WO2010036344A2

WO2010036344A2 - Compositions, oral care products and methods of making and using the same

Info

Publication number: WO2010036344A2
Application number: PCT/US2009/005302
Authority: WO
Inventors: James George Miller; Hans H. Hoefer; Werner Geurtsen; Peter Lucker; Matthias Wiens; Heinz C. Schroeder; Werner E. Mueller
Original assignee: Grace Gmbh & Co. Kg
Priority date: 2008-09-24
Filing date: 2009-11-20
Publication date: 2010-04-01
Also published as: WO2010036344A3; WO2010036344A8; EP2403473A2; CN102231975A

Abstract

Compositions and oral care products are disclosed. Methods of making and using compositions and oral care products are also disclosed.

Description

COMPOSITIONS, ORAL CARE PRODUCTS AND METHODS OF MAKING AND

USING THE SAME

FIELD OF THE INVENTION

[00011 The present invention is directed to biomineral compositions and oral care products. The present invention is further directed to methods of making and using biomineral compositions and oral care products.

BACKGROUND OF THE INVENTION

[0002] In recent years, biomolecules that are involved in natural processes of biomineral ization in the nanoscale range have become of increasing interest in nano(bio)technology. Examples of "nanotechnology in nature" are (i) the pearls that consist of calcium carbonate crystals (i.e., aragonite) or (ii) the shells of abalone-mussels that are built up from calcite crystals, which derive new, advantageous material properties by the incorporation of specific proteins, or (iii) the optical microlens system of the starfish Ophiocoma wendtii. In particular, a few marine or aquatic organisms, such as the diatom and the siliceous sponge, have the ability to create skeleton structures from biogenic silica.

[0003] Siliceous sponges have the surprising ability to form silica ("biosilica") under mild conditions, at room temperature, with an enzymatic mechanism as disclosed in Shimizu et al. Proc. Natl Acad. ScL USA 95, 6234-6238 (1998). In contrast, inorganic chemical methods for the production of silica require the use of elevated temperatures and more aggressive chemicals. The biosilica that is produced by sponges is used to create needle- like skeleton elements (i.e., "spicula") as disclosed, for example, in Mϋller et al., FEBS J. 272, 3838-3852 (2005), and Mϋller et al., Micron 37, 107- 120 (2006). The enzymes involved, namely, silicateins, may be cloned from marine siliceous sponge Suberites domuncula and a number of other sponges as disclosed in Krasko et al., Europ. J. Biochem. 267:4878-4887 (2000). Silicateins and their technical uses are described, for example, in PCT/US99/30601 , DE 1003 72 70 A 1, PCT/EPOl/084 23, EP 132 0624 and US 7, 169,589 B2 (i.e., silicatein-α and silicatein are mentioned); in DE 103 52 433.9 (silicatein-β is mentioned); as well as DE 102006001759.5 (i.e., four silicatein isoforms of Lubomirskia baicalensis are disclosed). [0004] By means of the above-mentioned enzymes, it is possible to produce biosilica on a nano scale at room temperature as disclosed, for example, in Schroder et al., J. Biol. Chem. 281 , 12001 - 12009 (2006), and Schroder et al., Naturwissenschafien 94, 339-359 (2007). The formation of biosilica by the immobilized protein may be demonstrated by means of various techniques including, but not limited to, "Scanning Force Microscopy" (SFM) and "Scanning Electron Microscopy" (SEM) as disclosed in Tahir et al., Chem. Commun. 2848-2849 (2004).

[00051 Silicatein has both anabolic (i.e., silica-polymerase) and catabolic (i.e., silica- esterase) activity, and consequently, makes possible the production of a flexible silica shell that, via the esterase activity of expressed silicateins, can be modified or perforated. The mechanism on which the silica-esterase activity of silicatein is based is shown in FIG. IA. The hydrolysis of oligo/polysilicate substrates is based on a nucleophilic attack of the hydroxyl group of the serine radical in the active center of the silicatein on one of the silicon atoms of the polymer substrate. In this case, the oxygen bond is cleaved in the polymer silicate. This reaction is facilitated by the formation of a hydrogen bridge bond between the imidazole-nitrogen of the histidine and the hydroxyl group of the serine in the active center, which leads to an increase of nucleophilicity of the serine-oxygen. The transient covalent bond between enzyme and substrate is hydrolyzed by water with release of silicic acid.

[0006] FIG. IB shows the mechanism of the silica-polymerase activity of silicatein. The histidine radical in the active center of the enzyme acts as an acid-base catalyst. The transfer of a proton from the orthosilicic acid to the imidazole-nitrogen results in the formation of a reactive silicate species that is able to attack a second orthosilicate molecule. The negatively-charged, pentavalent intermediate compound that is formed is then converted into disilicic acid (or higher oligomers) with release of water, which is formed by the transfer of a proton from the protonated imidazole ring of the histidine radical.

[0007] A number of publications disclose the use of silicateins to (i) form inorganic coatings on a variety of substrates including metals, metal oxides, plastics, carbon fibers, wool fibers, cotton fibers, drug additives (e.g., starch) , and semiconductor supports (see, for example, U.S. Patent Application Publication No. 2007/0280921 to Mϋller et al., paragraphs [01 15] to [01 19], and U.S. Patent No. 7, 169,589 to Mϋller et al., column 10, lines 55-59), (ii) modify an outer surface of stones (see, U.S. Patent No. 7,229,807 to Miiller et al., column 16, lines 6- 15), and (iii) form in vitro silica and silicone polymer networks (see, U.S. Patent No. 6,670,438 to Morse et al.).

[0008] Further, U.S. Patent Application Publication No. 2007/0238808 to Goldberg et al. discloses the use of a biocatalyst (e.g., a silicatein) to form dental restorative materials comprising a polymer matrix material and a biocatalyst (e.g., a silicatein) on and/or within the polymer matrix material, wherein the biocatalyst promotes the deposition of a mineral (e.g., silica) onto and/or within the polymer matrix material. The polymer matrix material is generally a thermosettable (i.e., hardenable) material (see, paragraph [0056]), and forms an integral component of the resulting dental material following polymerization and/or curing. The disclosed dental material may be used as a bonding agent between a restoration and a tooth structure as disclosed in paragraph [0089].

[0009] Efforts continue to develop additional uses of enzymes/biocatalysts, such as silicateins, as well as compositions containing such enzymes/biocatalysts. Further, efforts continue to develop biosilica products that utilize the catalytic properties of enzymes/biocatalysts, such as silicateins, and provide protection/restoration properties to teeth without forming organic polymeric -containing layers on the teeth.

SUMMARY OF THE INVENTION

[0010] The present invention relates to the discovery of compositions containing an enzyme and a precursor material that is catalyzed by the enzyme to form an inorganic coating material (e.g., silica). The compositions may be utilized in a variety of applications where an inorganic or mineral coating is desired, and are particularly useful as dental care compositions (i.e., compositions applied onto a tooth or teeth of an animal) capable of forming inorganic coatings on teeth of animals.

[001 1] In one exemplary embodiment, a composition of the present invention comprises (i) an enzyme, and (ii) a precursor material capable of being catalyzed with the enzyme to form an inorganic coating material. The composition may also include a protecting agent that chemically or physically protects the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material, or a an oral care component, such as a phosphorus-containing compound, a cleaning agent (e.g., a surfactant such as sodium lauryl sulfate), a polishing agent, an abrasive, an and- microbial material, a pH adjuster, a color-producing additive, a scent-producing additive, a fluoride-containing material, a production processing aid (e.g., polyethylene glycol), a thickening agent or mixtures thereof. The composition may also include accelerators, promoters, activators, or mixtures thereof, which serve to increase the rate of polymerization of the precursor material.

[0012] In another exemplary embodiment, the composition of the present invention comprises a oral care composition, wherein the oral care composition includes a mixture comprising an enzyme; a precursor material capable of reacting with the enzyme to form an inorganic coating material; and a protecting agent, the protecting agent separating the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material. The exemplary oral care composition may comprise or be utilized with any oral care materials or devices, including but not limited to, toothpaste, gum, mouthwash, teeth brushes, mouth guards or teeth trays, teeth strips, teeth alignment devices, food, drinks, lozengers, or other edible materials, and combinations thereof. In addition, the compositions of the present invention may be used with any oral care products for any animals. Such materials may comprise typical ingredients including, but are not limited to, a phosphorus-containing compound, a cleaning agent, an anti-microbial material, a pH adjuster, a color-producing additive, a scent-producing additive, a fluoride-containing material, a production processing aid, a thickening agent, a filler material, water or mixtures thereof. Desirably, the dental care composition comprises a coatable composition, namely, a composition having a viscosity that enables the composition to be coated onto a substrate using conventional coating methods.

[0013] In another exemplary embodiment, the composition of the present invention comprises an oral care composition, wherein the oral care composition comprises a toothpaste having an enzyme, and a precursor material capable of reacting with the enzyme to form an inorganic coating material. The toothpaste may further comprise additional ingredients such as those typically found in toothpastes, as well as those not typically found in toothpastes (e.g., an protecting agent such as polylactide, accelerators, promoters, activators, or mixtures thereof). Suitable additional ingredients may include, but are not limited to, an protecting agent that chemically or physically separates the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material, a phosphorus-containing compound, a cleaning agent, an anti-microbial material, a pH adjuster, a color-producing additive, a scent- producing additive, a fluoride-containing material, a production processing aid, a thickening agent, a filler material, and water.

[0014] In any of the disclosed compositions, including the oral care compositions of the present invention, the enzyme may comprise any enzyme that is capable of catalyzing the precursor material such as, for example, a silicatein, cathepsin L, papain, or the like. The precursor material may comprise, for example, a metal oxide such as silica, silicate, silicic acid, silicon alkoxides, or a compound having a general formula Si(Ri )_m(R2)_n(R3)_P(R4)q wherein each of Ri, R₂, R₃ and R₄ independently comprises H, OH, a Cl to C 12 alkane group, or a Cl to Cl 2 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures thereof. In an exemplary embodiment of the present invention, these materials may not toxic or harmful to any animals. In any of the disclosed compositions, such as oral care compositions of the present invention, the phosphorus-containing compound may comprise, for example, tetrasodium pyrophosphate, tetrapotasium pyrophosphate, disodium pyrophosphate, or a combination of two or more phosphates, while the cleaning agent may comprise, for example, a surfactant such as sodium lauryl sulfate. [0015] The present invention is also directed to methods of making compositions containing an enzyme, and a precursor material capable of reacting with the enzyme to form an inorganic coating material. In one exemplary embodiment, the method of making a composition comprises forming a mixture comprising (i) an enzyme, (ii) a precursor material capable of reacting with the enzyme to form an inorganic coating material, and (iii) at least one additional component including a protecting agent that chemically or physically separates the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material, or a component of an oral care composition. Additional components may include at least one accelerator, promoter, phosphorus-containing compound, cleaning agent, abrasive, polishing agent, anti-microbial material, pH adjuster, color-producing additive, scent-producing additive, fluoride-containing material, production processing aid, thickening agent, or mixtures thereof. [0016] In another exemplary embodiment, the method of making a composition comprises forming an oral care composition. An exemplary method of making an oral care composition comprises forming a mixture comprising an enzyme; a precursor material capable of reacting with the enzyme to form an inorganic coating material; and a protecting agent, the protecting agent separating the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material. The method of making an oral care composition may further comprise adding one or more additional ingredients to the mixture, wherein the one or more additional ingredients include, but are not limited to, at least one promoter, accelerator, phosphorus- containing compound, cleaning agent, polishing agent, abrasive, anti-microbial material, pH adjuster, color-producing additive, scent-producing additive, fluoride-containing material, production processing aid, thickening agent, filler material, water, and any combination thereof.

[0017] In another exemplary embodiment, the method of making a composition comprises forming an oral care composition, wherein the oral care composition comprises a toothpaste. A method of making a toothpaste comprises forming a mixture comprising an enzyme, and a precursor material capable of reacting with the enzyme to form an inorganic coating material. The method of making a toothpaste may further comprise adding at least one additional ingredient to the mixture, wherein the at least one additional ingredient may include, but is not limited to, protecting agent that chemically or physically separates the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material, phosphorus-containing compound, polishing agent, abrasive, promoter, accelerator, cleaning agent, anti-microbial material, pH adjuster, color-producing additive, scent-producing additive, fluoride-containing material, production processing aid, thickening agent, filler material, water, and any combination thereof.

[0018] The present invention is further directed to methods of forming a coating on a substrate. In one exemplary embodiment, the method of forming a coating on a substrate comprises coating at least a portion of the substrate with a mixture comprising an enzyme; a precursor material capable of reacting with the enzyme to form an inorganic coating; and a protecting agent that chemically or physically separates the enzyme from the precursor material so as to prevent premature reaction of the enzyme with the precursor material. The method of forming a coating on a substrate may further comprise one or more additional steps including, but not limited to, removing the protecting agent from the enzyme; bringing the enzyme into contact with the precursor material so as to initiate a reaction between the enzyme and the precursor material; and adding at least one additional reactive or non-reactive ingredient to the mixture, wherein the at least one additional reactive or non-reactive ingredient includes, but is not limited to, phosphorus-containing compound, promoter, accelerator, cleaning agent, anti-microbial material, pH adjuster, color-producing additive, scent-producing additive, fluoride-containing material, production processing aid, thickening agent, filler material, water, and any combination thereof. The substrate may comprise a variety of substrates including, but not limited to, a tooth, and a bone.

[0019] The present invention is further directed to inorganic coatings and coated substrates formed via the above-described methods. In one exemplary embodiment, the coating comprises biosilica and/or hydroxyl apatite, and the coated substrate comprises a tooth or bone at least partially coated with a coating comprising biosilica and/or hydroxyl apatite. The present invention may further be utilized to fill cracks or fissures in teeth and bone so as to repair the teeth or bone.

[0020] These and other features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.

BREF DESCRIPTION OF THE FIGURES

[0021 ] FIG. IA depicts the mechanism of silica-esterase activity for the enzyme silicatein;

[0022] FIG. IB depicts the mechanism of silica-polymerase activity for the enzyme silicatein;

[0023] FIGS. 2A-2D depict exemplary steps used to provide a protective inorganic coating layer on a tooth using the compositions of the present invention;

[0024] FIG. 3 depicts amplification of the cDNA that codes for the hydroxylapatite- binding silicatein-α protein formed in Example 2 below;

[0025] FIG. 4 depicts ligation of amplicon with the bacterial expression vector pTRC-His2 via T/A cloning as described in Example 2 below; 100261 FIGS. 5A-5D depict exemplary scanning electron microscope (SEM) images, at two different magnifications, of a tooth prior to coating and after coating with an inorganic coating material of the present invention as described in Example 5 below; and 100271 FIG. 6 depicts the EDX ("Energy Dispersive X-Ray") spectrum of the biosilica/bioapatite layer formed on a pig tooth as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

[0028J To promote an understanding of the principles of the present invention, descriptions of specific embodiments of the invention follow and specific language is used to describe the specific embodiments. It will nevertheless be understood that no limitation of the scope of the invention is intended by the use of specific language. Alterations, further modifications, and such further applications of the principles of the present invention discussed are contemplated as would normally occur to one ordinarily skilled in the art to which the invention pertains.

[0029] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an oxide" includes a plurality of such oxides and reference to "oxide" includes reference to one or more oxides and equivalents thereof known to those skilled in the art, and so forth.

[0030] "About" modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperatures, process times, recoveries or yields, flow rates, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that may occur, for example, through typical measuring and handling procedures; through inadvertent error in these procedures; through differences in the ingredients used to carry out the methods; and like proximate considerations. The term "about" also encompasses amounts that differ due to aging of a formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a formulation with a particular initial concentration or mixture. Whether modified by the term "about" the claims appended hereto include equivalents to these quantities.

[00311 As used herein, "metal oxides" is defined as binary oxygen compounds where the metal is the cation and the oxide is the anion. The metals may also include metalloids. Metals include those elements on the left of the diagonal line drawn from boron to polonium on the periodic table. Metalloids or semi-metals include those elements that are on this line. Examples of metal oxides include silica, alumina, titania, zirconia, etc., and mixtures thereof. Preferably, the metal oxides are amorphous, such as, for example, precipitated silica.

[0032] As used herein, the term "mineral" or "inorganic material" is defined as material that is comprised of metal oxides.

[0033] As used herein, the term "enzyme" is defined as material that is comprised of various proteins formed in plant or animal cells or made synthetically, that act as organic catalysts in initiating or speeding up certain chemical reactions.

[0034] The present invention is directed to compositions containing an enzyme and a precursor material that is capable of being catalyzed by the enzyme to form an inorganic coating material (e.g., silica). The present invention is further directed to methods of making compositions containing an enzyme, and a precursor material that is capable of being catalyzed by the enzyme to form an inorganic coating material. The present invention is even further directed to methods of forming a coating on a substrate, wherein the coating comprises (i) an enzyme, and a precursor material that is capable of being catalyzed by the enzyme to form an inorganic coating material, or (ii) the reaction product of a reaction between the enzyme and the precursor material (e.g., a reaction product in the form of an inorganic coating material). The present invention is even further directed to coatings and coated substrates, such as a coated tooth or bone.

[0035] The compositions of the present invention may provide one or more benefits and/or technical advantages that were not previously addressed in the art of compositions and coatings formed therefrom. For example, the disclosed compositions and resulting coatings formed therefrom do not require a polymeric matrix material or potentially toxic composition components (e.g., components that are unsuitable for use in human consumable products). Further, the resulting inorganic coating materials (e.g., a biosilica and/or hydroxyl-apatite) provide exceptional tensile strength, flexibility, capacity to self- repair, and adhesion to a given substrate (e.g., a tooth). Such coatings are almost indistinguishable from natural dentin/enamel regarding chemical composition and do not include potentially toxic polymeric matrix materials.

[0036] A description of exemplary compositions and composition components is provided below. /. Compositions

[0037] The compositions of the present invention may comprise a number of individual components. A description of individual components and combinations of individual components is provided below. Further, the compositions of the present invention may be presented in various forms. A description of types of compositions is also provided below.

A. Composition Components

[0038] The compositions of the present invention may comprise one or more of the following components.

1. Enzymes

[0039] The compositions of the present invention comprise at least one enzyme. Suitable enzymes include enzymes capable of catalyzing a precursor material that results in the formation of an inorganic nanolayer. Suitable enzymes include silica proteins, such as silicatein, and similar proteins, such as cathepsin, papain, or the like, and mutations thereof.

[0040] In some embodiments, the enzyme comprises a silicatein, such as silicatein-α, silicatein-β or silicatein-γ; a polypeptide that contains an animal, bacterial, plant or fungal silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ DD No. 3 below; or a polypeptide that is homologous thereto. In other embodiments, the enzyme comprises the above-described polypeptide that contains a silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ ID No. 3 and an oligo-glutamate sequence.

[0041 ] Suitable enzymes (e.g., silicatein-α, silicatein-β, or silicatein-γ from Suberites domuncula according to SEQ DZ⁾ No. 1 or SEQ ID No. 3, a polypeptide that is homologous thereto, namely, a polypeptide in the amino acid sequence of the silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown in, e.g., SEQ ID No. 1 or SEQ DD No. 3) may be extracted from an animal, bacterial, plant or fungus in a natural form, or may be produced synthetically or in that the polypeptide is present in a prokaryotic or eukaryotic cell extract or cell lysate. The cell extract or the lysate can be produced from a cell ex vivo or ex vitro, for example a recombinant bacterial cell or a sea sponge. The polypeptides may be purified according to conventional methods known in the prior art and can thus be free of other proteins. [0042] The properties of the cDNAs that code for the silicatein-α polypeptide or the silicatein-β polypeptide from 5. domuncula, as well as the polypeptides that are derived from the nucleotide sequence, are described in, for example, PCT/US99/30601 , DE 10037270 A 1 , PCT/EP01/08423, and DE 103 52 433.9, the subject matter of each of which is hereby incorporated by reference in its entirety.

[0043] The one or more enzymes are typically present in the compositions of the present invention in an amount greater than 0 weight percent (wt%) and up to about 50 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more enzymes in an amount ranging from about 0.0001 wt% to about 10 wt%, more typically, from about 0.001 wt% to about 5 wt%, and even more typically, from about 0.01 wt% to about 1 wt%, based on a total weight of the composition.

2. Precursor Materials

[0044] The compositions of the present invention also comprise at least one precursor material that is capable of being catalyzed by the enzyme to form an inorganic coating material.

[0045] In some embodiments, the precursor material comprises a metal oxide such as silica, silicate, silicic acid, silicon alkoxides, or a compound having a general formula Si(Ri )_m(R₂)_n(R3)p(R4)q wherein each of Ri, R₂, R₃ and R₄ independently comprises H, a Cl to Cl 2 alkane group, or a C l to C 12 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures thereof. Some exemplary precursor materials include, but are not limited to, silicic acids, monoalkoxysilane triols, dialkoxysilane diols, trialkoxysilanols, tetraalkoxysilanes, and chelate complexes of silicic acid. [0046] One or more precursor materials may be incorporated into the compositions of the present invention. The one or more precursor materials are typically present in the compositions of the present invention in an amount greater than 0 weight percent (wt%) and up to about 80 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more precursor materials in an amount ranging from about 0.01 wt% to about 30 wt%, more typically, from about 0.1 wt% to about 20 wt%, and even more typically, from about 1 wt% to about 10 wt%, based on a total weight of the composition.

3. Protecting Agents

[0047] The compositions of the present invention may further comprise at least one protecting agent that chemically or physically separates the enzyme from the precursor material within a given mixture so as to prevent premature reaction of the enzyme with the precursor material. The protecting agent may physically separate the enzyme, such as by encapsulating, isolating or containing the enzyme. The protecting agent may also chemically protect the enzyme, such as by intramolecular or intermolecular bonding, or steric hindrance, etc. Typically, when present, the one or more protecting agents are utilized to protect the enzyme, and not the precursor material; however, the one or more protecting agents may be utilized to protect the enzyme alone, the precursor material alone, or both the enzyme and the precursor material.

[0048] Suitable protecting agents include, but are not limited to, polylactide, polyvinyl alcohol, alginate, copolymers such as poly(lactide-co-ε-caprolactone). Desirably, the protecting agent is (i) water-soluble, (ii) degradable in the presence of saliva, or (ii) both (i) and (ii).

[0049] When present, the one or more protecting agents are typically present in the compositions of the present invention in an amount greater than 0 wt% and up to about 60 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more protecting agents in an amount ranging from about 0.1 wt% to about 30 wt%, more typically, from about 1 wt% to about 20 wt%, and even more typically, from about 2 wt% to about 15 wt%, based on a total weight of the composition.

4. Phosphorus-Containing Compounds

[0050] The compositions of the present invention may further comprise one or more phosphorus-containing compounds. Suitable phosphorus-containing compounds include, but are not limited to, salts of phosphoric acid, such as sodium phosphate, disodium phosphate, trisodium phosphate, tetrasodium phosphate, sodium metaphosphate, pentasodium triphosphate, sodium monofluoro-phosphate, monofluorophosphate, sodium dodecyl phosphate (sodium lauryl phosphate), calcium sodium phosphate, tetrapotassium phosphate, pentapotassium triphosphate, disodium pyrophosphate, tetrasodium pyrophosphate, tetrapotassium pyrophosphate, tin phosphate (stannous phosphate), and tin pyrophosphate (stannous pyrophosphate).

[0051 | When present, the one or more phosphorus-containing compounds are typically present in the compositions of the present invention in an amount greater than 0 wt% and up to about 90 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more phosphorus-containing compounds in an amount ranging from about 0.1 wt% to about 50 wt%, more typically, from about 1 wt% to about 30 wt%, and even more typically, from about 1 wt% to about 20 wt%, based on a total weight of the composition.

5. Promoting/Accelerating Agents

[0052] The compositions of the present invention may further comprise one or more promoting, accelerating or activating agents, which are capable of enhancing the enzymatic reaction. The agents for enhancing the enzymatic reaction may be those that activate the precursor material for nucleophilic attack by the enzyme. Suitable agents include, but are not limited to, various metals, metal salts, metal oxides, and mixtures thereof. Examples include Fe, Se, F, Al, salts and oxides thereof.

[0053] When present, the one or more promoting, accelerating or activating agents are typically present in the compositions of the present invention in an amount greater than 0 wt% and up to about 30 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more promoting, accelerating or activating agents in an amount ranging from about 0.001 wt% to about 20 wt%, more typically, from about 0.01 wt% to about 10 wt%, and even more typically, from about 0.1 wt% to about 5 wt%, based on a total weight of the composition.

6. Abrasive and Polishing Agents

[0054] The compositions of the present invention may further comprise one or more abrasive and polishing agents. The abrasive and polishing agents may remove any excess inorganic material

[0055] Suitable abrasive agents include metal oxide particles, such as silica gel, precipitated silica or fumed silica, or mixtures thereof.

[0056] Suitable polishing agents include metal oxide particles, such as colloidal silica. [0057] When present, the one or more abrasive and polishing agents are typically present in the compositions of the present invention in an amount greater than 0 wt% and up to about 30 wt% based on a total weight of the composition. In some exemplary embodiments, the compositions comprise one or more abrasive and polishing agents in an amount ranging from about 0.001 wt% to about 20 wt%, more typically, from about 0.01 wt% to about 10 wt%, and even more typically, from about 0.1 wt% to about 5 wt%, based on a total weight of the composition.

7. Additional Ingredients

[0058] The compositions of the present invention may further comprise one or more additional components. Suitable additional components for use in the compositions of the present invention include, but are not limited to, a cleaning agent, a tooth whitener, a breath freshener, a flavoring agent or sweetener, a fragrance-producing agent, a colorant, pH control agents, an antimicrobial agent, a foaming agent, a thickening agent, a bifunctional linking agent for binding an enzyme to another composition component (e.g., the protecting agent), a production processing aid, and combinations thereof. [0059] A number of commercially available cleaning agents may be ^ used in the compositions of the present invention. Suitable commercially available cleaning agents (i.e., surfactants) include, but are not limited to, sodium lauryl sulfate commercially available from Stepan (Northfield, IL).

[0060] Suitable flavoring agents include, but are not limited to, mint flavors (e.g., spearmint or peppermint flavor), orange flavor, cherry flavor, lime flavor, bubble gum flavor, etc. Suitable sweetening agents include, but are not limited to, glucose, sorbitol, sucralose, neo-tame, potassium acesulfame, etc. In one exemplary embodiment of the present invention, compositions of the present invention comprise sorbitol as the sweetener. '

[0061 ] Suitable fragrance-producing agents include, but are not limited to, lemon, orange, mint, pine, green-apple, eucalyptus, jasmine and various spa-like fragrances. [0062] Suitable colorants include, but are not limited to, FD&C Blue #1 , FD&C Green #3, FD&C Yellow #5 and FD&C Red #40. In one exemplary embodiment of the present invention, the compositions of the present invention comprise either a FD&C Blue #1 or FD&C Yellow #5.

[0063] Suitable pH control agents for use in the present invention include, but are not limited to, citric acid, phosphoric acid, sodium carbonate, sodium citrate, sodium benzoate, benzoic acid, sodium citrate, silver citrate, dihydrogen silver citrate, or a combination thereof. In one exemplary embodiment, the pH control agent comprises citric acid (i.e., 2-hydroxy- l ,2,3-propanetricarboxyIic acid), phosphoric acid, sodium benzoate, or any combination thereof.

L0064] Suitable thickening or gelling agent for use in the present invention include, but are not limited to, alkali metal starches, carboxycellulose compounds, cellulosic gums, xanthan gums, polyacrylic acid, and all other naturally-occurring gums and starches. As used herein, the above-mentioned thickening or gelling agents include both crosslinked and uncrosslinked versions of the thickening or gelling agents. For example, any of the above-mentioned starches, gums, cellulosic-based compounds, or polymers may be crosslinked via bonding forces within a given compound or polymer or via the addition of one or more crossl inking agents.

[0065] Suitable production processing aids include, but are not limited to, polyethylene glycols, alkali metal carbonates, alkali metal bicarbonates, malto-dextrins, etc. In one exemplary embodiment of the present invention, the compositions of the present invention comprise polyethylene glycol.

[0066] A number of commercially available additional components may be used in the present invention. Suitable commercially available additional components include, but are not limited to, foam enhancers such as foam enhancers commercially available from Stepan (Northfield, IL) under the trade designation NINOL^®; and teeth protecting agents such as sodium fluoride commercially available from Univar USA (Bellevue, WA). [0067] A number of commercially available thickening agents may be used in the present invention. Suitable commercially available thickening agents include, but are not limited to, silica gel commercially available from W. R. Grace & Co. -Conn.; xanthan gum commercially available from Sigma Aldrich (St. Louis, MO) and sold under the trade designation Sigma Aldrich G 1253; and hydroxypropyl methylcellulose commercially available from Dow Chemical (Midland, MI) and sold under the trade designation METHOCEL™ such as METHOCEL™ 40-202.

[0068] A number of commercially available processing aids may be used in the present invention. Suitable commercially available processing aids include, but are not limited to, CARBOWAX™ 8000 commercially available from Dow Chemical (Midland, MI), and PEG 3350 commercially available from Union Carbide (Houston, TX). [0069| Each additional component may be present in an amount ranging from greater than 0 to about 60 wt% based on a total weight of a given composition. In some exemplary embodiments, each additional component, when present, is present in a given composition in an amount ranging from about 0.01 wt% to about 50 wt%, more typically, from about 0.05 wt% to about 40 wt%, and even more typically, from about 0.1 wt% to about 30 wt%, based on a total weight of the composition.

B. Composition Forms

[0070J The compositions of the present invention may be formulated so as to have one or more of the following forms.

1. Powders

[0071] The compositions of the present invention may be formulated as a powder mixture.

2. Liquids and Suspensions

[0072] The compositions of the present invention may also be formulated as a solution, or as a dispersion or suspension having a liquid matrix and particulate material suspended within the liquid matrix.

3. Pastes and Creams

[0073] The compositions of the present invention may be formulated so as to form a paste such as a toothpaste or a cream.

4. Aerosols

[0074] The compositions of the present invention may be formulated so as to form an aerosol.

//. Methods of Making Compositions, Inorganic Coatings, and Coated Substrates [0075] The present invention is further directed to methods of making compositions containing an enzyme, and a precursor material capable of reacting with the enzyme to form an inorganic coating material. The method of making a composition may comprise forming a mixture comprising (i) an enzyme, and (ii) a precursor material capable of reacting with the enzyme to form an inorganic coating material. One or more additional composition components may be further added to the mixture including any combination of one or more components disclosed herein.

[0076] In one exemplary embodiment, the method of making a composition comprises forming a oral care composition, wherein the method comprises forming a mixture comprising an enzyme; a precursor material capable of reacting with the enzyme to form an inorganic coating material; and a protecting agent, the protecting agent chemically or physically protecting the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material. The method of making the oral care composition may further comprise adding at least one additional ingredient to the mixture, wherein the at least one additional ingredients includes, but is not limited to, an accelerator, a promoter, an activator, an abrasive, polishing agent, a tooth whitener, a breath freshener, a flavoring agent or sweetener, a fragrance-producing agent, a colorant, a pH control agent, an antimicrobial agent, a foaming agent, a thickening agent, a bifunctional linking agent for binding the enzyme to another composition component, a production processing aid, a filler material, a fluoride-containing material, a phosphorus- containing compound, a cleaning agent, water, and any combination thereof. The exemplary oral care composition may comprise or be utilized with any oral care materials or devices, including but not limited to, toothpaste, gum, mouthwash, teeth brushes, mouth guards or teeth trays, teeth strips, teeth alignment devices, food, drinks, lozengers, or other edible materials, and combinations thereof.

[0077] The method of making a composition may comprise forming an oral care composition, wherein the oral care composition comprises toothpaste. The method of making toothpaste may comprise forming a mixture comprising an enzyme, and a precursor material capable of reacting with the enzyme to form an inorganic coating material. The method of making a toothpaste may further comprise adding one or more additional ingredients to the mixture, wherein the one or more additional ingredients may include, but are not limited to, a protecting agent that chemically or physically separates the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material, a phosphorus-containing compound, a cleaning agent, a polishing agent, an abrasive, an accelerator, a promoter, an activator, an anti-microbial material, a pH adjuster, a color-producing additive, a scent-producing additive, a fluoride-containing material, a production processing aid, a thickening agent, a filler material, water, any other component disclosed herein, and any combination of the components disclosed herein.

[0078] In some embodiments, at least one protecting agent is added to the mixture, wherein the at least one protecting agent chemically or physically separates the enzyme from the precursor material within the mixture so as to prevent premature reaction of the enzyme with the precursor material. Typically, the at least one protecting agent encapsulates the one or more enzymes present in the mixture. However, in some embodiments, one or more protecting agents may be used to encapsulate the one or more enzymes, the one or more precursor materials, or both.

[00791 When one or more protecting agents are utilized, a method for chemical separation of the enzyme from the precursor material may comprise bonding one or more enzymes to one or more components in the composition, such as one or more protecting agents. Such bonding may be intramolecular, such as covalent or ionic bonds, or intermolecular, such as hydrogen or dipole to dipole (e.g., van der Waals) bonds. For example, an enzyme may be covalently bonded to a protecting agent or bonded to the protecting agent via hydrogen bonding. In other exemplary embodiments, a bifunctional linking agent may be used to bond one or more enzymes to one or more components in the composition, such as one or more protecting agents. The protecting agent may be chemically inert or reactive, and may be organic or inorganic. Protecting the enzyme or precursor may also be accomplished by modifying the pH.

[0080] In some embodiments, the method of making a composition may further comprise incorporating nanospheres into the composition, such as nanospheres formed from a biodegradable polymeric material such as polylactide. In this embodiment, one or more enzymes may be bonded to surfaces of the polylactide nanospheres either directly or via a bifunctional linker. For example, the bifunctional linker may comprise a polymer that contains nitrilotriacetic acid groups that bond to the enzyme via complexing to a polypeptide that is linked to a histidine tag, such as a polypeptide comprising an animal, bacterial, plant or fungal silicatein-α, silicatein-β or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown in, e.g., SEQ ID No. 1 or SEQ ID No. 3. In other embodiments, the enzyme is bonded to an inorganic material, such as a metal oxide (e.g., silica particles).

[0081 ] The disclosed methods of forming a composition may further comprise bringing the one or more enzymes and the one or more precursor materials into contact with one another so as to (i) react the enzyme(s) with the precursor material(s) and (ii) form an inorganic coating material. The step of bringing the one or more enzymes into contact with the one or more precursor materials may, for example, comprise chemically or physically displacing and/or removing a protecting agent from the enzyme or precursor material within the composition mixture. Displacing and/or removing the protecting agent may comprise, for example, physically displacing the protecting agent with mechanical action (e.g., via mechanical abrasion during a tooth brushing step) or removing the protecting agent by solubilizing and/or degrading the protecting agent (e.g., via contact with amylases in saliva during a tooth brushing step). Chemically removing the protecting agent from the enzyme or precursor may be performed by a reaction, by adjusting the temperature or the pH, or the like.

[0082] The present invention is also directed to methods of forming a coating and methods of forming coated substrates. In one exemplary embodiment, a method of forming a coating is disclosed, wherein the method comprises applying any of the above-described compositions onto a substrate. The resulting coated substrate comprises a substrate that is at least partially coated with a mixture (i.e., pre-reacted or post-reacted) as described above. The method of forming a coating may further comprise the step of bringing the one or more enzymes into contact with the one or more precursor materials either prior to or after applying the composition/mixture onto the substrate.

[0083] The methods of forming a coated substrate may further comprise one or more additional process steps. Suitable additional process steps include, but are not limited to, removing any unreacted mixture components from the coated substrate (e.g., via a rinsing or washing step), reapplying any of the above-described mixtures onto the coated substrate (i.e., the mixture being the same as or different from an original or previously applied composition/mixture), reacting one or more enzymes with one or more precursor materials within the subsequently applied composition/mixture, and repeating any of the above- mentioned steps.

[0084] Desirably, the resulting inorganic coating material, coating and coated substrate (i.e., the coating thereon) comprises (more desirably, "consists essentially of, and even more desirably, "consists of) amorphous silicon dioxide (silica), hydroxyl apatite, or a combination thereof.

[0085] In one embodiment, the method of forming a coated substrate comprises at least partially coating a tooth having an outer surface, an inner surface, or both. The method of forming a coated tooth results in a coating that at least partially covers the outer surface, the inner surface, or both the outer surface and the inner surface of the tooth. 10086] In any of the above-mentioned methods, the enzyme desirably comprises a silicatein, cathepsin, papain and/or a polypeptide having an oligo-glutamate sequence, and the precursor material desirably comprises a metal oxide such as silica, silicate, silicic acid, silicon alkoxides, and/or a compound having a general formula Si(Ri )_m(R₂)_n(R₃)p(R₄)_q wherein each of Ri, R₂, R₃ and R₄ independently comprises OH, H, a Cl to C12 alkane group, or a Cl to C12 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures of these materials. Typically, the enzyme includes a silicatein, such as silicatein-α, silicatein-β or silicatein-γ; a polypeptide that contains an animal, bacterial, plant or fungal silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ ID No. 3 below; or a polypeptide that is homologous thereto. In other embodiments, the enzyme comprises the above-described polypeptide that contains a silicatein-α, silicatein- β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ ID No. 3 and an oligo-glutamate sequence. Typically, the precursor material includes silicic acids, monoalkoxysilane triols, dialkoxysilane diols, trialkoxysilanols, tetraalkoxysilanes, and chelate complexes of silicic acid. [0087] Further, in any of the above-mentioned methods, the method may further comprise adding one or more phosphorus-containing compounds to the mixture. The one or more phosphorus-containing compounds may further react with the one or more enzymes to form hydroxyl apatite. In methods of forming coated substrates wherein the substrate is a tooth or teeth, the mixture desirably comprises one or more phosphorus-containing compounds. Suitable phosphorus-containing compounds include, but are not limited to, salts of phosphoric acid, such as sodium phosphate, disodium phosphate, trisodium phosphate, tetrasodium phosphate, sodium metaphosphate, pentasodium triphosphate, sodium monofluoro-phosphate, monofluorophosphate, sodium dodecyl phosphate (sodium lauryl phosphate), calcium sodium phosphate, tetrapotassium phosphate, pentapotassium triphosphate, disodium pyrophosphate, tetrasodium pyrophosphate, tetrapotassium pyrophosphate, tin phosphate (stannous phosphate), and tin pyrophosphate (stannous pyrophosphate).

[0088] The compositions of the present invention may further comprise at least one protecting agent that chemically or physically separates the enzyme from the precursor material within a given mixture so as to prevent premature reaction of the enzyme with the precursor material. The protecting agent may physically separate the enzyme, such as by encapsulating, isolating or containing the enzyme. The protecting agent may also chemically protect the enzyme, such as by intramolecular or intermolecular bonding, or steric hindrance, etc. Typically, when present, the one or more protecting agents are utilized to protect the enzyme, and not the precursor material; however, the one or more protecting agents may be utilized to protect the enzyme alone, the precursor material alone, or both the enzyme and the precursor material. An exemplary method for chemical separation of the enzyme from the precursor material may comprise bonding one or more enzymes to one or more components in the composition, such as one or more protecting agents. Such bonding may be intramolecular, such as covalent or ionic bonds, or intermolecular, such as hydrogen or dipole to dipole (e.g., van der Waals) bonds. For example, an enzyme may be covalently bonded to a protecting agent or bonded to the protecting agent via hydrogen bonding. In other exemplary embodiments, a bifunctional linking agent may be used to bond one or more enzymes to one or more components in the composition, such as one or more protecting agents. The protecting agent may be chemically inert or reactive, and may be organic or inorganic. Protecting the enzyme or precursor may also be accomplished by modifying the pH.

[00891 Suitable protecting agents include, but are not limited to, polylactide, polyvinyl alcohol, alginate, copolymers such as poly(lactide-co-ε-caprolactone. Desirably, the protecting agent is (i) water-soluble, (ii) degradable in the presence of saliva, or (ii) both (i) and (ii).

[00901 The compositions of the present invention may further comprise one or more promoting, accelerating or activating agents, which are capable of enhancing the enzymatic reaction, as well as other additional components as mentioned herein. ///. Applications/Uses

[0091 ] As discussed above, the compositions of the present invention may be utilized to form coatings on a substrate. Suitable substrates include, but are not limited to, teeth, bone, etc. In one exemplary embodiment, the compositions of the present invention are used to form bioactive oral care products that seal and protect hard dental tissue (enamel/dentin). The compositions produce an enzyme-catalyzed formation of a nanolayer that consists of amorphous silicon dioxide (biosilica), and thus, closes fissures in the enamel/dentin. For enzyme catalysis, the polypeptide that is used is desirably the above- described polypeptide that comprises an animal, bacterial, plant or fungal silicatein-α silicatein-β, or silicatein-γ domain having at least 25% sequence similarity to the sequence that is shown in, for example, SEQ ID No. 1 or SEQ ID No. 3.

|0092] In some exemplary embodiments, the compositions of the present invention are used to form bioactive oral care products that seal and protect hard dental tissue (enamel/dentin), wherein the compositions produce an enzyme-catalyzed formation of a nanolayer that consists of amorphous silicon dioxide (biosilica) and bioapatite. The bioapatite in combination with the biosilica closes fissures in the enamel/dentin of the tooth surface. The exemplary oral care composition may comprise or be utilized with any oral care materials or devices, including but not limited to, toothpaste, gum, mouthwash, teeth brushes, mouth guards or teeth trays, teeth strips, teeth alignment devices, food, drinks, lozengers, or other edible materials, and combinations thereof. In addition, the compositions of the present invention may be used with any oral care products for any animals.

[0093] FIGS. 2A-2D depict exemplary steps used to provide such a protective inorganic coatling layer on a tooth using the compositions of the present invention. As shown in FIG. 2A, toothpaste composition 10 comprising enzyme 20 (e.g., a silicatein), precursor material (e.g., silica) 22 and a phosphate-containing compound 25 may be delivered into the vicinity of enamel 11 with Fissures 13 and cavies 15 on tooth 12. In phase 1 , catalysed by enzyme 20, silicatein-formed silica building blocks form a nanoscale layer of biosilica 17 on a top surface 19 of enamel 11, partly or completely closing and sealing the caries contaminated fissures 13 as shown in FIG. 2B. The facultative aerobe bacteria forming caries 15 are cut off from both oxygen and glucose, and deteriorate over time. [0094] As shown in FIG. 2C, phosphate building blocks form bio hydroxyl-apatite 18 on biosilica layer 17 creating an enamel identical material in the form of layer 21. As shown in FIG. 2D, in the final phase, silica cleaning bodies 27, such as abrasive or polishing metal oxide particles (not shown in previous pictures), a possible component of toothpaste 10, may remove any surplus biohydroxyl apatite layer 21 from enamel 11, but leaves layer 19 of bio hydroxyl-apatite 18 deposited within fissures 13.

[0095] One advantage of the methods of the present invention is that it makes it possible to achieve a dense and tightly-adhering sealing of a tooth surface (e.g., top surface 19 of enamel 11) with a silica nanolayer (e.g., nanoscale layer of biosilica 17), which also results in closure of even the finest Fissures. The enzymatically-formed biosilica nanoparticles are sized so as to (i) penetrate the finest fissures, such as fissures having a smallest dimension of about 20 nanometers (nm), and (ii) deposit onto inner surfaces represented by side walls of the fissures (e.g., fissures 13).

[00961 In contrast to the enzymatically-formed biosilica nanoparticles formed in the present invention, silica particles, found as an abrasive in many toothpastes, are not capable of closing fine fissures due to their size, in particular, an average minimum dimension (e.g., a diameter) of at least 20 nm. Penetration of the biosilica nanoparticles and the subsequently formed apatite deposits, the fissures are closed, and caries-producing bacteria, such as the optionally anaerobic Staphylococcus mutans, are cut off from their needed supply of glucose oxygen.

[0097] The methods of the present invention may also be used to form silicified nanospheres (e.g., polylactide nanospheres) by means of the above-described enzymes and precursor materials. One or more of the above-described enzymes may be bonded to surfaces of a plurality of nanospheres (e.g., polylactide nanospheres), and deposited within the vicinity of a tooth or teeth. A separate composition component may comprise one or more precursor materials. The one or more precursor materials may be encapsulated by another material or otherwise separated from the enzyme-active nanoparticles so as to provide a time-release delay in contact between (i) the nanospheres (i.e., positioned within the finest fissures) and (ii) the one or more precursor materials. Upon reaction, silicification takes place either by (i) forming an inorganic coating layer or partial layer (e.g., a silica shell) on the surface of the nanospheres or (ii) by forming a silica core. As precursor materials, any of the above-described precursor materials (e.g., silicic acid, monoalkoxysilane triols, dialkoxysilanediols, trialkoxysilanols, tetraalkoxysilanes, chelate complexes of silicic acid) may be used.

[0098] In any of the above-described methods of using the compositions of the present invention, the polypeptide that is used for enzyme catalysis (and closing fissures) comprises an oligo-glutamate sequence, and the enzyme desirably comprises a silicatein, cathepsin, papain and/or a polypeptide having an oligo-glutamate sequence. Further, in any of the above described methods of using the compositions of the present invention, the precursor material that is used to form inorganic coating material (and close fissures) comprises a metal oxide such as silica, silicate, silicic acid, silicon alkoxides, and/or a compound having a general formula Si(Ri )_m(R₂)_n(R3)p(R₄)_q wherein each of Ri, R₂, R₃ and R₄ independently comprises OH, H, a C l to C 12 alkane group, or a Cl to C 12 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures of these materials. Typically, the enzyme includes a silicatein, such as silicatein-α, silicatein-β or silicatein-γ; a polypeptide that contains an animal, bacterial, plant or fungal silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ ED No. 3 below; or a polypeptide that is homologous thereto. In other embodiments, the enzyme comprises the above-described polypeptide that contains a silicatein-α, silicatein-β, or silicatein-γ domain that has at least 25% sequence similarity to the sequence that is shown as SEQ ID No. 1 or SEQ ID No. 3 and an oligo-glutamate sequence. Typically, the precursor material includes silicic acids, monoalkoxysilane triols, dialkoxysilane diols, trialkoxysilanols, tetraalkoxysilanes, and chelate complexes of silicic acid.

[0099] In any of the above-described methods of using the compositions of the present invention, the compositions may be formulated to be suitable for use as a oral care product in the form of a low-viscous liquid or as a highly viscous paste or cream. The compositions may be used for daily dental hygiene. Further, the compositions may be used in a modified dosage under a physician's supervision or may be utilized in modified form by a physician to repair teeth in out patient procedures. For example, the enzyme and precursor may be applied by the physician to patient's teeth without the use of a protecting material.

[0100] The present invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims. EXAMPLES

Test Methods

[0101 ] The following test methods were used in the examples below.

Determination of Silicatein Activity

[0102] To determine the enzyme activity of (recombinant) silicateins, an assay that is based on the measurement of polymerized and precipitated silica after hydrolysis and subsequent polymerization of tetraethoxysilane (TEOS) can be applied. In this connection, the enzyme is usually dissolved in 1 ml of an MOPS buffer (pH 6.8) and mixed with 1 ml of a 1 -4.5 mmol tetraethoxysilane solution. The enzymatic reaction is performed for various long periods, usually at room temperature.

[01031 To detect the silica products, the material is centrifuged off, washed with ethanol and air-dried. Then, the sediment is hydrolyzed with 1 M NaOH. In the solution that is produced, the released silicate is quantitatively measured with use of a molybdate- supported detection method (e.g., a silicon assay available from the Merck Company).

Determination of Silica Formation

[0104] The functional activity of the silicatein gene products, the silicatein-mediated formation, and physical-chemical characteristics of porous (bio)silica nanolayers may be analyzed by means of various methods (i.e., SEM, SDS-PAGE, molybdate assay of the silica formation in the presence of exogenic Na silicate or tetraethoxysilane, zymography, etc.).

[0105] The formation of biosilica is determined by coloring the forming silica particles with the fluorochrome Rhodamine 123 (i.e., incorporation of fluorochrome in the forming particles) or by means of the β-silicomolybdato-color system (i.e., silicate detecting reagent "Aquaquant kit") as disclosed in Cha et al., Proc. Natl. Acad. ScL USA 96:361 -365 (1999); and Krasko et al., Europ. J. Biochem. 267:4878-4887 (2000). [0106] In addition, the samples may be analyzed by means of EDX ("Energy Dispersive X-Ray") analysis.

Example 1 - Production of Recombinant Silicatein Polypeptides

[0107] The cDNAs for silicatein-α and silicatein-β were isolated from a cDNA library of the sponge Suberites domuncula, which was produced, for example, in a pBK-CMV vector (available from Stratagene (La Jolla, CA)). The open reading grids (ORF) corresponding to the mature proteins were obtained by means of polymerase chain reaction (PCR) with use of suitable primers.

[0108] The production of the recombinant proteins (silicatein-α: SEQ ED No. 1 ; silicatein- β: SEQ ID No. 3) was carried out in E. coli (although production in yeasts and mammal cells were other options). The respective cDNA were cloned in an expression vector, e.g., pQE-30. After transformation of E. coli, the expression of proteins was induced with IPTG (isopropyl-β-D-thiogalactopyranoside) (as described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York ( 1995). The purification of the recombinant proteins via the histidine tag was performed on a Ni-NTA matrix.

[0109] (It should be noted that between oligohistidine and silicatein, a sequence may be introduced that corresponds to the enterokinase gap site. The fusion protein can then be cleaved with enterokinase.)

[01 10] The production of recombinant silicatein-α in E. coli with use of the oligo-histidine expression vector pBAD/gDIA (commercially available from Invitrogen (Carlsbad, CA)), in which, based on the gene HI signal sequence, the recombinant protein was secreted into the periplasmatic space. The cDNA sequence that codes for the silicatein-α (i.e., SEQ ED No. 2 shown below) was amplified by means of PCR with use of the following primers (short form of silicatein-α): (i) Forward Primer: TAT CC ATG GAC TAC CCT GAA GCT GTA GAC TGG AGA ACC, and (ii) Reverse Primer: TAT T CTA GA A TTA TAG GGT GGG ATA AGA TGC ATC GGT AGC; and cloned in pBAD/gEHA (i.e., restriction nucleases for insertion in the expression vector: Ncol and Xbal). After transformation of E. coli XLl -Blue, the expression of the fusion protein was induced with L-arabinose. It should be noted that an insert that comprises the entire derived silicatein-α protein (long form) may also be used.

[01 1 I J A short and a long form of silicatein-β (cDΝA: SEQ ID No. 4; amino acid sequence derived therefrom: SEQ ED No. 3) was also expressed using an analogous procedure as described above.

Example 2 - Production of Recombinant Hydroxylapatite-Binding Silicatein-α Protein [01 12] Production of a recombinant silicatein, which can not only be purified by Ni-NTA metal affinity chromatography owing to the presence of a 6xHis tag, but also has hydroxylapatite-binding properties owing to the presence of an 8xGIu tag was prepared as follows. Oligonucleotides were utilized in the production of a modified silicatein-α amplicon with the forward primer comprising: 5' GAA GAG GAA GAG GAA GAG GAA GAG CCT GAA GCT GTA GAC TGG 3' (Underlined: HA-binding GIu tag; Double-underlined: beginning of the mature silicatein protein), and the reverse primer comprising 5' TAG GGT GGG ATA AGA TGC ATC GGT 3' (Underlined: the last codon that codes for mature silicatein-α). In the PCR step, amplification was successful at all annealing temperatures (i.e., 56°-60°C) as shown in FIG. 3.

[01 13] Ligation of the amplicon was conducted with bacterial expression vector pTRC- His2 (commercially available from Invitrogen (Carlsbad, CA)) via T/A cloning as shown in FIG. 4. Cloning of the construct was conducted in E. coli BL21 cells (commercially available from Invitrogen (Carlsbad, CA)). Ten colonies were isolated, each of which had integrated therein the correct construct. LO l 14] The final recombinant protein has the following appearance:

MΛLEEEEEEEEPEAVDWRTKGAVTAVKDOGDCGASYAFSAMGALEGAN ALAKG NAVSLSEQNΠDCSIPYGNHGCHGGNMYD AFL YVIANEGVDQDSAYPFVGKQSSC NYNSKYKGTSMSGMVSIKSGSESDLQAAVSNVGPVSVAIDGANSAFRFYYSGVY DSSRCSSSSLNHAMVVTGYGSYNGKKYWLAKNSWGTNWGNSGYVMMARNKY

NQCGIATDAS YYTLKGEFEA TVEQKLISEEDLNSA VDHHHHHH

(Tags are underlined; vector sequences are in cursive.)

[01 15] The individual clones were stimulated with 1 mmol of IPTG (2-8 hours) for synthesis of the recombinant protein.

Example 3 - Production of Various Composition Mixtures

[01 16] Composition mixture samples A-E were prepared as follows:

Composition 3A was prepared by encapsulating the enzyme silicatein-α formed in Example 1 in polylactide (source, city, state) so as to form spherical particles having an outer shell of polylactide and an inner core of the enzyme silicatein-α. Example 4 - Production of a Composition Mixture in the Form of a Toothpaste

[01 171 A composition suitable for use as a toothpaste was prepared by forming a mixture of the components shown in Table 1 below.

Table 1.Toothpaste Composition

[01 18] The resulting toothpaste had a mixture viscosity of about 200,000 cps after 24 hours measured with a Brookfield Viscosimeter, and a consistency similar to commercially available toothpastes. Example 5 - Production of a Coated Substrate

[0019] The toothpaste composition of Example 4 was applied onto the teeth of a pig by adding the teeth, about 0.5 grams (g) of the toothpaste composition of Example 4, and about 480 g of deionized water to an Erlenmeyer flask, and gently shaken for 8 hours at room temperature.

[0120] SEM imaging revealed that the enamel of the teeth was covered with a thin layer of biosilica/bioapatite as shown in FIGS. 5C-5D. FIGS. 5A-5B show a pig tooth at magnifications of 8500Ox and 20500Ox, respectively, prior to combining the tooth with the above-described toothpaste composition. FIGS. 5C-5D show the same pig tooth at magnifications of 8500Ox and 205000x, respectively, after the coating procedure described above. As shown in FIGS. 5C-5D, a coating partially covers the pig tooth outer surface. [0121 ] FIG. 6 depicts the EDX spectrum of the biosilica/bioapatite layer formed on the pig tooth as described above.

[0122] While the invention has been described with a limited number of embodiments, these specific embodiments are not intended to limit the scope of the invention as otherwise described and claimed herein. It may be evident to those of ordinary skill in the art upon review of the exemplary embodiments herein that further modifications, equivalents, and variations are possible. All parts and percentages in the examples, as well as in the remainder of the specification, are by weight unless otherwise specified. Further, any range of numbers recited in the specification or claims, such as that representing a particular set of properties, units of measure, conditions, physical states or percentages, is intended to literally incorporate expressly herein by reference or otherwise, any number falling within such range, including any subset of numbers within any range so recited. For example, whenever a numerical range with a lower limit, R_L, and an upper limit Ru, is disclosed, any number R falling within the range is specifically disclosed. In particular, the following numbers R within the range are specifically disclosed: R = R_L + k(Ru -R_L), where k is a variable ranging from 1 % to 100% with a 1 % increment, e.g., k is 1 %, 2%, 3%, 4%, 5%. ... 50%, 51 %, 52%. ... 95%, 96%, 97%, 98%, 99%, or 100%. Moreover, any numerical range represented by any two values of R, as calculated above is also specifically disclosed. Any modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. All publications cited herein are incorporated by reference in their entirety.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: an enzyme; a precursor material capable of being catalyzed by said enzyme to form an inorganic coating material; and a protecting agent, said protecting agent separating said enzyme from said precursor material within the mixture so as to prevent premature reaction of said enzyme with said precursor material.

2. The composition of Claim 1 , wherein said enzyme comprises a silicatein, cathepsin, papain and/or a polypeptide having an oligo-glutamate sequence.

3. The composition of Claim 1 or 2, wherein said precursor material comprises silica, silicate, silicic acid, silicon alkoxides, and/or a compound having a general formula Si(Ri )_m(R₂)n(R3)_P(R4)q wherein each of Ri, R₂, R₃ and R₄ independently comprises OH, H, a Cl to C12 alkane group, or a Cl to C12 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures of these materials.

4. The composition of any one of Claims 1 to 3, further comprising a phosphorus- containing compound.

5. The composition of Claim 4, wherein said enzyme, said precursor material and said phosphorus-containing compound react with one another to form hydroxyl apatite.

6. The composition of any one of Claims 1 to 5, wherein said phosphorus-containing compound comprises salts of phosphoric acid, such as sodium phosphate, disodium phosphate, trisodium phosphate, tetrasodium phosphate, sodium metaphosphate, pentasodium triphosphate, sodium monofluoro-phosphate, monofluorophosphate, sodium dodecyl phosphate (sodium lauryl phosphate), calcium sodium phosphate, tetrapotassium phosphate, pentapotassium triphosphate, disodium pyrophosphate, tetrasodium pyrophosphate, tetrapotassium pyrophosphate, tin phosphate (stannous phosphate), and tin pyrophosphate (stannous pyrophosphate).

7. The composition of Claim 1 , wherein said protecting agent chemically bonds to said enzyme or precursor material.

8. The composition of any one of Claims 1 to 7, wherein said protecting agent encapsulates said enzyme.

9. The composition of any one of Claims 1 to 8, wherein said protecting agent comprises polylactide.

10. The composition of any one of Claims 1 to 9, wherein said protecting agent further comprises a linking agent for binding said enzyme or precursor to said protecting agent.

1 1. The composition of any one of Claims 1 to 10 wherein said composition is an oral care composition comprising toothpaste, gum, mouthwash, teeth brushes, mouth guards or teeth trays, teeth strips, teeth alignment devices, food, drinks, lozengers, or other edible materials, and combinations thereof.

12. A coating comprising the composition of any one of Claims 1 to 1 1.

13. A coating comprising a reaction product formed from a reaction between reactive components within the composition of any one of Claims 1 to 1 1.

14. A substrate at least partially coated with the composition of any one of Claims 1 to 1 1.

15. A substrate at least partially coated with the coating of Claim 12 or 13.

16. The substrate of Claim 14 or 15, wherein said substrate comprises a tooth having an outer surface, an inner surface, or both.

17. The substrate of Claim 16, wherein said coating at least partially covers said outer surface, said inner surface, or both.

18. A method of forming a composition, said method comprising: forming a mixture comprising: an enzyme; a precursor material capable of being catalyzed by the enzyme to form an inorganic coating material; and a protecting agent that chemically or physically separates the enzyme from the precursor material so as to prevent premature reaction of the enzyme with the precursor material.

19. A method of forming an inorganic coating material on a tooth, said method comprising: mixing an enzyme and a precursor material capable of being catalyzed by the enzyme to form the inorganic coating material; and coating said tooth with the inorganic coating material to form an inorganic coating on at least a portion of said tooth.

20. The method of Claim 19, further comprising: a protecting agent that chemically or physically separates the enzyme from the precursor material so as to prevent premature reaction of the enzyme with the precursor material.

21. The method of any one of Claims 18 to 20, wherein the mixture further comprises a phosphorus-containing compound.

23. The method of any one of Claims 18 to 22, wherein the enzyme comprises a silicatein; the precursor material comprises silica, silicate, silicic acid, silicon alkoxides, and/or a compound having a general formula Si(Ri)_m(R₂)_n(R₃)p(R4)q wherein each of Ri, R₂, R₃ and R₄ independently comprises OH, H, a Cl to C 12 alkane group, or a Cl to Cl 2 alkoxy group; each of m, n, p and q is independently 0 or an integer ranging from 1 to 4; and (m + n + p + q) = 4, condensation products thereof, or mixtures of these materials.

24. The method of any one of Claims 19 to 23, further comprising: bringing the enzyme into contact with the precursor material so as to initiate a reaction between the enzyme and the precursor material.

25. The method of Claim 24, wherein the coating comprises hydroxyl apatite.