TW201521780A - System providing perhydrolase-catalyzed reaction - Google Patents

Abstract

Described herein are packages for storing and dispensing multi-part tooth whitening formulations, comprising a deformable material configured to form two or more sealed chambers, e.g., wherein the first chamber contains a low viscosity liquid solution comprising an enzyme having perhydrolytic activity, and the second chamber contains a peroxide source and a at least one acyl donor substrate. Particular multi-part tooth whitening formulations using this principle and methods of use thereof are also provided.

Description

System for catalytic reaction by hydrolase

The present invention relates to a package for storing and dispensing a multi-part whitening tooth formulation comprising a deformable material configured to form two or more sealed chambers, such as wherein the first groove is provided with a low viscosity containment A liquid solution of the hydrolyzed active enzyme, and the second tank contains a peroxide source and at least one sulfhydryl donor matrix. A special multi-part whitening tooth formula using this principle and its use are also provided.

It is often desirable to separate the formulation components prior to use, for example because the components may be extremely unstable if combined for long term storage. In such cases, it is desirable to be able to mix the ingredients in the active and easy manner when used in the point of use.

An example of a formulation of a whitening tooth formulation that may wish to separate the formulation components, including reactive components such as peroxides or peroxyacids or the like. For example, one might want to combine A+B or A+B+C to obtain an unstable bleach X, but leave A and B separate until the point in time. The difficulty is that it must be mixed quickly during use, and the bleach, X, must spread effectively on the tooth surface. Unfortunately, combining multiple colloids or other moderately viscous materials is often not an effective way to quickly mix chemicals; if typical consumers mix by hand, this will Lead to sample blocks that are easy to mix and difficult to mix. One person can easily see the problem by simply blending two sticky household paints together: instead of effectively mixing the two colors, the laminar flow creates an adjacent stripe color. In order to directly overcome this problem, more typical users are willing to work more time and blending time, and in which the reaction species X begins to decompose within a few minutes, these methods are not feasible.

Therefore, it is required that the product can allow the components to be efficiently and effectively combined in the present use.

The present invention provides a multi-tank system in which one tank contains a low viscosity liquid solution and the other tank contains a mixture of liquid, powder or powder which is separated by a fragile or tearable barrier so that When a tank is squeezed, the barrier is broken and the components in each tank can be mixed to form a solution, emulsion, suspension or extrudable gel which can be dispensed via the outlet of the second tank, wherein the low The viscosity liquid solution comprises a protein having perhydrolase activity, which contains a carbohydrate esterase family 7 signature motif, and other tanks containing a thiol donor, such as a carboxylic acid ester, and a peroxide. The source is such that upon mixing the contents of each of the tanks, the protein having perhydrolase activity catalyzes the reaction between the peroxide-derived peroxide and the thiol-providing donor to form a peroxyacid. Such peroxyacids are extremely effective in bleaching teeth when applied to teeth, thus achieving effective bleaching in a shorter period of time and at lower peroxide concentrations.

In a specific embodiment, a tank contains a low viscosity aqueous solution comprising a protein having perhydrolysis activity (ie, a family 7 carbohydrate esterase), and another tank containing a gelling agent, a peroxide, and a carboxyl group. Acid ester compounds, all in powder form, The peroxide and the carboxylic acid ester are reacted when the barrier is broken and the contents of the tank are allowed to mix, the reaction being catalyzed by a perhydrolase to form a peroxyacid, and the liquid The colloidal agent is formed in an extrudable colloid, and the extrudable colloid can be extruded and applied to the teeth, for example, using a sprocket or a toothpaste for a sufficient time, for example, 10-30 Minutes, while allowing teeth to bleach.

Further areas of applicability of the present invention will become apparent from the Detailed Description. The detailed description and specific examples are intended to be illustrative of the preferred embodiments of the invention

Brief description of the biological sequence

The following sequences are in accordance with 37 CFR §§ 1.821-1.825 ("Disclosure Requirements for Nucleotide Sequences and/or Amino Acid Sequences - Sequence Rules") and in accordance with World Intellectual Property Organization (WIPO) Guidelines ST.25 (2009) and Europe Sequence Listing Requirements in the Patent Convention (EPC) and Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5 (a-bis), and Chapter 208 and Appendix C of the Administrative Instructions. The symbols and formats used in the nucleotide and amino acid sequence data are in accordance with the rules set forth in 37 C.F.R.§ 1.822.

SEQ ID NO: 1 is the amino acid sequence of Thermotoga maritima C277S variant perhydrolase.

SEQ ID NO: 2 is an amino acid sequence comprising a fusion protein of Thermotoga maritima C277S variant perhydrolase coupled to a tooth-binding structural region (also known as "EZ-7" in International Patent Application Publication No. WO2012) /087970A2-Butterick et al.),

SEQ ID NO: 3 encoding from Bacillus subtilis (Bacillus subtilis) a nucleic acid sequence of ATCC ^® 31954 ^TM header to cephalosporin C acetyl Bacillus enzyme.

SEQ ID NO: 4 is the amino acid sequence from Bacillus subtilis ATCC ^® 31954 ^TM to the head cephalosporin C acetyl Bacillus enzyme.

SEQ ID NO: 5 is the amino acid sequence of the cephalosporin C deacetylase from Bacillus subtilis subsp. subtilis strain 168.

SEQ ID NO: 6 is the amino acid sequence from Bacillus subtilis ATCC ^® 6633 ^TM to the head cephalosporin C acetyl Bacillus enzyme.

SEQ ID NO: 7 is the head ATCC ^® 14580 ^TM Bacillus cephalosporin C acetyl enzyme to the amino acid sequence derived from Bacillus licheniformis (B. licheniformis).

SEQ ID NO: 8 is the amino acid sequence of the acetyl xylan esterase (acetyl xylan esterase) from B. pumilus PS213 .

SEQ ID NO: 9 is the amino acid sequence of the acetyl xylan esterase from the ATCC ^® 27405 ^TM thermophilic Clostridium (Clostridium thermocellum).

SEQ ID NO: 10 is the amino acid sequence of the beta xylan esterase from the cloned Thermotoga neapolitana .

SEQ ID NO: 11 is the amino acid sequence of the acetyl xylan esterase acetyl xylan esterase from Thermotoga marinum MSB8.

SEQ ID NO: 12 is the amino acid sequence of an acetyl xylan esterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 13 is the amino acid sequence of the cephalosporin C deacetylase from Bacillus halodurans C-125.

SEQ ID NO: 14 is the amino acid sequence of the cephalosporin C deacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 15 is the amino acid sequence of a cloned new Apollo Thermotoxin xylan esterase variant from U.S. Patent Application Publication No. 2010-0087529, the entire disclosure of which is incorporated herein by reference. Wherein the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 16 is the amino acid sequence of the Thermotoga maritima MSB8 acetyl xylan esterase variant from U.S. Patent Application Publication No. 2010-0087529, wherein the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 17 is the amino acid sequence derived from the Thermotoga lettingae xylan esterase variant from US Patent Application Publication No. 2010-0087529, wherein the Xaa residue at position 277 The base is Ala, Val, Ser, or Thr.

SEQ ID NO: 18 is the amino acid sequence of the Thermotoga sinensis xylan esterase variant from U.S. Patent Application Publication No. 2010-0087529, wherein the Xaa residue at position 277 is Ala, Val, Ser. , or Thr.

SEQ ID NO: 19 is the amino acid sequence of the Thermotoga RQ2 acetylene xylan esterase variant derived from "RQ2(a)" of U.S. Patent Application Publication No. 2010-0087529, wherein at position 277 The Xaa residue is Ala, Val, Ser, or Thr.

SEQ ID NO: 20 is the amino acid sequence of the Thermotoga RQ2 acetylene xylan esterase variant derived from "RQ2(b)" of U.S. Patent Application Publication No. 2010-0087529, wherein at position 278 The Xaa residue is Ala, Val, Ser, or Thr.

SEQ ID NO: 21 is the amino acid sequence of the Thermotoga xylan esterase of Letinger.

SEQ ID NO: 22 is the amino acid sequence of the Thermotoga sylvestre xylan esterase.

SEQ ID NO: 23 is the amino acid sequence of the first acetyl xylan esterase from "RQ2(a)" described by Thermotoxin RQ2.

SEQ ID NO: 24 is the amino acid sequence of the second acetyl xylan esterase from "RQ2(b)", which is described by Thermotoxin RQ2.

SEQ ID NO: 25 is the amino acid sequence of Thermoanearobacterium saccharolyticum cephalosporin C deacetylase.

SEQ ID NO: 26 is the amino acid sequence of the acetyl xylan esterase from Lactococcus lactis (GENBANK ^® registration number ABX75634.1) of.

SEQ ID NO: 27 is the amino acid sequence of the acetyl xylan esterase from Rhizobium corniculatus (Mesorhizobium loti) (GENBANK ^® registration number BAB53179.1) of.

SEQ ID NO: 28 is the amino acid sequence of the acetyl xylan esterase from Geobacillus stearothermophilus (Geobacillus stearothermophilus) (GENBANK ^® registration number AAF70202.1) of.

SEQ ID NOs 29-163 are amino acid sequences of peptides that have affinity for the oral surface.

SEQ ID NOs: 164-177 are the amino acid sequences of the peptide linker/spacer region.

SEQ ID NOs: 178-197 are a plurality of standard perhydrates comprising a perhydrolase coupled via a peptide linker to a binding structural region having affinity for the oral surface. Amino acid sequence of the enzyme-enhancing structure (see International Patent Application Publication No. WO2012/087970A2-Bartrick et al.).

Around 1‧‧

2‧‧‧first slot

3‧‧‧ Fragile seal

4‧‧‧second trough

5‧‧‧Scratched edges

6‧‧‧ nozzle

7‧‧‧first slot

8‧‧‧second trough

9‧‧‧ third slot

The invention will be more fully understood from the following detailed description and the accompanying drawings, in which: FIG. 1 is a <Desc/Clms Page number> And having a first tank (2) comprising a liquid component and a second tank (4) comprising a powder component separated by a frangible seal (3) such that when the first tank (2) is squeezed The frangible seal (3) ruptures and the liquid flows to the second tank (4) and mixes with the powder, which causes the mixture to then allow the mixture to pass from the nozzle by breaking the scored edge (5). (6) Distribute or squeeze out and distribute.

Figure 2 is a diagram illustrating another embodiment of the present invention which allows the components to be mixed just prior to use, as illustrated in Figure 1, but using a three-slot package of nozzles that are opened when the consumer dispenses the product. In this specific example, the package includes a first groove (7), a second groove (8), and a third groove (9) separated by a frangible seal (3), and a breakthrough tip is included The nozzle (6) that is dispensed after mixing.

The following description of the preferred embodiments is merely illustrative and is in no way intended to limit the invention, the application, or the application.

As used herein, the terms "substrate", "suitable substrate", "thiol donor", and "carboxylate matrix" are used interchangeably to mean: (a) one or more esters having the following structural formula Class [X] _m R ₅

Wherein X is an ester group of the formula R ₆ C(O)O; R ₆ is a C1 to C7 linear, branched or cyclic hydroxy moiety, optionally substituted by a hydroxy group or a C1 to C4 alkoxy group, wherein When R ₆ is C2 to C7, R ₆ optionally includes one or more ether linkages; R ₅ is a C1 to C6 straight chain, a branched or cyclic hydroxyl moiety or a cyclic five-membered heteroaryl or a six-membered aromatic ring. a hetero or heteroaryl moiety, optionally substituted by a hydroxy group; wherein each carbon atom in R ₅ independently comprises no more than one hydroxyl group or no more than one ester group, and wherein R ₅ Optionally comprising one or more ether linkages; m is an integer from the number of carbon atoms in 1 to R ₅ , one or more esters having a water solubility of 5 ppm at 25 ° C; or (b) one or more of the following structures Glyceryl ester

Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy group or a C1 to C4 alkoxy group and R ₃ and R ₄ are independently H or R ₁ C(O); or (c) One or more esters of the formula

Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight or branched alkyl group, an alkenyl group, an alkynyl group, Aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is from 1 to 10; or d) one or more acetylated monosaccharides, acetylated disaccharides, or acetaminolated polysaccharides; or (e) any combination of (a) to (d).

As used herein, the term "peracid" is synonymous with peroxyacid, peroxycarboxylic acid, peroxy acid, percarboxylic acid and Peroxoic acid.

As used herein, the term "peracetic acid" is abbreviated as "PAA" and is synonymous with peroxyacetic acid, ethane peroxyacid and all CAS registration numbers 79-21-0.

As used herein, the term "monoacetin" is synonymous with glycerol monoacetate, glycerin monoacetate, and glyceryl monoacetate.

As used herein, the term "diacetin" is synonymous with glycerol diacetate; glycerin diacetate, glyceryl diacetate, and all Synonyms of other CAS registration numbers 25395-31-7.

As used herein, the term "triacetin" is synonymous with glycerin triacetate; glycerol triacetate; glyceryl triacetate, 1, 2,3-Triethoxypropane; 1,2,3-propane triol triacetate and all other CAS registration numbers 102-76-1.

As used herein, the terms "sugar" and "saccharide" refer to mono-, di- and polysaccharides comprising at least one ethyl hydrazide group. Examples include, but are not limited to, glucose pentaacetic acid; xylose tetraacetic acid; acetylated xylan; acetylated xylan fragment; β-D-ribofuranosyl-1,2,3,5-tetraacetic acid; -O-acetamidine-D-galactose; and tri-O-acetamidine-gluconate.

As used herein, the terms "hydrocarbyl", "hydrocarbyl group", and "hydrocarbyl moiety" refer to a straight, branched or cyclic distribution of a carbon atom by means of a carbon-carbon mono, di or triple bond. And/or by ether bonds to replace one with a hydrogen atom. These hydrocarbyl groups can be aliphatic and/or aromatic. Examples of the hydrocarbyl group include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, pentyl, cyclopentyl, methylcyclopentyl. , hexyl, cyclohexyl, benzyl, and phenyl. In a preferred embodiment, the hydrocarbyl moiety is a linear, branched or cyclic distribution of carbon atoms which is bonded by a carbon-carbon single bond and/or by an ether linkage to be substituted with a hydrogen atom.

As used herein, 1,2-ethanediol; 1,2-propanediol; 1,3-propanediol; 1,2-butanediol; 1,3-butanediol; 2,3-butanediol; 1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol; 1,5-pentanediol; 1,6-pentanediol; 1,2-hexanediol; 5-hexanediol; 1,6-hexanediol; and the terms "monoester" and "diester" in the mixture, and the term "monoester" and "diester" means that the compound includes at least one ester group of the formula RC(O)O, wherein R is a C1 to C7 linear hydrocarbon Base part. In one embodiment, the carboxylate matrix is selected from the group consisting of propylene glycol diacetate (PGDA), ethylene glycol diacetate (EDGA), and mixtures thereof.

As used herein, the term "propylene glycol diacetate" is synonymous with 1,2-diethoxypropane, propylene diacetate, 1,2-propanediol diacetate, And synonymous with all other CAS registration numbers 623-84-7.

As used herein, the term "vinyl glycol diacetate" is synonymous with 1,2-diethoxymethoxyethane, vinyl diacetate, ethylene glycol diacetate, CAS. Synonyms in registration number 111-55-7.

As used herein, "appropriate enzymatic reaction mixture", "appropriate reaction component", "appropriate aqueous reaction mixture", "reaction mixture", and "component of peroxyacid production" mean the reactants and A substance and water that are in contact with each other by a hydrolase catalyst. The peroxyacid-producing component will comprise at least a hydrolytically active enzyme, wherein the perhydrolase is at least one CE-7 perhydrolase (any fusion protein type targeting the body surface), at least one suitable carboxylic acid Ester base, peroxide source, and water.

As used herein, the term "perhydrolysis" is defined as the reaction of a selected substrate with a peroxide to form a peroxyacid. Typically, the inorganic peroxide is combined with the selected substrate to form a peroxyacid in the presence of a catalyst. As used herein, the term "chemical perhydrolysis" includes a perhydrolysis reaction in which a matrix (peroxyacid precursor) is combined with a source of hydrogen peroxide, wherein the peroxyacid is formed in the absence of an enzyme catalyst. As used herein, the term "enzymatic perhydrolysis" includes a perhydrolysis reaction in which a carboxylate matrix (peroxyacid precursor; the "mercapto donor") is combined with a hydrogen peroxide source and water, The enzyme catalyst catalyzes the formation of peroxyacids.

As used herein, the term "perhydrolase activity" refers to a protein activity per unit mass (eg, milligrams) of protein, dry cell weight, or fixed catalyst weight.

As used herein, "one unit of enzyme activity" or "one unit of activity" or "U" is defined as the amount of perhydrolase activity required to produce 1 μmol of peroxyacid product per minute at a particular temperature.

As used herein, the terms "enzymatic catalyst" and "perhydrolase catalyst" refer to a catalyst comprising a perhydrolysis-active enzyme and the type thereof may be whole microbial cells, permeabilized microbial cells, one or A cellular component of a plurality of microbial cell extracts, a partially purified enzyme, or a purified enzyme. The enzyme catalyst can also be chemically modified (e.g., by pegylation or by reaction with a crosslinking reagent). The perhydrolase catalyst can also be immobilized on a soluble or insoluble support; see, for example, enzyme and cell immobilization; Gordon F. Bickstaff, editor; Hamana Publishing, Toto Watts, New Jersey, USA; 1997.

As used herein, "acetyl xylan esterase" refers to an enzyme (E.C. 3.1.1.72; AXEs) which catalyzes the deacetylation of acetylated xylan and other acetylated sugars.

As used herein, the terms "cephem C to acetylase" and "cephalosporin C hydrazine hydrolase" refer to an enzyme (EC 3.1.1.41) which catalyzes the cephalosporin. Deacetylation of sporecin C and 7-aminocephalosporin (Manji et al. (1995) Environmental Microbial Applications 61(6): 2224-2229). Amino acid sequences of many cephalosporin C deacetylases having significant perhydrolysis activity are provided herein.

As used herein, the term "Bacillus subtilis ATCC ^® 31954 ^TM " refers to a bacterial cell deposited with the National Stem Cell Resource Center (ATCC) having the international deposit registration number ATCC ^® 31954 ^TM . As defined herein, derived from Bacillus subtilis ATCC ^® 31954 ^TM having the amino acid sequences are significantly hydrolyzed by an enzyme activity in SEQ ID NO: 4 is provided (see, U.S. Patent Application Publication Number 2010-0041752).

As used herein, the term "H. thermophila MSB8" refers to a bacterial cell that has been reported to have acetyl sterol esterase activity (GENBANK ^® NP_227893.1; see U.S. Patent Application Publication No. 2008-0176299) . The amino acid sequence of an enzyme having perhydrolase activity from Thermotoga sinensis MSB8 is provided as SEQ ID NO: 11. The hydrolase of the Thermotoga sinensis MSB8 variant is provided as SEQ ID NOs: 1 and 16.

As used herein, "isolated nucleic acid molecule", "isolated polynucleotide", and "isolated nucleic acid fragment" are used interchangeably and refer to an RNA or DNA polymer, which is a single- or double- - Strands, optionally containing synthetic, unnatural or altered nucleotide bases. A DNA polymer type of isolated nucleic acid molecule can include one or more cDNA, genotypic DNA or synthetic DNA fragments.

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:

As used herein, "isolated nucleic acid molecule", "isolated polynucleotide", and "isolated nucleic acid fragment" are used interchangeably and refer to an RNA polymer or. As used herein, the terms "tag motif" and "identification motif" refer to conserved structures shared between families of enzymes having defined activities. The tag motif can be used in a defined family of matrices to define and/or identify a family of similar enzymes with similar enzymatic activity. The label motif can be a single continuous amino acid sequence or not A collection of continuous, conserved motifs that together form a label motif. Typically, the conservative mode system is represented by an amino acid sequence. In one embodiment, the perhydrolase enzyme used in the compositions and methods of the present invention comprises a CE-7 carbohydrate esterase tag motif.

As used herein, the term "sequence analysis software" refers to any computer algorithm or software program used to analyze nucleotide or amino acid sequences. "Sequence Analysis Software" is commercially available or independently developed. Typical sequence analysis software will include, but is not limited to, the GCG suite of programs (Wisconsin Software Pack Version 9.0, Accelrys Software, Inc., San Diego, Calif.), BLASTP, BLASTN, BLASTX (Ochu et al., Molecular Biology 215: 403-410 (1990)), and DNASTAR (DNASTAR, Inc. Madison South Park Road 1228, West Indiana 53715 United States), CLUSTALW (eg, version 1.83; Thompson et al, Nucleic Acids Research, 22(22): 4673-4680 (1994)), and the FASTA program incorporates the Smith Waltman algorithm (WR Pearson, Computer Methods Genomics Research, [Proc. Int. Symp.] (1994), date of the conference 1992, 111-20. Edit: Su Hai, Sandel. Issuer: Pu Nan, New York City, NY), Vector NTI (Informax, Bethesda, MD) and Sequencher v.4.05. In the context of this application, it should be understood that when using sequence analysis software for analysis, the results of the analysis will be based on the "initial value" of the referenced program unless otherwise stated. As used herein, "initial value" means any value or set of parameters set by the software manufacturer, the original loading of the software when first started.

The term "body surface" refers to any surface of the human body that serves as a beneficiary, such as a target for peroxyacid beneficiaries. The methods and compositions of the present invention relate to oral care applications and products. Therefore, the body surface includes the oral substance/surface. In one embodiment, the oral material comprises tooth enamel.

As used herein, the terms "whitening teeth" and "bleaching teeth" are used interchangeably and refer to improving the brightness of a tooth (eg, whitening).

As used herein, "intrinsic stains" on a tooth refers to the color caused by a color chromophore between the enamel and the associated dentin. The inner color of human teeth tends to be yellower with age, which is due to the thinning of the enamel and the gradual darkening of the yellow dentin. Removal of intrinsic staining typically requires the use of peroxides or other oxidizing agents that can penetrate the enamel and decolorize the inner chromophore.

Compared to intrinsic staining, "exogenous staining" is formed on the surface of the tooth when the exogenous chromogenic substance is bound to the enamel, and is usually attached to the surface of the tooth. Most people accumulate a degree of unsightly exogenous staining on their teeth over time. This dyeing process is motivated by (1) ingesting foods and beverages containing tannins such as coffee, tea, or red wine; (2) using tobacco products; and/or (3) exposing to certain cationic substances (eg , tin, iron, and chlorhexidine). These substances tend to adhere to the enamel hydroxyapatite structure, which causes discoloration of the teeth and a simultaneous reduction in tooth whiteness. After a few years, exogenous staining can penetrate the enamel layer and cause intrinsic staining.

As used herein, the term "destain" or "destaining" refers to the process of removing staining from the surface of the oral cavity. The staining can be intrinsic staining, exogenous staining, or a combination thereof.

As used herein, "effective amount of perhydrolase" refers to the amount of perhydrolase enzyme that must achieve the enzymatic activity required for a particular use. Such effective amounts are well established by those skilled in the art and are based on a number of factors, such as the particular enzyme variant employed.

As used herein, "peroxide source" and "source of peroxide" refer to a compound that provides hydrogen peroxide in an aqueous solution at a concentration of about 1 mM or greater, including, but not limited to, hydrogen peroxide. Hydrogen peroxide adducts (eg, urea-hydrogen peroxide adducts (urea peroxides)), perborates, and percarbonates. As defined herein, the peroxide source is in the form of a particulate particle in the whitening toothpaste of the present invention wherein the user hydrolyzes the particulate peroxide particles to release an effective amount of hydrogen peroxide. As defined herein, the concentration of hydrogen peroxide provided by the peroxide compound in the aqueous reaction formulation is initially at least 0.1 mM or greater upon binding of the reactive components. In one embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 0.5 mM. In one embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 1 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 10 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 100 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is at least 200 mM. In another embodiment, the hydrogen peroxide concentration in the aqueous reaction formulation is 500 mM or more. There are still other specific examples in which the hydrogen peroxide concentration in the aqueous reaction formulation is 1000 mM or more. In the formulation, the molar ratio of hydrogen peroxide to the enzyme substrate, such as triglyceride, (H ₂ O ₂ :matrix) may be from about 0.002 to 20, preferably from about 0.1 to 10, and most preferably about 0.1 to 1.

As used herein, the term "oligosaccharide" refers to a monosaccharide compound containing between 2 and at least 24 linked by a glycosidic linkage. The term "monosaccharide" refers to a compound of the formula (CH ₂ O) _n where n 3. The carbon skeleton is unbranched, except for one, each carbon atom contains a hydroxyl group, and the remaining carbon atoms are aldehydes or ketones at carbon atom 1. The term "monosaccharide" also refers to the form of a cyclic hemiacetal or hemi-ketal in the cell.

As used herein, the term "hydrolyzable adhesive" shall mean a hydrolyzable adhesive material. The hydrolyzable adhesive is substantially dry and non-sticky until hydrolyzed. Upon hydrolysis, the hydrolyzable adhesive becomes sufficiently viscous to adhere to the surface of the tooth.

As used herein, the term "effective amount" shall mean the quality of the material required to achieve the desired effect.

As used herein, the term "substantially non-adherent until hydrolyzed" shall mean a lack of adhesion strength sufficient to adhere to the surface of the tooth prior to hydrolysis.

By "sequence homology" is meant amino acid sequence homology using sequence alignment procedures, eg, ClustalW or BLAST, as generally described in Ochu SF, Gem W, Miller W, Myers EW , Lippmann DJ, "Basic Local Alignment Search Tool", Journal of Molecular Biology (1990) 215(3): 403-410, and Gu Yi M, Mai William H, Li W, Valentin F, History S, Pylon J, Lopez R, Nucleic Acids Research (2010) 38 Supplement: W695-9.

The thiol donor used in the present invention, for example, in the reaction with a peroxide to form a peroxyacid, is selected from one or more (i) C _2-18 carboxylic acids, such as C _2-6 carboxylic acid. An acid (eg, acetic acid), including a lower linear or branched alkyl carboxylic acid, optionally substituted with a hydroxy group and/or a C _1-4 alkoxy group; (ii) a hydrolyzable and acceptable ester thereof (eg, a single -, di-, and tri-glycerides and deuterated sugars) and (iii) mixtures thereof. For example, sulfhydryl donors include 1,2,3-triethoxypropane (sometimes referred to herein as triacetin or triacetin) and deuterated sugars, such as acetylated saccharides. In a particular embodiment, the ester in this use can be, for example, an ester having a water solubility of at least 5 ppm at 25 °C.

The thiol donor and/or enzyme may be optionally encapsulated. Various methods of encapsulation are well known in the art, both natural and synthetic. Modified starch and gum arabic Suitable because they are food grade, relatively inexpensive, dissolve quickly, and can absorb a relatively high degree of liquid oil. Any impact on the final viscosity needs to be considered.

In some embodiments, the particles include an anti-allergic agent that can desensitize or block dentinal tubules. In some embodiments, the anti-allergic agent is selected from the group consisting of a potassium ion source, a citrate, a stannous ion source, a basic amino acid, a clay, and combinations thereof. In some embodiments, the potassium ion source is an orally acceptable potassium salt and is present in an amount effective to reduce tooth sensitivity. In some embodiments, the potassium ion source is selected from the group consisting of potassium chloride, potassium nitrate, and combinations thereof. In some embodiments, the basic amino acid is arginine. In some embodiments, the basic amino acid is selected from the group consisting of arginine phosphate, arginine hydrogencarbonate, and arginine hydrochloride. In some embodiments, the citrate is calcium citrate.

CE-7 perhydrolase

The compositions and methods of the present invention include an enzyme having perhydrolysis activity which is structurally classified as a member of the esterase family 7 (CE-7 family) of a carbohydrate family enzyme (see Coutinho, PM, Hennessa, B). "Carbohydrate Active Enzymes: An Integrated Database Approach" Recent Developments in Carbo Bioengineering, HJ Gilbert, G. Davis, B. Hennessa and B. Svensson, (1999) UK Royal Society of Chemistry, Cambridge, pp. 3-12). The CE-7 family of enzymes has been shown to be particularly effective in producing peroxyacids from a variety of carboxylate matrices when combined with a source of peroxides (U.S. Patent Nos. 7,794,378; 7,951,566; 7,723,083; and 7,964,378 and U.S. Patent Application Publication No. 2008- 0176299, 2010-0087529, 2011-0081693, and 2011-0236335 - Dicosimo, et al; each incorporated herein by reference.

Members of the CE-7 family include cephalosporin C deacetylase (CAHs; E.C. 3.1.1.41) and acetyl xylan esterase (AXEs; E.C. 3.1.1.72). Members of the CE-7 esterase family share a conserved label motif (Wenson et al., J. Mol. Biol. 330: 593-606 (2003)). Perhydrolase enzymes include CE-7 tag motifs ("CE-7 perhydrolase") and/or substantially similar structures suitable for use in the compositions and methods described herein. Methods for identifying substantially similar biomolecules are well known in the art (e.g., sequence alignment protocols, nucleic acid hybridization, and/or the presence of a conserved label motif). In one aspect, the perhydrolase comprises an enzyme comprising a CE-7 tag motif and is at least 20%, preferably at least 30%, more preferably at least 33%, more preferably at least 40%, more preferably at least 42%, more preferably at least 50. %, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and most preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96% The 97%, 98%, or 99% amino acid sequence is identical to one of the sequences provided herein.

As used herein, the phrase "enzyme is structurally classified as CE-7 enzyme", "CE-7 perhydrolase" or "structurally classified as carbohydrate esterase family 7 enzyme" is used to refer to Hydrolytic activity, an enzyme structurally classified as CE-7 carbohydrate esterase. This family of enzymes can be defined by the presence of a tag motif (Wenson et al., supra). The CE-7 esterase tag motif includes three conserved motifs (numbers of residue positions are related to the reference sequence EQ ID NO: 1; Thermotoga sinensis C277S variant perhydrolase).

Arg118-Gly119-Gln120; Gly186-Xaa187- Ser188- Gln189-Gly190; and His303- Glu304.

Typically, Xaa at position 187 of the amino acid residue is glycine, alanine, valine, tryptophan, or threonine. Two of the three amino acid residues belonging to the catalytic triad are in bold. In one specific example, the amino acid residue The Xaa at base position 187 is selected from the group consisting of glycine, alanine, valine, tryptophan, and threonine.

Further analysis of the conserved motif in the CE-7 carbohydrate esterase family revealed the presence of other conserved motifs (LXD at amino acid positions 272-274 of SEQ ID NO: 1), which can be used to further define CE-7 a perhydrolase of the carbohydrate esterase family. In other embodiments, a tag motif as defined above may include other (fourth) conserved motifs as defined below: Leu272-Xaa273-Asp274.

Xaa at position 273 of the amino acid residue is typically isoleucine, valine, or methionine. The fourth region includes an aspartic acid residue (bold) belonging to the catalytic triad (Ser188-Asp274-His303).

The CE-7 perhydrolase may be a fusion protein version having at least one peptide component that has an affinity for at least one body surface. In one embodiment, all alignment methods used to confirm whether the target perhydrolase (fusion protein), including the CE-7 tag motif, will be based on a perhydrolase that does not contain a peptide component that has an affinity for the body surface. Amino acid sequence.

Many world-renowned alignment algorithms (i.e., sequence analysis software) can be used to align two or more amino acid sequences of enzymes that represent perhydrolase activity to determine if the enzyme includes the tag motif of the present invention. The aligned sequence is compared to a reference sequence (SEQ ID NO: 1) to determine the presence of the tag motif. In one embodiment, CLUSTAL alignment (eg, CLUSTALW) using a reference amino acid sequence (such as the perhydrolase sequence (SEQ ID NO: 1) used herein) is used to confirm that it belongs to the CE-7 esterase family. Hydrolase. The reference number of a conserved amino acid residue is based on the reference amino acid. The residues of the sequence number are identified as small insertions or deletions in the aligned sequence (e.g., typically 5 amino acids or less).

Examples of other suitable algorithms can be used to confirm that a sequence comprising a tag motif of the invention (when compared to a reference sequence) includes, but is not limited to, Nederman and Vaughan (Journal of Molecular Biology 48, 443-453 (1970) ); a global alignment tool) and Smith-Wattman (Journal of Molecular Biology 147:195-197 (1981); Local Alignment Tool). In one embodiment, the Smith-Wattman alignment is implemented using default parameters. Examples of suitable initial parameters include the use of the BLOSUM6 scoring matrix with a GAP open penalty = 10 and a GAP extension penalty = 0.5.

In one embodiment, a suitable perhydrolase, including an enzyme, comprising a CE-7 tag motif and at least 20%, preferably at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence is identical to SEQ ID NO: 1.

Examples of suitable CE-7 carbohydrate esterases having perhydrolysis activity include, but are not limited to, enzymes having amino acid sequences such as SEQ ID NOs: 1, and 4-28. In one embodiment, the enzyme comprises an amino acid sequence selected from the group consisting of 1, 10, 11, 15, and 16.

As used herein, the terms "CE-7 variant", "variant perhydrolase" or "variant" refer to a genetically engineered CE-7 perhydrolase that causes at least one amino acid to increase, decrease, and / or substitution, when the corresponding enzyme (typically wild-type enzyme) is compared to the derived variant; as long as the CE-7 tag motif and associated perhydrolysis activity are maintained. CE-7 variant perhydrolase enzymes can also be used in the compositions and methods of the present invention. Examples of CE-7 variants are provided as SEQ ID NOs: 1, 15, 16, 17, 18, 19, and 20. In one embodiment, the variant can include SEQ ID NOs: 1 and 16.

The skilled artisan will be aware that substantially similar CE-7 perhydrolase sequences (retained label motifs) can also be used in the compositions and methods of the present invention. In one embodiment, substantially similar sequences are defined by their ability to hybridize, under very stringent conditions, to nucleic acid molecules associated with the sequences exemplified herein. In another embodiment, the sequence alignment algorithm can define substantially similar enzymes based on the percentage of DNA or amino acid sequences provided by the present invention.

As used herein, a nucleic acid molecule "can hybridize" to another nucleic acid molecule, such as cDNA, genotype DNA, or RNA, when the single strand of the first molecule can be renatured at an appropriate temperature and ionic strength of the solution. When it comes to another molecule. Hybridization and washing conditions are well known and exemplified in Sambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of hybridization. Stringent conditions can be adjusted to screen for moderately similar molecules, such as homologous sequences from organisms that are distantly related, to very similar molecules, such as genes that replicate functional enzymes from closely related organisms. Post-hybrid cleaning typically determines stringent conditions. The preferred conditions are set to use a series of cleanings. Initially, 6X SSC, 0.5% SDS was used at room temperature for 15 minutes, then repeated with 2X SSC, 0.5% SDS at 45 °C for 30 minutes, and then repeated at 50 °C for 30 minutes with 0.2X SSC, 0.5% SDS. The preferred conditions of use are set to a higher temperature, wherein the cleaning is the same as above except that in the last two 30 minutes at 0.2X SSC, the cleaning temperature of 0.5% SDS is increased to 60 °C. Another preferred very stringent hybridization condition was set to 0.1X SSC, 0.1% SDS, 65 ° C and rinsed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS, and 65 °C.

Hybridization requires that two nucleic acids contain complementary sequences, although mismatching between bases may occur depending on the stringency of the hybridization. Appropriate stringency of hybrid nucleic acids Depending on the length and complementarity of the nucleic acid, variations thereof are well known to those skilled in the art. The greater the similarity or homology between the two nucleotide sequences, the greater the hybridization Tm value of the nucleic acid having the sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) is reduced in the following order: RNA: RNA, DNA: RNA, DNA: DNA. Hybrids of nucleotides greater than 100 in length, derived from the computational system Tm (Sambrook and Russell, as before). When hybridizing to shorter nucleic acids, i.e., oligonucleotides, the location of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (Sambrooke and Russell, as before). In one aspect, the hybridizable nucleic acid is at least about 10 nucleotides in length. Preferably, the hybrid nucleic acid has a minimum length of at least about 15 nucleotides, more preferably at least about 20 nucleotides in length, more preferably at least 30 nucleotides in length, and more preferably at least 300 nucleosides in length. The acid is preferably at least 800 nucleotides in length. Furthermore, those skilled in the art will know that the temperature and the salt concentration of the cleaning solution can be adjusted according to factors such as the length of the probe as needed.

As used herein, the term "percent identity" is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence association between polypeptide or polynucleotide sequences, as determined by the match between strings of such sequences. "Identity" and "similarity" can be easily calculated by known methods, including but not limited to those of the following: Computational Molecular Biology (Lisk, AM Editor) Oxford University Press, New York (1988); Biocomputing: Informatics and Genomics Project (Smith, DW, ed.) Academic Publishing, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, Editor) Shuman Inc., New Jersey (1994); Sequence Analysis of Molecular Biology (Feng Heine, G. Editor) Academic Publication (1987); and Introduction to Sequence Analysis (Jibikov, M. and Dick, J. Editor) Stoke pause Published, New York State (1991). Recognition and similar validation methods are compiled in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the MegAlign program sequence of the LASERGENE bioinformatics computing group (DNASTAR, Madison, West Indiana), the AlignX program of Vector NTI v.7.0 (Informax, Besse) Star, Maryland), or EMBOSS Open Software Suite (EMBL-EBI; Reith et al., Genetics Trends 16, (6): 276-277 (2000)). Multiple alignment of sequences can be performed using the following CLUSTAL alignment methods (eg CLUSTALW; eg version 1.83) (Higgins and Sharp, CABIOS, 5: 151-153 (1989); Higgins et al., Nucleic Acids Research. 22: 4673-4680 (1994) ); and Zinano et al., Nucleic Acids Research 31 (13): 3497-500 (2003)), available from the European Molecular Biology Laboratory via the European Institute for Bioinformatics, with initial parameters. Suitable parameters for CLUSTALW protein alignment include GAP extension penalty = 15, GAP extension = 0.2, matrix = Gonnet (eg, Gonnet 250), protein ENDGAP = -1, protein GAPDIST = 4, and KTUPLE = 1. In one embodiment, the fast or slow alignment uses an initial setting, with slow alignment being preferred. Alternatively, parameters using the CLUSTALW method (eg, version 1.83) can also be modified to use KTUPLE=1, GAP penalty=10, GAP extension=1, matrix=BLOSUM (eg, BLOSUM64), WINDOW=5, and TOP DIAGONALS SAVED =5.

In one aspect, the suitably isolated nucleic acid molecule encodes at least about 20%, preferably at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the polypeptide of the amino acid sequence identical to the amino acid sequence reported herein. In another aspect, the suitably isolated nucleic acid molecule encodes at least about 20%, preferably at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the polypeptide of the amino acid sequence identical to the amino acid sequence reported herein. Suitable nucleic acid molecules not only have the above homologs, but also typically encode polypeptides having a length of from about 210 to 340 amino acids, from about 300 to about 340 amino acids, preferably from about 310 to about 330 amino acids, And preferably from about 318 to about 325 amino acid lengths wherein each polypeptide is characterized by perhydrolysis activity.

Over-hydrolase

As used herein, the terms "target perhydrolase" and "enzymatically active enzyme" refer to a fusion protein comprising at least one perhydrolase (wild type or variant thereof) fused/coupled to at least one For the target surface, it is advisable to have a peptide component with affinity for the target body surface. The perhydrolase in the target perhydrolase can be any CE-7 carbohydrate esterase having perhydrolysis activity. The CE-7 perhydrolase can be confirmed by the presence of a CE-7 tag motif, which is aligned with the reference sequence EQ ID NO: 1, and the tag motif comprises: i) the RGQ motif corresponding to SEQ ID NO :1 position 118-120; ii) GXSQG motif corresponding to position 186-190 of SEQ ID NO: 1; and iii) HE motif corresponding to position 303-304 of SEQ ID NO: 1. position.

As used herein, a nucleic acid molecule can "hybridize" to another nucleic acid molecule, such as cDNA, genotype DNA, or when a single strand of the first molecule can be renatured to another molecule at the appropriate temperature and solution conditions, or RNA. In one embodiment, the perhydrolase may be those having at least about 20%, preferably at least 30%, of the amino acid sequence. 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 % is similar to any of the amino acid sequences reported herein (ie, SEQ ID NOs: 1, and 4-28).

In another embodiment, the fusion protein comprises a perhydrolase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, and 4-28.

As used herein, "peptide composition", "peptide composition with affinity for the oral surface", "oral association structure", and "OCBD" refer to the fusion protein component, which is A portion of a perhydrolase (including at least one polymer of two or more amino acids joined by a peptide bond); wherein the component has an affinity for the surface of the target oral cavity. In a preferred aspect, the OCBD has an affinity for tooth enamel.

In one embodiment, the peptide component having affinity for the body surface can be an antibody, a Fab antibody fragment, a single-chain variable fragment (scFv) antibody, a Camelidae antibody (Mudeman, S., molecule) Bioengineering Review (2001) 74:277-302), a non-antibody scaffold display protein (Hors et al., Protein Science (2006) 15(1): 14-27 and Bins, H. et al. 2005) Natural Biotechnology 23, 1257-1268 for a review of various scaffold-assisted approaches) or a single-chain polypeptide lacking an immunoglobulin population. On the other hand, the peptide component having affinity for oral tissues/surfaces (e.g., tooth enamel) is a single-stranded peptide lacking an immunoglobulin group.

The peptide component having affinity for the oral surface can be separated by a perhydrolase by any peptide linker. The particular peptide linker/spacer region is from 1 to 100 or from 1 to 50 amino acid lengths. In certain embodiments, the peptide spacer sequence region is from about 1 to about 25, from 3 to about 40, or from 3 to about 30 Amino acid length. In other embodiments, the spacer sequence region is from about 5 to about 20 amino acid lengths. Multiple peptide linkers can be used. In one embodiment, at least one peptide linker is present and can be repeated up to 10 fold.

In one embodiment, the fusion peptide comprises at least one oral surface-binding peptide selected from the group consisting of SEQ ID NOs: 178-197.

The peptide component having affinity for the oral surface can be isolated by CE-7 perhydrolase by any peptide linker. The particular peptide linker/spacer region is from 1 to 100 or from 1 to 50 amino acid lengths. In certain embodiments, the peptide spacer sequence region is from about 1 to about 25, from 3 to about 40, or from 3 to about 30 amino acid lengths. In other embodiments, the spacer sequence region is from about 5 to about 20 amino acid lengths. Multiple peptide linkers can be used. Examples of peptide linkers are provided as SEQ ID NOs: 164-177.

Thus, examples of the subject CE-7 perhydrolase can include, but are not limited to, any CE-7 perhydrolase having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, and 4-28 to The surface of the oral cavity has a peptide component of affinity. In a preferred embodiment, examples of the subject perhydrolase may include, but are not limited to, any CE-7 perhydrolase having an amino acid sequence selected from the group consisting of SEQ ID NOs 1, 4, 5, and 6. , 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27, and 28, which are coupled to One or more body surface-affinity peptides that have affinity for the oral surface (arbitrary via the peptide spacer sequence region). In a preferred embodiment, the subject perhydrolase comprises a CE-7 perhydrolase having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 and 16.

In one embodiment, the perhydrolase is a CE-7 perhydrolase in the form of a fusion protein of the general structural formula: PAH-[L]y-OCBD or OCBD-[L]y-PAH

Wherein PAH is an enzyme having perhydrolysis activity, for example, having a CE-7 tag motif, such as SEQ ID NO: 1, and OCBD is a peptide component having affinity for the oral surface; and L is any linker; And y is an integer from 0 to 10. In one embodiment, the linker (L) is present and is a peptide linker ranging from 1 to 100 amino acid lengths.

For example, SEQ ID NO: 2 is a fusion protein having a perhydrolase sequence SEQ ID NO: 1 coupled to the C-terminus of a structural region targeted for oral tissue.

The perhydrolase enzymes used in the products and methods of the invention may be free, protected (e.g., acetamidine), or salted.

In another embodiment, the target surface material is part of a package and is delivered to the oral cavity. The peptide component is selected for use as a substance such as a polymer, a plastic, and a film due to its affinity. The subject CE-7 perhydrolase fusion protein design allows control of the perhydrolase to be delivered or removed by the user by retaining it on a removable device, such as a dial or a patch.

Affinity

The peptide component having affinity for the oral surface includes a binding affinity of 10 ^-5 moles (M) or less to the oral surface. In a particular embodiment, the peptide component is one or more oral surface-binding peptides and/or binding structural regions having a binding affinity for tooth enamel of 10 ^-5 moles (M) or less. In certain embodiments, the binding peptide or structural region has a binding affinity value of 10 ^-5 M or less in the presence of at least about 50-500 mM salt. The term "binding affinity" refers to the strength of the interaction of a peptide with its interaction with the corresponding substrate. Binding affinity can be defined or measured with a combined solution of peptide dissociation constant ( "K _D"), or "MB _50" 's.

"K _D " corresponds to the peptide concentration, and the binding site on the target is occupied by half, that is, when the concentration of the target containing the peptide bond (bonding target substance) and the peptide-free bond When the target concentration is the same. The smaller the dissociation constant, the tighter the peptide is tied. For example, a peptide with a nanomolar (nM) dissociation constant is tighter than a peptide with a dissociation constant of micromolecules ( μM ). A specific embodiment of the present invention has a K _D value of 10 ^-5 or less.

"MB ₅₀ " refers to the 50% concentration of the maximum signal obtained by the ELISA-based binding assay when the signal is combined with the peptide. See, for example, Example 3 of U.S. Patent Application Publication No. 2005/022683, incorporated herein by reference. The MB ₅₀ line provides an indication of the strength of the binding interaction or affinity of the complex components. The lower the MB ₅₀ value, the stronger the interaction of the peptide with its corresponding substrate, that is, "the better." For example, a nanogram (nM) MB ₅₀ peptide is more tightly bound than a microgram ( μ M) MB ₅₀ peptide. A specific embodiment of the present invention has an MB ₅₀ value of 10 ^-5 M or less.

In certain embodiments, the binding affinity have affinity for peptides consisting of the parts for the oral surface, such as by ₅₀ MB or K _D measurement values, less than or equal to about 10 ^-5 M, less than or equal About 10 ^-6 M, less than or equal to about 10 ^-7 M, less than or equal to about 10 ^-8 M, less than or equal to about 10 ^-9 M, or less than or equal to about 10 ^-10 M.

In certain embodiments, the oral cavity surface - bound peptides and / or oral cavity surface - binding affinity binding zone structure, such as by ₅₀ MB or K _D measurement values, less than or equal to about 10 ^-5 M, Less than or equal to about 10 ^-6 M, less than or equal to about 10 ^-7 M, less than or equal to about 10 ^-8 M, less than or equal to about 10 ^-9 M, or less than or equal to about 10 ^-10 M .

As used herein, the term "strong affinity" means a binding affinity having a K _D or MB ₅₀ value of less than or equal to about 10 ^-5 M, preferably less than or equal to about 10 ^-6 M, more preferably less than Or equal to about 10 ^-7 M, more preferably less than or equal to about 10 ^-8 M, less than or equal to about 10 ^-9 M, or most preferably less than or equal to about 10 ^-10 M.

Enzyme powder

In some embodiments, the personal care composition can use an enzyme catalyst that stabilizes the enzyme powder form. A method of making and stabilizing a formulation comprising an enzyme powder is described in U.S. Patent Application Publication Nos. 2010-0086534 and 2010-0086535.

In one embodiment, the enzyme may be present in the enzyme powder in a range from about 5 weight percent (% by weight) to about 75% by weight, based on the dry weight of the enzyme powder. Preferably, the enzyme is present in the enzyme powder/spray dry mixture in a weight percentage ranging from about 10% to 50% by weight, and the enzyme is preferably in an enzyme powder/spray dry mixture in a weight percent range. 20% by weight to 33% by weight.

In one embodiment, the enzyme powder may further comprise an excipient. In one aspect, the amount of excipient is provided in the range of from about 95% to about 25% by weight, based on the dry weight of the enzyme powder. The preferred weight percent of the excipient in the enzyme powder ranges from about 90% to 50% by weight, and more preferably the excipient in the enzyme powder ranges from about 80% to about 67% by weight.

In one embodiment, the excipient used to prepare the enzyme powder can be an oligosaccharide excipient. In one embodiment, the oligosaccharide excipient has an average molecular weight of at least about 1250 and an average molecular weight of at least about 9000. In some embodiments, the oligosaccharide excipient has an average molecular weight of at least about 1700 and one point. The average amount of sub-quantities is at least about 15,000. Specific oligosaccharides may include, but are not limited to, maltodextrin, xylan, pullulan, mannan, fucoidan sulfate, galactomannan, chitosan, raffinose, Stachyose, pectin, insulin, fructan, melin fructan, amyloid pectin, sucrose, galactose, lactose, maltose, trehalose, cellobiose, Aspergillus trisaccharide, malt Trisaccharide, pine triose, maltotriulose, raffinose, canetriose, and mixtures thereof. In a preferred embodiment, the oligosaccharide excipient is maltodextrin. The oligosaccharide-based excipients may also include, but are not limited to, water-soluble nonionic cellulose ethers such as hydroxymethyl-cellulose and hydroxypropylmethylcellulose, and mixtures thereof. In still other embodiments, the excipient may be selected from, but not limited to, one or more compounds: trehalose, lactose, sucrose, mannitol, sorbitol, glucose, cellobiose, alpha-cyclodextrin. , pullulan, and carboxymethylcellulose.

Appropriate ester matrix / sulfhydryl donor

Suitable carboxylate matrices may include esters of the formula: (a) one or more esters of the formula [X] _m R ₅

Wherein X is an ester group of the formula R ₆ C(O)O; and R ₆ is a C1 to C7 linear, branched or cyclic hydroxy moiety, which is optionally substituted by a hydroxy group or a C1 to C4 alkoxy group, Wherein when R ₆ is C2 to C7, R ₆ optionally includes one or more ether linkages; and R ₅ is a C1 to C6 linear, branched, or cyclic hydroxyl moiety or a five member cyclic heteroaryl moiety or six members. a cyclic aryl or heteroaryl moiety, optionally substituted by a hydroxy group; wherein each carbon atom in R ₅ independently comprises no more than one hydroxy group or no more than one ester group or carboxylic acid a group, wherein R ₅ optionally includes one or more ether linkages; m is an integer from the number of carbon atoms in 1 to R ₅ , and one or more esters having a water solubility of at least 5 ppm at 25 ° C; or (b One or more glycerides having the following structural formula

Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy group or a C1 to C4 alkoxy group and R ₃ and R ₄ are independently H or R ₁ C(O); or (c One or more esters of the formula

Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy group or a C1 to C4 alkoxy group and R ₂ is a C1 to C10 linear or branched alkyl group, an alkenyl group, an alkynyl group, and an aromatic group. Alkyl, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is from 1 to 10; or (d One or more acetylated monosaccharides, acetylated disaccharides, or acetaminophen; or (e) any combination of (a) to (d).

Suitable matrices may also include one or more deuterated sugars selected from the group consisting of acetylated mono-, di-, and polysaccharides. In another embodiment, the deuterated sugar is selected from the group consisting of: acetylated xylan; acetylated xylan fragment; acetylated xylose (such as xylose tetraacetic acid); Glucose (such as α-D-glucose pentaacetate; β-D-glucose pentaacetate; 1-thio-β-D-glucose-2,3,4,6-tetraacetate); β-D-half Lactose pentaacetate; sorbitol pentaacetate; sucrose octaacetate; β-D-ribofuranosyl-1,2,3,5-tetraacetate; β-D-ribofuranosyl-1,2,3,4 -tetraacetate; tris-O-acetamidine-D-galactose; tris-O-acetamidine-D-glucoenose; β-D-furanosyltetraacetate, β-D-glucopyranose pentaacetic acid Ester; β-D-glucopyranose-1,2,3,4-tetraacetate; β-D-glucopyranose-2,3,4,6-tetraacetate; 2-acetamido-2 -deoxy-1,3,4,6-tetraacetyl-β-D-glucopyranose; 2-acetamido-2-deoxy-3,4,6-triethyl fluorene-1-chloro- α-D-glucopyranose; β-D-mannopyranomannose pentaacetate, and acetylated cellulose. In a preferred embodiment, the acetylated sugar is selected from the group consisting of β-D-ribofuranosyl-1,2,3,5-tetraacetate; tris-O-acetyl-D-galactose; Tri-O-acetam-D-glucoenose; sucrose octaacetate; and acetylated cellulose.

In another embodiment, other suitable substrates may also include 5-ethoxymethyl-2-furaldehyde; 3,4-diethoxy-1-butene; 4-ethyloxybenzoic acid Vanillin acetic acid; propylene glycol methyl ether acetate; methyl lactic acid; ethyl lactic acid; methyl alcohol; ethyl alcohol; methyl methoxy acetate; ethyl methoxy acetate; 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; and triethyl 2-acetamidine citrate.

In another embodiment, a suitable matrix is selected from the group consisting of: glyceryl monoacetate; glyceryl diacetate; glyceryl triacetate; glycerol monopropionate; glyceryl dipropionate; glyceryl tripropionate; Glyceryl butyrate; glyceryl dibutyrate; glycerol tributyrin Ester; glucose pentaacetate; xylose tetraacetic acid; acetylated xylan; acetylated xylan fragment; β-D-ribofuranosyl-1,2,3,5-tetraacetate; tri-O- Acetyl-D-galactose; tris-O-acetam-D-glucoenose; 1,2-ethanediol; 1,2-propanediol; 1,3-propanediol; 1,2-butanediol; , 3-butanediol; 2,3-butanediol; 1,4-butanediol; 1,2-pentanediol; 2,5-pentanediol; 1,5-pentanediol; 1,6 - pentanediol; 1,2-hexanediol; 2,5-hexanediol; monoesters or diesters of 1,6-hexanediol; and mixtures thereof. In another embodiment, the substrate is a C1 to C6 polyol comprising one or more ester groups. In a preferred embodiment, one or more of the hydroxyl groups on the C1 to C6 polyol are one or more ethoxylated groups (eg, 1,3-propanediol diacetate; 1,2-propanediol). Diacetate; 1,4-butanediol diacetate; 1,5-pentanediol diacetate, etc., substituted. In another embodiment, the substrate is propylene glycol diacetate (PGDA), vinyl glycol diacetate (EGDA), or a mixture thereof.

In another embodiment, a suitable matrix is selected from the group consisting of: monoacetin, diacetin, triacetin, glycerol monopropionate, dipropionate, tripropionate, single Butyric acid glyceride, dibutyric acid glyceride, and tributyrin. In still another aspect, the matrix is selected from the group consisting of diacetin and triacetin. In a preferred embodiment, the suitable substrate comprises triacetin.

In the case of an enzyme-catalyzed perhydrolysis reaction, the carboxylic acid ester is present in a concentration sufficient to produce the desired concentration of peroxyacid. The carboxylic acid ester does not need to be completely dissolved in the reaction formulation, but the solubility must be sufficient to allow the conversion of the ester to the corresponding peroxyacid by the perhydrolase catalyst. The carboxylic acid ester is present in the reaction formulation at a concentration of from 0.05% by weight to 40% by weight of the reaction formulation, preferably from 0.1% to 20% by weight of the reaction formulation, and more preferably from 0.5% by weight of the reaction formulation to A concentration of 10% by weight.

The peroxide source is provided as a granule and may include a hydrogen peroxide adduct (e.g., urea (urea)-hydrogen peroxide adduct (carbamide peroxide)) perborate, percarbonate and Oxide salt. The concentration of the peroxide compound may range from 0.0033 wt% to about 50 wt%, more preferably from 0.033 wt% to about 40 wt%, and more preferably from 0.1 wt% to about 30 wt%, of the reaction formulation. %.

Many perhydrolase catalysts (whole cells, permeabilized whole cells, and partially purified whole cell extracts) have been reported to have catalase activity (EC 1.11.1.6). Catalase is a reaction that catalyzes the conversion of hydrogen peroxide to oxygen and water. In one aspect, the perhydrolysis catalyst lacks catalase activity. On the other hand, a catalase inhibitor can be added to the reaction formulation. In this regard, the skilled artisan can adjust the concentration of the catalase inhibitor as needed. The catalase inhibitor concentration typically ranges from 0.1 mM to about 1 M; preferably from about 1 mM to about 50 mM; more preferably from about 1 mM to about 20 mM.

In another embodiment, the enzyme catalyst lacks significant catalase activity or can be designed to reduce or eliminate catalase activity. The catalase activity in the host cell can be down-regulated or eliminated by disrupting gene expression in response to catalase activity using well-known techniques including, but not limited to, transposon mutations, RNA Antisense expression, target mutations, and random mutations.

The peroxyacid concentration (e.g., peracetic acid) produced by the perhydrolysis reaction of at least one carboxylic acid ester is at least about 0.1 ppm, preferably at least 0.5 ppm, 1 ppm, 5 ppm, 10 ppm, 20 ppm, 100 ppm, 200 ppm, 300 ppm, 500 ppm, 700 ppm, 1000 ppm, 2000 ppm, 5000 ppm or 10,000 ppm of peroxyacid is preferably within 5 minutes from the start of the hydrolysis reaction within 10 minutes. It will be apparent that those skilled in the art can adjust the composition of the reaction to achieve the desired peroxyacid concentration.

In one aspect, the reaction time required to produce the desired peroxyacid concentration is no more than about 2 hours, preferably no more than about 30 minutes, more preferably no more than about 10 minutes, and most preferably about 5 minutes or less.

HPLC analysis of peroxyacid and hydrogen peroxide concentrations was measured.

A variety of analytical methods can be used to analyze the reactants and products, including, but not limited to, titration, high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), Capillary electrophoresis (CE), the analytical process is described in U.K. Kenny et al. (Analytical Chemistry, 69(17): 3623-3627 (1997)), and 2,2'-azo-bis(3) -Ethylbenzothiazoline-6-sulfonate (ABTS) analysis (U.French and other analysts, 122:567-571 (1997) and Danu and other material functions advanced, 20:392-398 (2010)), as explained in the example of this case.

In one embodiment, the invention provides a package of oral care products comprising a plurality of channels and designed to place the component parts in each of the cells and is non-reactive until the point of use. For example, the present invention provides a chemically stable structural package design that allows the enzymes that catalyze the whitening of tooth products to reach steady state kinetics in milliseconds after the components are exposed and mixed with one another. The contents of the container are dispensed via an opening device, such as via a nozzle with a removable lid or stopper or when the container is preferentially scored by a consumer to make it practicable, allowing the product to be clean and convenient via Shape the nozzle to dispense.

Each tank has a capacity to store, for example, a component of 0.1-30 grams. The oral care product is a whitening tooth product that provides a total amount of product delivered from each tank, such as between 1.0 and 5.0 grams, such as 1-2 grams to provide the desired benefit. The volume of the tank It is designed to accommodate a specific specific gravity component, for example, 1.0 to 1.1 and preferably a specific specific gravity range of 1.02 to 1.05.

In one embodiment, the package is manufactured using a thermoforming process using at least two flexible films having a thickness of from 50 microns to 500 microns and preferably 300 microns. The two films can be opaque, translucent or transparent and can be combined arbitrarily during assembly in a thermoforming process. Both materials provide water vapor barrier characteristics, such as less than 3% moisture loss over a three-year period, such as less than 1% water loss during the same period. The film also provides a fragrance barrier. The loss of scent can be determined by the gas chromatographic method and sensory evaluation.

The film is chemically resistant to the materials contained therein. For example, in one embodiment, they are resistant to 0.1% to 10% by weight hydrogen peroxide solution, such as up to 0.3% by weight hydrogen peroxide solution.

In one embodiment, one of the two flexible materials is a polymeric laminate and the inner layer of the laminate is selected to bond with the first flexible material, but when the pressure is applied by hand to a frangible seal The slots will be layered. The force required to break the seal is applied by hand and can vary between 2 inches-lbf and 5 inches-lbf.

After the frangible seal between the compartments breaks, the components in each of the slots will be in intimate contact with each other. The consumer is allowed to mix the individual components for a period of time to exceed the pre-steady state kinetic rate or lytic phase. The time of the pre-steady state kinetic or lytic phase is in milliseconds. This provides sufficient time to form and consume the enzyme-matrix intermediate until it reaches its steady state concentration. After reaching a steady state concentration, the consumer can destroy the previously scored portion of the multi-tank container and dispense the mixture to the crankset. The crankset is applied to the teeth for a period of 15 minutes to 45 minutes and provides effective whitening efficacy.

Thus, specific examples of the invention include, for example, packaging, oral care compositions, and methods of whitening teeth, such as:

1. A package 1 comprising a deformable material designed to form at least two seals having a first groove, a second groove, and any additional grooves that are blocked by one or more fragile or tearable barriers. Separation, wherein the first tank contains a low viscosity liquid solution comprising an enzyme having perhydrolysis activity, the enzyme-linked reference sequence EQ ID NO: 1 having a carbohydrate esterase family 7 (CE-7) tag motif The label motif comprises: i) a RGQ motif at a position corresponding to positions 118-120 of SEQ ID NO: 1; ii) a GXSQG motif at a position corresponding to positions 186-190 of SEQ ID NO: 1; Iii) the HE phantom is at a position corresponding to positions 303-304 of SEQ ID NO: 1; and the second tank is provided with at least one sulfhydryl donor matrix selected from the group consisting of: i) having the following structure Ester type [X] _m R ₅

Wherein X = an ester group of the formula R ₆ C(O)O R ₆ = a C1 to C7 linear, branched or cyclic hydroxy moiety, optionally substituted by a hydroxy group or a C1 to C4 alkoxy group Wherein R ₆ optionally includes one or more ether linkages, when R ₆ = C2 to C7; R ₅ = C1 to C6 straight chain, branched, or cyclic hydroxyl moiety or five member cyclic heteroaryl moiety or six a cyclic aryl or heteroaryl moiety, optionally substituted by a hydroxy group; wherein each carbon atom in R ₅ independently includes no more than one hydroxy group or no more than one ester group or carboxy group An acid group; wherein R ₅ optionally includes one or more ether linkages; m is an integer from the number of carbon atoms in 1 to R ₅ ; and wherein the ester has a water solubility of at least 5 ppm at 25 ° C; ii) has the following structure Glyceryl ester

Wherein R ₁ = C1 to C7 is a straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₃ and R ₄ are independently H or R ₁ C(O); iii) One or more esters of the formula wherein R ₁ is a C1 to C7 straight or branched alkyl group, any of which is hydroxy

Or a C1 to C4 alkoxy group substituted and R ₂ is a C1 to C10 straight or branched alkyl group, an alkenyl group, an alkynyl group, an aryl group, an alkylaryl group, an alkylheteroaryl group, a heteroaryl group, CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is from 1 to 10; and iv) an acetylated saccharide selected from the group consisting of acetylated monosaccharides, B a bismuth disaccharide, and an acetylated polysaccharide; and wherein the second or any additional tank contains a source of peroxide such that when one or more barriers in the tank rupture, such as when squeezing the first tank, The low viscosity liquid solution is mixed with a peroxide source and a sulfhydryl donor matrix, and the perhydrolysis-active lanthanide catalyzes the formation of a peroxide-derived peroxide and a ruthenium-based donor matrix. The reaction of peroxyacid; and the package has an opening means, such as a scribe line, lid or stopper to allow opening of the package to provide an outlet through which the mixture can be dispensed.

1.1. Package 1 wherein the enzyme having perhydrolysis activity comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1; and b) an amine having at least 80% amino acid sequence identical to SEQ ID NO: Base acid sequence.

Package 1 or 1.1, wherein the hydrolytically active enzyme further comprises a binding structural region fused to the N- or C-terminus of the enzyme, the binding structural region having an affinity for oral tissue or for whitening teeth or for whitening teeth.

1.2. Any of the preceding packages, wherein the binding structural region having affinity for oral tissue comprises an amino acid sequence selected from the group consisting of SEQ D NOs: 178-197.

1.3. Any of the foregoing packages, wherein the hydrolytically active enzyme has affinity for oral tissues and comprises an amino acid selected from the group consisting of SEQ ID NO: 2 and having at least 80% amino acid sequence identical to SEQ ID NO: Sequence amino acid sequence.

1.4. Any of the preceding packages, wherein the deformable material is plastic or aluminum.

1.5. Any of the foregoing packages, wherein the viscosity of the low viscosity liquid solution is sufficiently low to ensure effective mixing with the contents of the second tank, such as below 5,000 cps, such as below 500 cps.

1.6. The package of any of the preceding, wherein the low viscosity liquid solution comprises a buffer.

1.7. Any of the foregoing packages, wherein the thiol donor matrix is selected from the group consisting of (i) one or more C _2-18 carboxylic acids, such as C _2-6 carboxylic acids (eg, acetic acid), comprising a lower linear or branched alkyl group. a carboxylic acid, optionally substituted by a hydroxy group and/or a C _1-4 alkoxy group; (ii) one or more esters which are hydrolyzable and acceptable (eg, mono-, di-, and tri-glycerides and Deuterated sugars) and (iii) mixtures thereof.

1.8. Any of the foregoing packages, wherein the thiol donor matrix is selected from the group consisting of 1,2,3-triethoxypropane propane (sometimes referred to herein as triacetin or triacetin) and deuterated Sugars, such as acetylated sugar.

1.9. Any of the foregoing packages comprising a thiol donor matrix comprising an ester compound having a water solubility of at least 5 ppm at 25 °C.

1.10. The package of any of the preceding claims, wherein the source of peroxide is selected from the group consisting of solid peroxides and solid peroxide donors, and mixtures thereof, for example, selected from peroxide salts or complexes (eg, such as peroxidic phosphate). Ester, percarbonate, perborate, peroxyphenate, or potassium persulfate; for example, calcium peroxyphosphoric acid, sodium perborate, sodium carbonate peroxide, sodium peroxyphosphate, and potassium persulfate; Chlorate; urea peroxide; hydrogen peroxide polymer complex such as hydrogen peroxide-polyvinylpyrrolidone polymer complex; metal peroxide such as zinc peroxide and calcium peroxide; for example selected from peroxidation Urea, polyvinylpyrrolidone-hydrogen peroxide complex, sodium percarbonate, sodium perborate, and solid peroxides of metal peroxides such as zinc peroxide and calcium peroxide.

1.11. The above package, wherein the source of peroxide is urea peroxide.

1.12. The above package, wherein the source of peroxide comprises a hydrogen peroxide-polyvinylpyrrolidone complex.

1. 13. The package of any of the preceding claims, wherein the components in the tank are present at a dose sufficient to provide a bleaching dose effective to whiten the teeth upon mixing.

1. 14. The package of any of the preceding, wherein the second tank contains a powder type gelling agent.

1.15. The package of any of the preceding, wherein the gelling agent is selected from the group consisting of a carbomer gelling agent (eg, carbomer 971P), a polysaccharide gum such as xanthan gum, a modified edible starch, an animal or a fish. Gelatin, and cerium oxide.

1.16. The above package, wherein the gelling agent is a carbomer gelling agent.

1. The above package, wherein the second tank contains a powder type gelling agent which provides a viscosity of from 100,000 to 150,000 cps, such as about 125,000 cps, when used in combination with the contents of the first tank. For example, wherein the gelling agent is present in an amount of from 5% to 50% by weight of the final mixture of the first tank and the second tank contents.

1.18. The package of any of the preceding claims, wherein the first tank contains a low viscosity aqueous solution comprising a hydrolytically active enzyme and a buffer, and the second tank comprises a gelling agent, a peroxide source, and an acetylated compound, all of which are In a powder form, when the fragile barrier is broken and the contents of the two tanks are mixed, the peroxide and the acetamidine-containing compound can be reacted by catalyzing a protein having perhydrolase activity, and Forming peracetic acid in an extrudable colloid formed by a liquid and a gelling agent, and then the extrudable colloid can be extruded and applied to the teeth, for example, using a sprocket or a toothpick, sufficient Time, for example, 10-30 minutes, to bleach the teeth.

1.19. Any of the foregoing packages, further comprising an applicator such as a sprocket or a dental patch to apply a mixture of the first trough and the second trough contents to the teeth.

1.20. The aforementioned package wherein, when the mixture is to be dispensed, the opening of the second tank is in direct contact with the sprocket wheel and, therefore, the mixture is extruded to the sprocket wheel.

2. Composition 2, a multi-part oral care composition comprising a first portion that is physically separated from the second portion during storage and combined with the second portion just prior to use, eg, for 10 minutes of use Wherein the first portion comprises an enzyme having perhydrolysis activity, as described in any of the foregoing packagers, and a second portion comprising a source of peroxide and a carboxyl donor selected from the group consisting of a carboxylic acid and a mercapto compound, wherein The oxide source and the carboxyl group supply system are reacted in the presence of a perhydrolase to form a peroxyacid, such as a peroxide source and a carboxyl donor, as described in any of the foregoing packagers, eg,

2.1. The above composition, wherein the carboxyl group supply system is selected from the group consisting of C _2-18 carboxylic acids (eg, acetic acid), and hydrolyzable and acceptable esters thereof (eg, mono-, di-, and tri-glycerides) and Its mixture.

2.2. The aforementioned composition wherein the carboxyl donor is 1,2,3-triethoxypropane (sometimes referred to herein as triacetin or triacetin).

2.3. Any of the preceding compositions, wherein the source of peroxide is a solid peroxide selected from the group consisting of urea peroxide, polyvinylpyrrolidone-hydrogen peroxide complex, sodium percarbonate, sodium perborate, and metal. Oxides such as zinc peroxide and calcium peroxide.

2.4. Any of the preceding compositions, wherein the source of peroxide is urea peroxide.

2.5. Any of the preceding compositions, wherein the source of peroxide comprises a hydrogen peroxide-polyvinylpyrrolidone complex.

2.6. Any of the preceding compositions, wherein when packaged as described above, such as package 1.

3. A method of whitening teeth comprising the activation of the above two portions of the oral care composition (Method 3) by combining the two parts and mixing the effective amount thus obtained Application to the teeth, for example, using an applicator, such as a sprocket or a toothpaste, for a sufficient period of time, such as at least 10 minutes, such as 10-30 minutes, to whiten the teeth.

Peroxyacids are known to be effective antibacterial agents and bleaching agents. U.S. Patent No. 5,302,375 to U.S., D., discloses a whitening oral composition comprising peracetic acid dissolved in a carrier, wherein the peracetic acid is in situ in the carrier by combining water, acetaminosalicylic acid, And water soluble alkali metal hydrogencarbonate produced. U.S. Patent No. 5,279,816 to et al. discloses the use of a composition comprising peracetic acid to bleach dye or to decolorize teeth. U.S. Patent Nos. 6,221,341 and 7,189,385 to Montgomery, R., disclose a peroxyacid whitening tooth composition suitable for use in a whitening method. More particularly, the peracetic acid composition can be subjected to chemical perhydrolysis to produce peracetic acid by combining hydrogen peroxide precursor, glycerin acetate, and water.

Enzymatic perhydrolysis reactions are not illustrated in these references. U.S. Patent Application Publication 2009-0311198- number Comcast etc., based smegmatis had disclosed comprising the hydrolytic activity of Mycobacterium (M. smegmatis) to an oral composition for the enzyme bleaching teeth.

Many hydrolases and esterases, for example, lipases, serine hydrolases and carbohydrate esterases, catalytic perhydrolysis reactions, the reversible reaction of peroxyacids from carboxylic acids and hydrogen peroxide. Perhydrolases, esterases, and lipases typically contain a catalytic triad containing serine, glutamic acid (Glu) or aspartic acid (Asp), and histidine (His). Many perhydrolase enzymes (such as metal-free haloperoxidases) contain a Ser-His-Asp catalyzed triplet and catalyze the reversible reaction of peroxyacids from hydrogen peroxide and carboxylic acids. Without being bound by theory, it is generally believed that the hydrolysis reaction is carried out by an esterase-like mechanism in which a carboxylic acid reacts with an active point of serine to form a thiolase intermediate, which is then reacted with hydrogen peroxide to form Peroxyacid.

A variety of perhydrolase enzymes have been described in this art. The content of a specific variant of Bacillus subtilis Carlsberg protease in a body care product having been subjected to hydrolytic activity is disclosed in U.S. Patent 7,510,859-Wilande et al. The perhydrolase other than the specific variant protease has not been described, and there is no example to prove the enzymatic production method of peracid as a personal care benefit agent. U.S. Patent Application Publication No. 2008-0176783 A1; 2008-0176299 A1; 2009-0005590 A1; and 2010-0041752 A1-Decco, et al., disclose an enzyme whose structure is classified as carbohydrate esterase CE-7. Family (ie, cephalosporin C deacetylase [CAHs] and acetyl xylan esterase [AXEs]) characterized by significant perhydrolysis activity to base the carboxylate (suitable peroxide source) In the presence of hydrogen peroxide, it is converted to a peroxyacid sufficient to be used as a disinfectant and/or bleach. Some members of the carbohydrate esterase CE-7 family have been shown to have perhydrolysis activity, and once the reaction components are mixed, it is sufficient to produce 4000-5000 ppm from the alcohol, diol, and glycerol ester of glycerol in 1 minute. Peracetic acid is produced and is produced up to 9000 ppm between 5 minutes and 30 minutes (Dicosimo et al., US 2009-0005590 A1). U.S. Patent Application Publication No. 2010-0087529 A1 describes a variant CE-7 enzyme having improved perhydrolysis activity.

In one embodiment, the invention employs a perhydrolase enzyme comprising a catalytic structural region ("CE-7 perhydrolase") of a member of the carbohydrate esterase family 7 having perhydrolysis activity. Although CE-7 perhydrolase has excellent perhydrolysis activity, its use on personal cosmetic care products has not been disclosed in the aforementioned provisional application.

The thiol-based system of the present invention is selected from the group consisting of (i) one or more C _2-18 carboxylic acid esters, such as C _2-6 carboxylic acid esters, comprising a lower linear or branched alkyl carboxylic acid, any of which is hydroxy and/or Or a C _1-4 alkoxy group and (ii) a mixture thereof. For example, sulfhydryl donors include 1,2,3-triethoxypropane (sometimes referred to herein as triacetin or triacetin) and deuterated sugars, such as acetylated saccharides. In a particular embodiment, the esters for this purpose may, for example, be esters having a water solubility of at least 5 ppm at 25 °C.

The thiol donor and/or other materials may be optionally encapsulated. Various methods of encapsulation are well known in the art, both natural and synthetic. Modified starches and gum arabic are particularly suitable because they are food grade, relatively inexpensive, dissolve quickly, and are capable of adsorbing liquid oil to a relatively high degree. Any impact on the final viscosity needs to be considered.

As described above, the present invention may include a gelling agent such as a carbomer gelling agent (e.g., carbomer 971P), a polysaccharide gum such as xanthan gum, a modified edible starch, an animal or a base gelatin, and two Yttrium oxide. Adhesive formulations for use with tooth whitening agents are known in the art, as described in U.S. Patent Nos. 7,862,801; 5,746,598; 6,730,316; 7,128,899. The gelling agent can be used to thicken the whitening solution to such an extent that it does not run out of the tray or from the tooth to the soft tissue area. This allows the bleach to extend the time it takes to stay in contact with the teeth and protect the soft tissue. The use of a sprocket wheel and a viscous bleach allows the low concentration of bleach to effectively whiten human teeth over a period of 1-2 weeks and minimize the risk to the patient. The gelling agent used in this application should be selected and adjusted to provide a viscosity of from 100,000 to 150,000 cps, for example, about 125,000 cps.

In specific embodiments, the packaging of the previously described multi-part composition comprising carbomer gelling agent, e.g., one manufactured by Lubrizol of polyacrylic acid modified hydrophilic polymers such as Carbopol ^®. The carbomer can form a viscous colloid having a concentration of slightly more than 5% by weight.

All of the ingredients used in the formulations described herein should be orally acceptable. As used herein, "orally acceptable" means a component which is present in the formulation in an amount and form which does not render the formulation unsafe for use in the oral cavity.

In certain embodiments, the perhydrolysis-active enzyme comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1; and b) having at least 80% amino acid sequence identical to SEQ ID NO : 1 amino acid sequence.

In some embodiments, the perhydrolysis-active enzyme further comprises a binding structural region fused to the N- or C-terminus of the enzyme, the binding structural region having an affinity for the oral tissue.

In certain embodiments, the perhydrolysis-active enzyme has affinity for oral tissues and includes an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 2, and b) having at least 80% of an amine group The acid sequence is identical to the amino acid sequence of SEQ ID NO: 2.

In some embodiments, the immobilized enzyme is adsorbed to an insoluble material, locked in an insoluble bead, covalently bonded to the insoluble material via a chemical reaction, and is bonded by a peptide having affinity for the insoluble material. The structural regions are attached or embedded in an insoluble matrix.

Some specific examples provide a method for whitening teeth or treating gingivitis, plaque or bad breath, comprising preparing a liquid comprising a whitening agent according to the method of the patent application described later, and applying the liquid to the oral cavity, for example, by using the same The liquid is rinsed for 15 seconds to 1 minute and then the liquid is spit out.

In some embodiments, the product provides a bleaching agent in a mouthwash, wherein the bleaching agent is a peroxyacid which is formed by an enzymatic reaction of hydrogen peroxide and triacetin. In some embodiments, the two compositions - one comprising hydrogen peroxide and the other comprising triacetin - are stored in a mouthwash (first compartment). Some specific examples include - at the top of the bottle - the second compartment, which is attached, for example, using an insert or a screw-type joint. In some embodiments, the second compartment is, for example, a flow enthalpy, which contains an enzyme having perhydrolysis activity, which is immobilized to the surface of the carrier material. Such as hydroxyapatite or cellulose particles. In some embodiments, the second compartment is considered to be an active component of the mouthwash.

In some embodiments, hydrogen peroxide and triacetin are separated from the hydrolytically active enzyme. In some embodiments, the mixture flows through the hydrazine and is contacted with the surface having the hydrolytic activity on the surface, and the reaction is catalyzed to rapidly produce peroxyacid. In some embodiments - after use - the mixture is again separated by the enzyme.

Throughout the text, ranges are used in abbreviated form to describe values in each and every range. Any range of values can be selected as the terminal of the range. In addition, all of the references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between the disclosure of the present disclosure and the cited references, the invention is subject to the invention.

Unless otherwise indicated, it is to be understood that all percentages and quantities indicated herein and elsewhere refer to percentages by weight. The amount given is based on the amount of active material.

Example Example 1

In a one-two-slot package, 1.0 ml of a pH 7 phosphate buffer containing 0.04 mg of perhydrolase was separately stored with the multi-component powder. The multi-component powders are set forth in Tables 1A, 1B, and 1C and include encapsulated triacetin and perfume, particulate urea peroxide, and carbomer gelling agent. In the examples, the ratio of 1A:1B:1C was 92.3:1.7:6 in a uniformly stirred powder. The two tanks are separated by a watertight barrier that is impermeable to water, which is less strongly sealed than the periphery of the package (see Figure 1). When ready for use, the consumer squeezes the buffer/enzyme tank, which will destroy the fragile internal seal and push the buffer/enzyme into the powder tank. The powder is quickly mixed with the liquid and dissolved A source of peroxide containing starch adsorbed by triacetin and perfume, and, more slowly, hydrolyzing the gelling agent. After mixing these components for a few seconds, the colloid is effectively formed and can be applied to a sprocket. Approximately 0.5 grams of the newly formed gel was applied to the upper and lower delivery devices at a dose of 4.3 mg of urea peroxide (equivalent to 1.5 mg of hydrogen peroxide), 10 mg of triacetin, and 0.01 mg of hydrolase.

Opening the hole in the package, through a pre-screening opening (see Figure 1), the user can apply the gel to the sprocket wheel and then wear the sprocket wheel for 20-30 minutes. Alternatively, the gel can be applied to an elastic patch, such as a non-porous elastomeric polyethylene or a slowly dissolvable film.

Example 2

An exemplary perhydrolase enzyme is immobilized on a solid permeable substrate. The matrix is loaded into a syringe and a solution comprising hydrogen peroxide and triacetin is propelled into the matrix to form and dispense peracetic acid (PAA).

The prototype enzyme substrate was prepared by using 0.1 gram of hydroxyapatite powder with 1500 ml of 5 micrograms of enzyme having a hydrolysis zone and a hydroxyapatite binding structure zone at 37 ° C in 10 mM phosphate buffer pH 7.2. Cultivate for 1 hour. The powder was then washed with 3 x 1 ml of 10 mM phosphate buffer, each time down, the liquid was removed, resuspended in buffer buffer, and repeated. The powder was then resuspended in 500 ml of 10 mM phosphate buffer and filled into 3 ml syringes (5 micron membranes) with a 25 m syringe filter and excess liquid was dispensed via a filter.

500 ml of reaction buffer including 100 mM phosphate buffer, 100 mM triacetin, and 100 mM hydrogen peroxide were loaded into the syringe, dispensed via a filter, and collected. 90 ml of the product was collected, and then the reaction was stopped with 40 ml of 1.3 MH ₃ PO ₄ . The resulting mixture was then diluted 1:10 in phosphate buffer and added to the detection reagent, incubated for 10 minutes and read at A ₄₀₅ nm. The proportion of PAA produced is measured. The enzyme-free process was then repeated as a control group.

By this method, approximately 900 ppm of PAA was produced, compared to 32 ppm without perhydrolase. The reactant is contacted with the immobilized enzyme for less than 60 seconds:

Repeat the experiment, there are three enzymes, no enzyme three times; let the contact enzyme not more than 15 seconds. PAA is produced with a stable concentration of about 300-350 ppm of enzyme and no enzyme of about 65 ppm. The results are illustrated in Table 2B below.

Similar results were obtained using a larger (10 ml) syringe:

Thus, the reaction in the presence of the immobilized enzyme can be reproduced, performed rapidly and efficiently, and the resulting PAA concentration is many times the concentration required to kill the bacteria, and will be sufficient to whiten the teeth.

<110> American Palm Company

<120> System for catalytic reaction by hydrolase

<130> 9434-00-WO-OC

<150> US 61/577,529

<151> 2011-12-19

<160> 197

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<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 34

<210> 35

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 35

<210> 36

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 36

<210> 37

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 37

<210> 38

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 38

<210> 39

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 39

<210> 40

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 40

<210> 41

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 41

<210> 42

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 42

<210> 43

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 43

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 44

<210> 45

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 45

<210> 46

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 46

<210> 47

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 47

<210> 48

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 48

<210> 49

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 49

<210> 50

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 50

<210> 51

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 51

<210> 52

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 52

<210> 53

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 53

<210> 54

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 54

<210> 55

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 55

<210> 56

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 56

<210> 57

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 57

<210> 58

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 58

<210> 59

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 59

<210> 60

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 60

<210> 61

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 61

<210> 62

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 62

<210> 63

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 63

<210> 64

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 64

<210> 65

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 65

<210> 66

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 66

<210> 67

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 67

<210> 68

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 68

<210> 69

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 69

<210> 70

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 70

<210> 71

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 71

<210> 72

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 72

<210> 73

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 73

<210> 74

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 74

<210> 75

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 75

<210> 76

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 76

<210> 77

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 77

<210> 78

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 78

<210> 79

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 79

<210> 80

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 80

<210> 81

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 81

<210> 82

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 82

<210> 83

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 83

<210> 84

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 84

<210> 85

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 85

<210> 86

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 86

<210> 87

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 87

<210> 88

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 88

<210> 89

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 89

<210> 90

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 90

<210> 91

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 91

<210> 92

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 92

<210> 93

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 93

<210> 94

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 94

<210> 95

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 95

<210> 96

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 96

<210> 97

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 97

<210> 98

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 98

<210> 99

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 99

<210> 100

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 100

<210> 101

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 101

<210> 102

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 102

<210> 103

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 103

<210> 104

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 104

<210> 105

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 105

<210> 106

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 106

<210> 107

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 107

<210> 108

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 108

<210> 109

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 109

<210> 110

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 110

<210> 111

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 111

<210> 112

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 112

<210> 113

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 113

<210> 114

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 114

<210> 115

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 115

<210> 116

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 116

<210> 117

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 117

<210> 118

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 118

<210> 119

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic oral surface binding peptide

<400> 119

<210> 120

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 120

<210> 121

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 121

<210> 122

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 122

<210> 123

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 123

<210> 124

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 124

<210> 125

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 125

<210> 126

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 126

<210> 127

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 127

<210> 128

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 128

<210> 129

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 129

<210> 130

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 130

<210> 131

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 131

<210> 132

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 132

<210> 133

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 133

<210> 134

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 134

<210> 135

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 135

<210> 136

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 136

<210> 137

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 137

<210> 138

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 138

<210> 139

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 139

<210> 140

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 140

<210> 141

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 141

<210> 142

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 142

<210> 143

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 143

<210> 144

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 144

<210> 145

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 145

<210> 146

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 146

<210> 147

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 147

<210> 148

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 148

<210> 149

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 149

<210> 150

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 150

<210> 151

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 151

<210> 152

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 152

<210> 153

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 153

<210> 154

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 154

<210> 155

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 155

<210> 156

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 156

<210> 157

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 157

<210> 158

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 158

<210> 159

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 159

<210> 160

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 160

<210> 161

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 161

<210> 162

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 162

<210> 163

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 163

<210> 164

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure - caspace 3 cleavable linker

<400> 164

<210> 165

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 165

<210> 166

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 166

<210> 167

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 167

<210> 168

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 168

<210> 169

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 169

<210> 170

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 170

<210> 171

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 171

<210> 172

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 172

<210> 173

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 173

<210> 174

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 174

<210> 175

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 175

<210> 176

<211> 37

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 176

<210> 177

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 177

<210> 178

<211> 431

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 178

<210> 179

<211> 353

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 179

<210> 180

<211> 359

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 180

<210> 181

<211> 375

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 181

<210> 182

<211> 431

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 182

<210> 183

<211> 359

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 183

<210> 184

<211> 386

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 184

<210> 185

<211> 387

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 185

<210> 186

<211> 386

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 186

<210> 187

<211> 387

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 187

<210> 188

<211> 382

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 188

<210> 189

<211> 383

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 189

<210> 190

<211> 390

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 190

<210> 191

<211> 391

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 191

<210> 192

<211> 426

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 192

<210> 193

<211> 372

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 193

<210> 194

<211> 429

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 194

<210> 195

<211> 375

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 195

<210> 196

<211> 418

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 196

<210> 197

<211> 363

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic structure

<400> 197

Claims (15)

A package comprising at least two sealing grooves formed by a deformable material, the package comprising: a first groove; a second groove; and any additional groove, wherein the groove is weakened or tearable by one or more Separating the barrier, wherein the first tank contains a low viscosity liquid solution comprising a hydrolytically active enzyme having a carbohydrate esterase family 7 (CE-7) signature motif, The alignment reference sequence EQ ID NO:1, the label motif comprising: i) RGQ motif at a position corresponding to positions 118-120 of SEQ ID NO: 1; ii) GXSQG motif corresponding to SEQ ID NO: 1 position 186-190; and iii) HE phantom at a position corresponding to positions 303-304 of SEQ ID NO: 1; and the second groove includes at least one sulfhydryl donor matrix, the matrix Selected from the group consisting of: i) esters of the formula: [X] _m R ₅ wherein X = an ester group of the formula R ₆ C(O)O R ₆ = C1 to C7 straight chain, branched or cyclic hydrocarbon moiety , any hydroxy group or a C1 to C4 substituted alkoxy group, wherein R ₆ comprises any one or more ether bond, R ₆ = C2 to C7 to time R ₅ = C1 to C6 straight, branched or cyclic moiety or hydroxyl five bicyclic heteroaryl cyclic moiety or six aryl or heteroaryl moieties, any hydroxy groups are substituted; wherein the R Each of the carbon atoms in ₅ independently includes no more than one hydroxyl group or no more than one ester group or a carboxylic acid group; wherein R ₅ optionally includes one or more ether linkages; m is from 1 to R An integer of the number of carbon atoms in ₅ ; and wherein the ester has a water solubility of at least 5 ppm at 25 ° C; ii) a glyceride having the following structural formula Wherein R ₁ = C1 to C7 is a straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₃ and R ₄ are independently H or R ₁ C(O); iii) One or more esters of the formula Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight or branched alkyl group, an alkenyl group, an alkynyl group, Aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is from 1 to 10; An acetylated saccharide selected from the group consisting of acetylated monosaccharide, acetylated disaccharide, and acetylated polysaccharide; and wherein the second or any additional tank contains a peroxide source such that the tank The low viscosity liquid solution is mixed with the peroxide source and the sulfhydryl donor matrix when one or more of the barriers are broken by squeezing the first groove, and the perhydrolysis-active enzyme is catalyzed by the peroxide source Forming a peroxyacid by reaction between the released peroxide and the sulfhydryl donor matrix; and the package has an opening means, such as a scribe line, lid or stopper to allow opening of the package to provide an outlet through which the mixture can pass Export to distribute. The package of claim 1, wherein the perhydrolysis-active enzyme comprises an amino acid sequence selected from the group consisting of: a) SEQ ID NO: 1; and b) having at least 80% amino acid sequence identical to SEQ ID NO: amino acid sequence of 1. The enzymatically active enzyme further comprises a binding structural region fused to the N- or C-terminus of the enzyme, the binding structural region having an affinity for the oral tissue, as in the packaging of claim 1 or 2. A package according to any of the preceding claims, wherein the binding structural region has an affinity for oral tissue comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 178-197. A package according to any of the preceding claims, wherein the hydrolytically active enzyme has affinity for oral tissues and comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, and having at least 80% amino acid sequence identical to SEQ ID NO: amino acid sequence of 2. A package according to any of the preceding claims, wherein the material in the second tank is in a powder form. A package according to any of the preceding claims, wherein the source of peroxide is selected from the group consisting of urea peroxide, polyvinylpyrrolidone-hydrogen peroxide complex, sodium percarbonate, sodium perborate, and metal peroxides, such as peroxidation. A solid peroxide source of zinc and calcium peroxide. A package according to any of the preceding claims, wherein the second tank contains a gelling agent such that upon mixing and formation of the peroxyacid, the liquid and the gelling agent form an extrudable colloid, including the peroxyacid, and then The extrudable colloid is extruded and applied to the tooth surface to allow sufficient time to whiten the teeth. A package according to any of the preceding claims, wherein the gelling agent is in a powder form. The package of claim 9, wherein the gelling agent is selected from the group consisting of a carbomer gelling agent, a polysaccharide gum, a modified edible starch, an animal or fish base gelatin, cerium oxide, and the like. Or a combination of multiple. A package according to any of the preceding claims, wherein a. the low viscosity aqueous solution further comprises a buffer, and b. the second tank contains a gelling agent, a peroxide source, and a compound containing acetamidine, all In a powder form such that when the fragile barrier is pressurized to allow mixing of the contents of the two tanks, the peroxide and the acetamidine-containing compound are reacted in the presence of an enzyme having perhydrolysis activity to form peracetic acid. In an extrudable colloid formed by a liquid and a gelling agent. A multi-part oral care composition comprising a first portion that is physically separated from the second portion during storage and combined with the second portion prior to use, wherein the first portion comprises an enzyme having perhydrolysis activity, The enzyme has a carbohydrate esterase family 7 (CE-7) tag motif aligned with a reference sequence EQ ID NO: 1, the tag motif comprising: i) an RGQ motif corresponding to SEQ ID NO: 1 Positions 118-120; ii) GXSQG motif at a position corresponding to positions 186-190 of SEQ ID NO: 1; and iii) HE motif, corresponding to positions 303-304 of SEQ ID NO: And the second portion comprises a source of peroxide and at least one sulfhydryl donor matrix selected from the group consisting of: i) an ester having the following structural formula [X] _m R ₅ wherein X = formula R ₆ C (O) O ester group R ₆ = C1 to C7 linear, branched or cyclic hydroxyl moiety, optionally substituted by a hydroxyl group or a C1 to C4 alkoxy group, wherein R ₆ optionally includes one or more ether bond, R ₆ = C2 to C7 to time; R ₅ = C1 to C6 linear, branched, or cyclic hydrocarbon moiety or five cyclic heteroaryl moieties Six cyclic aromatic moiety or heteroaromatic moiety, any of which is substituted with a hydroxyl group; wherein each of the carbon atoms R ₅ is based on independent includes no more than one hydroxyl group or no more than one ester group a carboxylic acid group; wherein R ₅ optionally includes one or more ether linkages; m is an integer from the number of carbon atoms in from 1 to R ₅ ; and wherein the ester has a water solubility of at least 5 ppm at 25 ° C; a glyceride having the following structural formula Wherein R ₁ = C1 to C7 is a straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₃ and R ₄ are independently H or R ₁ C(O); iii) One or more esters of the formula Wherein R ₁ is a C1 to C7 straight or branched alkyl group, which is optionally substituted by a hydroxy or C1 to C4 alkoxy group and R ₂ is a C1 to C10 straight or branched alkyl group, an alkenyl group, an alkynyl group, Aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n , or (CH ₂ CH(CH ₃ )-O) _n H and n is from 1 to 10; The acetylated saccharide is selected from the group consisting of acetylated monosaccharide, acetylated disaccharide, and acetylated polysaccharide; and wherein the peroxide source and at least one thiol donor substrate are hydrolyzed The reaction is carried out in the presence of an active enzyme to form a peroxyacid. The oral care composition of claim 12, wherein the peroxide source is selected from the group consisting of urea peroxide, polyvinylpyrrolidone-hydrogen peroxide complex, sodium percarbonate, sodium perborate, and metal peroxide. Solid peroxide source. The oral care composition of claim 12 or 13, wherein the peroxide source is urea peroxide. A method of whitening teeth comprising: a. packaging according to item 1 of the claim or a portion of the oral care composition according to claim 12 activated by combining substances respectively loaded in different tanks or portions; and b. An effective amount of the mixture so obtained is applied to the teeth for a sufficient period of time to whiten the teeth.