Sigma Product #255599) His6 peptide (21st Century Biochemicals Product #HIS6-0400) Distilled water pH 5 and 10 2 mL microtubes Pipette Pipette Tips Plate Reader 96-well Plate Oven Rocker Glidden Vinyl Latex Paint (Cat #AF 1424)

[0456] Films were prepared by adding 93 mg/ml His6 peptide into water and mixing into a Glidden coating at a ratio of 1 part water to 7 parts coating. The coating was mixed well until the peptide was evenly dispersed. A draw down bar was used to make films on a polypropylene block at 8 mils thickness. The films were allowed to cure for 48 hours before use. The control films were made in the same manner without the added peptide.

[0457] 3 cm.sup.2 pieces of film were cut from the polypropylene blocks. Individual coupons were placed into labeled 2 mL microtubes. Each of the coupons was tested in triplicate. 1 ml 200 mM nickel sulfate was added into each microtube. The microtubes were placed on a rocker overnight. The liquid was removed from the microtubes and add distilled water (pH 5) into each microtube. The microtubes were placed on a rocker overnight. The liquid was removed (leave the films behind) from the microtubes and place into new microtubes. The microtubes were placed in an oven at 70 C. to remove all of the water. Once the microtubes were dry, 200 ul water (pH 10) was added into each tube, and the water transferred into 96-well plate and read the absorbance at 340 nm. The concentration of nickel was determined using a standard curve. This procedure was repeated 100 mM copper sulfate using an absorbance of 450 nm, and with 42 mM cobalt chloride hexahydrate using an absorbance of 414 nm, to determine the concentration of those metals.

[0458] The recorded absorbances were as follows:

TABLE-US-00060 TABLE 52 Bound Metal Absorbances Metal Nm Control Film H6 Peptide Film Nickel sulfate 340 0.2648 0.4159 Copper sulfate 450 0.1303 0.1939 Cobalt chloride hexahydrate 414 0.1382 0.1527

[0459] Using the standard curves for each metal, the following molarities were calculated:

TABLE-US-00061 TABLE 53 Standard Curves Control Film (mM) H6 Peptide Film (mM) Nickel sulfate 77 131 Copper sulfate 60 102 Cobalt chloride hexahydrate 30 35

[0460] Standard curves are shown below.

TABLE-US-00062 TABLE 54 Standard Curves (Note: 200 L well volume used) Nickel sulfate Copper sulfate Cobalt chloride hexahydrate pH 10 pH 10 pH 10 mM Abs 340 nm mM Abs 450 nm mM Abs 414 nm 200 0.6101 100 0.1944 42 0.1878 180 0.5405 90 0.1745 38 0.1582 160 0.4914 80 0.1609 34 0.1473 140 0.4482 70 0.1462 29 0.1344 120 0.3897 60 0.1300 25 0.1195 100 0.3302 50 0.1162 21 0.1061 80 0.2744 40 0.1019 17 0.0927 60 0.2213 30 0.0878 13 0.0833 40 0.1479 20 0.0716 8 0.0671 20 0.1117 10 0.0593 4 0.0546 0 0.0423 0 0.0362 0 0.0368

[0461] The Glidden films with the metal binding peptide bound more nickel, copper and cobalt than in the control films. There was stronger nickel and copper binding in the films. The cobalt chloride hexahydrate did not show as much binding as the other metals which may be due to the fact that the cobalt complex is larger than the other metals that were tested. Distilled water at pH 5 was used to wash the films was to optimize the release of metal peptides from the free films back into solution. At lower pH values, there is more competition at the binding sites of the peptide between hydrogen ions and the metals.

[0462] Example 37This Example describes the contemplated ability of a coating to bind silver by the incorporation of a metal binding peptide.

[0463] The materials that may be used are shown on the Table below.

TABLE-US-00063 TABLE 55 Materials Silver nitrate Sodium chloride Distilled water pH 12 2 mL microtubes Pipette Pipette Tips Plate Reader 96-well Plate Rocker Glidden Vinyl Latex Paint (Cat #AF 1424)

[0464] Films may be prepared by adding 93 mg/ml His6 peptide into water and mix into a Glidden coating at a ratio of 1 part water to 7 parts coating. The peptide and coating may be mixed well until the peptides will be evenly dispersed. A draw down bar may be used to make films on a polypropylene block at 8 mils thickness. The films may be allowed to cure for 48 hours before use. Control films may be made in the same manner without the added peptide.

[0465] 3 cm.sup.2 pieces of film may be cut from the polypropylene blocks. Individual coupons may be placed into labeled 2 mL microtubes. Each of the coupons may be tested in triplicate. 1 ml 20 mM nickel sulfate (pH 12) may be added into each microtube. The microtubes may be placed on a rocker overnight. 100 ul of solution may be taken from each tube and added into 96-well plate. 100 ul 0.2 mg/ml NaCl (pH 12) may be added into each well.

[0466] It is contemplated that the addition of NaCl with the presence of silver nitrate will cause a brown precipitate to appear. It is contemplated that the solution that will be exposed to the control free films will show more brown precipitate in solution as opposed to the films containing the metal binding peptide. Thus, there will be more silver nitrate attached to the films containing the peptide as opposed to the control films.

[0467] Example 38This Example describes an assay to detect an incorporated proteinaceous molecule in a coating.

[0468] The stain for a proteinaceous molecule used was Coomassie R250. The Coomassie R250 characteristics included the development of intensely colored complexes with proteins, and can determine as little as 0.5 g/cm.sup.2 of protein present in a gel matrix. An anion of Coomassie Brilliant Blue formed in the acidic staining medium combines with the protonated amino groups of proteins by electrostatic interaction; though the resulting complex is reversible under the proper conditions.

[0469] Films prepared as described above with His peptide or peptide free controls were stained with Coomassie R250 in 40% methanol, 10% acetic acid, 50% water for 1 hr. The films were then washed with 40% methanol, 10% acetic acid, 50% water for 40 min. The films were then stored in water. The films prepared with peptide stained darker than the controls, indicating peptide was incorporated into the material.

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