EXHIBIT A
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`

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`US. Patent Application
`RACT-00200
`
`Express Mail No. EJ756342393US
`
`APPLICATION FOR UNITED STATES OF AMERICA LETTERS PATENT
`
`for
`
`TITLE:
`
`BIOLOGICAL ACTIVE COATING COMPONENTS, COATINGS,
`
`AND COATED SURFACES
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`10
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`APPLICANT: C. Steven McDaniel
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`CITIZENSHIP: USA
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`RESIDENCE: Austin, Texas
`
`ASSIGNEE:
`
`REACTIVE SURFACES, LTD.
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`15
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`US. Patent Application
`RACT-00200
`
`TABLE OF CONTENTS
`
`Title Page
`Table of Contents
`
`' Priority Statement
`Background of the Invention
`A.
`Field of the Invention
`
`Description of the Related Art
`B.
`Summary of Invention
`Detailed Description of the Invention
`A.
`Biomolecules
`
`B.
`
`Enzymes
`1.
`Preferred Enzymes
`a.
`OPH
`
`b.
`
`Paraoxonase
`
`c.
`d.
`
`Carboxylases
`OPAAs, Prolidases, Aminopeptideases and
`PepQ
`Squid-Type DFPases
`e
`Mazur—Type DFPases
`f.
`Other Phosphoric Triester Hydrolases
`9.
`Functional Equivalents of Wild-Type Enzymes
`a.
`OPH Functional Equivalents
`b.
`Paraoxonase Functional Equivalents
`c.
`Squid—type DFPase Functional Equivalents
`’ Combinations of Biomolecules
`
`2.
`
`3.
`
`Recombinantly Produced Enzymes
`1.
`General Expression Vector Components and Use
`2.
`Prokaryotic Expression Vectors and Use.
`Host Cells
`
`Production of Expressed Proteinaceous Molecules
`Processing of Expressed Proteinaceous Molecules
`1.
`Cell Permeabilization/Disruption
`2.
`Sterilization
`
`Concentrating a Biomolecule Composition
`3.
`Drying a Biomolecule Composition
`4.
`Resuspending Biomolecule Composition
`5.
`Temperatures
`6.
`Other Processing Steps
`7.
`Coatings
`1.
`Paints
`
`2.
`
`Clear-coatings
`a.
`Varnishes
`
`b.
`c.
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`d.
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`e.
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`Lacquers
`Shellacs
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`Stains
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`Water repellent-coatings
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`C.
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`D.
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`E.
`F.
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`G.
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`Coating Categories by Use
`a.
`Architectural Coatings
`(1) Wood Coatings
`(2) Masonry Coatings
`(3)
`Artist’s Coatings
`industrial Coatings
`(1)
`Automotive Coatings
`(2)
`Can Coatings
`(3)
`Sealant Coatings
`(4)
`Marine Coatings
`Specification Coatings
`(1)
`Pipeline Coatings
`(2)
`Traffic Marker Coatings
`(3)
`Aircraft Coatings
`(4)
`Nuclear Power Plant Coatings
`Coating Components
`1.
`Binders
`
`b.
`
`0.
`
`a.
`
`Oil-based binders
`
`(1)
`(2)
`
`Oils
`Alkyd Resins
`(i)
`Oil Length Alkyd-Binders
`(ii)
`High Solid Alkyd Coatings
`(iii)
`Uralkyd Coatings
`‘
`(iv) Water-Borne Alkyd Coatings
`Oleoresinous Binders
`(3)
`Fatty Acid Epoxy Esters
`(4)
`Polyester Resins
`Modified Cellulose Binders
`Polyamide‘and amidoamine binders
`Amino Resins
`
`-
`
`Urethane Binders
`
`(1) Water-Borne Urethanes
`(2)
`Urethane Powder Coatings
`Phenolic Resins
`(1)
`Resole
`(2)
`Novolak
`Epoxy Resins
`(1)
`Ambient Condition Curing Epoxies
`(2)
`Bake Curing Epoxies
`(3)
`Electrodeposition Epoxies
`(4)
`Powder Coating Epoxies
`(5)
`Cycloaliphatic Epoxies
`Polyhydroxyether Binders
`Acrylic Resins
`(1)
`Thermoplastic Acrylic Resins
`(2) Water-Borne Thermoplastic Acrylic
`
`'
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`b.
`c.
`d.
`e.
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`f.
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`g.
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`h.
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`i.
`j.
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`US. Patent Application
`RACT-00200
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`(3)
`
`Coatings
`(i)
`Architectural Coatings
`(ii)
`industrial Coatings
`Thermosetting Acrylic Resins
`(i)
`Acrylic-Epoxy Combinations
`(ii)
`Acrylic-Amino Combinations
`(iii)
`Acrylic-Urethane Combinations
`(iv) Water-Borne Thermosetting
`Acrylics
`Polyvinyl Binders
`(1)
`Plastisols and Organisols
`(2)
`Powder Coatings
`(3) Water-Borne Coatings
`Rubber Resins
`(1)
`Chlorinated Rubber Resins
`(2)
`Synthetic Rubber Resins
`Bituminous Binders
`
`Polysulfide Binders
`Silicone Binders
`
`k.
`
`|.
`
`m.
`
`n.
`o.
`
`(2)
`
`2. Liquid Components
`a.
`Solvents, Thinners, and Diluents
`(1)
`Hydrocarbons
`(i)
`Aliphatic Hydrocarbons
`(ii)
`Cycloaliphatic Hydrocarbons
`(iii)
`Terpene Hydrocarbons
`(iv)
`Aromatic Hydrocarbons
`Oxygenated Solvents
`(i)
`Alcohols
`(ii)
`Ketones
`(iii)
`Esters
`(iv)
`Glycol Ethers
`(v)
`Ethers
`Cholrinated Hydrocarbons
`(3)
`Nitrated Hydrocarbon
`(4)
`(5) Miscellaneous Organic Liquids
`Plasticizers
`
`b.
`
`3.
`
`Water-Borne Coatings
`c.
`Colorants
`a.
`Pigments
`(1)
`Corrosion Resistance Pigments
`(2)
`camouflage Pigments
`(3)
`Color Property Pigments
`(i)
`Black Pigments
`(ii)
`Brown Pigments
`(iii) White Pigments
`(iv)
`Pearlescent Pigments
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`US. Patent Application
`RACT-00200
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`Violet Pigments
`(v)
`Blue Pigments
`(vi)
`(vii) Green Pigments
`(viii) Yellow Pigments
`(ix)
`Orange Pigments
`(x)
`Red Pigments
`(xi) Metallic Pigments
`Extender Pigments
`
`(4)
`Dyes
`b.
`Coating Additives
`a.
`Preservatives
`b.
`Wetting Additives and Dispersants
`(1) Wetting Additives
`(2)
`Dispersants
`Buffers
`Rheology Modifiers
`Defoamers
`Catalysts
`(1)
`Driers
`(2)
`Acids
`(3)
`Bases
`(4)
`Urethane Catalysts
`Antiskinning Agent
`Light Stabilizers
`Corrosion Inhibitors
`Dehydrators
`Electrical Additives
`
`c.
`d.
`e.
`f.
`
`g.
`h.
`i
`j
`k.
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`L.
`Anti—Insect Additives
`209
`Coating Preparation
`Empirically Determining the Properties of Biomolecule,
`Coatings and/or Film
`214
`Preferred Use of the Invention
`227
`Combinations of Decontamination Compositions and
`Methods
`Removing a Coating of Film
`
`228
`230
`
`Example 1
`Assay for Active Phosphoric Triester Hydrolase Expression in Cells
`Example 2
`,'
`Preparation of Enzyme Powder
`Example 3
`Two-Pack OPH Paint Coating: OPH Powder and Latex Paint
`Example 4
`Application of OPH Paint to a Surface
`Example 5
`Buffered Enzyme Paint
`Example 6
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`4.
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`5.
`6.
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`7.
`8.
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`9.
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`US. Patent Application
`RACT-00200
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`NATO Demonstration of Soman Detoxification Using OPH—Painted
`Surfaces
`-
`
`Example 7
`Aberdeen Proving Ground
`Example 8
`NATO Protocols for Organophosphorus CWA Decontamination
`A.
`Coated Surface
`
`B.
`
`C.
`
`D.
`
`Contamination
`
`Incubation
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`Decontamination
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`Sampling
`Analysis
`
`E.
`F.
`Example 9
`Large-Scale Batch Fermentation of Produce OPH
`Example 10
`Large-Scale Fed-Batch Fermentation to Produce OPH
`Example 11
`Exterior Gloss Alkyd Paint Formulation
`Example 12
`Ranges
`Example 13
`Elastomers
`
`Example 14
`Fillers and Filed Polymers
`Example 15
`Adhesives and Sealants
`
`Example 16
`Textiles
`
`Example 17
`Waxes
`
`V
`Example 18
`Additional OPAAs
`
`Example 19
`Dowel Assay-Paraoxonase
`References
`
`Claims
`
`Abstract
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`This application claims the benefit of Provisional Patent Application
`Entitled “Bioactive Protein Paint Additive, Paint, and Painted Various,” Ser. No.
`60/409,102, filed September 9, 2002, incorporated herein in its entirety by
`reference.
`‘
`
`US. Patent Application
`RACT-00200
`
`BACKGROUND OF THE INVENTION
`
`A.
`
`Field of the Invention
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`The present invention relates generally to the field of biological molecules
`as components of coatings conferring an activity or other advantage to the
`coating proteinaceous molecule related to the biological molecule. More
`specifically, the present invention relates to proteins as such components of
`coatings.
`In one specific regard, the present invention relates to protein
`compositions capable of organophosphorus detoxification, and methods of
`reducing organophosphorus compoUnds on surfaces. More specifically, the
`present invention relates to coatings such as paints that degrade
`organophosphorus compounds such as pesticides and chemical warfare agents.
`The present invention further relates to paint and coating compositions and
`methods of their use to detoxify organophosphorus chemical warfare agents.
`
`.
`
`B.
`
`Description of the Related Art
`
`Organophosphorus compounds (“organophosphate compounds” or “OP
`compounds”) and organosulfur (“08”) compounds are used extensively as
`insecticides and are highly toxic to many organisms, including humans. OP
`compounds function as nerve agents. The primary effects of exposure to these
`agents are very similar, including inhibition of acetylcholinesterase and
`butyrylcholinesterase, with the subsequent breakdown of the normal operation of
`the autonomic and central nervous systems (Gallo and Lawryk, 1991).
`
`Over 40 million kilograms of OP pesticides are used in the United States
`annually (Mulchandani, A. et al., 1999a). The number of people accidentally
`poisoned by OP pesticides has been estimated to be upwards of 500,000
`persons a year (LeJeune, K. E. et al., 1998). Depending on the toxicity to the
`organism (e.g., humans), repeated, prolonged and/or low-dose exposure to an
`OP compound can cause neurotoxicity and delayed cholinergic toxicity. High-
`dose exposure can produces a fatal response (Tuovinen, K. et al., 1994).
`
`Arguably of greater danger to humans, however, is the fact that some of
`the most toxic OP compounds are used as chemical warfare agents (“CWA”).
`Chemical warfare agents are classified into G agents, such as GD (“soman”), GB
`(“sarin”), GF (“cyclosarin”) and GA (“tabun”), and the methyl phosphonothioates,
`commonly known as V agents, such as VX and Russian VX (“R-VX” or “VR”).
`The most important CWAs are as follows: tabun (O-methyl
`dimethylamidophosphorylcyanide), which is the easiest to manufacture; sarin
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`US. Patent Application
`RACT-00200
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`(“isopropyl methylphosphonofluoridate”), which is a volatile substance mainly
`taken up through inhalation; soman (“pinacolyl methylphosphonofluoridate"), a
`moderately volatile substance that can be taken up by inhalation or skin contact;
`cyclosarin (“cyclohexyl methylphosphonofluoridate”), a substance with low
`volatility that is taken up through skin contact and inhalation of the substance as
`a gas or aerosol; and VX (“O-ethyl S—diisopropylaminomethyl
`methylphosphonothioate") and its isomeric analog R-VX [“O-isobutyl S-(2-
`diethylamino)-methylphosphonothioate, R-VX or VR”], both of which can remain
`on material, equipment and terrain for long periods, such as weeks, with R-VX
`being an especially persistent substance. All CWAs are colorless liquids with
`volatility varying from VX to sarin. VX is an involatile oil-like liquid, while sarin is
`a water-like, easily volatilized liquid. By addition of a thickener (e.g., a variety of
`carbon polymers), soman or other more volatile agents may be made to be less
`volatile and more persistent.
`‘
`
`The CWAs are extremely toxic and have a rapid effect. Such agents enter
`the body through any of the following manners:
`inhalation, direct contact to the
`skin with a gas or with a contaminated surface, or through ingestion of
`contaminated food or drink. The poisoning effect takes longer when the agents
`enter through the skin, but is much faster when they are inhaled because of the
`rapid diffusion in the blood from the lungs. These toxins are fat-soluble and can
`penetrate the skin, but take longer to reach the deep blood vessels. Because of
`this, the first symptoms may not appear for 20-30 minutes after initial contact with
`a contaminated surface. This increases the danger for personnel entering a
`contaminated area, because the contamination may not be detected for 30
`minutes or more (depending on concentrations) after the contaminated area is
`entered.
`
`The first and most important method of protection from nerve agents is to
`prevent exposure. For military personnel and other first responders, masks and
`full body protective gear are available, but this equipment has certain drawbacks.
`lmpermeable suits and even some air permeable suits are bulky and hot. The
`equipment inhibits free movement and tasks are harder and take longer to
`complete.
`In addition to those factors, hard physical work in these suits this may
`cause heat stress or even collapse. There may also be long delays before
`decontamination can be completed so the protective gear must be worn for long
`periods. This makes for a marginally acceptable first defense against a chemical
`warfare agent attack. Decontamination is also time-consuming so the equipment
`must often be destroyed and new equipment provided.
`It is also difficult to
`provide everyone with such protective equipment in the general population, and
`the effectiveness of such equipment diminishes during use. Tasks requiring
`detailed work using fingers and hands such as keystrokes on a keyboard, or
`pushing buttons on phones or equipment can be severely hampered by such
`bulky protective gear.
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`U.S. Patent Application
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`In addition to direct contact with a gaseous agent during an attack,
`surfaces that are exposed to the gas retain their toxicity for long periods of time.
`The OP nerve agents are soluble in materials such as paint, plastics, and rubber,
`allowing agents to remain in those materials and be released over long time
`periods. Nerve agents with thickening agents are even more persistent and
`difficult to decontaminate from a painted surface such as a wall, vehicle, or even
`a computer keyboard.
`It is understood that on painted metal surfaces, soman
`may persist for from’one to five days, and that the less volatile VX may persist for
`12 to 15 days. Under certain environmental conditions, OP compounds have
`been shown to persist indefinitely. On surfaces that are convoluted such as the
`surface of a military vehicle, the hidden surfaces that are less exposed to the
`environment can be especially difficult to decontaminate. Decontamination also
`requires detection, which is often not possible, and so resources and time may
`be wasted treating uncontaminated surfaces.
`
`Historically, most approaches to chemical agent decontamination have
`focused on the treatment of surfaces after chemical exposure, whether real or
`merely suspected, has occurred. There are several current methods of
`decontamination of surfaces. One method is post-exposure washing with hot
`water with or without addition of detergents or organic solvents, such as caustic
`solutions (e.g., D82, bleach) or foams (e.g., Eco, Sandia, Decon Green).
`Additional types of methods are anapplication of use of intensive heat and
`carbon dioxide applied for sustained periods, and incorporation of oxidizing
`materials (e.g., TiOz and porphyrins) into coatings that, when exposed to
`sustained high levels of UV light, degrade chemical agents (Buchanan, J.
`H et ai., 1989; Fox, M. A., 1983). Chemical agent resistant coatings (“CARCs”)
`have been developed to withstand repeated decontamination efforts with such
`caustize and organic solvents. However, the resulting “decontaminated”
`materials are often still contaminated. Moreover, many decontamination
`procedures aerosolize contaminants on surfaces to be cleaned.
`In addition, it is
`often hard to clean certain kinds of surfaces such as those with rough texture, or
`with deep crevasses and other hard to reach areas that must often “self-
`decontaminate.”
`
`Although each of these approaches can be effective under specific
`conditions, a number of additional limitations exist. Caustic solutions degrade
`surfaces, create personnel handling and environmental risks, and require
`transport and mixing logistics. Additionally, alkaline solutions, such as a
`bleaching agent, is both relatively slow in chemically degrading VX CPS and can
`produce decontamination products nearly as toxic as the OP itself (Yang, Y.-
`C. et ai., 1990). While foams may have both non-specific biocidal and chemical
`decontamination properties, they require transport and mixing logistics, may have
`personnel handling and environmental risks, and are not effective on sensitive
`electronic equipment or interior spaces. CARCs have been shown to become
`porous after sustained UV light exposure that can create a sponge effect that
`may actually trap chemical agents and delay decontamination. Moreover, these
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`approaches are not well suited for decontamination of convoluted surfaces.
`Decontamination with heat and carbon dioxide presents logistical requirements
`and does not allow rapid reclamation of equipment. UV-based approaches can
`be costly and have logistical requirements, including access to UV-generating
`equipment and power, as well as the production of toxic byproducts of
`degradation (Yang, Y.-C. et al., 1992; Buchanan, J. H. et al., 1989; Fox, M. A.,
`1983)
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`One attempted solution to the problem of surface contamination has been
`to provide paints with shedding (“chalking") properties such as an acrylic surface
`that may shed, or at least not be penetrated by a CWA, making decontamination
`easier. This has been unsatisfactory solution, however, because the area
`remains contaminated and there is no way to know if the surface is or is not
`poisonous.
`In addition, shedding coatings over existing painted surfaces require
`additional materials and labor over a single coating. Shedding may or may not
`occur over timeframes necessary to protect personnel from residual nerve agents
`on contaminated surfaces, and in many instances may require washing despite
`the shedding characteristic.
`~
`
`Various enzymes have been identified that detoxify OP compounds, such
`as organophosphorus hydrolase (“OPH”), organophosphorus acid anhydrolase
`(“OPAA”), and DFPase, which detoxifies 0,0-diisopropyl phosphorofluoridate
`(“DFP”). A number of civilian (e.g., Texas A&M University, private sector), and
`military laboratories [e.g., the Army research facilities at Edgewood (SBCCOM)]
`have worked on enzyme-based detection or decontamination systems for OP
`compounds. Various approaches taken in such laboratories include dispersion
`systems or immobilization systems of one or more OP degrading enzymes for
`use in detection or decontamination of OP compounds, as well as for
`convenience of handling of the enzyme preparation.
`
`Sensors of OP compounds using an OP compound degrading enzyme
`have been described primarily forthe detection of OP pesticides. OP compound
`sensors have been described that detect pH changes upon OP compound
`degradation using recombinant Escherichia coli cells expressing OPH
`cryoimmobilized in poly(vinyl)alcohol gel spheres (Rainina, E. I. et al., 1996).
`Endogenously expressed OPH from whole Flavobacterium sp. cells or cell
`membranes have been described as immobilized to glass membrane using
`poly(carbamoyl sulfonate) and poly(ethylene_imine) to produce a sensor of pH
`changes due to OP compound degradation (Gaberlein, S. et al., 2000a). OP
`compound sensors have been described that detect pH changes upon OP
`compound degradation using recombinant Escherichia coli cells, expressing
`OPH cytosolically or at the cell surface, that were fixed behind a polycarbonate
`membrane (Mulchandani, A. et al., 1998a; Mulchandani, A. et al., 1998b). An OP
`compound sensor has been described that detects optical changes upon OP
`compound degradation using recombinant Escherichia coli cells, expressing
`OPH at the cell surface, that were admixed in low melting point agarose and
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`applied to membrane that was affixed to a fiber optic sensor (Mulchandani,
`A. et al., 19986).
`
`An OP compound sensor has been described that detects pH changes
`upon OP compound degradation using purified OPH chemically cross-linked with
`bovine serum albumin by glutaraldehyde on an electrode’s glass membrane and
`covered with a dialysis membrane (Mulchandani, P. et al., 1999). Such
`chemically cross-linked OPH has been placed on a nylon membrane, and the
`membrane affixed to a fiber optic sensor to detect optical changes upon OP
`compound degradation (Mulchandani, A. et al., 1999a). Purified OPH has been
`immobilized by glutaraldehyde to glass-beads having aminopropyl groups in the
`construction of an OP compound degradation sensor (Mulchandani,
`P. et al., 2001a). An OP compound sensor has been described that detects
`optical changes upon OP compound degradation using recombinant Moraxella
`sp. cells, expressing OPH at the cell surface, that were admixed in 75% (w/w)
`graphite powder and 25% (w/w) mineral oil and placed into an electrode cavity
`(Mulchandani, P. et al., 2001b). Purified OPH was attached to silica beads by
`glutaraldehyde or N-y-maleimidobutyrylozy succinimide ester linkages, and the
`beads placed as a layer on a glass slide to construct a sensor (Singh, A.
`K. et al., 1999). Purified OPH has been labeled with fluorescein isothiocyanate
`and absorbed to poly(methyl methacrylate) beads that were placed on a nylon
`membrane to construct a sensorthat detects OP compound cleavage by
`decreased fluorescence (Rogers, K. R. et al., 1999). Purified OPH has been
`immobilized by placement within a poly(carbamoyl sulfonate) prepolymer that
`Was allowed to polymerize on a heat-sealing film in the construction of a sensor
`(Gaberlein, S. et al., 2000b). A purified fusion protein comprising OPH and a
`FLAG octapeptide sequence was immobilized to magnetic particles (Wang,
`J. et al., 2001 ). Additional sensors using OPH have been described
`(Mulchandani, A. et al., 2001).
`
`Different OP compound degrading enzyme compositions have been -
`described, primarily for the detoxification of OP pesticides (Chen, W. and
`Mulchandani, A., 1998; LeJeune, K. E. et al., 1998a). A parathion hydrolase
`enzyme degrading cell extract has been immobilized onto silica beads and
`porous glass (Munnecke, D. M., 1979; Munnecke, D. M., 1978). OPH has also
`been immobilized onto porous glass and silica beads (Caldwell, S. R. and
`Raushel, F. M., 1991b). Purified OPH has been mixed with fire fighting foams in .
`an attempt to create a readily dispersible decontamination composition (LeJeune,
`K. E., and Russell, A. J., 1999; LeJeune, K. E. et al., 1998b). Purified OPH has
`been incorporated into micelles in an OP compound degradation device
`(Komives, C. et al., 1994). Purified OPH has been encapsulated in a liposome
`for use in OP compound degradation (Pei, L. et al., 1994; Petrikovics,
`I. et al., 1999). OPH enzyme supported by glass wool in a biphasic solvent and
`gas phase reactor for OP compound detoxification has been described (Yang,
`F. et al., 1995). Purified OPH has also been immobilized onto trityl agarose and
`nylon (Caldwell, S. R. and Raushel, F. M., 1991a). Recombinant Escherichia coli
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`U.S. Patent Application
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`cells co-expressing OPH and a surface expressed cellulose-binding domain have
`been immobilized to cellulose supports (Wang, A. A. et al., 2002). Partly purified
`OPH, acetylcholinesterase or butyrylcholinesterase has been incorporated into
`polyurethane foam sponges (Havens, P. L. and Rase, H. F., 1993; Gordon, R.
`K. et al., 1999). Partly purified or purified OPH has been incorporated into solid
`polyurethane foam (LeJeune, K. E. and Russell, A. J., 1996; LeJeune, K.
`E. et al., 1997; LeJeune, K. E. et al., 1999). Recombinant Escherichia coli cells
`expressing OPH have been immobilized in a poly(vinylalcohol) cryogel (Hong, M.
`S. et al., 1998; Efremenko, E. N. et al., 2002; Kim, J.-W. et al., 2002). Purified
`OPH has been immobilized in polyethylene glycol hydrogels (Andreopoulos, F.
`M. et al., 1999). Recombinant Escherichia coli expressing OPH at the cell
`surface has been immobilized to polypropylene fabric by absorption of the cells
`to the fabric (Mulchandani, A. et al., 1999b). Purified OPH was immobilized to
`mesoporous silica by Tris-(methoxy)carboxylethylsilane or Tris-
`(methoxy)aminopropylsilane (Lei, C. et al., 2002). A fusion protein comprising
`OPH and a cellulose-binding domain has been immobilized to cellulose supports
`(Richins, R. D. et al., 2000). Sonicated Escherichia coli cells expressing a fusion
`protein comprising OPH, a green fluorescent protein, and a polyhistidine
`sequence as an affinity tag, have been attached to a nickel-iminodiacetic acid-
`agarose bead resin (Wu, C.-F. et al., 2002). A fusion protein comprising OPH
`and a polyhistidine sequence as an affinity tag has been attached to a chitosan
`film (Chen, T. et al., 2001). A purified fusion protein comprising an elastin-like
`polypeptide and OPH has shown to reversibly bind to the hydrophobic surface of
`polystyrene plates at temperatures above 37°C (Shimazu, M. et al., 2002).
`
`in addition to OPH, other OP compound enzyme compositions have been
`described. Purified OPAA has been encapsulated in a liposome for use in OP
`compound degradation (Petrikovics, I. et al., 2000a; Petrikovics, I. et al., 2000b).
`Purified OPAA has been mixed with firefighting foams, detergents, and a skin
`care lotion in an attempt to create a readily dispersible decontamination
`composition (Cheng, T.-C. et al., 1999). Purified squid-type DFPase has been
`encapsulated in erythrocytes for use in OP compound degradation (McGuinn, W.
`D. et al., 1993). Purified squid-type DFPase has been coupled to agarose beads
`(Hoskin, F. C. G. and Roush, A. H., 1982). Purified squid-type DFPase has also
`been incorporated into a polyurethane matrix (Drevon, G. F. et al., 2002; Drevon,
`G. F. et al., 2001; Drevon, G. F. and Russell, A. J., 2000).
`
`US. Patent Publication no. US 2002/0106361 A1 discusses a marine anti-
`
`fungal enzyme for use in a marine coating. However, the substrate for the
`enzyme was incorporated into the marine coating, and the enzyme was in a
`marine environment as the organism from which it was obtained.
`immobilized
`enzymes in an latex are discussed in the April, 2002 edition of “Emulsion
`Polymer Technologies,” by the Paint Research Association website
`http://www.pra.org.uk/publications/emulsion/emulsion highlights-2002.htm.
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`US. Patent Application
`RACT-00200
`
`However, to date, there has been limited success in using these and other
`approaches to harness the potential of these enzymes in systems that can be
`readily and cost effectively used in field-based military or civilian applications.
`Thus, despite the current understanding of the various OP compound degrading
`compositions and techniques, whether based on caustic chemicals or enzymes,
`there is a clear and present need for compositions and methods that can readily
`be used in OP compound degradation. This is particularly true for the
`detoxification of OP chemical warfare agents.
`In particular, compositions and
`methods are needed that will detoxify surfaces contaminated with OP
`compounds.
`
`SUMMARY OF THE INVENTION
`
`The present invention provides compositions and methods for their use as
`- components of surface treatments such as coatings. More specifically, the
`present invention provides compositions and methods for incorporating biological
`molecules into coatings in a manner to retain biological activity conferred by such
`biological molecule.
`
`The present invention provides compositions and methods capable of
`effective decontamination of OP compounds, as well as prophalactic protection
`of buildings, equipment, and personel that contact such objects, from OP
`compounds, including CWAs. As they relate to detoxification of CPS, the
`compositions and methods disclosed herein differ substantially from prior efforts,
`which focus on enzymatic detoxification of chemical compounds by application of
`a decontamination composition or method to the site of contamination after
`contact with the chemical compound. The preferred embodiments of the present
`invention represent a paradigm shift in chemical decontamination. They
`demonstrate usable compositions and methods for prophylactic protection of a
`site prior to contact with a chemical by prior application of a protective coating
`comprising an enzyme composition of the present invention. While prophylactic
`treatment with the coatings of the invention are a preferred embodiment, such a
`coating can also be used to coat a surface after contamination occurs. A
`preferred coating comprises a paint. Specifically, a paint comprising a preferred
`enzyme composition of the present invention degrades an organophosphorus
`compound, including a chemical warfare agent, into a significantly less toxic
`compound. Another preferred coating comprises a clear coat, a textile treatment,
`a wax, elastomer, or a sealant.
`
`Further, the present disclosure is the first composition of which Applicant
`is aware that comprises a bioactive molecule such as an enzyme composition
`that retains activity after being admixed with paint.
`In addition, it still retains
`activity after the paint is applied to a surface, and renders the surface bioactive.
`
`In light of these and additional disclosures herein, it is now possible to
`produce paints and other coatings that detoxify chemical compounds for
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`U.S. Patent Application
`RACT-00200
`
`extended periods. Remarkably, a preferred enzyme composition of the present
`invention remained stable in the coating for an extended period of time (e.g.,
`months) at ambient conditions.
`It is contemplated that the extended period of
`activity may further comprise time periods in excess of a year.
`In particular,
`stabilized embodiments of the enzyme composition are designed to enhance the
`life time of the biomolecule composition in a coating and on a surface.
`In a
`preferred embodiment, the invention comprises at least one polymer-based
`compound that provides prophylactic protection and continuous detoxification of
`an organophosphorus nerve agent.
`
`In certain embodiments, it is contemplated that the compositions and
`methods of the present invention may be used to produce self-decontaminating
`surfaces that remain active for extended periods.
`In a specific aspect, it is
`contemplated that such a self-decontaminating surface will not need additional
`decontamination compositions or methods to effect decontamination, thereby
`minimizing logistical requirement.
`In a particular aspect, it is contemplated that
`the compositions and methods of the present invention may be easily applied in
`advance of, during or after exposure to a chemical compound. _In another
`preferred aspect, the compositions and methods of the present invention will be
`used in conjunction with existing other decontamination compositions and
`methods.
`In a preferred facet, the compositions and methods of the present
`invention are applied in advance of, during or after a field-based military
`operation to protect troops against a chemical warfare agent.
`
`A recombinant phosphoric triester hydrolase may be produced using
`specific expression vectors in a variety of host cells.
`In the practice of the
`present invention, any of the described cells, nucleic acid sequences, genes,
`gene fragments, vectors and transcriptional or translational signals may be used,
`or any others that are known in the art.
`In preferred facets, the enzyme is grown
`in bacterial, fungal, plant (e.g., corn), or insect cells.
`In preferred embodiments,
`an expression vector includes an opd DNA fragment in the correct orientation
`and reading frame with respect to the promoter sequence to allow translation of
`the opd gene or gene fragment.
`In a specific aspect, the expression vector
`produces heterologous expression of the opd gene or gene fragment.
`In
`particularly preferred embodiments of the composition, the enzyme is an OPH
`enzyme.
`In other preferred embodiments the enzyme is an OPAA enzyme.
`In
`other preferred embodiments, the enzyme is a DFPase. Examples of the cloning
`and expression of exemplary opd gene and gene fragments are described
`(McDaniel, S. et al.,1988; McDaniel, 1985; Wild, J. R. et al., 1986; each
`incorporated herein in its entirety by reference).
`
`In general embodiments, a biomolecule of the present invention refers to a
`compound comprising one or more chemical moieties normally produced by a
`living organism such as an amino acid, a nucleic acid, a sugar, a lipid, or a
`combination thereof. The invention provides a coating comprising a biomolecule
`composition, wherein the biomolecule composition comprises an active
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`U.S. Patent Application
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`biomolecule. A biomolecule typically has a function in or upon a living organism,
`such as binding another molecule, catalyzing a chemical reaction, or a
`combination thereof. Specific examples of such activity by a biomolecule include
`an antibody binding an antigen, a cell receptor binding a ligand, an enzyme
`binding a substrate, a transport protein may bind a ligand, etc.
`In some aspects,
`binding a ligand may be a desired activity such as, for example, to sequester an
`undesired molecule, such as a toxin, to the biomolecule. Often, a biomolecule’s
`activity further comprises a specific chemical reaction in addition to a
`physical/chemical affinity for another molecule. For example, an enzyme may
`accelerate a chemical reaction upon the bound substrate, a cell receptor may
`change conformation a

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