RU2793140C2 - Enzymatic formation of peracid for application in oral cavity care products - Google Patents

Abstract

FIELD: personal hygiene methods and products.

SUBSTANCE: method of oral care including: 1) providing a kit of reactional components, comprising: at least one substrate selected from a group consisting of compound esters or glycerides or compound esters with a determined structure or acetylated saccharides selected from acetylated mono- di- and polysaccharides; a source of peroxy group oxygen and an enzymatic catalyst having a perhydrolytic activity, representing a hydride protein with SEQ ID NO: 425-430, 438-459, 461-464, 466, 467 or 479; 2) combination of the said reaction components under suitable reaction conditions and preparing thereby at least one peracid; and 3) bringing the oral cavity surface in contact with at least one peracid due to which the oral cavity surface receives a peracid-based beneficial effect such as clarifying, teeth whitening, decontamination, decolorization, deodorization, reduction or elimination of biofilm or a combination thereof. Product for hygiene of oral cavity comprises the said enzymatic catalyst, substrate and source of peroxy group oxygen and also a carrier vehicle acceptable for the mouth. A composition is proposed comprising the said enzymatic catalyst, substrate and source of peroxy group oxygen that when mixed produce a peracid for treatment or prevention of dental caries, gingivitis, oral candidiasis or periodontitis.

EFFECT: stimulation of perhydrolytic activity on the surface of oral cavity.

11 cl, 25 tbl, 17 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US Provisional Application No. 61/424903, filed December 20, 2010, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the field of personal care products containing at least one peracid as a useful oral care agent. The peracid is enzymatically produced in the presence of at least one suitable carboxylic acid ester substrate and a peroxy oxygen source. In particular, an enzyme catalyst having perhydrolytic activity is used to produce a peracid benefit agent for use in an oral care product. The perhydrolytic enzyme may be in the form of a fusion protein ("targeted perhydrolase") engineered to contain at least one peptide component with an affinity for the oral surface such that the enzymatically produced peracid is produced at or near the desired surface.

BACKGROUND OF THE INVENTION

Peroxycarboxylic acids ("peracids") are effective antimicrobial agents. Methods for cleaning, decontaminating and/or sanitizing hard surfaces, foodstuffs, living plant tissues, and medical devices against the growth of unwanted microorganisms have been described (e.g., US Pat. 6,545,047; US Pat. 6,183,807; US Pat. 6,518,307; US Pat. . Peracids have also been reported to be useful in the preparation of brightening compositions for laundry detergent applications (eg, US Pat. No. 3,974,082; US Pat. No. 5,296,161; and US Pat. No. 5,364,554).

Oral compositions containing peracid have also been disclosed. US Pat. No. 5,302,375 to Viscio, D., discloses oral tooth whitening compositions containing peracetic acid dissolved in a medium, wherein the peracetic acid is produced in situ by combining water, acetylsalicylic acid and a water-soluble alkali metal percarbonate. US Pat. No. 5,279,816 to Church et al. discloses the use of a composition containing peracetic acid to whiten discolored or discolored teeth. US Pat. Nos. 6,221,341 and 7,189,385 to Montgomery, R. disclose peroxy acid dentifrice compositions suitable for use in a method of teeth whitening. More specifically, a peracetic acid composition is prepared by combining a hydrogen peroxide precursor, an acetic ester of glycerol, and water to form peracetic acid by chemical perhydrolysis. Enzymatic perhydrolysis has not been described.

US Patent Application Publication No. 2009-0311198 to Concar et al. discloses an oral composition containing a M. smegmatis enzyme with perhydrolytic tooth whitening activity. The use of perhydrolase CE-7 to produce a peracid benefit agent is not disclosed. Concar et al. also silent about the use of targeted perhydrolytic enzyme in the composition for caring for the oral cavity.

The inclusion of specific Carlsberg variant subtilisin proteases having perhydrolytic activity in a body care product is disclosed in US Pat. No. 7,510,859 to Wieland et al. Perhydrolytic enzymes other than specific variant proteases are not described, nor are there any working examples demonstrating the enzymatic production of peracid as a useful personal care agent.

US Patent Application Publication Nos. 2008-0176783 A1; 2008-0176299 A1; 2009-0005590 A1 and 2010-0041752 A1, DiCosimo et al. disclosed enzymes structurally classified as members of the CE-7 family of carbohydrate esterases (i.e., cephalosporin C deacetylase [CAH] and acetylxylanesterase [AXE]), which are characterized by significant perhydrolytic activity in relation to the conversion of carboxylic acid ester substrates (in the presence of a suitable a peroxy oxygen source such as hydrogen peroxide) into peroxycarboxylic acid at concentrations suitable for use as a disinfectant and/or brightening agent. Some members of the CE-7 family of carbohydrate esterases have been shown to have sufficient perhydrolytic activity to produce 4000-5000 ppm. peracetic acid from acetylesters of alcohols, diols and glycerols in 1 minute and up to 9000 ppm. over a period of 5 to 30 minutes after mixing the reaction components (DiCosimo et al., U.S. 2009-0005590 A1). US Patent Application Publication No. 2010-0087529 A1 describes variant CE-7 enzymes having increased perhydrolytic activity. Although CE-7 perhydrolases have excessive perhydrolytic activity, their use in personal care products has not yet been disclosed. In this regard, a problem to be solved is the preparation of personal care compositions and methods involving the use of at least one CE-7 perhydrolase to produce a peracid benefit agent.

Peracids are strong oxidizing agents that can react with a number of materials, including materials that are not targeted for the desired beneficial effect. In this regard, certain personal care applications may benefit by being able to target/focus the peracid benefit agent to a desired body surface by localizing peracid generation at or near the desired target body surface. Enzymatic production of the peracid may have a beneficial effect by targeting the perhydrolase to body surfaces. An additional beneficial effect can be achieved by targeting the perhydrolase to the delivery material so as to limit the concentration of the enzyme and the exposure to the consumer.

Oral care compositions and/or methods have been reported for treating an oral surface in need of care with an enzyme associated with the oral material. US Pat. No. 4,138,476 to Simonson et al. discloses a method for treating plaque comprising the use of a glucan-degrading enzyme covalently bonded via a complexing agent to a phosphate carrier group with affinity for the tooth surface. The enzymatic breakdown of glucan deposits is believed to aid in the dissolution and dispersion of plaque material.

U.S. Patent Application Publication No. 2005-0158253, U.S. Patent 6,830,745 to Budny et al., discloses a two-component composition containing a fixing enzyme complex for enzymatic degradation of biofilm structures and a second fixing enzyme component capable of acting directly on bacteria. Biofilm degrading enzymes are enzymes that directly degrade exopolysaccharide backbone structures.

US Pat. No. 5,871,714 to Budny, J., discloses a bacterial growth/colony control (eg, plaque reduction) composition containing an enzyme that degrades plaque matrix associated with a latching molecule. The use of targeted perhydrolase is not disclosed.

United States Patent 5,490,988 and EP 0,479,600 to Beggs et al. disclose the use of antibody fragments as target site binding agents, wherein the therapeutic agent is coupled via an additional peptide attached to the antibody fragment to attach the therapeutic agent to the target site. Disclosed is an oral care product containing a modified antibody fragment having an affinity for an antigenic component of bacteria in plaque to deliver a therapeutic agent. The therapeutic agent may be a cytotoxic agent produced by an enzyme or combination of enzymes, such as an oxidase combined with a peroxidase to form halides. The use of a targeted perhydrolase to produce a peracid benefit agent is not described.

European patent EP 0450800 B1, Beggs et al., discloses the use of two different enzymes working together to act on substances found in the oral microflora. The first enzyme forms an intermediate which is used as a substrate for the second enzyme to produce an agent active on the target in the mouth. Each enzyme is attached to an antibody or antibody fragment that has an affinity for a target surface in the mouth, and when applied, the enzymes bind to the target site in close proximity to each other. Illustrated is the combination with glucose oxidase to produce hydrogen peroxide, which can then be converted by peroxidase, in the presence of halide or thiocyanate, to produce hypohalogenite or hypothiocyanate, respectively. The use of a targeted perhydrolase to produce a peracid benefit agent is not described.

EP 0451972 B1, Beggs et al., describes a product containing two enzymes, wherein the product contains a first enzyme to form an active agent against the target and a second enzyme to form an intermediate which is a substrate for the first enzyme; wherein said product further comprises binding agents (ie, antibody or antibody fragment) attached or attached to both enzymes to bind the enzymes to each other, resulting in a complex that binds to the target cell. Illustrated is an oxidase (capable of producing hydrogen peroxide) coupled to a peroxidase that catalyzes the formation of a hypohalogen or hypothiocyanate active agent.

European patent EP 0453097 B1, Beggs et al., describes the delivery of an active agent to a target site using a plurality of antibodies or antibody fragments that can self-assemble to form a bridge between the agent and the target site. The active agent is glucose oxidase or a combination of glucose oxidase and peroxidase. The use of a targeted perhydrolase to produce a peracid benefit agent is not described.

The use of antibodies, antibody fragments (F _ab ), a single strand fused to an antibody containing a variable region (scFc), Camelidae antibodies, and skeletal display proteins as affinity peptide materials may not be suitable for some personal applications. hygiene due to their size and cost. As such, there remains a need for certain low cost cosmetic applications to use shorter, less expensive affinity peptide materials for targeted delivery of a beneficial agent.

The use of shorter, higher affinity peptides for targeting a cosmetic benefit to a body surface has been described (US Patent Nos. 7220405; 7309482; 7285264 and 7807141; US Patent Application Publication Nos. 2005-0226839 A1; 2007-0196305 A1; 2006 0199206 A1; 2007-0065387 A1; 2008-0107614 A1; 2007-0110686 A1; 2006-0073111 A1; 2010-0158846; 2010-0158847 and 2010-0247589; and published PCT applications WO2008/054746; WO2004/04799; 073368). US Pat. No. 7,807,141 to Huang et al. discloses peptide oral care reagents suitable for binding an oral benefit to a tooth surface. The use of a peptide material having an affinity for the oral surface to bind an active CE-7 perhydrolase (ie "targeted perhydrolases") producing a peracid benefit agent has not been described.

In this regard, an additional problem to be solved is to provide compositions and methods suitable for targeting the enzymatic production of peracid to the surface of the oral cavity.

BRIEF DESCRIPTION OF THE PRESENT INVENTION

Methods and compositions containing components for the enzymatic production and delivery of a peracid beneficial agent to the surface of the oral cavity are proposed.

In one embodiment, oral care compositions and methods are provided that use CE-7 perhydrolase to enzymatically produce a peracid benefit agent for use in oral hygiene applications such as oral surface cleaning, tooth whitening, decontamination. , bleaching, deodorizing, treating dental caries, preventing dental caries, reducing mouth bacteria associated with dental caries, and treating or removing biofilms in the mouth (eg, plaque).

In one embodiment, a method is provided, including:

1) providing a set of reaction components that contains:

a) at least one substrate selected from the group consisting of:

i) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

ii) glycerides with the structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

iii) one or more esters of the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; And

iv) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides;

b) a source of peroxy oxygen; And

c) an enzyme catalyst having perhydrolytic activity, said catalyst comprising an enzyme having the signature motif CE-7, which is aligned with the reference sequence of SEQ ID NO: 2 using CLUSTALW, said signature motif containing:

i) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

ii) a GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

iii) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2; And

2) combining the reaction components (1) under suitable reaction conditions, whereby at least one peracid is enzymatically obtained;

3) contacting the oral surface with at least one peracid, whereby the oral surface receives a peracid-based benefit selected from the group consisting of brightening, teeth whitening, decontamination, discoloration, deodorization, biofilm reduction or elimination, or their combinations.

In one embodiment, the oral surface is tooth enamel, plaque on teeth, soft tissue in the oral cavity (eg, gums, tongue), or biofilm in the oral cavity (eg, plaque in the mouth).

In another embodiment, compositions and methods are provided comprising the use of a fusion protein (i.e., "targeted perhydrolase") comprising a perhydrolytic enzyme and a peptide component with affinity for the oral surface, the two components optionally being separated by a peptide spacer.

In one embodiment, a method is provided, including:

1) providing a set of reaction components that contains:

a) at least one substrate selected from the group consisting of:

i) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

ii) glycerides with the structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

iii) one or more esters of the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; And

iv) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides;

b) a source of peroxy oxygen; And

c) an enzyme catalyst having perhydrolytic activity, said enzyme catalyst comprising a fusion protein with the following general structure:

PAH-[L] _y -OCBD

or

OCBD-[L] _y -PAH,

Where

PAH is an enzyme with perhydrolytic activity;

OCBD is a peptide component with an affinity for the oral surface; And

L is an optional peptide linker varying in length from 1 to 100 amino acids; And

y is 0 or 1;

2) combining the reaction components (1) under suitable reaction conditions, whereby at least one peracid is enzymatically obtained;

3) contacting the oral surface with at least one peracid, whereby the oral surface receives a peracid-based benefit selected from the group consisting of brightening, teeth whitening, decontamination, discoloration, deodorization, biofilm reduction or elimination, or their combinations.

The fusion protein may contain a perhydrolytic enzyme selected from the group consisting of lipases, proteases, esterases, acyltransferases, arylesterases, carbohydrate esterases, and combinations thereof.

In one embodiment, the fusion protein contains perhydrolytic arylesterase (ArE) from Mycobacterium smegmatis . In another embodiment, the fusion protein comprises a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Mycobacterium smegmatis arylesterase S54V shown in SEQ ID NO: 460.

In one embodiment, the fusion protein contains a perhydrolytic esterase from Pseudomonas fluorescens . In another embodiment, the fusion protein contains a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Pseudomonas fluorescens esterase shown in SEQ ID NO: 477.

In another embodiment, the fusion protein contains a perhydrolytic enzyme with an amino acid sequence selected from the group consisting of SEQ ID NOs: 424, 425, 426, 427, 428, 429, 430, 437, 438, 439, 440, 441, 442, 443 , 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 476 , 477, 478 and 479.

In another embodiment, the fusion protein comprises a CE-7 perhydrolase with a CE-7 signature motif that aligns to a reference sequence of SEQ ID NO: 2 with CLUSTALW, said signature motif comprising:

i) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

ii) a GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

iii) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2.

In another embodiment, the oral surface affinity peptide component is preferably a single chain peptide comprising at least one oral surface binding peptide. In a further embodiment, the oral surface binding peptide is a peptide having an affinity for tooth enamel, dental plaque, and both tooth enamel and dental plaque.

In another embodiment, a fusion protein is provided having the following general structure:

PAH-[L] _y -OCBD

or

OCBD-[L] _y -PAH,

Where

1) PAH is an enzyme with perhydrolytic activity;

2) OCBD is a peptide component with affinity for the oral surface;

3) L is an optional peptide linker varying in length from 1 to 100 amino acids; And

4) y is 0 or 1.

In another embodiment, a fusion protein is provided having the general structure:

PAH-[L] _y -OCBD

or

OCBD-[L] _y -PAH,

Where

a) PAH is a carbohydrate esterase CE-7 with perhydrolytic activity; while the RAS has the signature motif CE-7, which is aligned with the reference sequence SEQ ID NO: 2 using CLUSTALW, and the specified signature motif contains:

i) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

ii) a GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

iii) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2; And

b) OCBD is a peptide component with affinity for the oral surface;

c) L is an optional peptide linker varying in length from 1 to 100 amino acids; And

d) y is 0 or 1.

In another embodiment, an oral care product is provided comprising:

1) an enzyme catalyst containing any of the above perhydrolytic fusion proteins;

2) at least one substrate selected from the group consisting of:

a) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

b) glycerides with the structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

c) one or more esters with the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; And

d) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides;

3) a source of oxygen of the peroxy group; And

4) an orally acceptable carrier medium.

In another embodiment, an oral care product is provided comprising:

1) an enzyme catalyst having perhydrolytic activity, said catalyst comprising an enzyme having the signature motif CE-7, which is aligned with the reference sequence of SEQ ID NO: 2 using CLUSTALW, said signature motif containing:

a) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

b) GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

c) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2; And

2) at least one substrate selected from the group consisting of:

a) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

b) glycerides with the structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

c) one or more esters with the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; And

d) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides;

3) a source of oxygen of the peroxy group; And

4) an orally acceptable carrier medium.

In another embodiment, there is provided an isolated polypeptide having an affinity for the oral surface, said polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421 and 422.

In another embodiment, the use of a CE-7 carbohydrate esterase having perhydrolytic activity is also provided in an oral care product to provide an effective concentration of at least one peracid to brighten, whiten, decontaminate, bleach, deodorize, or remove biofilm from oral material. / surface of the oral cavity.

In another embodiment, the use of a peracid formation composition comprising:

a) an enzyme catalyst having perhydrolytic activity, said catalyst comprising an enzyme having the signature motif CE-7, which is aligned with the reference sequence of SEQ ID NO: 2 using CLUSTALW, said signature motif containing:

i) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

ii) a GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

iii) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2; And

b) at least one substrate selected from the group consisting of:

1) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl fragment, or a five-membered cyclic heteroaromatic or six-membered cyclic aromatic or heteroaromatic fragment, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

2) glycerides with structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

3) one or more esters with the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H and n is 1-10; And

d) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides; And

c) a source of peroxy oxygen;

resulting in the formation of peracid with simultaneous or stepwise (but not in a specific order) mixing (a), (b) and (c); for the treatment, prevention of dental caries, gingivitis, oral candidiasis or periodontitis.

In another embodiment, the use in an oral product of a fusion protein containing the following general structure is provided, which includes:

PAH-[L] _y -OCBD

or

OCBD-[L] _y -PAH,

Where

1) PAH is an enzyme having perhydrolytic activity with an amino acid sequence that has at least 95% amino acid identity with SEQ ID NO: 460;

2) OCBD is a peptide component with affinity for the oral surface;

3) L is a peptide linker varying in length from 1 to 100 amino acids; And

4) y is 0 or 1.

Some of the ester substrates described herein (Table 20) were particularly susceptible to chemical perhydrolysis when reacted with hydrogen peroxide to form peracetic acid. In another embodiment, a personal care product is provided comprising a peracid precursor selected from the group consisting of 1,2,3,5-tetra-O-acetylribofuranose; 1,2,3,4-tetra-O-acetylribofuranoses; 2-acetamido-2-deoxy-1,3,4,6-tetraacetyl-β-D-glucopyranose; β-D-glucopyranose 1,2,3,4-tetraacetate; 2,3,4,6-tetraacetyl-β-D-glucopyranoses; 1,3,4,6-tetra-O-acetylmannopyranose and α-D-mannopyranose pentaacetate. In a preferred embodiment, the personal care product is an oral care product.

In another embodiment, a method is also provided, including:

a) providing a set of reaction components that contains

i) a peracid precursor selected from the group consisting of 1,2,3,5-tetra-O-acetylribofuranose; 1,2,3,4-tetra-O-acetylribofuranoses; 2-acetamido-2-deoxy-1,3,4,6-tetraacetyl-β-D-glucopyranose; β-D-glucopyranose 1,2,3,4-tetraacetate; 2,3,4,6-tetraacetyl-β-D-glucopyranoses; 1,3,4,6-tetra-O-acetylmannopyranose and α-D-mannopyranose pentaacetate; And

ii) a peroxy oxygen source;

b) bringing into contact the surface of the body with an effective amount of peracetic acid formed by combining a set of reactive components in the presence of water; as a result, peracetic acid provides a beneficial effect on the surface of the body.

In a preferred aspect, the body surface in the above method is tissue in the oral cavity, such as teeth and/or gums.

BRIEF DESCRIPTION OF BIOLOGICAL SEQUENCES

The following sequences correspond to §§ 1.821-1.825 37 C.F.R. ("Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence Disclosures - Sequence Rules") and is consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (2009) and European Patent Convention (EPC) Sequence Listing Requirements and Rules 5.2 and 49.5 (a-bis) of the Patent Cooperation Treaty (PCT) and Section 208 and Appendix C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data follow the rules set out in 37 C.F.R. § 1.822.

SEQ ID NO: 1 is the nucleic acid sequence encoding cephalosporin C deacetylase from Bacillus subtilis ^ATCC® 31954™.

SEQ ID NO: 2 is the amino acid sequence of cephalosporin C deacetylase from Bacillus subtilis ^ATCC® 31954™.

SEQ ID NO: 3 is the nucleic acid sequence encoding cephalosporin C deacetylase from Bacillus subtilis subsp. subtilis, strain 168.

SEQ ID NO: 4 is the amino acid sequence of cephalosporin C deacetylase from Bacillus subtilis subsp. subtilis, strain 168.

SEQ ID NO: 5 is the nucleic acid sequence encoding cephalosporin C deacetylase from B. subtilis ^ATCC® 6633™.

SEQ ID NO: 6 is the amino acid sequence of cephalosporin C deacetylase from B. subtilis ^ATCC® 6633™.

SEQ ID NO: 7 is the nucleic acid sequence encoding cephalosporin C deacetylase from B. licheniformis ^ATCC® 14580™.

SEQ ID NO: 8 is a derived amino acid sequence of cephalosporin C deacetylase from B. licheniformis ^ATCC® 14580™.

SEQ ID NO: 9 is the nucleic acid sequence encoding B. pumilus PS213 acetylxylanesterase.

SEQ ID NO: 10 is a derived amino acid sequence of B. pumilus PS213 acetylxylanesterase.

SEQ ID NO: 11 is the nucleic acid sequence encoding acetylxylanesterase from Clostridium thermocellum ^ATCC® 27405™.

SEQ ID NO: 12 is a derived amino acid sequence of acetylxylanesterase from Clostridium thermocellum ^ATCC® 27405™.

SEQ ID NO: 13 is a nucleic acid sequence encoding acetylxylanesterase from Thermotoga neapolitana .

SEQ ID NO: 14 is the amino acid sequence of acetylxylanesterase from Thermotoga neapolitana .

SEQ ID NO: 15 is the nucleic acid sequence encoding acetylxylanesterase from Thermotoga maritima MSB8.

SEQ ID NO: 16 is the amino acid sequence of acetylxylanesterase from Thermotoga maritima MSB8.

SEQ ID NO: 17 is a nucleic acid sequence encoding acetylxylanesterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 18 is a derived amino acid sequence of acetylxylanesterase from Thermoanaerobacterium sp. JW/SL YS485.

SEQ ID NO: 19 is the nucleic acid sequence of cephalosporin C deacetylase from Bacillus sp . NRRL B-14911. It should be noted that the nucleic acid sequence encoding cephalosporin C deacetylase from Bacillus sp . NRRL B-14911 according to ^GENBANK® with accession number ZP_01168674 appears to encode a 15 amino acid long N-terminal complement, which is likely incorrect based on sequence alignments with other cephalosporin C deacetylases and comparison of the reduced length (340 amino acids) compared to the observed length of other CAH enzymes (typically 318-325 amino acids long; see US Patent Application Publication US-2010-0087528-A1; incorporated herein by reference). In this regard, the nucleic acid sequence provided herein encodes a cephalosporin C deacetylase sequence from Bacillus sp . NRRL B-14911 without the N-terminal 15 amino acids listed under ^GENBANK® accession number ZP_01168674.

SEQ ID NO: 20 is a derived amino acid sequence of cephalosporin C deacetylase from Bacillus sp . NRRL B-14911 encoded by the nucleic acid sequence of SEQ ID NO: 19.

SEQ ID NO: 21 is the nucleic acid sequence encoding cephalosporin C deacetylase from Bacillus halodurans C-125.

SEQ ID NO: 22 is a derived amino acid sequence of cephalosporin C deacetylase from Bacillus halodurans C-125.

SEQ ID NO: 23 is the nucleic acid sequence encoding cephalosporin C deacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 24 is a derived amino acid sequence of cephalosporin C deacetylase from Bacillus clausii KSM-K16.

SEQ ID NO: 25 is the nucleic acid sequence encoding cephalosporin C deacetylase (CAH) from Bacillus subtilis ^ATCC® 29233™.

SEQ ID NO: 26 is a derived amino acid sequence of cephalosporin C deacetylase (CAH) from Bacillus subtilis ^ATCC® 29233™.

SEQ ID NO: 27 is the derived amino acid sequence of the Thermotoga neapolitana acetylxylanesterase variant from US Patent Application Publication No. 2010-0087529 (incorporated herein by reference), wherein the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 28 is the derived amino acid sequence of the Thermotoga maritima MSB8 acetylxylanesterase variant from US Patent Application Publication No. 2010-0087529, wherein the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 29 is the derived amino acid sequence of the Thermotoga lettingae acetylxylanesterase variant from US Patent Application Publication No. 2010-0087529, where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 30 is the derived amino acid sequence of the Thermotoga petrophila acetylxylanesterase variant from US Patent Application Publication No. 2010-0087529, where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 31 is a derived amino acid sequence of the Thermotoga sp. acetylxylanesterase variant. RQ2 derived from "RQ2(a)" of US Patent Application Publication No. 2010-0087529, where the Xaa residue at position 277 is Ala, Val, Ser, or Thr.

SEQ ID NO: 32 is a derived amino acid sequence of the Thermotoga sp. acetylxylanesterase variant. RQ2 derived from "RQ2(b)" of US Patent Application Publication No. 2010-0087529, where the Xaa residue at position 278 is Ala, Val, Ser, or Thr.

SEQ ID NO: 33 is a derived amino acid sequence of Thermotoga lettingae acetylxylanesterase.

SEQ ID NO: 34 is a derived amino acid sequence of Thermotoga petrophila acetylxylanesterase.

SEQ ID NO: 35 is the derived amino acid sequence of the first acetylxylanesterase fromThermotogasp. RQ2, described herein as "RQ2(a)".

SEQ ID NO: 36 is the derived amino acid sequence of the second acetylxylanesterase fromThermotogasp. RQ2, described herein as "RQ2(b)".

SEQ ID NO: 37 is a codon-optimized nucleic acid sequence that encodes Thermoanearobacterium saccharolyticum cephalosporin C deacetylase.

SEQ ID NO: 38 is a derived amino acid sequence of Thermoanearobacterium saccharolyticum cephalosporin C deacetylase.

SEQ ID NO: 39 is the nucleic acid sequence encoding acetylxylanesterase from Lactococcus lactis (accession number ^GENBANK® EU255910).

SEQ ID NO: 40 is the amino acid sequence of acetylxylanesterase from Lactococcus lactis (accession number ^GENBANK® ABX75634.1).

SEQ ID NO: 41 is the nucleic acid sequence encoding acetylxylanesterase from Mesorhizobium loti (accession number ^GENBANK® NC_002678.2).

SEQ ID NO: 42 is the amino acid sequence of acetylxylanesterase from Mesorhizobium loti (accession number ^GENBANK® BAB53179.1).

SEQ ID NO: 43 is the nucleic acid sequence encoding acetylxylanesterase from Geobacillus stearothermophilus (GENBANK® accession number AF038547.2).

SEQ ID NO: 44 is the amino acid sequence of acetylxylanesterase from Geobacillus stearothermophilus (GENBANK® accession number AAF70202.1).

SEQ ID NO: 45 is a nucleic acid sequence encoding a variant acetylxylanesterase (variant “A3”) with the following amino acid sequence substitutions relative to wild type Thermotoga maritima acetylxylanesterase: (F24I/S35T/Q179L/N275D/C277S/S308G/F317S).

SEQ ID NO: 46 is the amino acid sequence of variant acetylxylanesterase "A3".

SEQ ID NO: 47 is the nucleic acid sequence encoding the N275D/C277S variant acetylxylanesterase.

SEQ ID NO: 48 is the amino acid sequence of the N275D/C277S variant acetylxylanesterase.

SEQ ID NO: 49 is the nucleic acid sequence encoding the C277S/F317S variant acetylxylanesterase.

SEQ ID NO: 50 is the amino acid sequence of the C277S/F317S variant acetylxylanesterase.

SEQ ID NO: 51 is the nucleic acid sequence encoding the S35T/C277S variant acetylxylanesterase.

SEQ ID NO: 52 is the amino acid sequence of the S35T/C277S variant acetylxylanesterase.

SEQ ID NO: 53 is the nucleic acid sequence encoding the Q179L/C277S variant acetylxylanesterase.

SEQ ID NO: 54 is the amino acid sequence of the Q179L/C277S variant acetylxylanesterase.

SEQ ID NO: 55 is the nucleic acid sequence encoding the 843H9 variant acetylxylanesterase with the following amino acid sequence substitutions relative to wild type Thermotoga maritima acetylxylanesterase: (L8R/L125Q/Q176L/V183D/F247I/C277S/P292L).

SEQ ID NO: 56 is the amino acid sequence of the 843H9 variant acetylxylanesterase.

SEQ ID NO: 57 is the nucleic acid sequence encoding the 843F12 variant acetylxylanesterase with the following amino acid sequence substitutions relative to wild type Thermotoga maritima acetylxylanesterase: K77E/A266E/C277S.

SEQ ID NO: 58 is the amino acid sequence of the 843F12 variant acetylxylanesterase.

SEQ ID NO: 59 is the nucleic acid sequence encoding the 843C12 variant acetylxylanesterase with the following amino acid sequence substitutions relative to wild-type Thermotoga maritima acetylxylanesterase: F27Y/I149V/A266V/C277S/I295T/N302S.

SEQ ID NO: 60 is the amino acid sequence of the 843C12 variant acetylxylanesterase.

SEQ ID NO: 61 is the nucleic acid sequence encoding the 842H3 variant acetylxylanesterase with the following amino acid sequence substitutions relative to wild-type Thermotoga maritima acetylxylanesterase: L195Q/C277S.

SEQ ID NO: 62 is the amino acid sequence of the 842H3 variant acetylxylanesterase.

SEQ ID NO: 63 is the nucleic acid sequence encoding the 841A7 variant acetylxylanesterase with the following amino acid sequence substitutions relative to wild type Thermotoga maritima acetylxylanesterase: Y110F/C277S.

SEQ ID NO: 64 is the amino acid sequence of the 841A7 variant acetylxylanesterase.

SEQ ID NOs: 65-221, 271 and 368 are a non-limiting listing of amino acid sequences of peptides with affinity for hair.

SEQ ID NO: 217-269 are the amino acid sequences of peptides with skin affinity.

SEQ ID NO: 270-271 are the amino acid sequences of peptides with affinity for the nail.

SEQ ID NO: 272-382 are the amino acid sequences of peptides with affinity for the oral surface. SEQ ID NOs: 272-291 and 312-382 have an affinity for dental plaque. SEQ ID NO: 272-291 and 292-311 have an affinity for tooth enamel.

SEQ ID NO: 383-396 are the amino acid sequences of the peptide linkers/spacers.

SEQ ID NO: 397 is the nucleic acid sequence of the expression plasmid pLD001.

SEQ ID NO: 398 is the nucleic acid sequence of the sequencing primer.

SEQ ID NO: 399-410 are the amino acid sequences of the enamel-binding and plaque-binding peptides from Example 2.

SEQ ID NO: 411 is the amino acid sequence of the tooth binding peptides, DenP03 with a C-terminal lysine, as shown in Table 4.

SEQ ID NO: 412-422 are the amino acid sequence of tooth enamel binding peptides and dental plaque binding peptides with C-terminal lysine as shown in Table 4.

SEQ ID NO: 423 is the amino acid sequence of the HC263 peptide.

SEQ ID NO: 424 is the amino acid sequence of the variant Thermotoga maritima C277S, also referred to herein as the "EZ-1" enzyme.

SEQ ID NOs: 425-430 and 437-467 and 479 are the amino acid sequences of various perhydrolase constructs as disclosed in Table 5 and/or Table 6.

SEQ ID NOs: 431-436 and 468-475 are the amino acid sequences of the various targeting sequences disclosed in Example 4.

SEQ ID NO: 476 is the amino acid sequence of the variant HTS-007-D5 Thermotoga maritima with the following substitutions: C277T/R296P.

SEQ ID NO: 477 is the amino acid sequence of Pseudomonas fluorescens esterase having perhydrolytic activity (U.S. Patent 7,384,787; variant "L29P". It should be noted that the substitution numbering is taken from the referenced patent, which does not include the original methionine residue. SEQ ID NO: 477 contains a substitution L29P at residue position number 30 because the original methionine is included in this sequence).

SEQ ID NO: 478 is the amino acid sequence of arylesterase wild typeMycobacterium smegmatis (US patent 7754460).

DETAILED DESCRIPTION OF THE PRESENT INVENTION

A number of terms and abbreviations are used in this disclosure. Unless expressly stated otherwise, the following definitions apply.

As used herein, the singular form of the element and component of the present invention is intended to be non-limiting as to the number of examples (ie, instances) of the element and component. Thus, the singular forms should be read as including at least one, and the singular form of an element or component also includes the plural, unless the numbers are clearly intended to mean the singular.

As used herein, the term "comprising" means the presence of the stated features, integers, steps, or components as set forth in the claims, but in such a way that it does not exclude the presence of one or more additional features, integers, steps, components, or groups of them. . The term "comprising" is meant to include the embodiments encompassed by the terms "substantially consisting of" and "consisting of". Similarly, the term "essentially consisting of" is intended to include the embodiments encompassed by the term "consisting of".

As used herein, the term "approximately", modifying the amount of ingredient or reagent used, refers to variation in the numerical amount that can occur, for example, as a result of standard measurement procedures and fluid handling procedures used to prepare concentrates or ready-to-use solutions for practice; as a result of an inevitable error in these procedures; as a result of differences in the production, source and purity of the ingredients used to prepare the compositions or to carry out the methods; etc. The term "approximately" also covers amounts that differ due to different equilibrium states for the composition obtained from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents of these amounts.

If available, all ranges are inclusive and combinable. For example, when referring to the range "1 to 5", said range should be considered to include the ranges "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1 -3 and 5" etc.

As used herein, the term "contacting" refers to placing the composition in contact with the target surface of the body for a period of time sufficient to achieve the desired result (binding to the target surface, peracid based effects, etc.). In one embodiment, "contacting" may refer to contacting a composition containing (or capable of producing) an effective concentration of a peracid upon contact with a target body surface for a period of time sufficient to achieve the desired result. In another embodiment, "contacting" may also refer to placing at least one component of a personal care composition, such as one or more enzymatic perhydrolysis reaction components, in contact with a target body surface. Contacting includes spraying, handling, dipping, rinsing, pouring or pouring, mixing, combining, brushing, coating, applying, attaching, and other means of interacting a peracid solution or composition containing an effective concentration of peracid, solution or composition that forms an effective the concentration of peracid or component of the composition that forms an effective concentration of peracid, with the surface of the body.

As used herein, the terms "substrate", "suitable substrate", and "carboxylic acid ester substrate" refer interchangeably to specifically:

(a) one or more esters with the structure

[X] _m R ₅ ,

Where

X is an ester group with the formula R ₆ C(O)O;

R ₆ is a C1-C7 linear, branched or cyclic hydrocarbyl moiety, optionally substituted with a hydroxyl group or a C1-C4 alkoxy group, while R ₆ optionally contains one or more ether linkages, where R ₆ is C2-C7;

R ₅ is a C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a cyclic five-membered heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety optionally substituted with a hydroxyl group; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group, and where R ₅ optionally contains one or more ester bonds;

m is an integer ranging from 1 to the number of carbons in R ₅ ,

wherein said one or more esters have a solubility in water of at least 5 ppm. at 25°C; or

(b) one or more glycerides with the structure

,

,

where R ₁ is straight chain or branched C1-C7 alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O); or

(c) one or more esters of the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; or

(d) one or more acetylated monosaccharides, acetylated disaccharides or acetylated polysaccharides; or

(e) any combination of (a)-(d).

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

As used herein, the term "peracetic acid" is abbreviated "PAA" and is synonymous with peroxyacetic acid, ethaneperoxic acid, and all other synonyms of CAS Registry Number 79-21-0.

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

As used herein, the term "diacetin" is synonymous with glycerol diacetate, glycerol diacetate, glyceryl diacetate, and all other synonyms of CAS Registry Number 25395-31-7.

As used herein, the term "triacetin" is synonymous with glycerol triacetate, glycerol triacetate, glyceryl triacetate, 1,2,3-triacetoxypropane, 1,2,3-propanetriol, and all other synonyms of CAS Registry Number 102-76-1.

As used herein, the term "monobutyrin" is synonymous with glycerol monobutyrate, glycerol monobutyrate, and glyceryl monobutyrate.

As used herein, the term "dibutyrin" is synonymous with glycerol dibutyrate and glyceryl dibutyrate.

As used herein, the term "tributyrin" is synonymous with glycerol tributyrate, 1,2,3-tributyrylglycerol, and all other synonyms of CAS 60-01-5.

As used herein, the term "monopropionin" is synonymous with glycerol monopropionate, glycerol monopropionate, and glyceryl monopropionate.

As used herein, the term "dipropionin" is synonymous with glycerol dipropionate and glyceryl dipropionate.

As used herein, the term "tripropionin" is synonymous with glyceryl tripropionate, glycerol tripropionate, 1,2,3-tripropionoglycerol, and all other synonyms of CAS Registry Number 139-45-7.

As used herein, the terms "acetylated sugar" and "acetylated saccharide" refer to mono-, di-, and polysaccharides containing at least one acetyl group. Examples include, but are not limited to, glucose pentaacetate, xylose tetraacetate, acetylated xylan, acetylated xylan moieties, β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactate; and tri-O-acetylglucal.

As used herein, the terms "hydrocarbyl", "hydrocarbyl group" and "hydrocarbyl moiety" mean a straight chain, branched or cyclic grouping of carbon atoms connected by single, double or triple carbon-carbon bonds and/or ether bridges and respectively substituted by hydrogen atoms. Such hydrocarbyl groups may be aliphatic and/or aromatic. Examples of hydrocarbyl groups 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 fragment is a straight chain, branched or cyclic grouping of carbon atoms connected by single carbon-carbon bonds and/or ether bridges and respectively substituted with hydrogen atoms.

As used herein, the terms "monoesters" and "diesters" of 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, 2,5-hexanediol, 1,6-hexanediol and mixtures thereof refer to said compounds containing at least one ester group with the formula RC(O)O, where R is a C1-C7 linear hydrocarbyl moiety. In one embodiment, the carboxylic acid ester substrate 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-diacetoxypropane, propylene diacetate, 1,2-propanediol diacetate, and all other synonyms of CAS Registry Number 623-84-7.

As used herein, the term "ethylene glycol diacetate" is synonymous with 1,2-diacetoxyethane, ethylene diacetate, glycol diacetate, and all other synonyms of CAS Registry Number 111-55-7.

As used herein, the terms "suitable enzyme reaction mixture", "components suitable for in situ peracid production", "suitable reaction components", "suitable aqueous reaction mixture", "reaction mixture" and "peracid generating components" refer to materials and water in which the reagents and the perhydrolytic enzyme catalyst are brought into contact. The peracid generating components will include at least an enzyme having perhydrolytic activity, where preferably the perhydrolytic enzyme is at least one CE-7 perhydrolase (optionally in the form of a body surface targeting fusion protein), at least one substrate based on a carboxylic acid ester, a peroxy oxygen source, and water (an aqueous solution containing a peroxy oxygen source, such as hydrogen peroxide). In one embodiment, any perhydrolytic enzyme that does not belong to the class of CE-7 carbohydrate esterases will, with reservation, be used in the form of a fusion protein with at least one peptide component having an affinity for a target surface, preferably the oral surface.

As used herein, the term "perhydrolysis" is defined as the reaction of a selected substrate with a peroxide to form a peracid. Typically, the inorganic peroxide is reacted with the chosen substrate in the presence of a catalyst to form the peroxycarboxylic acid. As used herein, the term "chemical perhydrolysis" includes perhydrolysis reactions in which a substrate (peroxycarboxylic acid precursor) is combined with a source of hydrogen peroxide, whereby the peroxycarboxylic acid is formed in the absence of an enzyme catalyst. As used herein, the term "enzymatic perhydrolysis" includes perhydrolysis reactions in which a carboxylic acid ester substrate (peracid precursor) is combined with a source of hydrogen peroxide and water, whereby an enzymatic catalyst catalyzes the formation of peracid.

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

Used in this document, the expression "one unit of enzymatic activity" or "one unit of activity" or "Unit." defined as the amount of perhydrolase activity required to produce 1 µmol peroxycarboxylic acid product per minute at a given temperature.

As used herein, the terms "enzyme catalyst" and "perhydrolase catalyst" refer to a catalyst comprising an enzyme having perhydrolytic activity, and which can be in the form of a whole cell of a microorganism, a permeabilized cell(s) of a microorganism(s), one or more cellular components of an extract of microorganism cells, a partially purified enzyme or a purified enzyme. The enzyme catalyst may also be chemically modified (such as by pegylation or by reaction with cross-linking reagents). The perhydrolase catalyst can also be immobilized on a soluble or insoluble support using well known methods, see, for example, Immobilization of Enzymes and Cells ; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997. In one embodiment, the perhydrolase catalyst can be non-covalently or covalently immobilized in or on an oral hygiene strip (eg, tooth whitening strip) or tray. The immobilized enzyme can be directly bonded to a polymeric substrate and/or component in an oral hygiene strip or mouthguard (e.g., titanium dioxide, hydroxyapatite, an orally acceptable adhesive, polyethylene, polypropylene, etc.) In a further embodiment, non-covalent immobilization with strip or cap can be achieved by using a peptide binding domain having a strong affinity for the material in or on the strip or cap (eg, a perhydrolytic enzyme-containing fusion protein coupled via an optional peptide spacer to the peptide binding domain). In another embodiment, the mouthguard is a deformable mouthguard. In another additional embodiment, the perhydrolase catalyst is immobilized in or on a deformable cap after dental impression formation.

As used herein, the expression "acetylxylanesterase" refers to an enzyme (EC 3.1.1.72; AXE) that catalyzes the deacetylation of acetylated xylans and other acetylated saccharides. Some of the enzymes illustrated herein, classified as acetylxylanesterases, are listed as having significant perhydrolytic activity.

As used herein, the terms "cephalosporin C deacetylase" and "cephalosporin C acetylhydrolase" refer to an enzyme (E.C. 3.1.1.41) that catalyzes the deacetylation of cephalosporins such as cephalosporin C and 7-aminocephalosporanic acid (Mitsushima et al., ( 1995) Appl Env Microbiol 61(6):2224-2229). This document provides the amino acid sequences of some cephalosporin C deacetylases that have significant perhydrolytic activity.

As used herein, the term " Bacillus subtilis ^ATCC® 31954™" refers to a bacterial cell deposited with the American Type Culture Collection (ATCC) with International Accession Number ^ATCC® 31954™. The enzyme described herein with significant perhydrolase activity from B. subtilis ^ATCC® 31954™ is shown as SEQ ID NO: 2 (See US Patent Application Publication No. 2010-0041752).

As used herein, " Thermotoga maritima MSB8" refers to a bacterial cell reported to have acetylxylanesterase activity ( ^GENBANK® NP_227893.1; see US Patent Application Publication No. 2008-0176299). The amino acid sequence of an enzyme having perhydrolase activity from Thermotoga maritima MSB8 is given as SEQ ID NO: 16.

As used herein, the terms "isolated nucleic acid molecule", "isolated polynucleotide", "isolated nucleic acid fragment" will be used interchangeably and refer to an RNA or DNA polymer that is single or double stranded, optionally containing synthetic, non-natural or altered nucleotide bases. . An isolated nucleic acid molecule in the form of a DNA polymer may consist of one or more segments of cDNA, genomic DNA, or synthetic DNA.

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

Amino acid three-letter
Abbreviation Single letter
Abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine gly G Histidine His H Isoleucine ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serene Ser S Threonine Thr T Tryptophan trp W Tyrosine Tyr Y Valine Val V Any amino acid or which is designated in this document xaa X

For example, it is well known in the art that changes in a gene that result in a chemically equivalent amino acid at a given site but do not affect the functional properties of the encoded protein are common. In the context of the present invention, substitutions are defined as changes within one of the following five groups:

1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gln;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic non-polar residues: Met, Leu, Ile, Val (Cys); And

5. Large aromatic residues: Phe, Tyr and Trp.

Thus, the codon for the amino acid alanine, a hydrophobic amino acid, can be replaced by a codon that codes for a different, less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine or isoleucine). Similarly, changes that result in the replacement of one negatively charged residue by another (such as aspartic acid for glutamic acid) or one positively charged residue by another (such as lysine for arginine) can also be expected to give a functionally equivalent product. In many cases, it is expected that nucleotide changes that result in changes in the N-terminal and C-terminal portions of a protein molecule will not result in changes in protein activity. Each of the proposed modifications is within the skill of the ordinary person skilled in the art, as well as determining the retention of the biological activity of the encoded products.

As used herein, the terms "signature motif" and "diagnostic motif" refer to conserved structures that are common to members of a family of enzymes that have a particular activity. The signature motif can be used to define and/or identify a family of structurally related enzymes that have similar enzymatic activity against a particular family of substrates. A signature motif can be a single sequence of contiguous amino acids or a collection of non-contiguous conservative motifs that together form a signature motif. Typically, conservative(e) motif(s) is(s) amino acid sequence. In one embodiment, the perhydrolytic enzyme contains the CE-7 carbohydrate esterase signature motif.

As used herein, the term "optimized codon", as it refers to genes or coding regions of nucleic acid molecules for transformation of different hosts, refers to changing codons in a gene or coding regions of nucleic acid molecules to reflect typical codon usage in a host organism. without changing the polypeptide that is encoded by DNA.

As used herein, "synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are cross-linked or annealed to form gene segments, which are then enzymatically assembled to build the entire gene. "Chemically synthesized", in relation to the DNA sequence, means that the nucleotide components have been assembled in vitro . Manual chemical synthesis of DNA can be performed using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available devices. Accordingly, genes can be tailored for optimal gene expression based on optimization of nucleotide sequences to reflect codon shift in the host cell. One skilled in the art will appreciate the likelihood of successful gene expression if the codon usage is biased towards host-preferred codons. Determination of preferred codons can be based on browsing genes derived from the host cell where sequence information is available.

As used herein, "gene" refers to a nucleic acid molecule that expresses a specific protein, including regulatory sequences before (5' non-coding sequences) and after (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene that occurs naturally with its own regulatory sequences. "Fusion gene" refers to any gene that is not a native gene, including regulatory and coding sequences that do not occur together in nature. Accordingly, the hybrid gene may contain regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences that are derived from the same source but are arranged in a different order than naturally occurring. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is not normally found in the host organism, but which is introduced into the host organism by gene transfer. The foreign genes may comprise native genes inserted into a non-native organism or hybrid genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

As used herein, "coding sequence" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within or downstream (3' non-coding sequences) of a coding sequence and which affect transcription, RNA processing or stability, or translation of an associated coding sequence. The regulatory sequences may include promoters, translational leader sequences, PHA processing sites, an effector-binding site, and a stem-loop structure.

As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid molecule so that the function of one affects the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence, ie. the coding sequence is under the transcriptional control of a promoter. Coding sequences can be operably linked to regulatory sequences in either sense or antisense orientation.

As used herein, the term "expression" refers to the transcription and persistent accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecule of the present invention. Expression can also refer to the translation of an mRNA into a polypeptide.

As used herein, "transformation" refers to the transfer of a nucleic acid molecule into the genome of a host organism, resulting in genetically stable inheritance. In the present invention, the genome of the host cell includes chromosomal and extrachromosomal (eg, plasmid) genes. Host organisms containing transformed nucleic acid molecules are referred to as "transgenic", "recombinant", or "transformed" organisms.

As used herein, the term "sequence analysis software" refers to any computer algorithm or software that is suitable for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Exemplary sequence analysis software will include, but is not limited to, GCG (Wisconsin Package, Version 9.0, Accelrys Software Corp., San Diego, CA), BLASTP, BLASTN, BLASTX (Altschul et al ., J. Mol. Biol. 215:403-410 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, WI 53715 USA), CLUSTALW (e.g. version 1.83; Thompson et al. , Nucleic Acids Research, 22( 22):4673-4680 (1994)), and the FASTA program including the Smith-Waterman algorithm (WR Pearson, Comput . Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20 Editor(s): Suhai, Sandor Publisher: Plenum, New York, NY), Vector NTI (Informax, Bethesda, MD) and Sequencher v. 4.05. In the context of this application, it should be understood that if sequence analysis software is used for the analysis, then the results of the analysis will be based on the "default values" of the specified program, unless otherwise specified. As used herein, "default values" will mean any set of values or options set by the software manufacturer that is initially loaded by the software when it is first run.

The term "body surface" refers to any surface of the human body that can be targeted by a beneficial agent, such as a peracid beneficial agent. The present methods and compositions are directed to oral hygiene applications and products. In this regard, the surface of the body includes the material/surface of the oral cavity. In one embodiment, the oral material is tooth enamel, dental plaque, soft tissues such as the cheeks, tongue, and gums, and biofilms in the oral cavity (eg, plaque in the mouth).

As used herein, the term "biological contaminants" refers to one or more unwanted and/or pathogenic biological entities, including, but not limited to, microorganisms, spores, viruses, prions, and mixtures thereof. In one embodiment, a method is provided for enzymatically obtaining an effective concentration of at least one peracid useful for reducing and/or eliminating the presence of biological contaminants.

As used herein, the term "decontaminate" refers to a method of destroying or preventing the growth of biological contaminants. As used herein, the term "decontaminant" refers to an agent that decontaminates by destroying, neutralizing, or inhibiting the growth of biological contaminants, which may include biological contaminants in the human oral cavity, such as microorganisms associated with dental caries, gingivitis, oral candidiasis, or periodontitis. . Used in this document, the term "decontamination" refers to the act or method of decontamination. As used herein, the term "antiseptic" refers to a chemical agent that inhibits the growth of disease-carrying microorganisms. In one aspect, the biological contaminants are pathogens.

Used in this document, the term "disinfection" means or refers to the restoration or preservation of health, usually by removing, preventing or controlling an agent that may be harmful to health. Used in this document, the term "disinfect" means the implementation of disinfection. As used herein, the term "sanitizer" means a disinfectant. Used in this document, the term "disinfection" refers to the act or method of disinfection.

Used in this document, the term "biocide" refers to a chemical agent, usually a broad spectrum, which inactivates or destroys microorganisms. A chemical that exhibits the ability to inactivate or kill microorganisms is described as having "biocidal" activity. Peracids may have biocidal activity. Typical alternative biocides may include, for example, chlorine, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, acrolein, amines, chlorinated phenolics, copper salts, organosulfur compounds, and quaternary ammonium salts.

As used herein, the phrase "minimum biocidal concentration" refers to the minimum concentration of a biocidal agent that, over a specified contact time, will produce the desired lethal, irreversible reduction in the viable population of the target microorganism. Efficacy can be measured by the log ₁₀ reduction in viable microorganisms after treatment. In one aspect, the targeted reduction in viable microorganisms after treatment is at least a 3-log ₁₀ reduction, more preferably at least a 4-log ₁₀ reduction, and most preferably at least a 5-log ₁₀ reduction. In another aspect, the minimum biocidal concentration is at least 6-log ₁₀ reduction in viable microorganism cells.

As used herein, "cleaning compositions" and "cleaning compositions" refer to compositions that find use in removing unwanted compounds from teeth (mouth rinses, toothpastes, etc.). The term encompasses any materials/compounds chosen for the specific type of cleaning composition required and product form (e.g., liquid, paste, gel, emulsion, granule, or spray formulation), provided the composition is compatible with perhydrolase and other enzyme(s). used(s) in the composition.

As used herein, "oral cleaning compositions" refers to dentifrices, toothpastes, tooth gels, tooth powders, mouthwashes, mouth sprays, mouth gels, chewing gums, lozenges, sachets, tablets, biogels. , prophylactic pastes, solutions for therapeutic and prophylactic dental care, etc. Oral care compositions that find use in combination with the perhydrolases of the present invention are well known in the art (see, for example, US Pat. in full).

As used herein, "pharmaceutically acceptable" means that the drugs, drug products, and/or inert ingredients that the term describes are suitable for use in contact with tissues in humans and other animals without undue toxicity, incompatibility, instability, irritation, allergic reaction, and etc. according to an acceptable benefit/risk ratio.

As used herein, personal care products means products used in cleaning, brightening, and/or disinfecting hair, skin, scalp, and teeth, including, but not limited to, shampoos, body lotions, shower gels, topical moisturizers, toothpaste, mouth rinses, mouthwashes, anti-cavity rinses, and/or other topical cleansers. In some particularly preferred embodiments, these products are used in humans, while in other embodiments, these products find applications in other animals (eg, veterinary applications).

As used herein, the terms "teeth whitening" and "teeth whitening" are used interchangeably to refer to brightening (eg, whitening) a tooth or teeth. The term is intended to cover any method suitable for whitening teeth, including the present invention, as well as chemical treatments, mild acid treatments, abrasive teeth whitening, and laser tooth whitening. In particularly preferred embodiments, the present invention provides perhydrolase and perhydrolase-containing compositions suitable for tooth whitening.

As used herein, "intrinsic stains" in teeth refer to the development of color from chromogens in the enamel and underlying dentine. The intrinsic color of human teeth tends to become more yellow with age due to thinning of the enamel and darkening of the underlying yellow dentin. Removal of the characteristic stain usually requires the use of peroxides or other oxidizing agents that penetrate the enamel and discolour the internal chromogens.

In contrast to internal stains, "external stains" form on the surface of teeth when external chromogenic materials bind to the enamel, usually in plaque that naturally coats the teeth. Most people accumulate some level of unsightly extrinsic stains on their teeth over time. This staining process is stimulated by factors such as: (1) the ingestion of tannin-containing foods and drinks such as coffee, tea, or red wine; (2) use of tobacco products; and/or (3) exposure to certain cationic substances (eg, tin, iron, and chlorhexidine). These substances tend to adhere to the hydroxyapatite structure of the enamel, resulting in discoloration and a concomitant decrease in tooth whiteness. After a few years, external stains can penetrate the enamel layer and lead to the formation of internal stains.

As used herein, the term "deodorization" means the elimination or prevention of an unpleasant odor.

As used herein, the term "discolor" or "bleaching" refers to the process of removing a stain from the surface of the oral cavity. The spot(s) may be inner spots, outer spots, or a combination thereof.

As used herein, the term "improved performance" of a perhydrolase-containing composition is defined as an increase in the clearance of bleach-sensitive stains compared to other compositions, as measured by standard methods in the dental art. In specific embodiments, the implementation of the perhydrolase of the present invention provides improved efficiency of oxidation and removal of colored spots. In further embodiments, the perhydrolase of the present invention provides improved stain removal and/or bleaching performance.

As used herein, "effective amount of perhydrolase enzyme" refers to the amount of perhydrolase enzyme required to achieve the enzymatic activity desired in a particular application. Such effective amounts will be readily determined by one of ordinary skill in the art based on many factors such as the particular enzyme variant used, the cleaning application, the particular cleaning composition composition and whether a liquid or non-liquid (e.g., emulsion) composition is desired, etc. .

As used herein, the terms "peroxy oxygen source" refer to compounds capable of providing hydrogen peroxide at a concentration of approximately 1 mM or more when in aqueous solution, including, but not limited to, hydrogen peroxide, hydrogen peroxide adducts (e.g., hydrogen peroxide adducts and urea (carbamide peroxide)), perborates and percarbonates. Described herein, the concentration of hydrogen peroxide provided by the connection with the oxygen of the peroxy group in the aqueous reaction composition is initially at least 0.1 mm or more when combining the reaction components. In one embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 0.5 mm. In one embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 1 mM. In another embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 10 mM. In another embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 100 mm. In another embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 200 mm. In another embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 500 mm or more. In another embodiment, the concentration of hydrogen peroxide in the aqueous reaction composition is at least 1000 mm or more. The molar ratio of hydrogen peroxide to enzyme substrate, e.g., triglyceride, (H ₂ O ₂ :substrate) in the aqueous reaction composition may be from about 0.002 to 20, preferably from about 0.1 to 10, and most preferably from about 0.5 to 5 .

Used in this document, the term "oligosaccharide" refers to compounds containing from 2 to at least 24 monosaccharide units connected by glycosidic bonds. The term "monosaccharide" refers to a compound with the empirical formula (CH ₂ O) _n where n≥3, the carbon skeleton is unbranched, every carbon atom except one contains a hydroxyl group, and the remaining carbon atom is aldehyde or ketone at carbon 1 The term "monosaccharide" also refers to intracellular cyclic hemiacetal or hemiketal forms.

Used in this document, the term "filler" refers to an inactive substance used as a carrier for the active ingredients in the composition. The excipient can be used to stabilize the active ingredient in the formulation, such as the storage stability of the active ingredient. Fillers are also sometimes used to bulk up formulations that contain active ingredients. The "active ingredient" described herein may be an enzyme having perhydrolytic activity, a peracid produced by a perhydrolytic enzyme under suitable reaction conditions, or a combination thereof.

The term "substantially free of water" will refer to the concentration of water in the formulation that does not adversely affect the storage stability of the enzyme or enzyme powder in the presence of a carboxylic acid ester. The carboxylic acid ester may contain a very low concentration of water, for example, triacetin typically contains from 180 ppm. up to 300 ppm water. In one embodiment, the perhydrolytic enzyme is stored in a carboxylic acid ester substrate that is substantially free of water. In a further embodiment, "substantially free of water" may mean less than 2000 ppm, preferably less than 1000 ppm, more preferably less than 500 ppm. and even more preferably less than 250 ppm. water in a formulation containing an enzyme (or enzyme powder) and a carboxylic acid ester. In one embodiment, the perhydrolytic enzyme can be stored in an aqueous solution if the generating system is designed so that the enzyme is stable in an aqueous solution (eg, the solution does not contain a significant concentration of a carboxylic acid ester substrate that can be hydrolyzed by the enzyme upon storage). In one embodiment, the perhydrolytic enzyme can be stored in a mixture containing a carboxylic acid ester substrate that is substantially free of water and one or more buffers (e.g., sodium and/or potassium bicarbonate, citrate, acetate, phosphate, pyrophosphate , methylphosphonate, succinate, malate, fumarate, tartrate and maleate).

Enzymes with perhydrolytic activity

Enzymes having perhydrolytic activity may include certain enzymes classified as lipases, proteases, esterases, acyltransferases, aryltransferases, carbohydrate esterases, and combinations, provided that the enzyme has perhydrolytic activity against one or more of the substrates present. Examples may include, but are not limited to, perhydrolytic proteases (a variant of Carlsberg's subtilisin; US Pat. No. 7,510,859), perhydrolytic arylesterases ( Pseudomonas fluorescens; SEQ ID NO: 477; US Pat. and 478; US Pat. No. 7,754,460; WO2005/056782; and EP1689859 B1) and carbohydrate perhydrolase esterases. In a preferred aspect, the carbohydrate perhydrolytic esterase is CE-7 carbohydrate esterase.

In one embodiment, suitable perhydrolases may include enzymes containing an amino acid sequence with at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to any of the amino acid sequences provided herein, provided that perhydrolases other than CE-7 are limited to targeted perhydrolase applications (ie, perhydrolytic enzymes not belonging to the CE-7 family of carbohydrate esterases are used in the form of a fusion protein containing at least one peptide targeting domain).

In another embodiment, suitable perhydrolases may include enzymes containing an amino acid sequence of at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with SEQ ID NOs: 460, 477, and 478, provided that perhydrolases other than CE-7 are limited to targeted perhydrolase applications ( ie, perhydrolytic enzymes not belonging to the CE-7 family of carbohydrate esterases are used in the form of a fusion protein containing at least one peptide targeting domain). It is understood that comparisons of percent identity and sequence alignments used to identify substantially similar perhydrolytic enzymes are made against the portion of the fusion protein containing the perhydrolytic enzyme (ie, targeting domains and linkers are not included).

In another embodiment, the fusion protein comprises a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Mycobacterium smegmatis arylesterase S54V shown in SEQ ID NO: 460.

In one embodiment, the fusion protein contains a perhydrolytic esterase from Pseudomonas fluorescens . In another embodiment, the fusion protein contains a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Pseudomonas fluorescens esterase shown in SEQ ID NO: 477.

In another embodiment, the fusion protein comprises a perhydrolytic enzyme with an amino acid sequence selected from the group consisting of SEQ ID NOs: 424, 425, 426, 427, 428, 429, 430, 437, 438, 439, 440, 441, 442, 443 , 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 476 , 477, 478 and 479.

In another embodiment, substantially similar perhydrolytic enzymes may include enzymes encoded by polynucleotide sequences that hybridize under very stringent hybridization conditions (0.1X SSC, 0.1% SDS, 65°C and washed with 2X SSC, 0.1% SDS with followed by a final wash with 0.1X SSC, 0.1% SDS, 65°C) with polynucleotide sequences encoding the present perhydrolytic enzymes, provided that perhydrolases other than CE-7 are limited to targeted perhydrolase applications (i.e., perhydrolytic enzymes not belonging to the CE-7 family of carbohydrate esterases are used in the form of a fusion protein containing at least one peptide targeting domain).

Perhydrolases CE-7

In a preferred embodiment, the oral care compositions and method comprise enzymes having perhydrolytic activity that are structurally classified as members of the carbohydrate family esterases, enzyme family 7 (CE-7 family) (see, Coutinho, PM, Henrissat, B. “Carbohydrate-active enzymes: an integrated database approach” in Recent Advances in Carbohydrate Bioengineering , HJ Gilbert, G. Davies, B. Henrissat and B. Svensson eds., (1999) The Royal Society of Chemistry, Cambridge, pp. 3- 12.). The CE-7 family of enzymes has been shown to be particularly effective in producing peroxycarboxylic acids from a range of carboxylic acid ester substrates when combined with a peroxy oxygen source (U.S. Patents 7,794,378, 7,951,566, 7,723,083, and 7,964,378 and U.S. Patent Application Publication Nos. 2008- 0176299, 2010-0087529, 2011-0081693 and 2011-0236335, DiCosimo et al ., each of which is incorporated herein by reference).

Members of the CE-7 family include cephalosporin C deacetylases (CAH, E.C. 3.1.1.41) and acetylxylanesterases (AXE; EC 3.1.1.72). Members of the CE-7 esterase family share a conserved signature motif (Vincent et al ., J. Mol. Biol ., 330:593-606 (2003)). Perhydrolases containing the signature motif CE-7 ("CE-7 perhydrolases") and/or a substantially similar structure are suitable for use in the compositions and methods described herein. Means for detecting substantially similar biological molecules are well known in the art (eg, protocols for sequence alignment, nucleic acid hybridization, and/or the presence of a conserved signature motif). In one aspect, the perhydrolase comprises an enzyme characterized by the signature motif CE-7 and 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%, 97%, 98%, or 99% amino acid identity with one of the sequences provided herein.

As used herein, the phrase "enzyme structurally classified as CE-7 enzyme", "CE-7 perhydrolase" and "structurally classified as carbohydrate esterase family enzyme 7" will be used in reference to enzymes having perhydrolytic activity that are structurally classified as carbohydrate esterase CE -7. An enzyme family can be identified by the presence of a signature motif (Vincent et al., supra). The signature motif for esterases CE-7 includes three conservative motifs (numbering of residue positions relative to the reference sequence SEQ ID NO: 2; CE-7 perhydrolase from B. subtilis ATCC ^® 31954™):

a) Arg118-Gly119-Gln120;

b) Gly179-Xaa180 -Ser181 -Gln182-Gly183; And

c) His298 -Glu299.

Typically, Xaa at amino acid residue position 180 is glycine, alanine, proline, tryptophan, or threonine. Two of the three amino acid residues belonging to the catalytic triad are shown in bold. In one embodiment, Xaa at amino acid residue position 180 is selected from the group consisting of glycine, alanine, proline, tryptophan, and threonine.

Further analysis of conserved motifs in the CE-7 family of carbohydrate esterases reveals the presence of an additional conserved motif (LXD at amino acid positions 267-269 in SEQ ID NO: 2) that can be used to further define a perhydrolase belonging to the CE-7 family of carbohydrate esterases. In a further embodiment, the signature motif defined above may include an additional (fourth) conservative motif defined as follows:

Leu267-Xaa268- Asp269 .

Xaa at amino acid residue position 268 is typically isoleucine, valine, or methionine. The fourth motif includes an aspartic acid residue (in bold) belonging to the catalytic triad ( Ser181-Asp269-His298 ).

The CE-7 perhydrolases may be in the form of fusion proteins with at least one peptide component having an affinity for at least one surface of the body. In one embodiment, all alignments used to determine if a targeted perhydrolase (fusion protein) contains the CE-7 signature motif will be based on the amino acid sequence of the perhydrolytic enzyme without the peptide component having an affinity for the body surface.

A number of well known global alignment algorithms (ie, sequence analysis software) can be used to align two or more amino acid sequences representing enzymes having perhydrolase activity to determine if the enzyme contains a true signature motif. The aligned sequence(s) is compared to a reference sequence (SEQ ID NO: 2) to determine the existence of a signature motif. In one embodiment, a CLUSTAL alignment (such as CLUSTALW) with a reference amino acid sequence (as used herein, the perhydrolase sequence (SEQ ID NO: 2) from Bacillus subtilis ^ATCC® 31954™) is used to detect perhydrolases belonging to the CE-7 family of esterases. . The relative numbering of conservative amino acid residues is based on the numbering of the reference amino acid sequence residues in order to take into account small insertions or deletions (eg, typically five amino acids or less) within the aligned sequence.

Examples of other suitable algorithms that can be used to identify sequences containing the present signature motif (when compared to a reference sequence) include, but are not limited to, Needleman and Wunsch (J. Mol. Biol. 48, 443-453 (1970); tool global alignment) and Smith-Waterman (J. Mol. Biol. 147, 195-197 (1981); local alignment tool). In one embodiment, the Smith-Waterman alignment is performed using the default settings. An example of suitable default parameters includes applying the BLOSUM62 substitution matrix with GAP open penalty = 10 and GAP extension penalty = 0.5.

Comparison of the overall percent identity among perhydrolases shows that enzymes that share up to 33% amino acid identity with SEQ ID NO: 2 (while retaining the signature motif) exhibit significant perhydrolase activity and are structurally classified as CE-7 carbohydrate esterases. In one embodiment, suitable perhydrolases include enzymes containing the signature motif CE-7 and characterized by 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 identity with SEQ ID NO: 2.

Examples of suitable CE-7 carbohydrate esterases having perhydrolytic activity include, but are not limited to, enzymes with an amino acid sequence such as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 , 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62 , 64, 424, 437, and 476. In one embodiment, the enzyme comprises an amino acid sequence selected from the group consisting of 14, 16, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437 and 476.

As used herein, the term "CE-7 variant", "variant perhydrolase", or "variant" will refer to CE-7 perhydrolases with a genetic modification that results in at least one amino acid addition, deletion, and/or substitution compared to with the appropriate enzyme (usually a wild-type enzyme) from which the variant was derived; provided that the signature CE-7 motif and associated perhydrolytic activity are preserved. Variant CE-7 perhydrolases can also be used in the present compositions and methods. Examples of CE-7 variants are given as SEQ ID NOs: 27, 28, 29, 30, 31, 32, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437 and 476. In one embodiment, the variants may include SEQ ID NOs: 27, 28, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437, and 476.

One of skill in the art will appreciate that substantially similar CE-7 perhydrolase sequences (retaining signature motifs) can also be used in the present compositions and methods. In one embodiment, substantially similar sequences are determined by their ability to hybridize under high stringency conditions to nucleic acid molecules linked to the sequences illustrated herein. In another embodiment, sequence alignment algorithms can be used to determine essentially similar enzymes based on percent identity with DNA sequences or amino acid sequences recited herein.

As used herein, a nucleic acid molecule "can hybridize" with another nucleic acid molecule, such as cDNA, genomic DNA, or RNA, if one strand of the first molecule can complementarily combine with the other molecule under appropriate conditions of temperature and ionic strength of the solution. Hybridization and wash conditions are well known and are illustrated in Sambrook, J. and Russell, D., T. Molecular Cloning: A Laboratory Manual , Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the "stringency" of the hybridization. Stringent conditions can be adjusted to select from moderately similar molecules, such as homologous sequences from distantly related organisms, to highly similar molecules, such as genes that copy functional enzymes from closely related organisms. The stringency conditions are usually determined by post-hybridization washes. One set of preferred conditions involves a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeating with 2X SSC, 0.5% SDS at 45°C for 30 min, and then repeating two times with 0.2X SSC, 0.5% SDS at 50°C for 30 min. In a more preferred set of conditions, higher temperatures are used where the washes are identical to those described above, except that the temperature of the final two 30 minute washes in 0.2X SSC, 0.5% SDS was raised to 60°C. Another preferred set of extremely stringent hybridization conditions is 0.1X SSC, 0.1% SDS, 65°C and washes with 2X SSC, 0.1% SDS followed by a final wash of 0.1X SSC, 0.1% SDS, 65°C.

Hybridization requires two nucleic acids to contain complementary sequences, although base mismatches are possible depending on the stringency of the hybridization. The appropriate stringency of hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids with such sequences. The relative stability (corresponding to a larger T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids larger than 100 nucleotides in length, equations have been derived to calculate T m (Sambrook and Russell, formerly ). For hybridizations with shorter nucleic acids, i.e. oligonucleotides, the position of mismatches becomes more important, and the length of an oligonucleotide determines its specificity (Sambrook and Russell, formerly ). In one aspect, the hybridizable nucleic acid is at least 10 nucleotides in length. Preferably, the minimum length for a hybridizable nucleic acid is at least 15 nucleotides in length, more preferably at least about 20 nucleotides in length, even more preferably at least 30 nucleotides in length, even more preferably at least 300 nucleotides in length. and most preferably at least 800 nucleotides in length. In addition, one skilled in the art will appreciate that the temperature and salt concentration of the wash solution can be adjusted as necessary according to factors such as, for example, the length of the probe.

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 sequence comparison. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by matching between strands of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignments and percent identity calculations can be made using the Megalign program from the LASERGENE bioinformatics data calculation software package (DNASTAR Inc., Madison, WI), the AlignX program from Vector NTI, version 7.0 (Informax, Inc., Bethesda, MD), or the EMBOSS open source software package (EMBL-EBI; Rice et al., Trends in Genetics 16, (6):276-277 (2000)). Multiple sequence alignment can be performed using a Clustal alignment method (such as CLUSTALW; eg version 1.83) (Higgins and Sharp, CABIOS, 5:151-153 (1989); Higgins et al., Nucleic Acids Res. 22:4673-4680 ( 1994); and Chenna et al., Nucleic Acids Res 31 (13):3497-500 (2003)), available from the European Molecular Biology Laboratory through the European Bioinformatics Institute) with default parameters. Suitable parameters for CLUSTALW protein alignments include GAP Existence penalty = 15, GAP extension = 0.2, template = Gonnet (e.g., Gonnet250), protein ENDGAP = -1, protein GAPDIST = 4, and KTUPLE = 1. In one embodiment, fast or slow leveling is applied with default settings, where slow leveling is preferred. Alternatively, the parameters used in the CLUSTALW method (e.g., version 1.83) can be modified to also use KTUPLE = 1, GAP PENALTY = 10, GAP extension = 1, matrices = BLOSUM (e.g., BLOSUM64), WINDOW = 5, and TOP DIAGONALS SAVED = 5.

In one aspect, suitable isolated nucleic acid molecules encode a polypeptide with an amino acid sequence that is 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% identical to the amino acid sequences described in this document. In another aspect, suitable isolated nucleic acid molecules encode a polypeptide with an amino acid sequence that is 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% identical to the amino acid sequences described herein, with the proviso that the polypeptide retains the signature motif CE- 7. Suitable nucleic acid molecules not only have the homologies described above, but also typically encode a polypeptide having about 210-340 amino acids in length, from about 300 to about 340 amino acids, preferably from about 310 to about 330 amino acids, and most preferably from about 318 to approximately 325 amino acids in length, where each polypeptide is characterized as having perhydrolytic activity.

Targeted perhydrolases

As used herein, the terms "targeted perhydrolase" and "targeted enzyme having perhydrolytic activity" will refer to fusion proteins containing at least one perhydrolytic enzyme (wild-type or variant) fused/fused to at least one peptide component, having an affinity for a target surface, preferably a target surface of an organism. The perhydrolytic enzyme in the targeted perhydrolase can be any perhydrolytic enzyme and can include lipases, proteases, esterases, acyltransferases, arylesterases, carbohydrate esterases, and combinations, so long as the enzyme has perhydrolytic activity on one or more of the substrates present. Examples may include, but are not limited to, perhydrolytic proteases (eg, subtilisin variant, US Pat. No. 7,510,859), perhydrolytic esterases (eg, Pseudomonas fluorescens ; US Pat. No. 7,384,787; SEQ ID NO: 477), and perhydrolytic arylesterases (eg, Mycobacterium smegmatis ; 7754460; WO2005/056782; and EP1689859 B1; SEQ ID NOs: 460 [S54V variant] and 478 [wild type]).

In one embodiment, the fusion protein contains a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Mycobacterium smegmatis arylesterase S54V shown in SEQ ID NO: 460.

In one embodiment, the fusion protein contains a perhydrolytic esterase from Pseudomonas fluorescens . In another embodiment, the fusion protein contains a perhydrolytic enzyme with an amino acid sequence that is at least 95% identical to the Pseudomonas fluorescens esterase shown in SEQ ID NO: 477.

In another embodiment, the fusion protein comprises a perhydrolytic enzyme with an amino acid sequence selected from the group consisting of SEQ ID NOs: 424, 425, 426, 427, 428, 429, 430, 437, 438, 439, 440, 441, 442, 443 , 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 476 , 477, 478 and 479.

As used herein, the terms “peptide component”, “oral surface affinity peptide component”, and “OCBD” will refer to the fusion protein component that is not part of a perhydrolytic enzyme containing at least one polymer of two or more amino acids. , connected by a peptide bond; where the component has an affinity for the target surface of the oral cavity.

In one embodiment, the peptide component having an affinity for the body surface may be an antibody, a Fab fragment of an antibody, a single chain variable fragment (scFv) antibody, a Camelidae antibody (Muyldermans, S., Rev. Mol. Biotechnol. 2001) 74:277- 302), a non-antibody scaffold display protein (Hosse et al ., Prot. Sci . (2006) 15(1): 14-27 and Binz, H. et al. (2005) Nature Biotechnology 23, 1257-1268 for an overview of various scaffold approaches), or a single chain polypeptide without an immunoglobulin fold. In another aspect, the peptide component having an affinity for a body surface is a single chain peptide without an immunoglobulin fold (i.e., a body surface binding peptide or body surface binding domain comprising at least one body surface binding peptide having affinity for the oral surface). In a preferred embodiment, the peptide component is a single chain peptide containing one or more body surface-binding peptides with oral surface affinity.

The peptide component with affinity for the oral surface can be separated from the perhydrolytic enzyme using an optional peptide linker. Certain peptide linkers/spacers are 1 to 100 or 1 to 50 amino acids in length. In some embodiments, the peptide spacers are about 1 to about 25, 3 to about 40 amino acids, or 3 to about 30 amino acids in length. In other embodiments, spacers are present that are from about 5 to about 20 amino acids in length. Multiple peptide linkers may be used. In one embodiment, at least one peptide linker is present and may be repeated up to 10 times.

Peptides previously defined as having an affinity for one surface of the body may have an equal affinity for the surface of the oral cavity. In this regard, the hybrid protein may contain at least one, previously described as having an affinity for another surface of the body, such as hair (SEQ ID NOS 65-221, 271 and 368); skin (SEQ ID NO: 217-269) or nail (SEQ ID NO: 270-271). In one embodiment, the fusion protein comprises at least one oral surface binding peptide from the group consisting of SEQ ID NOs: 272-382 and 399-422. In one embodiment, the fusion peptide comprises at least one oral surface-binding peptide selected from the group consisting of SEQ ID NOs: 272-382, 399-410, and 412-422; where SEQ ID NO 272-291 and 312-382 have an affinity for plaque on the teeth; SEQ ID NOs: 292-311 have an affinity for tooth enamel, and SEQ ID NOs 399-410 and 412-422 have an affinity for tooth enamel or dental plaque. Some of the body surface-binding peptides may have a strong affinity for several body surfaces and, therefore, can be used to target perhydrolytic enzymes to various body surfaces. In another embodiment, the fusion peptide may include any body surface-binding peptide designed to have an electrostatic attraction to a target body surface (e.g., a body surface-binding peptide designed to electrostatically bind to a target body surface).

In another embodiment, the target surface is a material that is part of the packaging process and/or method of delivery to the oral cavity. The peptide component is selected for its affinity for the material or materials used, such as polymers, plastics and films. The targeted perhydrolase fusion protein design provides for the controlled delivery and removal of the perhydrolase from the user by retaining it on a removable device such as, but not limited to, a mouthguard or mouth strip.

Targeted CE-7 perhydrolases

In a preferred embodiment, the "targeted perhydrolase" is a targeted CE-7 carbohydrate esterase having perhydrolytic activity. As used herein, the terms "targeted CE-7 perhydrolase" and "targeted CE-7 carbohydrate esterase" will refer to fusion proteins containing at least one CE-7 perhydrolase (wild-type or variant perhydrolase) fused/fused to at least with one peptide component having an affinity for the target surface, preferably the target surface of the organism. The peptide component having an affinity for the body surface may be any of those described above. In a preferred aspect, the peptide component of the targeted CE-7 perhydrolase is a single chain peptide without immunoglobulin fold (i.e., a body surface binding peptide or body surface binding domain containing at least one body surface binding peptide having affinity for the oral surface). In a preferred embodiment, the peptide component is a single chain peptide containing one or more body surface-binding peptides with oral surface affinity.

The peptide component with affinity for the oral surface can be separated from the CE-7 perhydrolase using an optional peptide linker. Certain peptide linkers/spacers are 1 to 100 or 1 to 50 amino acids in length. In some embodiments, the peptide spacers are about 1 to about 25, 3 to about 40 amino acids, or 3 to about 30 amino acids in length. In other embodiments, spacers are present that are from about 5 to about 20 amino acids in length. Multiple peptide linkers may be used.

In this regard, examples of targeted CE-7 perhydrolases may include, but are not limited to, any of the CE-7 perhydrolases with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437 and 476, coupled to a peptide moiety having an affinity for the oral surface. In a preferred embodiment, examples of targeted perhydrolases may include, but are not limited to, any of the CE-7 perhydrolases with an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437, and 476, coupled to one or more body surface-binding peptides with oral surface affinity (optionally via a peptide spacer).

In another embodiment, targeted CE-7 perhydrolases may contain peptides previously identified as having an affinity for one body surface and may have an equal affinity for the oral surface. In this regard, the hybrid protein may contain at least one previously described as having an affinity for another surface of the body, such as hair (SEQ ID NOS: 65-221, 271 and 368); skin (SEQ ID NO: 217-269) or nail (SEQ ID NO: 270-271). In one embodiment, the fusion protein comprises at least one oral surface-binding peptide from the group consisting of SEQ ID NOs: 272-382 and 399-422. In one embodiment, the CE-7 perhydrolase fusion peptide comprises at least one oral surface binding peptide selected from the group consisting of SEQ ID NOs: 272-382, 399-410, and 412-422; where SEQ ID NOs 272-291 and 312-382 have an affinity for dental plaque, SEQ ID NOs: 292-311 have an affinity for tooth enamel, and SEQ ID NOs 399-410 and 412-422 have an affinity for tooth enamel or plaque teeth. Some of the body surface-binding peptides may have a strong affinity for multiple body surfaces and therefore can be used to target perhydrolytic enzymes to various body surfaces. In another embodiment, the CE-7 perhydrolase fusion peptide may include any body surface binding peptide designed to have an electrostatic attraction to a target body surface (e.g., a body surface binding peptide designed to electrostatically bind to a target body surface).

In another embodiment, the target surface is a material that is part of the packaging and or oral delivery process. The peptide component is selected for its affinity for the material or materials used, such as polymers, plastics and films. The CE-7 targeted perhydrolase fusion protein design provides for controlled delivery and removal of the perhydrolase from the user by retaining it on a removable device such as a mouthguard or mouth strip.

Peptides with affinity for the surface of the body

Single-chain peptides without immunoglobulin folds that are capable of binding to the oral surface are referred to as "oral surface-binding peptides" (OCBP) and may include, for example, peptides that bind to the tooth surface (tooth-binding peptides), peptides that have an affinity for soft tissue, such as gums, or peptides that have an affinity for an orally acceptable material that is safe for use in the oral cavity. Tooth-binding peptides can include peptides having an affinity for tooth enamel ("enamel-binding peptides") and peptides having an affinity for dental plaque ("dental plaque-binding peptides").

This document provides a non-limiting list of peptides having an affinity for at least one surface of the body, including those that have an affinity for hair (hair-binding peptides with an amino acid sequence selected from the group consisting of SEQ ID NO: 65-221, 271 and 368), skin (skin-binding peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 217-269), and nails (nail-binding peptides comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 270-271). Examples of peptides having affinity for the oral surface (oral-binding peptides) contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 272-382 and 399-422. In a preferred aspect, peptides having affinity for oral surfaces are selected from the group consisting of SEQ ID NOs: 272-382, 399-410 and 412-422; where SEQ ID NO 272-291 and 312-382 have an affinity for plaque on the teeth; SEQ ID NOs: 292-311 have an affinity for tooth enamel, while SEQ ID NOs: 399-410 and 412-422 have an affinity for tooth enamel or dental plaque.

In one embodiment, the peptide, which may also have an affinity for the oral surface, may include one or more of SEQ ID NOs: 65-382, 399-410, and 412-422. Preferably, the peptides used in the present compositions and methods are selected from the group consisting of SEQ ID NOs: 272-382, 399-410 and 412-422. In another embodiment, the oral surface-binding peptides may include skin-binding peptides for certain oral surfaces (eg, gums). In another embodiment, the fusion peptide may include any body surface-binding peptide designed to have an electrostatic attraction to a target body surface (e.g., a body surface-binding peptide designed to electrostatically bind to a target body surface).

In another embodiment, the present compositions and methods comprise at least one oral surface-binding peptide with an amino acid sequence selected from the group consisting of SEQ ID NOs: 399-410 and 412-422.

In some embodiments, the oral surface binding domains consist of oral surface binding peptides that are up to about 60 amino acids in length. In one embodiment, the oral surface-binding peptides are 5 to 60 amino acids in length. In other embodiments, the surface-binding peptides are 7 to 50 amino acids in length, or 7 to 30 amino acids in length. In still other embodiments, such oral surface-binding peptides are provided that are 7 to 27 amino acids in length.

While hybrid peptides containing oral surface-binding peptides are certain embodiments of the present invention, in other embodiments of the present invention, multiple oral surface-binding peptides may advantageously be used. Set inclusion, i.e. two or more oral surface-binding peptides can yield a peptide component that is, for example, even more reliable than such binding elements that include a single oral surface-binding peptide. In some embodiments, the oral surface binding domains (i.e., multiple, i.e., two or more oral surface binding peptides) comprise 2 to about 50 or 2 to about 25 oral surface binding peptides. oral cavity. Other embodiments include those oral surface binding domains that include 2 to about 10 or 2 to 5 oral surface binding peptides.

Multiple binding elements (ie, oral surface binding peptides or oral surface binding domains) can be connected together directly, or they can be connected together using peptide spacers. Certain peptide spacers/linkers are 1 to 100 or 1 to 50 amino acids in length. In some embodiments, the peptide spacers are about 1 to about 25, 3 to about 40, or 3 to about 30 amino acids in length. In other embodiments, spacers are provided that are 1 to about 20, or about 5 to about 20 amino acids in length.

The oral surface binding domains and the shorter oral surface binding peptides of which they are composed can be identified by any number of methods known to those skilled in the art, including, for example, any known biopanning techniques, such as , phage display, bacterial display, yeast display, ribosome display, mRNA display, and combinations thereof. Typically, a random or nearly random (if there is a standard error) polypeptide library is biopanned against a target body surface to determine which peptides within the library have affinity for the target body surface.

The generation of random peptide libraries is well known and can be accomplished using a variety of techniques including bacterial display (Kemp, DJ; Proc. Natl. Acad. Sci. USA 78(7):4520-4524 (1981) and Helfman et al ., Proc Natl Acad Sci USA 80(1):31-35, (1983)), yeast display (Chien et al ., Proc Natl Acad Sci USA 88(21):9578-82 (1991)), combinatorial solid phase peptide synthesis (US patent No. 5449754, US patent No. 5480971, US patent No. 5585275, US patent No. 5639603) and phage display technology (US patent No. 5223409, US patent No. 5403484, US patent No. 5571698, US patent No. 5837500); ribosome display (US Pat. No. 5,643,768; US Pat. No. 5,658,754 and US Pat. No. 7,074,557) and mRNA display technology (PROFUSION™; see US Pat. Nos. 6,258,558; 6,518,018; 6,281,344; 6,214,553; ; 6416950; 6429300; 7078197 and 6436665).

Binding affinity

The peptide component having an affinity for the oral surface has a binding affinity for the oral surface of 10 ^-5 molar (M) or less. In certain embodiments, the peptide component is one or more oral surface binding peptides and/or domain(s) having a binding affinity for human hair, skin, nail, or oral cavity of 10 ^-5 molarity (M) or less. In some embodiments, the binding peptides or domains will have a binding affinity value of 10 ^-5 molar (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 the binding peptide with its respective substrate in the case of the surface of the human oral cavity (gums, teeth, etc.). Binding affinity can be determined or measured in terms of the dissociation constant (“K _D ”) of the binding peptide, or “MB ₅₀ ”.

“K _D ” corresponds to the concentration of the peptide at which the binding site on the target is half occupied, i.e. when the concentration of target with bound peptide (bound target material) is equal to the concentration of target with no bound peptide. The lower the dissociation constant, the stronger the peptide binds. For example, a peptide with a nanomolar (nM) dissociation constant binds more strongly than a peptide with a micromolar (μM) dissociation constant. Certain embodiments of the present invention will have a K _D value of 10 ^-5 or less.

“MB ₅₀ ” refers to the concentration of the binding peptide that gives a signal that is 50% of the maximum signal obtained in the ELISA binding assay. See, for example, Example 3 of US Patent Application Publication No. 2005/022683; which is incorporated herein by reference. MB ₅₀ gives an indication of the strength of the binding interaction or the binding affinity of the components of the complex. The lower the MB ₅₀ value, the stronger, i. "better", the interaction of the peptide with its respective substrate. For example, a nanomolar (nM) MB ₅₀ peptide binds more strongly than a micromolar (μM) MB ₅₀ peptide. Certain embodiments of the present invention will have an MB ₅₀ value of 10 ^-5 M or less.

In some embodiments, a peptide component having an affinity for the oral surface may have a binding affinity, which is measured using K values_D or MB₅₀, less than or equal to approximately 10^-5 M less than or equal to approximately 10^-6 M less than or equal to approximately 10^-7 M less than or equal to approximately 10^-8 M less than or equal to approximately 10^-9 M or less than or equal to approximately 10^-10 M.

In some embodiments, oral surface binding peptides and/or oral surface binding domains may have a binding affinity as measured by K _D or MB ₅₀ values less than or equal to about 10 ^-5 M, less than or 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" will refer to a binding affinity having a K _D or MB ₅₀ value 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.

Multicomponent systems for the production of peroxycarboxylic acid

The design of systems and methods for separating and combining multiple active components is known in the art and will primarily depend on the physical form of the individual reactive components. For example, in systems with multiple active liquid media (liquid-liquid), multi-chamber containers with a dispenser or two-phase systems are commonly used (for example, US patent application No. 2005/0139608; US patent No. 5398846; US patent No. 5624634; US patent 6391840; EP 0807156B1; US Patent Application Publication No. 2005/0008526 and PCT Publication No. WO 00/61713), such as those found in certain bleaching applications where the desired bleaching agent is obtained by mixing reactive liquid media. Other forms of multicomponent systems used to prepare peroxycarboxylic acid may include, but are not limited to, those designed for one or more solid components or combinations of solid-liquid components, such as powders (for example, US patent No. 5116575), multilayer tablets (eg, US Pat. No. 6,210,639), water-soluble pouches with multiple components (eg, US Pat. No. 6,995,125), and solid agglomerates that react upon addition of water (eg, US Pat. No. 6,319,888).

In another embodiment, the carboxylic acid ester in the first component is selected from the group consisting of monoacetin, diacetin, triacetin, and combinations thereof. In another embodiment, the carboxylic acid ester in the first component is an acetylated saccharide. In another embodiment, the enzyme catalyst in the first component may be a particulate solid, liquid, or gel. In another embodiment, the first reaction component may be a solid tablet or powder.

Peroxycarboxylic acids are quite reactive and generally decrease in concentration over time. This is especially true of commercial preformed compositions of peroxycarboxylic acids, which often lack long-term stability. Aqueous solutions of preformed peroxycarboxylic acids can also present some packaging and/or transport difficulties, especially when transporting large containers and/or highly concentrated peroxycarboxylic acid solutions over long distances. In addition, solutions of preformed peroxycarboxylic acids may not be able to provide the desired concentration of peroxycarboxylic acid for a particular intended use. In this regard, it is highly desirable to store the various reaction components separately, especially as far as liquid formulations are concerned.

The use of multicomponent systems for the formation of peroxycarboxylic acids has been reported, containing two or more components that are combined to obtain the desired peroxycarboxylic acid. The individual components must be safe to handle and stable over extended periods of time (ie as measured by the concentration of peroxycarboxylic acid obtained by mixing). In one embodiment, the storage stability of a multicomponent peroxycarboxylic acid enzymatic formation system can be measured based on the stability of the enzymatic catalyst.

This document provides personal care products containing a multicomponent composition for the formation of peroxycarboxylic acids, which use an enzyme catalyst to quickly obtain an aqueous solution of peroxyacid with the desired concentration of peroxycarboxylic acid. Mixing can be done immediately before use and/or at the site of use ( in situ ). In one embodiment, the personal care product formulation will consist of at least two components that remain separated prior to use. Mixing the components quickly forms an aqueous peracid solution. Each component is designed so that the resulting aqueous peracid solution contains an effective concentration of peracid suitable for the intended end use. The composition of the individual components must be designed to (1) provide stability during long-term storage and/or (2) provide the ability to enhance the formation of a suitable aqueous reaction composition containing peroxycarboxylic acid.

A multi-component composition may have at least two essentially liquid components. In one embodiment, the multi-component formulation may be a two-component formulation that contains a first liquid component and a second liquid component. The use of the terms "first" or "second" liquid component is relative, provided that the two different liquid components containing said ingredients remain separated until use. At a minimum, the peroxycarboxylic acid multicomponent formulation comprises (1) at least one enzyme catalyst having perhydrolysis activity, wherein said at least one enzyme is preferably classified as a CE-7 esterase, (2) a carboxylic acid ester substrate, and (3 ) a source of oxygen peroxy group and water, where the composition enzymatically produces the necessary peracid as a result of the combination of components.

The type and amount of different ingredients used in two-component formulations must be carefully selected and balanced to ensure (1) the storage stability of each component, especially the perhydrolysis activity of the enzyme catalyst, and (2) physical characteristics that enhance solubility and/or the ability to effectively form the desired aqueous peroxycarboxylic acid solution (e.g., ingredients that increase the solubility of the ester-based substrate in the aqueous reaction mixture and/or ingredients that modify the viscosity and concentration of at least one of the liquid components [i.e., at least one a co-solvent that does not have a significant, adverse effect on enzymatic perhydrolysis activity]).

Various methods have been disclosed to improve the performance and/or catalytic stability of peracid enzymatic formation systems. US Patent Application Publication No. 2010-0048448 A1 describes the use of at least one co-solvent to improve the solubility and/or mixing characteristics of certain ester substrates. A co-solvent may also be used in the present personal care compositions and methods. In one embodiment, the component containing the carboxylic acid ester substrate and the perhydrolase catalyst contains an organic solvent with a log P value of less than about 2, where log P is defined as the logarithm of the partition coefficient of the substance between octanol and water, expressed as P = [dissolved substance] _octanol / [solute] _water . Several co-solvents with a log P value of 2 or less have been described that do not have a clear adverse effect on enzymatic activity. In another embodiment, the co-solvent is from about 20% to about 70% by weight in the reaction component containing the carboxylic acid ester substrate and the enzyme. The reaction component containing the carboxylic acid ester substrate and the enzyme may optionally contain one or more buffers (for example, sodium and/or potassium salt of bicarbonate, citrate, acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate, fumarate, tartrate and maleate).

US Patent Application Publication No. 2010-0086534 A1 describes the use of a two-component system, wherein the first component contains a liquid carboxylic acid ester formulation and a solid enzyme powder; where the specified enzyme powder contains a composition of (a) at least one CE-7 esterase with perhydrolytic activity, and (b) at least one oligosaccharide filler; and the second component contains water with a peroxy oxygen source and a hydrogen peroxide stabilizer. In the present personal care compositions and methods, a two-component composition similar to the system described in US 2010-0086534 A1 can be used. In this regard, the oligosaccharide filler can be used to help stabilize the enzymatic activity. In one embodiment, the oligosaccharide excipient may have a number average molecular weight of at least about 1250 and a weight average molecular weight of at least about 9000. In another embodiment, the oligosaccharide excipient may have a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15,000. In another embodiment, the oligosaccharide is maltodextrin.

U.S. Patent Application Publication No. 2010-0086535-A1 also describes a two-component system wherein the first component comprises a composition of a liquid carboxylic acid ester and a solid enzyme powder, said composition comprising (a) an enzyme powder containing at least one esterase CE- 7 having perhydrolytic activity and at least one oligosaccharide excipient and at least one surfactant; and (b) at least one buffer, wherein in a preferred embodiment the buffer is added as a separated (ie, separate from the enzyme powder) insoluble component to the carboxylic acid ester substrate; and the second component contains water with a peroxy oxygen source and a hydrogen peroxide stabilizer. In the present personal care compositions and methods, a two-component composition similar to the system described in US 2010-0086535 A1 can be used. In one embodiment, the excipient may be an oligosaccharide excipient that has a number average molecular weight of at least about 1250 and a weight average molecular weight of at least about 9000. In another embodiment, the oligosaccharide excipient may have a number average molecular weight of at least about 1700 and a weight average molecular weight at least about 15,000. In another embodiment, the oligosaccharide is maltodextrin. In a further embodiment, the optional pH buffer is a bicarbonate buffer. In yet another embodiment, the hydrogen peroxide stabilizer is ^TURPINAL® SL.

Enzyme powders

In some embodiments, personal care compositions can use an enzyme catalyst in the form of a stabilized enzyme powder. Methods for preparing and stabilizing formulations containing enzyme powder are described in US Patent Application Publication Nos. 2010-0086534 and 2010-0086535.

In one embodiment, the enzyme may be present in the enzyme powder in an amount ranging from about 5 weight percent (wt.%) to about 75 wt.% of the dry weight of the enzyme powder. A preferred weight percent range of enzyme in the enzyme powder/spray dried mixture is from about 10 wt.% to 50 wt.%, and a more preferred range of weight percent enzyme in the enzyme powder/spray dried mixture is from 20 wt.% to 33 wt.%.

In one embodiment, the enzyme powder may further include a filler. In one aspect, the filler is provided in an amount ranging from about 95 wt.% to about 25 wt.% based on the dry weight of the enzyme powder. A preferred weight % range of excipient in enzyme powder is from about 90 wt % to 50 wt %, and a more preferred weight % range of excipient in enzyme powder is from about 80 wt % to 67 wt %.

In one embodiment, the excipient used to prepare the enzyme powder may be an oligosaccharide excipient. In one embodiment, the oligosaccharide excipient has a number average molecular weight of at least about 1250 and a weight average molecular weight of at least about 9000. In some embodiments, the oligosaccharide excipient has a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15,000. Specific oligosaccharides may include, but are not limited to, maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, insulin, levan, graminan, amylopectin, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, nigerotriose, maltotriose , melecytose, maltotriulose, raffinose, ketose and mixtures thereof. In a preferred embodiment, the oligosaccharide bulk is maltodextrin. Oligosaccharide-based fillers may also include, but are not limited to, water-soluble non-ionic cellulose ethers such as hydroxymethylcellulose and hydroxypropylmethylcellulose and mixtures thereof. In yet another additional embodiment, the filler can be selected from, but not limited to, one or more of the following compounds: trehalose, lactose, sucrose, mannitol, sorbitol, glucose, cellobiose, α-cyclodextrin, and carboxymethylcellulose.

The compositions may contain at least one optional surfactant, where preferably the presence of at least one surfactant. Surfactants may include, but are not limited to, ionic or non-ionic surfactants or wetting agents such as ethoxylated castor oil, polyglycolized glycerides, acetylated monoglycerides, sorbitol fatty acid esters, poloxamers, polyoxyethylene esters of sorbitol, and fatty acids, polyoxyethylene derivatives, monoglycerides or their ethoxylated derivatives, diglycerides or their polyoxyethylene derivatives, sodium docusate, sodium lauryl sulfate, cholic acid or its derivatives, lecithins, phospholipids, block copolymers of ethylene glycol and propylene glycol and non-ionic organosilicones. Preferably, the surfactant is a polyoxyethylene sorbitan fatty acid ester, with polysorbate 80 being more preferred.

In the case where the composition contains an enzyme powder, the surfactant used to prepare the powder may be present in an amount ranging from about 5 wt.% to 0.1 wt.% by weight of the protein present in the enzyme powder, preferably from about 2 wt.% to 0.5 wt.% by weight of the protein present in the powder of the enzyme.

The enzyme powder may additionally contain one or more buffers (for example, sodium and/or potassium salts of bicarbonate, citrate, acetate, phosphate, pyrophosphate, methylphosphonate, succinate, malate, fumarate, tartrate, and maleate) and an enzyme stabilizer (for example, ethylenediaminetetraacetic acid, ( 1-hydroxyethylidene)bisphosphonic acid)).

Spray drying of the formulation to form the enzyme powder is carried out, for example, as described generally in Spray Drying Handbook, ^5th ed., K. Masters, John Wiley & Sons, Inc., NY, NY 1991) and PCT Patent Publication No. WO 97 /41833 and WO 96/32149, Platz, R. et al.

In general, spray drying consists of combining a highly dispersed liquid and a sufficient volume of hot air to produce evaporation and drying of liquid droplets. Typically, the initial reaction mixture is sprayed into a stream of warm filtered air, which evaporates the solvent and carries the dried product to the receiver. The exhaust air is then removed with the solvent. Those skilled in the art will appreciate that several different types of devices may be used to obtain the desired product. For example, industrial spray dryers manufactured by Buchi Ltd. (Postfach, Switzerland) or GEA Niro Corp. (Copenhagen, Denmark) will efficiently produce particles of the required size. Additionally, it will be understood that such spray dryers, and in particular their atomizers, can be modified or adapted for specific applications, such as spraying two solutions simultaneously using dual nozzle technology. More specifically, a water-in-oil emulsion can be sprayed from one nozzle, and a solution containing an anti-adhesive such as mannitol can be simultaneously sprayed from a second nozzle. In other cases, it may be useful to move a solution of the initial reaction mixture through a custom-designed nozzle using a high pressure liquid chromatography (HPLC) pump. Provided that microstructures are obtained having the correct morphology and/or composition, the choice of device is not critical and may be obvious to one skilled in the art in view of the teachings of this document.

The temperature at both the inlet and outlet of the gas used to dry the nebulized material is such that it does not decompose the enzyme in the nebulized material. Such temperatures are usually determined experimentally, although, in general, the inlet temperature will be in the range from about 50°C to about 225°C, while the outlet temperature will be in the range from about 30°C to about 150°C. C. Preferred settings include spray pressures ranging from about 20 to 150 psi (0.14 MPa - 1.03 MPa) and preferably from about 30-40 to 100 psi (0.21-0.28 MPa to 0.69 MPa) . Typically, the spray pressure used will be one of the following (MPa): 0.14, 0.21, 0.28, 0.34, 0.41, 0.48, 0.55, 0.62, 0.69, 0.76, 0.83 or higher.

In one embodiment, "substantially retains its enzymatic activity" means that the enzyme powder or carboxylic acid ester enzyme powder formulation retains at least about 75 percent of the enzymatic activity of the enzyme in the enzyme powder or enzyme powder formulation after prolonged storage at ambient temperature and/or after a short period of storage at elevated temperature (above ambient temperature) in a carboxylic acid ester/enzyme powder formulation compared to the initial enzymatic activity of the enzyme powder prior to preparation of the carboxylic acid ester/enzyme powder formulation. enzyme powder. An extended storage period is a period of time from about one year to about two years at ambient temperature. In one embodiment, the short storage period at elevated temperature is the time period from when the carboxylic acid ester/enzyme powder formulation is prepared at 40°C to about eight weeks at 40°C. In another embodiment, the elevated temperature is in the range from about 30°C to about 52°C. In a preferred embodiment, the elevated temperature is in the range of about 30°C to about 40°C.

In some embodiments, the enzyme powder retains at least 75 percent of the enzyme activity after eight weeks of storage at 40° C. in a carboxylic acid ester-enzyme powder formulation compared to the enzyme powder's initial enzyme activity prior to preparation of the enzyme powder formulation. carboxylic acid ester and enzyme powder, at 40°C. In other embodiments, the enzyme powder retains at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 percent enzymatic activity of at least one enzyme after eight weeks of storage at 40°C in a formulation consisting of a carboxylic acid ester and enzyme powder compared to the initial enzymatic activity of the enzyme powder prior to formulation, consisting of a carboxylic acid ester and an enzyme powder, at 40°C. Preferably, the perhydrolysis activity is measured as described in Examples 8-13 of US Patent Application Publication No. 2010-0086510; but any method for measuring perhydrolysis activity can be used.

Further improvement in enzyme activity within the indicated time periods can be achieved by adding a buffer having a buffering capacity in the pH range of about 5.5 to about 9.5 to a formulation of carboxylic acid ester and spray-dried enzyme powder as described in the publication US Patent Application No. 2010-0086534. A suitable buffer may include, but is not limited to, sodium salt, potassium salt, or mixtures of sodium or potassium salts of bicarbonate, pyrophosphate, phosphate, methylphosphonate, citrate, acetate, malate, fumarate, tartrate, maleate, or succinate. Preferred buffers for use in a formulation consisting of a carboxylic acid ester and a spray-dried enzyme powder include sodium salt, potassium salt, or mixtures of sodium or potassium salts of bicarbonate, pyrophosphate, phosphate, methylphosphonate, citrate, acetate, malate, fumarate, tartrate, maleate or succinate.

In embodiments where a buffer may be present in the carboxylic acid ester and enzyme powder formulation, the buffer may be present in an amount in the range of from about 0.01 wt% to about 50 wt% by weight of the carboxylic acid ester in the formulation consisting from a carboxylic acid ester and an enzyme powder. The buffer may be present in a more preferred range of from about 0.10% to about 10% by weight of the carboxylic acid ester in the composition of the carboxylic acid ester and enzyme powder. Additionally, in these embodiments, the comparison between enzyme perhydrolysis activity is determined against an enzyme powder that retains at least 75 percent of the perhydrolysis activity of at least one enzyme after eight weeks of storage at 40° C. in a formulation consisting of a carboxylic acid ester, buffer , having a buffering capacity in the pH range of about 5.5 to about 9.5, and an enzyme powder compared to the initial perhydrolysis activity of the enzyme powder prior to formulation, consisting of a carboxylic acid ester, a buffer having a buffering capacity in the pH range of about 5.5 to about 9.5, and enzyme powder.

It is contemplated that the dried enzyme powder is stored as a formulation with an organic compound that is a substrate for at least one enzyme, such as triacetin. In the absence of added hydrogen peroxide, triacetin is normally hydrolyzed in aqueous solution by carbohydrate esterase CE-7 to form diacetin and acetic acid, and the formation of acetic acid results in a decrease in the pH of the reaction mixture. One condition for long-term storage stability of the enzyme in triacetin is that there is no significant reaction of triacetin with any water that might be present in triacetin; the specification for water content in one commercial triacetin (supplied by Tessenderlo Group, Brussels, Belgium) is 0.03 wt% water (300 ppm). Any hydrolysis of triacetin that occurs during storage of the enzyme in triacetin will produce acetic acid, which may result in decreased activity or inactivation of CE-7 perhydrolases; perhydrolases are generally inactive at or below pH 5.0 (See U.S. Patent Application Publication No. 2009-0005590, DiCosimo, R. et al .). The excipient selected for use according to the present application must ensure the stability of the enzyme in the organic substrate for the enzyme under conditions where acetic acid can be formed due to the presence of low concentrations of water in the composition. The dried enzyme powder can be stored as a formulation in an organic compound that is a substrate for at least one enzyme, where the formulation additionally contains an excipient and one or more buffers (e.g., sodium and/or potassium salts of bicarbonate, citrate, acetate, phosphate, pyrophosphate , methylphosphonate, succinate, malate, fumarate, tartrate and maleate).

Suitable reaction conditions for the enzymatically catalyzed production of peracid from carboxylic acid esters and hydrogen peroxide

To obtain an effective concentration of the desired peracid(s) in the present personal care compositions and methods, one or more enzymes with perhydrolytic activity. The desired peroxycarboxylic acid can be prepared by reacting carboxylic acid esters with a source of peroxy oxygen, including but not limited to hydrogen peroxide, zinc peroxide, sodium peroxide, urea peroxide, calcium peroxide, sodium perborate, sodium percarbonate, or hydrogen peroxide complexes, in the presence of an enzyme catalyst. with perhydrolytic activity.

The perhydrolytic enzyme in the targeted perhydrolase can be any perhydrolytic enzyme and can include lipases, proteases, esterases, acyltransferases, arylesterases, carbohydrate esterases, and combinations, so long as the enzyme has perhydrolytic activity on one or more of the substrates present. Examples may include, but are not limited to, perhydrolytic proteases (subtilisin variant, US Pat. No. 7,510,859), perhydrolytic esterases ( Pseudomonas fluorescens ; US Pat. No. 7,384,787; "L29P" variant of SEQ ID NO: 477), and perhydrolytic arylesterases ( Mycobacterium smegmatis ; US Pat. No. 7754460; WO2005/056782; and EP1689859 B1; SEQ ID NOs: 460 [S54V variant] and 478 [wild type]).

In one embodiment, the enzyme catalyst comprises at least one enzyme having perhydrolase activity, wherein said enzyme is structurally classified as a member of the CE-7 family of carbohydrate esterases (CE-7; see Coutinho, P.M., and Henrissat, B., formerly). In another embodiment, the perhydrolase catalyst is structurally classified as cephalosporin C deacetylase. In another embodiment, the perhydrolase catalyst is structurally classified as acetylxylanesterase.

In one embodiment, the perhydrolase catalyst contains an enzyme having perhydrolysis activity and having a signature motif comprising:

a) an RGQ motif that aligns with amino acid residues 118-120 of SEQ ID NO: 2;

b) a GXSQG motif that aligns with amino acid residues 179-183 of SEQ ID NO: 2; And

c) an HE motif that aligns with amino acid residues 298-299 of SEQ ID NO: 2.

In a preferred embodiment, alignment with the reference SEQ ID NO: 2 is performed using CLUSTALW.

In a further embodiment, the CE-7 signature motif may further comprise an additional (i.e., fourth) motif, designated as the LXD motif, at amino acid residues 267-269 when aligned with the reference sequence of SEQ ID NO: 2 using CLUSTALW.

In another embodiment, the perhydrolase catalyst contains an enzyme having perhydrolase activity, said enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437 and 476.

In another embodiment, the perhydrolase catalyst contains an enzyme having perhydrolase activity, said enzyme having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 424, 437, and 476, wherein said enzyme may have one or more additions, deletions, or substitutions, provided that the signature motif is conserved and perhydrolase activity is preserved.

As described above, the CE-7 perhydrolase may be a fusion protein having a first portion that contains the CE-7 perhydrolase and a second portion that contains a peptide moiety that has an affinity for a target body surface such that the perhydrolase is "targeted" at the desired body surface. In one embodiment, the CE-7 perhydrolase (as determined by the presence of the CE-7 signature motif) can be fused to any peptide moiety/coupler capable of targeting the enzyme to an organism's surface. In one aspect, the peptide component with affinity for the oral surface may include antibodies, antibody fragments (F _ab ), as well as single-chain variable fragments (scFv; a hybrid of heavy (V _H ) and light chain (V _L ) immunoglobulin variable regions), single domain camel antibodies, scaffold display proteins and single chain affinity peptides without immunoglobulin fold. Compositions containing antibodies, antibody fragments and other immunoglobulin-derived binding elements, as well as large frame display proteins, are often not economically viable. In this regard, and in a preferred aspect, the peptide component/binding element is a single chain affinity peptide without an immunoglobulin fold and/or an immunoglobulin domain. Short single chain peptides that bind to an organism surface can be generated empirically (eg, positively charged polypeptides targeting negatively charged surfaces) or generated by biopanning the target organism surface. Methods for detecting/producing affinity peptides using a variety of display technologies (eg, phage display, yeast display, bacterial display, ribosome display, and mRNA display) are well known in the art. Individual oral surface-binding peptides can be connected to each other via optional spacers/linkers to form larger binding "domains" (also referred to herein as binding "hands") to improve attachment/localization of the perhydrolytic enzyme to the target oral surface. mouth.

The fusion proteins may also include one or more peptide linkers/spacers separating the CE-7 perhydrolase enzyme, the oral surface binding domain, and/or various oral surface binding peptides (e.g., if multiple oral surface binding peptides , are linked to form a larger domain that binds to the target oral surface). Multiple peptide linkers/spacers may be present and the number of linkers may be repeated up to 10 times. A non-limiting list of illustrative peptide spacers is given as the amino acid sequences of SEQ ID NOs: 383-396 and those shown in Table 5.

Suitable peptides with affinity for the oral surface are described hereinbefore. Methods for detecting additional oral surface-binding peptides using any of the "display" technologies described above are well known and can be used to detect additional oral surface-binding peptides.

Suitable carboxylic acid ester substrates may include esters with the following formulae:

(a) one or more esters with the structure

[X] _m R ₅ ,

Where

X is an ester group with the formula R ₆ C(O)O;

R ₆ is a C1-C7 linear, branched or cyclic hydrocarbyl moiety optionally substituted with a hydroxyl group or a C1-C4 alkoxy group, where R ₆ optionally contains one or more ether linkages, where R ₆ is C2-C7;

R ₅ is a C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety optionally substituted with a hydroxyl group; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group, and where R ₅ optionally contains one or more ether linkages;

m is an integer ranging from 1 to the number of carbons in R ₅ ,

wherein said one or more esters have a solubility in water of at least 5 ppm. at 25°C; or

(b) one or more glycerides with the structure

,

,

where R ₁ is straight chain or branched C1-C7 alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O); or

(c) one or more esters of the formula

,

,

where R ₁ is straight chain or branched C1-C7 alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 straight or branched alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; or

(d) one or more acetylated monosaccharides, acetylated disaccharides or acetylated polysaccharides; or

(e) any combination of (a)-(d).

Suitable substrates may also include one or more acylated saccharides selected from the group consisting of acylated mono-, di- and polysaccharides. In another embodiment, the acylated saccharides are selected from the group consisting of acetylated xylan; fragments of acetylated xylan; acetylated xylose (such as xylosetetraacetate); acetylated glucose (such as α-D-glucose pentaacetate; β-D-glucose pentaacetate; 1-thio-β-D-glucose-2,3,4,6-tetraacetate); β-D-glucose pentaacetate; sorbitol hexaacetate; sucrose octaacetate; β-D-ribofuranose-1,2,3,5-tetraacetate; β-D-ribofuranose-1,2,3,4-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; β-D-xylofuranose tetraacetate; α-D-glucopyranose pentaacetate; β-D-glucopyranose-1,2,3,4-tetraacetate; β-D-glucopyranose-2,3,4,6-tetraacetate; 2-acetamido-2-deoxy-1,3,4,6-tetracetyl-β-D-glucopyranose; 2-acetamido-2-deoxy-3,4,6-triacetyl-1-chloride-α-D-glucopyranose; α-D-mannopyranosapentaacetate and acetylated cellulose. In a preferred embodiment, the acetylated saccharide is selected from the group consisting of β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; sucrose octaacetate and acetylated cellulose.

In another embodiment, additional suitable substrates may also include 5-acetoxymethyl-2-furaldehyde; 3,4-diacetoxy-1-butene; 4-acetoxybenzoic acid; vanillin acetate; propylene glycol methyl ether acetate; methyl lactate; ethyl lactate; methyl glycolate; ethyl glycolate; methyl methoxyacetate; ethyl methoxyacetate; methyl 3-hydroxybutyrate; ethyl 3-hydroxybutyrate; and triethyl 2-acetyl citrate.

In another embodiment, suitable substrates are selected from the group consisting of monoacetin; diacetin; triacetin; monopropionin, dipropionin, tripropionin, monobutyrin; dibutyrin; tributyrin; glucose pentaacetate; xylose tetraacetate; acetylated xylan; fragments of acetylated xylan; β-D-ribofuranose-1,2,3,5-tetraacetate; tri-O-acetyl-D-galactal; tri-O-acetyl-D-glucal; monoesters or diesters of 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; 2,5-hexanediol; 1,6-hexanediol and mixtures thereof. In another embodiment, the substrate is a C1-C6 polyol containing one or more ester groups. In a preferred embodiment, one or more hydroxyl groups on the C1-C6 polyol are substituted with one or more acetoxy groups (such as 1,3-propanediol diacetate; 1,2-propanediol diacetate; 1,4-butanediol diacetate; 1,5-pentanediol diacetate, etc.). d.). In a further embodiment, the substrate is propylene glycol diacetate (PGDA), ethylene glycol diacetate (EGDA), or mixtures thereof.

In a further embodiment, suitable substrates are selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, dipropionin, tripropionin, monobutyrin, dibutyrin and tributyrin. In another aspect, the substrate is selected from the group consisting of diacetin and triacetin. In the most preferred embodiment, a suitable substrate contains triacetin.

In a preferred embodiment, the carboxylic acid ester is a liquid substrate selected from the group consisting of monoacetin, diacetin, triacetin, and combinations (ie, mixtures) thereof. The carboxylic acid ester is present in the reaction composition at a concentration sufficient to obtain the required concentration of peroxycarboxylic acid by enzymatically catalyzed perhydrolysis. The carboxylic acid ester does not need to be completely soluble in the reaction composition, but has sufficient solubility to allow the ester to be converted by the perhydrolase catalyst to the corresponding peroxycarboxylic acid. The carboxylic acid ester is present in the reaction composition at a concentration of from 0.05 wt.% to 40 wt.% of the reaction composition, preferably at a concentration of from 0.1 wt.% to 20 wt.% of the reaction composition, and more preferably at a concentration of from 0, 5 wt.% to 10 wt.% of the reaction composition.

The peroxy oxygen source may include, but is not limited to, hydrogen peroxide, hydrogen peroxide adducts (eg, hydroperite (carbamide peroxide) adduct), perborate salts, percarbonate salts, and peroxide salts. The concentration of the peroxy oxygen compound in the reaction composition may vary from 0.0033 wt.% to about 50 wt.%, preferably from 0.033 wt.% to about 40 wt.%, more preferably from 0.1 wt.% to about 30 wt. .%.

The oxygen source of the peroxy group (ie, hydrogen peroxide) can also be formed enzymatically with an enzyme capable of producing an effective amount of hydrogen peroxide. For example, various oxidases can be used in the present compositions and methods to produce an effective amount of hydrogen peroxide, including, but not limited to, glucose oxidase, lactose oxidase, carbohydrate oxidase, alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, and amino acid oxidase.

Many perhydrolase catalysts (whole cells, permeabilized whole cells and partially purified whole cell extracts) have been reported to have catalase activity. Catalases catalyze the conversion of hydrogen peroxide into oxygen and water. In one aspect, the perhydrolysis catalyst does not have catalase activity. In another aspect, a catalase inhibitor can be added to the reaction composition. One of skill in the art can adjust the concentration of the catalase inhibitor as needed. The concentration of the catalase inhibitor 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 does not have significant catalase activity or can be designed to reduce or eliminate catalase activity. Catalase activity in a host cell can be suppressed or eliminated by disrupting the expression of the gene(s) responsible for catalase activity using well-known technologies, including, but not limited to, transposon mutagenesis, antisense RNA expression, site-directed mutagenesis, and random mutagenesis. In a preferred embodiment, the gene(s) encoding(e) endogenous catalase activity is suppressed or disrupted (ie, knocked out). As used herein, a "disturbed" gene is one in which the activity and/or function of the protein encoded by the modified gene is no longer present. Means of disrupting a gene are well known in the art and may include, but are not limited to, insertions, deletions, or mutations of a gene, so long as the activity and/or function of the corresponding protein is no longer present. In a further preferred embodiment, the source organism is an E. coli source organism containing the disrupted catalase gene selected from the group consisting of kat G and kat E (see US Patent Application Publication No. 2008-0176299). In another embodiment, the source organism is an E. coli strain that has suppression and/or aberration in both the kat G and kat E catalase genes.

The concentration of catalyst in the aqueous reaction composition depends on the specific catalytic activity of the catalyst and is chosen to obtain the required reaction rate. The weight of catalyst in perhydrolysis reactions generally ranges from 0.0001 mg to 10 mg per ml of total reaction volume, preferably from 0.001 mg to 2.0 mg per ml. The catalyst can also be immobilized on a soluble or insoluble support using methods well known to those skilled in the art, see, for example, Immobilization of Enzymes and Cells ; Gordon F. Bickerstaff, Editor; Humana Press, Totowa, NJ, USA; 1997. The use of immobilized catalysts allows the recovery and reuse of the catalyst in subsequent reactions. The enzyme catalyst may be in the form of whole microbial cells, permeabilized microbial cells, extracts of microbial cells, partially purified or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peroxycarboxylic acid formed from the combination of chemical perhydrolysis and enzymatic perhydrolysis of the carboxylic acid ester is sufficient to provide an effective concentration of peroxycarboxylic acid for the selected personal care application. In another aspect, the present methods provide combinations of enzymes and enzyme substrates to obtain the desired effective concentration of peroxycarboxylic acid, where in the absence of an added enzyme, there is a significantly lower concentration of peroxycarboxylic acid produced. Although in some cases significant chemical perhydrolysis of the enzyme substrate may occur as a result of the direct chemical reaction of the inorganic peracid with the enzyme substrate, the concentration of peroxycarboxylic acid formed to obtain an effective concentration of peroxycarboxylic acid in the required applications may not be sufficient, and a significant increase in the total concentration of peroxycarboxylic acid achieved by adding an appropriate perhydrolase catalyst to the reaction composition.

The concentration of peroxycarboxylic acid (e.g., peracetic acid) formed from the perhydrolysis 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 peracids within 10 minutes, preferably within 5 minutes, after the start of the perhydrolysis reaction. The product formulation containing the peroxycarboxylic acid may optionally be diluted with water or a solution predominantly comprised of water to produce a formulation with the desired lower concentration of peroxycarboxylic acid based on the intended application. It is understood that one skilled in the art can adjust the reaction components and/or the amounts of diluent to obtain the desired concentration of peracid for the selected personal care product.

In one aspect, the reaction time required to form the desired peracid concentration is no more than about two hours, preferably no more than about 30 minutes, more preferably no more than about 10 minutes, and most preferably no more than about 5 minutes or less. In other aspects, the surface of the oral cavity is brought into contact with peroxycarboxylic acid, formed in accordance with the methods described herein, within 5 minutes after combining the reaction components. In one embodiment, the target oral surface is contacted with a peroxycarboxylic acid produced by the method described herein for from about 5 minutes to about 168 hours after these reaction components are combined, or for from about 5 minutes to about 48 hours, or within about 5 minutes to 2 hours after the combination of these reaction components, or any such time interval within the values specified in this document.

The peracid formed according to the methods described herein is used in personal care products/personal care applications where the peracid is brought into contact with the target oral surface to produce a peracid based oral benefit. In one embodiment, the method for producing a peracid for a target body surface is carried out in situ.

The reaction temperature can be selected to control both the reaction rate and the stability of the activity of the enzyme catalyst. It is understood that for certain personal care applications, the temperature of the target body surface (eg, 37° C. inside the mouth) may be the reaction temperature. The reaction temperature may vary from slightly above the freezing point of the reaction composition (about 0°C) to about 95°C, with a preferred range of 5°C to about 75°C and a more preferred reaction temperature range of about 5°C to about 55°C. C.

The pH of the final reaction composition containing peroxycarboxylic acid is from about 2 to about 9, preferably from about 3 to about 8, more preferably from about 5 to about 8, even more preferably from about 5.5 to about 8, and even more preferably from about 6.0 to about 7.5. The pH of the reaction and the final reaction composition can optionally be controlled by the addition of a suitable buffer, including, but not limited to, phosphate, pyrophosphate, bicarbonate, acetate, or citrate. The buffer concentration, if used, is typically 0.1 mM to 1.0 M, preferably 1 mM to 300 mM, most preferably 10 mM to 100 mM.

In another aspect, the enzymatic perhydrolysis reaction composition may contain an organic solvent that acts as a dispersant to increase the degree of dissolution of the carboxylic acid ester in the reaction composition. Such solvents include, but are not limited to, propylene glycol methyl ether, acetone, cyclohexanone, diethylene glycol butyl ether, tripropylene glycol methyl ether, diethylene glycol methyl ether, propylene glycol butyl ether, dipropylene glycol methyl ether, cyclohexanol, benzyl alcohol, isopropanol, ethanol. , propylene glycol and mixtures thereof.

One-Step Application vs. Multi-Step Application

Typically, the minimum set of reactive components for enzymatic production of a peracid benefit will include (1) at least one enzyme having perhydrolytic activity as described herein, such as CE-7 perhydrolase (optionally in the form of a targeted fusion protein), (2) at least one suitable carboxylic acid ester substrate; and (3) a peroxy oxygen source.

The peracid-forming reactive components of the personal care composition can be stored separately prior to use. In one embodiment, the peracid-forming components are combined and then brought into contact with a target body surface, whereby the peracid-based beneficial agent exerts a beneficial effect on the body surface. The components can be combined and then brought into contact with the target body surface, or can be combined on the target body surface. In one embodiment, the peracid-forming components are combined to form the peracid in situ.

You can also use multi-stage application. One or two separate components of the peracid forming system composition (i.e. sequential application of at least one of the three main reaction components to the body surface) can be brought into contact with the oral surface prior to application of the remaining components necessary for the enzymatic production of the peracid. In one embodiment, the perhydrolytic enzyme is contacted with the oral surface prior to contacting the oral surface with the carboxylic acid ester substrate and/or peroxy oxygen source (i.e., "two step application"). In one embodiment, the enzyme having perhydrolytic activity is a targeted perhydrolase that is applied to the oral surface prior to combining the remaining components needed to enzymatically produce the peracid.

In a preferred embodiment, the enzyme having perhydrolytic activity is a "targeted CE-7 perhydrolase" (i.e., a CE-7 fusion protein) which is applied to the oral surface prior to being combined with the remaining components needed to enzymatically produce the peracid. (i.e. two-stage method of application). The targeted perhydrolase is brought into contact with the oral surface under suitable conditions to promote non-covalent binding of the fusion protein to the oral surface. An optional rinsing step can be used to remove excess and/or unbound fusion protein before combining the remaining reaction components.

In a further embodiment, a perhydrolytic enzyme (optionally in the form of a fusion protein targeted to the oral surface) and a carboxylic acid ester are applied to the target oral surface prior to the addition of the peroxy oxygen source.

In a further embodiment, a perhydrolytic enzyme (optionally in the form of a fusion protein targeting the oral surface) and a peroxy oxygen source (e.g., an aqueous solution containing hydrogen peroxide) are applied to the oral surface prior to addition of the carboxylic acid ester substrate.

In a further embodiment, a carboxylic acid ester substrate and a peroxy oxygen source (e.g., an aqueous solution containing hydrogen peroxide) is applied to the oral surface prior to addition of the perhydrolytic enzyme (optionally in the form of a fusion protein targeted to the oral surface).

In yet another embodiment, any of the compositions or methods described herein may be included in a kit for carrying out the present invention. The kits may contain materials and reagents to enable enzymatic production of the peracid. A typical kit contains a substrate, a peroxy oxygen source, and an enzyme catalyst having perhydrolytic activity, where the enzyme catalyst may optionally be targeted to the oral surface. Other components of the kit may include, without limitation, one or more of the following: sample tubes, solid supports, guidance material, and other solutions or other chemicals suitable for the enzymatic production of peracids, such as suitable components or carriers.

Compositions for oral care

Orally acceptable ingredients/carriers

The present compositions and methods may also include orally acceptable carriers as well as additional ( ie , in addition to the peracid-based benefit) oral health benefits. As used herein, the term "oral benefit" is a general term applied to a compound or substance that provides a desired/beneficial effect or property to the surface of the mouth. In one embodiment, the oral surface benefit may include (in addition to the peracid benefit) coloring agents including, but not limited to, white pigments such as titanium dioxide and white minerals such as hydroxyapatite or zircon. In another embodiment, useful oral agents may also include whitening agents and additional enzymes such as, for example, oxidases, peroxidases, proteases, lipases, glycosidases, esterases, and polysaccharide hydrolases. In another aspect, useful agents may include antiplaque agents, tooth stain removers, and antimicrobials. Antimicrobial agents may include, but are not limited to, antimicrobial peptides, magainins, cecropins, microbiocides, triclosan, chlorhexidine, cetylpyridinium chloride, quaternary ammonium compounds, chlorxylenol, chlorooxyethanol, phthalic acid and its salts, thymol, and combinations thereof. Useful oral agents may also include anti-caries agents such as sodium fluoride or sodium monofluorophosphate, and flavoring agents such as oil of wintergreen, peppermint or spearmint, or methyl salicylate, eucalyptol or vanillin. Useful oral agents may also include refrigerants such as succinate refrigerants and saliva inducing agents, among others. As used herein, the term "salivating agent" refers to a material that promotes increased salivation in the consumer when present in an oral composition. In one embodiment, the benefit agent is an orally acceptable material suitable for use in oral care products. In another embodiment, an orally acceptable benefit is used to cosmetically improve the appearance of teeth.

A non-limiting list of compounds commonly used in an orally acceptable carrier medium are described by White et al ., US Pat. No. 6,740,311; Lawler et al ., US Pat. No. 6,706,256; Fuglsang et al ., US Pat. No. 6,264,925; and Ibrahim et al . in US Patent Application Publication No. 2005-0069501, each of which is incorporated herein by reference in their entirety. For example, an oral composition may contain one or more of the following: abrasives, surfactants, antioxidants, chelating agents, fluoride sources, thickeners, buffering agents, solvents, wetting agents, carriers, bulking agents, antiplaque agents, antistaining agents, antimicrobial agents, anticaries agents, anti-inflammatory agents, desensitizers, sweeteners, flavoring agents, breath fresheners, refrigerants, nutrients and salivary agents.

It is understood that the components in the mixture are chosen such that the oral composition retains the ability to enzymatically form the desired peracid benefit agent. Suitable mixtures of oral hygiene systems disclosed herein can be determined by one of ordinary skill in the art by simple experimentation. The total concentration of beneficial oral agents with an oral composition may be from about 0.001% to about 90% by weight based on the total weight of the oral composition.

Oral care compositions may include, but are not limited to, toothpaste, tooth cream, tooth gel or tooth powder, mouthwash, breath freshener, and dental floss. Additional embodiments include the use of the reaction components in a paste or gel that is applied to a cavity environment using a mouthguard. One or more reactive components may also be initially applied to a plastic strip that is adhered to the enamel to deliver one or more reactive components forming a peracid benefit agent. In the case of applying a perhydrolase fusion to a delivery device such as a strip, the perhydrolase fusion may be designed to include binding elements with an affinity for the material of the strip to aid in the application and retention of the perhydrolase on the strip during use and removal of the device after use.

Peracid oral care products to reduce oral disease-associated microbes or remove unwanted biofilm

Peracid oral care products can be used to reduce oral bacteria associated with dental caries (such as Streptococcus mutans ), gingivitis, oral candidiasis, or periodontitis. Peracid oral care products can be used to reduce or remove biofilm(s) in the mouth.

In one embodiment, the use of an enzyme having perhydrolytic activity in an oral care product is provided to provide an effective concentration of at least one peracid to brighten, whiten, decontaminate, bleach, deodorize, or remove biofilm from an oral surface.

In one embodiment, the enzyme having perhydrolytic activity is a targeted perhydrolase and may include lipases, proteases, esterases, acyltransferases, arylesterases, carbohydrate esterases, and combinations, provided that the enzyme has perhydrolytic activity against one or more of the substrates present. Examples may include, but are not limited to, perhydrolytic proteases (e.g. subtilisin variant, US Pat. No. 7,510,859), perhydrolytic esterases (e.g., Pseudomonas fluorescens ; US Pat. No. 7,384,787; SEQ ID NO: 477), perhydrolytic arylesterases (e.g., Mycobacterium smegmatis ; patent US Pat.

In another embodiment, the use of a CE-7 carbohydrate esterase having perhydrolytic activity is provided in an oral care product to provide an effective concentration of at least one peracid provided to brighten, whiten, decontaminate, decolorize, deodorize, or remove biofilm from a cavity surface. mouth.

In another embodiment, the use of the following peracid formation composition is provided, comprising:

a) an enzyme catalyst having perhydrolytic activity, said enzyme catalyst comprising an enzyme having a CE-7 signature motif that aligns to the reference sequence of SEQ ID NO: 2 with CLUSTALW, said signature motif comprising:

i) RGQ motif at positions corresponding to positions 118-120 of SEQ ID NO: 2;

ii) a GXSQG motif at positions corresponding to positions 179-183 of SEQ ID NO: 2; And

iii) HE motif at positions corresponding to positions 298-299 of SEQ ID NO: 2; And

b) at least one substrate selected from the group consisting of:

1) esters with the structure

[X] _m R ₅ ,

where X = ester group with the formula R ₆ C(O)O;

R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7;

R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds;

M is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And

where these esters have a solubility in water of at least 5 ppm. at 25°C;

2) glycerides with structure

,

,

where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O);

3) one or more esters with the formula

,

,

where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, straight chain heteroaryl, or branched chain, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )O) _n H, and n is 1-10; And

4) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides; And

c) a source of peroxy oxygen;

resulting in the formation of peracid when mixing (a), (b) and (c); wherein the peracid formulation is used to treat or prevent dental caries, gingivitis, oral candidiasis, or periodontitis.

HPLC method for determining the concentration of peroxycarboxylic acid and hydrogen peroxide

A number of analytical methods can be used in the present methods for the analysis of reagents and products, including, but not limited to, titration, high performance liquid chromatography (HPLC), gas chromatography (GC), mass spectroscopy (MS), capillary electrophoresis (CE), analytical method , described by U. Pinkernell et al ., ( Anal. Chem., 69(17): 3623-3627 (1997)), and analysis using 2,2'-azino-bis-(3-ethylbenzotazoline)-6-sulfonate (ABTS) (U. Pinkernell et. al. Analyst , 122: 567-571 (1997) and Dinu et. al . Adv. Funct. Mater. , 20: 392-398 (2010)), which are described in the present examples.

Determination of the minimum biocidal concentration of peroxycarboxylic acids

Certain personal care applications may be associated with the removal of unwanted microbes, such as those associated with bodily conditions, fungal infections and dental caries, among others. In this regard, it may be necessary to measure the minimum biocidal concentration for the targeted application related to personal care. The method described by J. Gabrielson, et al . ( J. Microbiol. Methods 50: 63-73 (2002)), can be used to determine the minimum biocidal concentration (MBC) of peroxycarboxylic acids or hydrogen peroxide and enzyme substrates. The assay method is based on inhibition of XTT reduction, where XTT is ((2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-5-[(phenylamino)carbonyl]-2H-tetrazolium, internal salt, single sodium salt) is a redox dye that indicates microbial respiration activity by the change in optical density (OD) measured at 490 nm or 450 nm However, there are a number of other available methods for testing the activity of disinfectants and antiseptics, including, but not limited to, determination of viable bacteria by culture, determination of the number of microorganisms by direct counting under a microscope, dry weight, density measurements, logarithm of the absorbance and bioluminescence (see, for example, Brock, Semour S., Disinfection, Sterilization, and Preservation , 5 ^th edition, Lippincott Williams & Wilkins, Philadelphia, PA, USA; 2001).

Recombinant microbial expression

The genes and gene products of the discussed sequences can be obtained in heterologous host cells, in particular in microbial host cells. Preferred heterologous host cells for the expression of the genes and nucleic acids in question are microbial hosts, which can be found in fungal or bacterial families, and which grow over a wide range of temperatures, pHs, and solvent tolerances. For example, it is contemplated that any bacteria, yeast, and filamentous fungus can respectively act as a host for the expression of the present nucleic acid molecules. The perhydrolase can be expressed intracellularly, extracellularly, or both intracellularly and extracellularly, where extracellular expression is an easier release of the desired protein from the fermentation product than methods for isolating the protein produced by intracellular expression. The apparatuses of transcription, translation and protein biosynthesis remain unchanged in regard to the cellular raw materials used for the formation of cell biomass; functional genes will be expressed independently. Examples of host strains include, but are not limited to, bacterial, fungal, and yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Phaffia, Kluyveromyces, Candida, Hansenula, Yarrowia, Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium , Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena , Thiobacillus, Methanobacterium, Klebsiella and Myxococcus. In one embodiment, the bacterial host strains include Escherichia , Bacillus , Kluyveromyces , and Pseudomonas . In a preferred embodiment, the bacterial host cell is Bacillus subtilis or Escherichia coli .

A wide range of simple or complex carbohydrates, organic acids and alcohols or saturated hydrocarbons, such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts, the form and amount of nitrogen, phosphorus, sulfur, oxygen, carbon or any trace trace element, including any inorganic ion. Growth rate regulation can be influenced by adding, or not adding, specific regulatory molecules to the culture, and these are not generally considered as sources of nutrients or energy.

Vectors or cassettes suitable for transforming suitable host cells are well known in the art. Typically, the vector or cassette contains sequences that control transcription and translation of the corresponding gene, a selectable marker, and sequences that allow for autonomous replication or integration into the chromosome. Suitable vectors contain the 5' region of the gene, which carries elements that control the initiation of transcription, and the 3' region of the DNA fragment, which controls the termination of transcription. Most preferably, both control regions are derived from genes that are homologous to the transformed host cell and/or native to the source organism, although such control regions need not be derived in this manner.

The initiation control regions or promoters that are suitable for directing the expression of the coding region of the present cephalosporin C deacetylase in the desired host cell are diverse and well known to those skilled in the art. In fact, any promoter capable of driving these genes is suitable for the present invention, including but not limited to the CYC1 , HIS3 , GAL1 , GAL10 , ADH1 , PGK , PHO5 , GAPDH , ADC1 , TRP1 , URA3 , LEU2 , ENO , TPI promoters (suitable for expression in Saccharomyces ); AOX1 (suitable for expression in Pichia ); and lac , araB , tet , trp , lPL , lPR , T7 , tac , and trc (suitable for expression in Escherichia coli ), as well as amy , apr , npr , and various phage promoters suitable for expression in Bacillus .

Termination control regions can also be derived from various genes native to the preferred host cell. In one embodiment, the inclusion of a terminating control region is optional. In another embodiment, the fusion gene includes a termination control region derived from a preferred host cell.

Industrial production

A number of cultivation techniques can be used to prepare the perhydrolase catalyst. For example, large-scale production of a particular gene product overexpressed in a recombinant microbial host can be done in batch, semi-batch, and continuous culture techniques. Batch and semi-batch culture methods are conventional and well known in the art, and examples can be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989) and Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36: 227-234 (1992).

Industrial production of the desired perhydrolase catalyst can also be carried out using continuous culture. Continuous cultures are an open system in which a specific culture medium is continuously added to the bioreactor and an equal amount of the conditioned medium is simultaneously removed for processing. Continuous cultures typically maintain cells at a constant high density in the liquid phase, in which the cells are predominantly in the exponential growth phase. Alternatively, continuous culture can be practiced using immobilized cells where carbon and nutrients are continuously added and valuable products, by-products or waste products are continuously removed from the cell mass. Cell immobilization can be performed using a wide range of solid supports, consisting of natural or synthetic materials.

Isolation of the desired perhydrolase catalysts from batch fermentation, semi-batch fermentation, or continuous culture can be accomplished using any of the methods known to those skilled in the art. For example, in intracellular production of an enzyme catalyst, the cell mass is separated from the culture medium by centrifugation or membrane filtration, optionally washed with water or an aqueous buffer with the required pH, then a suspension of the cell mass in an aqueous buffer with the required pH is homogenized to obtain a cell extract containing the necessary enzyme catalyst. The cell extract may optionally be filtered through a suitable filter aid, such as celite or silica, to remove cell debris prior to a heat treatment step to precipitate the unwanted protein from the enzyme catalyst solution. The solution containing the desired enzyme catalyst can then be separated from the precipitated cell debris and protein by membrane filtration or centrifugation, and the resulting partially purified enzyme catalyst solution can be concentrated by further membrane filtration, then optionally mixed with an appropriate carrier (e.g., maltodextrin , phosphate buffer, citrate buffer or mixtures thereof) and spray dried to give a solid powder containing the desired enzyme catalyst.

When an amount, concentration, or other value or parameter is given as either a range, or a preferred range, or a list of most preferred values and less preferred values, this should be understood, in particular, as the disclosure of all ranges formed from any pair of any upper limit of the range or the preferred value and any lower range limit or preferred value, whether or not the ranges are disclosed separately. Where a range of numerical values is set forth herein, unless otherwise indicated, the range is meant to include its extreme points and all integers and fractional values within the range. The scope is not intended to be limited to the specific values mentioned in the range definition.

GENERAL WAYS

The following examples are provided to demonstrate the preferred aspects of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the examples are derived from techniques that work well in the practice of the present invention, and therefore can be considered as establishing preferred methods for its practical implementation. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made to the specific embodiments that are disclosed and still obtain a similar or similar result without deviating from the intent and scope currently disclosed. ways and examples.

All reagents and materials were obtained from DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD), TCI America (Portland, OR), Roche Diagnostics Corporation (Indianapolis, IN), Thermo Scientific (Pierce Protein Research Products) ( Rockford, IL) or Sigma/Aldrich Chemical Company (St. Louis, MO) unless otherwise noted.

The following abbreviations in the description correspond to units, procedures, properties, or compounds as follows: “sec” or “s” means second(s), “min” means minute(s), “h.” or “hour” means hour(s), “µl” means microliter(s), “mL” means milliliter(s), “l” means liter(s), “mM” means millimole, “M” means mole, “ mmol” means millimoles, “ppm” means ppm, “wt” means weight, “wt%” means weight percentage, “g” means gram, “mg” means milligram, “mcg” means microgram, “ng” means nanogram, “ g ” means attractive force , “HPLC” means high performance liquid chromatography, “dd H ₂ O” means distilled or deionized water, “dcw” means cell dry weight, “ATCC” or “ATCC®” means American Type Culture Collection (Manassas, Vancouver), “ed .” means unit(s) of perhydrolase activity, “rpm” means revolutions per minute, “Tg” means glass transition temperature, and “EDTA” means ethylenediaminetetraacetic acid.

Expression vector pLD001

Plasmid pLD001 (SEQ ID NO: 397) has previously been mentioned as a suitable expression vector for E. coli (See US Patent Application Publication No. 2010-0158823 A1 to Wang et al .; incorporated herein by reference).

The pLD001 vector was derived from the commercially available pDEST17 vector (Invitrogen, Carlsbad, CA). It includes sequences derived from the commercially available vector pET31b (Novagen, Madison, Wisconsin), which encodes an enzymatic ketosteroid isomerase (KSI) fragment. The KSI fragment was included as a fusion component in order to aid in the resolution of peptides into insoluble inclusion bodies in E. coli . The KSI coding sequence from pET31b was modified using standard mutagenesis techniques (QuickChange II, Stratagene, La Jolla, CA) to insert three additional Cys codons, in addition to the one Cys codon that occurs in the wild-type KSI sequence. In addition, all Asp codons in the coding sequence were changed to Glu codons. Plasmid pLD001 set forth in SEQ ID NO: 397 was constructed using standard recombinant DNA techniques well known to those skilled in the art.

EXAMPLE 1

EFFECTIVENESS OF PERACETIC ACID AS A TOOTH WHITENING AGENT

This example describes the use of peracetic acid to achieve a whitening effect on models with painted enamel surfaces. Enamelled bovine incisors were obtained from SE Dental (Baton Rouge, Louisiana). The teeth were sectioned and cut into pieces of enamel approximately 7 mm on each side using a Dremel® circular saw (Robert Bosch Power Tool Corporation; Chicago, Illinois) with a diamond blade. Enamel bars were cleaned and lightly varnished to remove chips from the surface. The enamel was pre-treated with a mixture of coffee and tea for 1-5 days in order to stain to a color similar to stained human teeth.

Each block of enamel was hydrated in water for at least 1 hour before use. Substrate color measurements were obtained prior to immersion in test solutions. Peracetic acid solutions were prepared from a 32% stock solution in 500 mM sodium phosphate buffer, pH 7.2. A solution of 2.5% H ₂ O ₂ was also prepared in the same buffer. A plurality of enamel blocks were exposed to each solution for 1 min, followed by additional immersions for 5 min, 10 min, 15 min and 30 min. For each treatment, a fresh solution of peracetic acid and hydrogen peroxide was prepared from stock solutions. After each treatment, the enamel blocks were rinsed with water and measured using a Konica-Minolta 2600d spectrophotometer. The whiteness index was determined for each sample listed in Tables 1 and 2.

The whiteness index (WI) is defined by the International Commission on Illumination (CIE) and described in ASTM method E313-05 and is calculated for incident beam D65/10 as:

WI=Y+800*(0.3138-x)+1700*(0.3310-y),

where Y, x and y, respectively, are the luminance factor and chromaticity coordinates for the enamel substrate.

Table 1
Comparison of peracetic acid bleaching with hydrogen peroxide on painted bovine enamel Sample Whiteness index ∆WI 0 min 1 min 6 min 16 min 31 min 61 min Buffer -135.1 -136.4 -131.6 -135.7 -129.2 -124.0 11.1 2.5% H ₂ O ₂ -127.5 -127.2 -124.5 -118.4 -103.6 -84.9 42.6 0.5% PAA -129.1 -111.6 -80.7 -56.5 -44.5 -38.3 90.8

table 2
Comparison of peracetic acid bleaching with hydrogen peroxide on stained bovine enamel at various concentrations Sample Whiteness index ∆WI 0 min 1 min 6 min 16 min 31 min 61 min Buffer -100.8 -98.6 -100.0 -97.1 -89.5 -84.0 16.8 2.5% H ₂ O ₂ -93.8 -92.6 -86.4 -79.5 -68.3 -56.4 37.4 0.05% PAA -103.4 -97.1 -93.1 -70.1 -59.0 -34.7 68.7 0.2% PAA -90.6 -85.5 -67.0 -47.5 -31.7 -16.2 74.4 0.5% PAA -97.5 -86.6 -64.1 -46.4 -32.7 -24.7 72.8 1% PAA -102.8 -89.3 -36.0 -20.6 -5.1 97.7

A change in the whitening index to a more positive value indicated a whitening effect. Visual inspection of the samples also showed a marked whitening effect for the peracetic acid treated samples compared to the buffered and hydrogen peroxide controls. These data demonstrate that peracetic acid is an effective whitening agent and provides superior performance over hydrogen peroxide at lower concentrations.

EXAMPLE 2

Selection of enamel and plaque binding peptides using standard biopanning

The purpose of this example was to determine phage peptides that bind to tooth enamel and pellicle using standard phage display based biopanning.

Enamelled bovine incisors were obtained from SE Dental (Baton Rouge, Louisiana). The teeth were cut into approximately 5 mm squares and varnished to remove chips from the surface. The enamel blocks were sterilized before use. The enamel blocks were immersed in the well plate containing the molding material so that only the surface of the enamel in the cell was exposed. Plaque was formed on additional enamel blocks by fixing the blocks on a wax fixation for incubation in the mouth for 30 minutes to obtain a surface covered with plaque. Plaque enamel substrates were brushed with a 1:2 slurry of ^{Colgate® MaxFresh®} toothpaste (Colgate-Palmolive, New York, NY) and re-incubated for an additional 30 minutes. Some of the blocks were removed from the wax and dipped into the well plate, while others were re-brushed before being dipped into the well plate. The immersion method provided for the contact of the solution with only the enamel and plaque-coated enamel surface.

The substrates were then incubated in blocking buffer for 1 hour at room temperature (~22°C; 1 mg/mL bovine serum albumin in phosphate buffered saline pH 7.2 (Pierce BupH™ No. 28372) with 0.1% Tween ^® 20 (PBST) followed by 2 washes with PBST Phage libraries containing random peptide inserts ( ^10-11 CFU) 15 to ²⁰ amino acids in length were added to each well. Whole saliva UV and 1 mg/ml BSA in 0.1% ^Tween® 20 (PBST) After 30 minutes incubation at 37° C. with shaking at 50 rpm, unbound phages were removed by fluid aspiration from each well followed by 6 washes with 1 .0 ml PBST.

The enamel blocks were then transferred to a clean tube and 1 ml of elution buffer consisting of 1 mg/ml BSA in 0.2 M glycine-HCl, pH 2.2 was added to each well and incubated for 10 min to elute bound phages. Then 167 μl of neutralization buffer consisting of 1M Tris-HCl, pH 9.1 was added to each well. The phage particles that were in the elution buffer as well as on the enamel blocks were amplified by incubation with 20 ml of diluted E. coli ER2738 cells from an overnight culture diluted 1:100 in LB medium at 37°C for 4, 5 hours After this time, the cell culture was centrifuged for 2 minutes and the top 15 ml of the supernatant was transferred to an unused tube, 2.5 ml of PEG/NaCl (20% polyethylene glycol-800, 2.5 M sodium chloride) was added, and phages were allowed to settle overnight at 4°C. The pellet was collected by centrifugation at 10,000× g at 4°C and the resulting pellet was resuspended in 1 ml PBS. This was the first cycle of the amplified stock solution. The amplified stock solution of phages from the first cycle was then titrated according to the standard protocol. For subsequent cycles of biopanning, more than 2×10 ¹¹ CFU of the stock phage solution from the previous cycle was used. Each additional cycle after the first also included an additional wash with whole human saliva (treated with UV for 2 hours at room temperature), two washes with carbon buffer pH 9.4 (Pierce BupH™ carbonate-bicarbonate buffer #28382), 2 washes with 50 mM phosphate buffer pH 2.5 followed by 2 washes with normal PBST.

After cycle 3 of biopanning and each subsequent cycle, 95 randomly selected individual phage plaques were isolated and single-stranded phage genomic DNA was generated using the Illustra Templiphi 500 Amplification Kit (GE Healthcare, Piscataway, NJ) and sequenced on sequencing equipment. DuPont Sequencing Facility using -96 gIII sequencing primer (5'-CCCTCATAGTTAGCGTAACG-3'; SEQ ID NO: 398). The detected peptide was located immediately after the gene III signal peptide. Based on the peptide sequences, 12 candidate phages were identified for subsequent binding analysis, which are shown in Table 3.

Table 3
Sequences of peptides that bind to tooth enamel and bind to plaque sequence ID Subsequence SEQID NO: P301 SNATMYNIQSHSHHQ 399 P302 QAAQVHMMQHSRPTT 400 P303 HDPYTMKSALRQSTS 401 P304 DLGTFPNRTLKMAAH 402 P305 DTIHPNKMKSPSSPL 403 P306 GSNHLPSTVPRLTV 404 P307 SNPIPNFAHDLRHSKYNS 405 P308 TKPPRTPTANTSRPHHNF 406 P309 ANSGFPIWLQKYPWSEVQQE 407 P310 ATPRLTPEAHHKAGNWYAS 408 P311 ATPSQHRYGLMQNHAPNGIE 409 P312 GMGSEVLSQYPQAPVG 410

EXAMPLE 3

Characterization of Tooth-Binding Enamel Candidates

The purpose of this example is to confirm the binding of the compositions of peptides on the surface of the enamel using synthetically obtained peptides.

A total of 11 synthetic peptides were made using sequences taken from Table 3. The peptides were obtained from SynBioSci Corp. (Livermore, CA) with biotin labeled lysine at the C-terminus.

Enamel substrates were prepared as described in Example 2. Each substrate was incubated for 1 hour at room temperature (~22°C) with 1 ml of blocking buffer consisting of 1 mg/ml BSA in PBST (Pierce BupH™ No. 28372 with 0.1% ^Tween® 20). The blocking buffer was removed by aspiration of fluid from each well. The tube was rinsed 2 times with wash buffer consisting of PBST. The wells were filled with 500 μl of a 20 μM peptide solution, which was diluted in blocking buffer. Samples were incubated for 30 min with slow shaking at 37°C. Non-binding peptides were removed by washing 6 times with PBST. Then 500 μl horseradish peroxidase/streptavidin conjugate (Pierce No. 22127) diluted 1:1000 in PBST was added and incubated for 1 hour at room temperature (~22°C). The conjugate solution was removed and the enamel blocks were washed 4 times with PBST.

Each enamel substrate was removed from the well and washed again in a 15 ml test tube with 10 ml PBST. Each enamel substrate was then immersed in a clean well plate, leaving only the enamel surface exposed. 200 µl of QuantaBlu™ substrate solution (Thermo-Fisher, Rockford, IL; no. 1856187) was added directly to each enamel block. The solution was incubated for 20 min at room temperature. 200 µl QuantaBlu™ stop solution (Thermo Fisher) was added. After mixing, 200 μl of the solution was transferred to a clean dark 96-well microcentrifuge plate. Plate fluorescence was measured at 325 nm excitation and 420 nm emission without threshold wavelength using a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA). The resulting fluorescence values are shown in Table 4. An assay of 11 plaque/enamel binding candidates was compared to the known DenP03 binding peptide. Each sequence was tested with enamel substrates in triplicate.

Table 4
ELISA results of a synthetic peptide on bovine enamel for binding candidates obtained from biopanning Peptide ID Amino acid sequence Medium fluorescence 325/420 SEQID NO Peptide absent --- 494.2 --- DenP03 TTYHYKNIYQESYQQRNPAVK(biotin) 3448.3 411 DenP301 SNATMYNIQSHSHHQK(biotin) 1098.4 412 DenP302 QAAQVHMMQHSRPTTK(biotin) 608.6 413 DenP303 HDPYTMKSALRQSTSK(biotin) 948.6 414 DenP304 DLGTFPNRTLKMAAHK(biotin) 642.8 415 DenP305 DTIHPNKMKSPSSPLK(biotin) 581.2 416 DenP306 GSNHLPSTVPRLTVK(biotin) 1300.0 417 DenP307 SNPIPNFAHDLRHSKYNSK(biotin) 861.6 418 DenP308 TKPPRTPTANTSRPHHNFK(biotin) 12302.6 419 DenP309 ANSGFPIWLQKYPWSEVQQEK(biotin) 1729.5 420 DenP311 ATPSQHRYGLMQNHAPNGIEK(biotin) 795.3 421 DenP312 GMGSEVLSQYPQAPVGK(biotin) 2301.0 422

EXAMPLE 4

Construction of perhydrolase and perhydrolase hybrids

This example describes the construction of an expression system to produce enamel-targeted perhydrolases with enamel-binding sequences.

Genes encoding hybrids of an enzyme having perhydrolytic activity ("perhydrolase") with enamel-binding domains were constructed with the polynucleotide sequence of the various enzymes listed in Table 5 fused at the 3' end to a nucleotide sequence encoding various amino acid flexible linkers; each linker is further fused to an enamel-binding domain or non-binding control sequences as described in Table 6. The genes were codon-optimized for expression in E. coli and synthesized using DNA2.0 (Menlo Park, CA). The coding sequences were cloned into plasmids after the T7 promoter (expression vector pLD001 (SEQ ID NO: 397)) or the pBAD promoter, between the Nde I and Asc I restriction sites, to obtain plasmids. For expression of the fusion protein, plasmids were transferred into the appropriate expression host: E. coli strain BL21AI (Invitrogen, Carlsbad, Calif.) for constructs driven by the T7 promoter, or into an AraBAD derivative of E. coli MG1655 for constructs driven by the pBAD promoter.

The non-targeted perhydrolase variants listed in Table 5 were cloned in a similar manner. The production and recombinant expression of Thermotoga maritima variants has been previously described by DiCosimo et al . in US Patent Application Publication No. 2010-0087529; included in this document by reference.

Additional CE-7 hydrolases fromLactococcus lactis (acetylxylanesterase; SEQ ID NO: 40),Mesorhizobium loti (acetylxylanesterase; SEQ ID NO: 42) andBacillus pumilus (acetylxylanesterase; SEQ ID NO: 10) was cloned in a similar manner. Cloning and expression of CE-7 perhydrolases fromLactococcus lactis,Mesorhizobium loti AndBacillus pumilushave been previously described DiCosimoet al. in US Patent Application Publication No. 2011-0081693 and US Patent No. 7951566; each of which is incorporated herein by reference.

Perhydrolytic enzymes not belonging to the CE-7 family of perhydrolases were also cloned in a similar manner. Mycobacterium smegmatis aryl esterase (“ArE”; wild-type sequence is SEQ ID NO: 478; S54V variant is given as SEQ ID NO: 460) having perhydrolytic activity is described in US Pat. No. 7,754,460. ; SEQ ID NO: 477) having perhydrolytic activity is described in US Pat. No. 7,384,787.

Table 5
Description and sequences for perhydrolase constructs Enzyme ID Abbreviated description Subsequence
(SEQ ID NO:) wt T. maritima wild type MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNICPPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKG (SEQ ID NO: 16) C277S (EZ-1) T. maritima C277S MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNI S PPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKG (SEQ ID NO: 424) C277T (EZ-12) T. maritima C277T MAFFDLPLEELKKYRPERYEEKDFDEFWEETLAESEKFPLDPVFERMESHLKTVEAYDVTFSGYRGQRIKGWLLVPKLEEEKLPCVVQYIGYNGGRGFPHDWLFWPSMGYICFVMDTRGQGSGWLKGDTPDYPEGPVDPQYPGFMTRGILDPRTYYYRRVFTDAVRAVEAAASFPQVDQERIVIAGGSQGGGIALAVSALSKKAKALLCDVPFLCHFRRAVQLVDTHPYAEITNFLKTHRDKEEIVFRTLSYFDGVNFAARAKIPALFSVGLMDNI T PPSTVFAAYNYYAGPKEIRIYPYNNHEGGGSFQAVEQVKFLKKLFEKG (SEQ ID NO: 437) HTS-007-D5 T. maritima C277T/R296P Maffdlpleelkkyrperyeekdfdefweetlaesekfpldpvfermeshlktveaydvtfsgyrgqrikgwllvpkleeeklpcvvqyigynggrgfphdwlfwpsmgyicfvmdtrgqgsgwlkgdtpdypegpvdpqypgfmtrgildprtyyyrrvftdavraveaaasfpqvdqeriviaggsqgggialavsalskkakallcdvpflchfrravqlvdthpyaeitnflkthrdkeeivfrtlsyfdgvnfaarakipalfsvglmdnitppstvfaaynyyagpkeipiypynnhegggsfqaveqvkflkklfekg (SEQ ID NO: 476) bpu B. pumilus wild type Mqlfdlsleelkkykpkktarpdfsdfwkksleelrqveaeptlesydypvkgvkvyrltyqsfghskiegfyavpdqtgphpalvrfhgynasydggihdivnwalhgyatfgmlvrgqggsedtsvtpgghalgwmtkgilskdtyyyrgvyldavraleviqsfpevdehrigviggsqggalaiaaaalsdipkvvvadypylsnferavdvaleqpyleinsyfrrnsdpkveekafetlsyfdlinlagwvkqptlmaiglidkitppstvfaaynhletdkdlkvyryfghefipafqteklsflqkhlllst
(SEQ ID NO: 10) mlo M. loti wild type Mpfpdliqpelgayvssvgmpddfaqfwtstiaearqaggevsivqaqttlkavqsfdvtfpgygghpikgwlilpthhkgrlplvvqyigygggrglaheqlhwaasgfayfrmdtrgqgsdwsvgetadpvgstssipgfmtrgvldkndyyyrrlftdavraidallgldfvdperiavcgdsqgggislavggidprvkavmpdvpflcdfpravqtavrdpyleivrflaqhrekkaavfetlnyfdcvnfarrskapalfsvalmdevcppstvygafnayagektiteyefnnheggqgyqerqqmtwlsrlfgvg (SEQ ID NO: 42) Lla L. lactis wild type Mtkinnwqdyqgsslkpedfdkfwdekinlvsnhqfefelieknlsskvvnfyhlwftaidgakihaqlivpknlkekypailqfhgyhcdsgdwvdkigivaegnvvlaldcrgqgglsqdniqtmgmtmkglivrgidegyenlyyvrqfmdlitatkilsefdfvdetnisaqgasqggalavacaalsplikkvtatypflsdyrkayelgaeesafeelpywfqfkdplhlredwffnqleyidiqnlaprikaeviwilggkdtvvppitqmaaynkiqskkslyvlpeygheylpkisdwlrenq
(SEQ ID NO: 40) Are M. smegmatis S54V Makrilcfgdsltwgwvpvedgapterfapdvrwtgvlaqqlgadfevieeglvarttniddptdprlngasylpsclathlpldlviimlgtndtkayfrrtpldialgmsvlvtqvltsaggvgttypapkvlvvsppplapmphpwfqlifeggeqktellarvysalasfmkvpffdagsvistdg NOvdgihfteannrdv6 ID: Pfl P. fluorescens L29P Mstfvakdgtqiyfkdwgsgkpvlfshgwpldadmweyqmeylssrgyrtiafdrrgfgrsdqpwtgndydtfaddiaqliehldlkevtlvgfsmgggdvaryiarhgsarvaglvllgavtplfgqkpdypqgvpldvfarfktellkdraqfisdfnapfyginkgqvvsqgvqtqtlqiallaslkatvdcvtafaetdfrpdmakidvptlvihgdgdqivpfettgkvaaelikgaelkvykdaphgfavthaqqlnedllaflkr (SEQ ID NO: 477)

Table 6
Perhydrolase constructs with targeting sequences developed for use in oral hygiene construct ID
(SEQ ID NO:) Abbreviated description Targeting sequence ^a
fusion protein
(SEQ ID NO:) EZ-1
(SEQ ID NO: 424) C277S N/A EZ-2
(SEQ ID NO: 425) C277S-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-3
(SEQ ID NO: 426) C277S-link2-H6 GS HHHHHH
(SEQ ID NO:432) EZ-4
(SEQ ID NO: 427) C277S-link1-(GK) ₅ GPGSGGAGSPGSAGGPGS GKGKGKGKGK
(SEQ ID NO:433) EZ-5
(SEQ ID NO: 428) C277S-link1-(GK) ₅ -H6 GPGSGGAGSPGSAGGPGS GKGKGKGKGKHHHHHH
(SEQ ID NO:434) EZ-7
(SEQ ID NO: 429) C277S-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-9
(SEQ ID NO: 430) C277S-link1-H6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-12
(SEQ ID NO: 437) C277T N/A EZ-14
(SEQ ID NO: 438) C277T-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-15
(SEQ ID NO: 439) C277T-link1-H6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-16
(SEQ ID NO: 440) C277T-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-17
(SEQ ID NO:441) C277T-link2-H6 GS HHHHHH
(SEQ ID NO:432) EZ-18
(SEQ ID NO:442) C277T-link1-(GK) ₅ -H6 GPGSGGAGSPGSAGGPGS GKGKGKGKGKHHHHHH
(SEQ ID NO:434) EZ-19
(SEQ ID NO: 443) C277S-EPEPE-link1-EPEPE-CXH201-H6 EPEPE GPGSGGAGSPGSAGGPGS EPEPEWKTKKILLSRTRRIMRQVVRSVMHKIWHHHHHH
(SEQ ID NO: 468) EZ-20
(SEQ ID NO: 444) C277S-EPEPEPEPEPE-link1-CXH201-H6 EPEPEPEPEPE GPGSGGAGSPGSAGGPGS WKTKKILLSRTRRIMRQVVRSVMHKIWHHHHHH
(SEQ ID NO: 469) EZ-21
(SEQ ID NO:445) C277S-EPEPE-link1-EPEPE-CXHG2-H6 EPEPE GPGSGGAGSPGSAGGPGS EPEPEPLWRRITKRKLVRPVATLMWYWFTSKRHHHHHH
(SEQ ID NO: 470) EZ-22
(SEQ ID NO: 446) C277S-EPEPEPEPEPE-Link1-CXHG2-H6 EPEPEPEPEPE GPGSGGAGSPGSAGGPGSP LWRRITKRKLVRPVATLMWYWFTSKRHHHHHH
(SEQ ID NO: 471) EZ-23
(SEQ ID NO: 447) C277S-EPEPE-Link1-EPEPE-CXH104-H6 EPEGPGSGGAGSPGSAGGPGSEPERMLSRILRMFVRILKRERLSQVRGLFVHHHHHH
(SEQ ID NO: 472) EZ-24
(SEQ ID NO: 448) C277S-EPEPEPEPEPE-Link1-CXH104-H6 EPEPEPE GPGSGGAGSPGSAGGPGS RMLSRILRMFVRILKRERLSQVRGLFVHHHHHH
(SEQ ID NO: 473) EZ-25
(SEQ ID NO: 449) C277S-EPEPE-Link1-EPEPE-CXHG102-H6 EPEPEPE GPGSGGAGSPGSAGGPGS EPEPEPELRFLARRFLKLRRARKWWNAWKVWVTRHHHHHH
(SEQ ID NO: 474) EZ-26
(SEQ ID NO: 450) C277S-EPEPEPEPEPE-Link1-CXHG102-H6 EPEPEPEPEPEPE GPGSGGAGSPGSAGGPGS LRFLARRFLKLRRARKWWNAWKVWVTRHHHHHH
(SEQ ID NO: 475) EZ-27
(SEQ ID NO: 451) bpu-link1-h6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-28
(SEQ ID NO: 452) Bpu-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-29
(SEQ ID NO: 453) Bpu-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-30
(SEQ ID NO: 454) Mlo-link1-H6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-31
(SEQ ID NO: 455) Mlo-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-32
(SEQ ID NO: 456) Mlo-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-33
(SEQ ID NO: 457) Lla-link1-H6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-34
(SEQ ID NO: 458) Lla-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-35
(SEQ ID NO: 459) Lla-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-36
(SEQ ID NO: 460) M. smegmatis ArE S54V N/A EZ-37
(SEQ ID NO: 461) ARE-link1-H6 GPGSGGAGSPGSAGGPGS HHHHHH
(SEQ ID NO:436) EZ-38
(SEQ ID NO: 462) ArE-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-39
(SEQ ID NO: 463) ArE-link1-(GK) ₅ -H6 GPGSGGAGSPGSAGGPGS GKGKGKGKGKHHHHHH
(SEQ ID NO:434) EZ-40
(SEQ ID NO: 464) ArE-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-41
(SEQ ID NO: 465) Pfl-link1-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) EZ-42
(SEQ ID NO: 466) Pfl-link1-(GK) ₅ -H6 GPGSGGAGSPGSAGGPGS GKGKGKGKGKHHHHHH
(SEQ ID NO:434) EZ-43
(SEQ ID NO: 467) Pfl-link1-DenP308-H6 GPGSGGAGSPGSAGGPGS TKPPRTPTANTSRPHHNF GSGGGGSP HHHHHH
(SEQ ID NO:435) EZ-44
(SEQ ID NO: 479) Pfl-link1-HC263-H6 GPGSGGAGSPGSAGGPGS PSAQSQLPDKHSGLHERAPQRYGPEPEPEPEPIPEPPPKEAPVVIEKPKPKPKPKPKPPAHDHKNQKETHQRHAA GSGGGGSP HHHHHH
(SEQ ID NO:431) a = flexible linkers in italics.

Example 5

Obtaining hybrid proteins

This example describes the expression and purification of perhydrolases in the presence and absence of targeting sequences for binding to the oral surface.

The strains were grown in 1 L of self-inducing medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 50 mM Na ₂ HPO ₄ , 50 mM KH ₂ PO ₄ , 25 mM (NH ₄ ) ₂ SO ₄ , 3 mm MgSO ₄ , 0.75% glycerol, 0.075% glucose and 0.05% arabinose) containing 50 mg/l spectinomycin at 37°C for 20 hours with 200 rpm rocking. The production of an untargeted perhydrolase has previously been described in US Patent Application Publication No. 2010-0087529 by DiCosimo et al . The production of targeted perhydrolases was carried out according to a similar protocol. Cells were harvested by centrifugation at 8000 rpm and washed by resuspension of the cell pellet in 20 ml of 50 mM potassium phosphate buffer, pH 7.1, containing 1 mM DTT. The solution was again centrifuged at 8000 rpm, the supernatant was removed and the pellet was redispersed in phosphate buffer containing DTT. The solution was then homogenized for 30 seconds to disperse the precipitate (Brinkman homogenizer model PCU11). The cells were then lysed by passing the solution through a French Press (SLM Instruments) at 13,000 psi (~89.6 MPa). The solution was passed through the press two additional times to achieve complete lysis. The cell solution was then transferred to a conical tube and centrifuged at 8500 rpm for 5 min. For T. maritima constructs, the supernatant was removed and heated at 80°C for 30 min. The solution was centrifuged again and the supernatant was transferred to a clean vessel.

For non-thermophilic enzymes, heat treatment was not used to purify the enzymes from contaminating cellular components. Instead, samples were purified using a His6 tag fused to the C-terminus of the enzymes by metal complexing chromatography using Co-NTA agarose (HisPur Cobalt Resin, Thermo Scientific, product number: 89965). Typically, cell extracts were loaded onto a 5-10 ml Co-NTA agarose column equilibrated with 4 volumes of equilibrium buffer (10 mM Tris HCl, pH 7.5, 10% glycerol, 1 mM imidazole, and 150 mM NaCl). The amount of each extract loaded onto the column was adjusted to contain 5 to 10 mg of perhydrolase hybrid per ml of Co-NTA agarose beads. The resin was washed with two volumes of a bed of equilibrium buffer and eluted with two volumes of elution buffer (10 mM Tris HCl, pH 7.5, 10% glycerol, 150 mM imidazole, 500 mM NaCl). Fractions were collected and the presence of purified full length proteins was confirmed by PAGE.

To obtain constructs EZ-19-EZ-26 after cell preparation, cells were harvested by centrifugation at 8000 rpm and washed by resuspension of the cell pellet in 20 ml of 50 mM potassium phosphate buffer, pH 7.2. The solution was again centrifuged at 8000 rpm, the supernatant was removed and the pellet was redispersed in phosphate buffer. The solution was then homogenized for 30 seconds to disperse the precipitate (Brinkman homogenizer model PCU11). The cells were then lysed by passing the solution through a French Press (SLM Instruments) at 13,000 psi (~89.6 MPa). The solution was passed through the press two additional times to achieve complete lysis. The cell solution was then transferred to a conical tube and centrifuged at 8500 rpm for 5 min. Insoluble lysate pellets were dissolved in sarcosyl buffer (50 mM phosphate buffer at pH 7.2, 2% ^Triton® -X100 and 1.5% sarcosyl) with 3 ml buffer per 50 ml cell lysate pellet. The solution was centrifuged and the supernatant was transferred to a new tube. Fusion proteins were purified using the HisPur ^™ Cobalt Resin kit from Thermo Scientific (Rockford, IL).

The yield according to such production and purification instructions is typically 2-10 mg protein per ml with a hybrid perhydrolase purity of 90% to 75% protein as assessed by polyacrylamide gel electrophoresis (PAGE) analysis. Total protein was quantified by bicinchoninic acid (BCA) assay (Thermo Scientific) using bovine serum albumin solution as a standard.

Example 6

Binding of enamel-targeted perhydrolase hybrid to hydroxyapatite

This example describes the binding of perhydrolase to hydroxyapatite particles. Hydroxyapatite is an effective enamel simulant.

The perhydrolase enzymes listed in Table 6 were evaluated for binding to hydroxyapatite. A dispersion of hydroxyapatite nanoparticles (Aldrich 677418) was prepared at 0.5% solids in 10 mM phosphate buffer at pH 7.2. The enzyme stock solution was added to the hydroxyapatite dispersion to a final concentration of 10 µm and incubated for 30 min in a micro centrifuge tube with gentle shaking. Each sample was centrifuged for 5 min at 10,000 rpm. The supernatant was removed and additional buffer was added. The particles were resuspended and transferred to a new tube. The method was repeated two more times. Primary enzyme injection samples, supernatant removed from the particles in each wash step, and final particles were prepared for SDS-PAGE analysis by combining 60 µl of sample, 20 µl of LDS buffer and 8 µl of reconstituting agent (Nu-PAGE). Samples were heated at 85°C for 10 min. 25 µm of each sample was loaded onto a 4-12% BisTris gel (Invitrogen) and run at 115 volts for 1.5 h. Remove the gel and stain with a 1:1 diluted Simply Blue dye solution (Invitrogen) overnight and wash off the dye for 4 hours in deionized water. The gel was analyzed and the determination of the enzyme in each fraction was evaluated by the presence of the band. The results of the analysis are presented in Table 7 indicating the relative intensity of each band in the gel compared to the enzyme input (input = 1) to determine the binding strength between the hydroxyapatite surface and each enzyme.

Table 7
Retention of perhydrolase on hydroxyapatite Enzyme Input Unrelated Wash 1 Flushing 2 HAP particles EZ-1 1 0.98 0.01 0.0 0.01 EZ-2 1 0.5 0.0 0.0 0.5 EZ-3 1 0.8 0.05 0.05 0.1 EZ-4 1 0.5 0.05 0.05 0.4 EZ-5 1 0.2 0.05 0.05 0.7 EZ-7 1 0.5 0.01 0.0 0.5 EZ-9 1 0.9 0.01 0.00 0.05

The data in Table 7 demonstrates that the targeting sequence perhydrolase fusions persisted on the hydroxyapatite after washes while the untargeted perhydrolase did not.

Example 7

Quantitative determination of enzymatic perhydrolase activity in solution and in the state associated with hydroxyapatite

This example describes a method for detecting and quantifying perhydrolase by its perhydrolase activity using triacetin and hydrogen peroxide to form peracetic acid.

Determination of peracetic acid was carried out in accordance with the method described by Pinkernell et. al . ( Analyst , 1997, 122, 567), using a colorimetric determination of the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS) with peracetic acid. After the formation of peracetic acid by adding triacetin and hydrogen peroxide, 90 μl of the solution was added to 10 μl of 0.1 MH ₃ PO ₄ in a well plate. 50 µl 1 M acetic acid, 50 µl 0.5 g/l ABTS and 50 µl 0.002 g/l KI were added. The solution was allowed to develop for 5 minutes. The absorbance of the solution was measured at 405 nm using a microplate reader. The concentration of peracetic acid was calculated based on the calibration curve, built simultaneously with the use of a solution of the reagent peracetic acid.

Enzymatic activity in solution was measured by preparing a solution of 0.625 μg/ml of each enzyme in 50 mM phosphate buffer, pH 7.2. 10 µl of the enzyme solution was mixed with 90 µl of buffer, 30 µl of 3% triacetin in water, 30 µl of 30 mM H ₂ O ₂ . The solution was incubated for 5 min. A 90 μl aliquot was taken for determination by ABTS oxidation as described above. The results are shown in Table 8.

Table 8
Determination of Peracetic Acid by ABTS Oxidation for Enzyme with Triacetin and Hydrogen Peroxide in Solution Enzyme ID Average absorption, 405 nm
co-substrate Enzyme missing 0.000 EZ-1 1,830 EZ-2 1.577 EZ-3 1.751 EZ-5 1.658 EZ-7 1.619

The perhydrolase activity of the hybrids after binding to the surfaces of the HAP discs was determined using a similar ABTS method. Hydroxyapatite discs (HiMed Inc, Old Bethpage, NY; 5 mm diameter x 1.8 mm thick) were incubated in 20 μM enzyme solution (50 mM potassium phosphate buffer, pH 7.2) for 60 min followed by 6- washing multiple times (50 mM potassium phosphate buffer, pH 7.2). The absorbed enzyme disks were transferred to new wells and 200 µl of phosphate buffer (10 mM, pH 7.2), 30 µl of 3% triacetin (final concentration 0.346%) and 30 µl of 30 mM H ₂ O ₂ (final concentration 3.46) were added. mM). The solution was allowed to incubate at room temperature for 5 minutes. 90 μl of the solution was pipetted into a new well containing 10 μl of 100 mM H ₃ PO ₄ . 50 µl acetic acid, 50 µl KI, 50 µl ABTS were added as described above. The solution was developed for 5 min at room temperature and read at A405 nm. The results are shown in Table 9.

Table 9
Determination of peracetic acid using ABTS with hydroxyapatite-related enzyme Enzyme Average absorption after 5 minutes Average absorption
minus background PAA conc.
µm per disk Enzyme missing, HAP missing 0.775 0 0 Control, no enzyme 0.840 0.065 1.2 EZ-1 0.901 0.126 3.5 EZ-5 2.154 1.379 49.7 EZ-7 1.45 1,450 23.5

This experiment demonstrates that EZ-5 and EZ-7 are active enzymes when bound to hydroxyapatite and produce peracetic acid when triacetin and hydrogen peroxide are added. The low value of peracetic acid determined with EZ-1 was consistent with other results of the observation that EZ-1 does not bind to hydroxyapatite (Example 6) and is not present on the surface, forming peracetic acid.

Example 8

Quantitative determination of enzymatic perhydrolase activity in solution and in hydroxyapatite-bound state for additional CE-7 perhydrolase constructs

This example describes the perhydrolytic activities of perhydrolase CE-7 and corresponding fusion proteins in solution and when coupled to hydroxyapatite from Thermotoga maritima , Bacillus pumilus (Bpu) , Mesorhizobium loti (Mlo), and Lactobacillus lactis (Lla) using triacetin and hydrogen peroxide to produce peracetic acid. acids.

The perhydrolase enzymes listed in Table 6 were evaluated for binding activity in solutions. A method for measuring perhydrolase activity in solution was described in Example 7 by preparing a 500 µl solution containing 0.5 µM of each enzyme, 100 mM triacetin and 100 mM H ₂ O ₂ in 100 mM phosphate buffer, pH 7.2. The solution was stirred and incubated for 10 min at 37°C. A 10 μl aliquot was taken and diluted for determination as described in Example 7. The concentration of peracetic acid was determined using a calibration curve built using a stock solution of peracetic acid (Aldrich). The experiment was also carried out in the absence of an enzyme as a control.

Table 10
Peracetic acid obtained in solution during 10 minutes of reaction with various CE-7 perhydrolase hybrids Enzyme ID Description [PAA] (ppm) Enzyme missing --- 378.9 EZ-7 C277S-DenP308 4877.2 EZ-27 bpu-h6 2332.9 EZ-28 BPU-HC263 2512.7 EZ-29 Bpu-DenP308 2273.5 EZ-30 Mlo-H6 468.8 EZ-31 Mlo-HC263 434.9 EZ-32 Mlo-DenP308 497.7 EZ-33 Lla-H6 1062.5 EZ-34 Lla-HC263 786 EZ-35 Lla-DenP308 998

To evaluate the binding of these constructs to hydroxyapatite, 33 mg of hydroxyapatite particles (Macro-prep TYPE I ceramic hydroxyapatite, 80 μm, BioRad, Hercules, CA) were washed with 10 mM phosphate buffer, pH 7.2. After the supernatant was removed, the enzyme stock solution was added to the hydroxyapatite dispersion to a final concentration of 10 µm and incubated for 30 min in a micro centrifuge tube with gentle shaking. Each sample was centrifuged for 1 min at 10,000 rpm. The supernatant was removed and additional buffer was added. The particles were resuspended and transferred to a new tube. The method was repeated two more times. The activity of the hydroxyapatite bound enzyme was measured by adding 500 μl of a solution containing 100 mM triacetin and 100 mM H ₂ O ₂ in 100 mM phosphate buffer, pH 7.2. The solution was incubated for 30 min at 37ºC. An aliquot was taken and mixed with H ₃ PO ₄ and appropriately diluted for determination by ABTS oxidation as described in Example 7. Experiments were performed on a different day with no enzyme as control for each experiment. The results are shown in Table 11.

Table 11
Peracetic acid formation in CE-7 perhydrolase hybrids associated with hydroxyapatite Enzyme ID Description Peracetic acid (ppm) Enzyme missing 118.8 EZ-9 C277S-H6 1885.1 EZ-27 bpu-h6 634.9 EZ-30 Mlo-H6 658.5 EZ-33 Lla-H6 359.1 Enzyme missing thirty EZ-2 C277S-HC263 3666.4 EZ-28 BPU-HC263 534.2 EZ-31 Mlo-HC263 241.4 EZ-34 Lla-HC263 242.2 Enzyme missing 113.9 EZ7 C277S-DenP308 4453.7 EZ-29 Bpu-DenP308 1299.8 EZ-32 Mlo-DenP308 419.3 EZ-35 Lla-DenP308 260

The experiments demonstrated that all CE-7 perhydrolase hybrids from the Bpu, Mlo and Llo enzyme exhibited significant perhydrolase activity in solution as well as after binding to hydroxyapatite surfaces compared to the control without enzyme. All three CE-7 perhydrolase hybrids from B. pumilus have higher enzymatic activity compared to hybrid enzymes from M. loti or L. lactis. Targeted EZ-29 from B. pumilus showed higher binding activity compared to untargeted EZ-27.

Example 9

Quantitative determination of enzymatic activity in solution and bound to hydroxyapatite for targeted C277S perhydrolase hybrids with CXH peptides

This example describes CE-7 C277S and hybrids with CXH peptides and their perhydrolase activities using triacetin and hydrogen peroxide to produce peracetic acid in solution and when coupled to hydroxyapatite. Hydroxyapatite is an effective enamel simulant.

The perhydrolase enzymes EZ-19-EZ-26 listed in Table 6 were evaluated for enzymatic activity in solution. A method for measuring perhydrolase activity in solution was described in Example 7. Enzymatic activity in solution was measured by preparing 500 μl of a sarcosyl buffer solution of 0.5 μM of each enzyme in 100 mM triacetin and 100 mM H ₂ O ₂ and 10 mM phosphate buffer, pH 7 ,2. The solution was incubated for 10 min at 37°C. After stopping the reaction by mixing with H ₃ PO _4, a 10 µl aliquot was taken for appropriate dilution and then determined by ABTS oxidation as described in Example 7. The results are shown in Table 12.

The same perhydrolase hybrid enzymes were also evaluated for binding to hydroxyapatite. Water-hydrated hydroxyapatite discs (5 mm diameter x 1.8 mm thick, from HiMed Inc.) were used. The disks were equilibrated in 10 mM phosphate buffer at pH 7.2 for 10 min. 200 µl of enzyme solution was added to hydroxyapatite discs to a final concentration of 10 µM in 10 mM phosphate buffer and incubated for 30 min in a micro centrifuge tube with gentle shaking. The supernatant was removed and used for activity assays of unbound enzymes. The discs were transferred to a new tube and rinsed with phosphate buffer. The method was repeated two more times. Enzymatic activity of hydroxyapatite discs with bound enzymes was measured by adding 500 μl of a solution containing 100 mM triacetin, 100 mM H ₂ O ₂ and 100 mM phosphate buffer, pH 7.2, to the disc and incubating for 30 min at 37°C. After stopping the reaction by mixing with H ₃ PO _{4 ,} a 10 μl aliquot was taken for appropriate dilution and then determined by ABTS oxidation as described in Example 7. Experiments were carried out for several days with no enzyme solution used as control every day. The results after subtracting the control without enzyme are shown in Table 12.

Table 12
Peracetic acid derived from C277s and CXH perhydrolase fusion proteins in solution and when bound to hydroxyapatite discs Enzyme ID Peracetic acid (ppm) in solution On the surface of hydroxyapatite Supernatant Disk EZ-7 5471 4864 2017 EZ-19 4088 1665 3136 EZ-20 2265 1229 2795 EZ-21 2764 1581 3109 EZ-22 2305 477 2495 EZ-23 2212 1712 4463 EZ-24 2951 719 3453 EZ-25 1703 1029 1791 EZ-26 2273 1636 1849

The experiments demonstrated that all variants of C277S with CXH targeting sequences are active and form significant amounts of peracetic acid in solution and when bound to hydroxyapatite.

Example 10

Quantitative determination of enzymatic perhydrolase activity in solution and in the state associated with hydroxyapatite for constructs that do not code for CE-7 perhydrolase

The purpose of this example is to demonstrate the use of targeted or non-targeted arylesterase enzyme variants from M. smegmatis and perhydrolase variants from P. fluorescens shown in Table 6 to produce peracetic acid in solution and when coupled to hydroxyapatite.

For arylesterase constructs, enzymatic activity in solution was measured by preparing a 1 ml solution of 0.5 μM of each enzyme, 100 mM triacetin and 100 mM H ₂ O ₂ in 100 mM phosphate buffer, pH 7.2. The solution was stirred and incubated for 30 min at 37°C. As described in Example 7, the reaction was stopped by removing a portion of H ₃ PO ₄ and a 10 μl aliquot was taken and diluted for determination by ABTS oxidation.

To evaluate binding and activity on hydroxyapatite, arylesterase enzymes were exposed to hydroxyapatite particles as described in Example 8 with 33 mg of buffer-washed HAP particles (Macro-prep ceramic Type I hydroxyapatite, 80 µm size, BioRad, Hercules, CA) with using a 10 μM solution in 10 mM phosphate buffer. After centrifugation and removal of the enzyme solution, the particles were washed with phosphate buffer by centrifugation and removal of the supernatant. 200 μl of a solution containing 100 mM triacetin, 100 mM H ₂ O ₂ and 100 mM phosphate buffer, pH 7.2, was added to the particles and incubated at 37°C for 30 min. As described in Example 7, the reaction was stopped by removing a portion of H ₃ PO ₄ and a 10 μl aliquot was taken, diluted for determination by ABTS oxidation. The results of the analyzes, both for the solution and for the state associated with the surface, are shown in Table 13.

Table 13
Peracid Formation in Solution and Surface-Bound State for Arylesterase Samples M. smegmatis Enzyme ID Description
(SEQID NO.) Peracetic acid (ppm) in solution On hydroxyapatite Enzyme missing 158 323 EZ-36 Are
(SEQ ID NO: 460) 2697 452 EZ-37 ArE-H6
(SEQ ID NO: 461) not measured 927 EZ-39 ArE-(GK) ₅ H6
(SEQ ID NO: 463) 1366 3553 EZ-40 ArE-DenP308-H6
(SEQ ID NO: 464) 3605 2476

For constructs P. fluorescens activity of the solution was measured by mixing 1 ml of a solution containing 2 μm of the enzyme, 100 mm H ₂ O ₂ in 1 M sodium acetate buffer, pH 5.5. The solution was incubated at 37°C for 30 min. As described in Example 7, the reaction was stopped by removing a portion of H ₃ PO ₄ and a 10 μl aliquot was taken and diluted for determination by ABTS oxidation. To assess binding and activity on hydroxyapatite, P. fluorescens enzymes were exposed to 20 μM hydroxyapatite particles in 10 mM phosphate buffer as described in Example 8 with 100 mg buffer-washed HAP particles (Macro-prep type I ceramic hydroxyapatite, 80 µm size, BioRad, Hercules, CA). After centrifugation and removal of the enzyme solution, the particles were washed with phosphate buffer by centrifugation and removal of the supernatant. 200 μl of a solution containing 300 mM H ₂ O ₂ in 1 M sodium acetate buffer, pH 5.5, was added to the particles and incubated at 37°C for 10 min. As described in Example 7, the reaction was stopped by removing a portion of H ₃ PO ₄ and a 10 μl aliquot was taken and diluted for determination by ABTS oxidation. The results of the analyzes, both for the solution and for the state associated with the surface, are shown in Table 14.

Table 14
Peracid Formation in Solution and Surface-Bound State for Perhydrolase Samples P. fluorescens Enzyme ID Description
(SEQID NO.) Peracetic acid (ppm) in solution On hydroxyapatite Enzyme missing 9 17 EZ-41 Pfl-link1-H6
(SEQ ID NO: 465) not measured 18 EZ-42 Pfl-link1-(GK) ₅ -H6
(SEQ ID NO: 466) 64 63 EZ-43 Pfl-link1-DenP308-H6
(SEQ ID NO: 467) 79 68 EZ-44 Pfl-link1-HC263-H6
(SEQ ID NO: 479) 58 97

These experiments demonstrate that perhydrolases from families other than the CE-7 family are active in solution and when bound to hydroxyapatite in constructs comprising a targeting sequence.

Example 11

Quantitative determination of the perhydrolytic activity of enamel-targeted variant perhydrolase hybrids C277S and C277T in solution and bound to bovine enamel

This example describes the binding of perhydrolase fusion proteins to bovine enamel and the measurement of enzymatic activity in solution and upon binding to bovine enamel.

The perhydrolase enzymes listed in Table 5 were evaluated for enzymatic activity in solution as described in Example 7. Enzymatic activity in solution was measured by preparing 500 μl of a solution of 0.5 μM of each enzyme in 100 mM triacetin and 100 mM H ₂ O ₂ and 10 mM phosphate buffer, pH 7.2. The solution was incubated for 10 min at 37°C. After stopping the reaction by mixing an aliquot with H ₃ PO ₄ , a 10 μl aliquot was taken for the appropriate dilution and then determined by ABTS oxidation as described in Example 7. The results are shown in Table 15.

Perhydrolase enzymes were also evaluated for binding to bovine enamel substrates. Enamel substrates were prepared as described in Examples 2 and 3. Each enamel block was hydrated in water overnight at room temperature (~22°C) before use. The enamel blocks were then equilibrated with 10 mM potassium phosphate buffer, pH 7.2, for 10 minutes. Enamel substrates were washed 3 times with buffer. Enamel wells were filled with 500 µl of 10 µM enzyme solutions, which were diluted in 10 mM phosphate buffer. Samples were incubated for 30 min with slow shaking at 37°C. Non-binding enzymes were removed by washing 4 times with phosphate buffer. Each block of enamel was then immersed in a gel-filled 24-well plate, leaving only the top surface of the enamel exposed. The perhydrolase activity of the enamel-associated enzyme was measured as described in Example 8. 100 μl of reaction mixtures (100 mM phosphate buffer, pH 7.2, and 100 mM H ₂ O ₂ and 100 mM triacetin) were added to the top of the enamel and incubated for 30 min at 37°C. A 90 µl aliquot was taken for ABTS oxidation determination as described in Example 8. The results are shown in Table 15. Each sample data point represents the average of 3 independent enamel blocks.

Table 15
Peracetic acid formed in solution and upon binding to the surface of bovine enamel, for constructs T. maritima Enzyme ID Description
(SEQ ID NO:) Peracetic acid (ppm) in solution on enamel Enzyme missing 114 570 EZ-1 C277S
(SEQ ID NO: 424) 4270 628 EZ-2 C277S-HC263-H6
(SEQ ID NO: 425) 4761 2549 EZ-3 C277S-link2-H6
(SEQ ID NO: 426) 5187 733 EZ-5 C277S-(GK) ₅ H6
(SED ID NO: 428) 3956 1344 EZ-7 C277S-DenP308-H6
(SEQ ID NO: 429) 5519 1040 EZ-9 C277S-H6
(SEQ ID NO: 430) 5499 612 EZ-12 C277T
(SEQ ID NO: 437) 4918 612 EZ-16 C277T-HC263-H6
(SEQ ID NO: 440) 6496 1832 EZ-17 C277T-link2-H6
(SEQ ID NO: 441) 5360 616 EZ-14 C277T-(GK) ₅ H6
(SEQ ID NO: 438) 4689 1418 EZ-18 C277T-DenP308-H6
(SEQ ID NO: 442) 5537 1158 EZ-15 C277T-H6
(SEQ ID NO: 439) 5921 631

This experiment demonstrates that hybrids generated with sequence variants C227S and C277T from T. maritima are active in solution and when bound to bovine enamel in constructs including the targeting sequence and produce sufficient levels of peracetic acid to whiten teeth.

EXAMPLE 12

PERHYDROLYTIC ENZYMES PERHYDROLYTIC ENZYMES EFFICIENCY IN ONE-STEP APPLICATION

The purpose of this example is to demonstrate the tooth whitening effect of enzymatically formed peracetic acid in a single step application and in comparison to the performance achieved with chemically prepared peracetic acid. Two methods have been developed to use the perhydrolytic enzyme system (perhydrolase CE-7) to achieve the target level of tooth whitening. The first method (referred to herein as the "one-step approach") involves combining varying amounts of at least one CE-7 perhydrolase with triacetin (an example of a suitable ester substrate) and hydrogen peroxide to form peracetic acid using stained enamel model substrates. . The bovine enamel cutters were prepared as described in Example 1. The colored bovine enamel blocks were hydrated in water at least 1 hour prior to use to stabilize the color of the substrate. The color of each enamel block was measured after hydration before the start of the experiment. Three enamel samples were processed for each type of solution. The solutions used were a buffer-only control, 2.5% H ₂ O ₂ , 1% peracetic acid, a perhydrolase formulation containing 10 μM EZ-1 (C277S; SEQ ID NO: 424), 100 mM triacetin, and 250 mM H ₂ O ₂ and an enzyme-free control of 100 mM triacetin and 250 mM H ₂ O ₂ . All solutions were prepared in 500 mM sodium phosphate buffer, pH 7.2. A high buffer strength was needed to maintain a neutral pH in a 1% peracetic acid solution. All solutions were freshly prepared for each treatment point. The enzyme-triacetin-H ₂ O ₂ combination and the triacetin-H ₂ O ₂ combination were mixed just prior to exposure to the enamel. After each stage of processing, each block of enamel was rinsed with water and measured. A total treatment time of 61 minutes was used. with exposure times of 1 min, 5 min, 10 min, 15 min and 30 min. The concentration of peracetic acid in the solution at 1 min, 10 min and 30 min after the treatments was assessed using the ABTS oxidation colorimetric determination. The results are shown in Tables 16 and 17.

Table 16
Clarification efficiency of the perhydrolase system in 1-step application on painted bovine enamel Whiteness index Sample 0 min 1 min 6 min 16 min 31 min 61 min ∆WI Buffer -136.9 -134.9 -130.9 -130.4 -125.5 -121.2 15.6 _{H2O2 _} -149.3 -146.3 -142.5 -134.7 -125.2 -103.1 46.2 Triacetin/H ₂ O ₂ -144.1 -137.4 -138.2 -125.3 -111.0 -80.9 63.2 1% PAA -145.7 -119.6 -73.4 -62.9 -59.0 -59.4 86.3 EZ-1/
Triacetin/H ₂ O ₂ -141.2 -115.6 -54.0 -37.3 -32.4 -27.9 113.3

Determination of the concentration of peracetic acid (PAA) in the reaction mixtures using a colorimetric method was carried out in accordance with the method described by Dinu et. al . ( earlier ). A solution of reagents was prepared from 1 mM 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonate (ABTS), 50 μM potassium iodide in 125 mM potassium citrate buffer pH 5.0. 25 µl of sample was mixed with 975 µl of this assay reagent and left to incubate for 5 minutes. The solution was analyzed for absorbance at 405 nm using a microplate reader. Comparison of relative amounts of peracetic acid in solution was determined by direct comparison of absorbance values.

Table 17
Estimate the concentration of peracetic acid in the solution at the end of the indicated treatment time. Each sample was a 1:100 dilution in ABTS reagent measured at 405 nm Absorption, 405 nm Sample 1 min 10 min 30 min Buffer 0.055 0.059 0.055 _{H2O2 _} 0.056 0.060 0.054 Triacetin/H ₂ O ₂ 0.061 0.069 0.052 1% PAA 2.579 1.863 2.874 EZ-1/Triacetin/H ₂ O ₂ 1.245 1.330 0.735

The data in Table 16 confirms that the enzyme-ester-peroxide composition is effective in whitening teeth. The data in Table 17 demonstrates that peracetic acid is formed by the action of a combination of the enzyme EZ-1 (C277S; SEQ ID NO: 424), triacetin and H ₂ O ₂ . A comparison of the absorbance data for the chemical peracetic acid also shows that, surprisingly, the enzymatic clarification system performs better with lower detectable peracetic acid. A low level of peracetic acid was also produced over time for enzyme-free triacetin and H ₂ O ₂ , which was detected at a 1:10 dilution in the ABTS reagent (not included in the Table). This results in detectable clarification performance, but at a slower rate compared to enzyme catalyzed high peracetic acid production.

EXAMPLE 13

PERHYDROLYTIC ENZYME ENAMEL-TARGETED EFFICIENCY IN ONE-STEP APPLICATION

The purpose of this example is to demonstrate the tooth whitening effect of enzymatically produced peracetic acid in a single step application with targeted CE-7 perhydrolase and compared to the performance achieved with chemically produced peracetic acid.

The bovine enamel cutters were prepared as described in Example 1. The colored bovine enamel blocks were hydrated in water at least 1 hour prior to use to stabilize the color of the substrate. The color of each enamel block was measured after hydration before the start of the experiment. For each type of solution, two enamel samples were processed. The solutions used were a buffer-only control, 0.1% peracetic acid, a perhydrolase formulation containing 0.52 μM EZ-7 (C277S with an enamel-binding domain; SEQ ID NO: 429), 100 mM triacetin, and 32. 6 mM H ₂ O ₂ . All solutions were prepared in 100 mM sodium phosphate buffer, pH 7.2. All solutions were freshly prepared for each treatment point. The enzyme-triacetin-H ₂ O ₂ combination was mixed just prior to exposure to the enamel. After each processing step, each block of enamel was rinsed with water and measured. The 30 min treatment periods were repeated 4 times for each set of enamel substrates. The concentration of peracetic acid in the solution after 30 minutes of treatment was evaluated by colorimetric determination of the oxidation of ABTS. The results are shown in Tables 18 and 19.

Table 18
Lightening efficiency of targeted perhydrolase in 1-step application on stained bovine enamel. Data averaged from two substrates Whiteness index Sample 0 min 30 min 60 min 90 min 120 min ∆WI Buffer -133.5 -128.7 -123.4 -123.0 -127.8 5.8 0.1% PAA -125.8 -101.9 -91.9 -80.2 -70.8 55.0

Table 19
Estimate the concentration of peracetic acid in the solution at the end of the indicated treatment time. Each sample was a 1:10 dilution in ABTS reagent,
measured at 405 nm Absorption, 405 nm [PAA] (ppm) Sample Tooth 1 Tooth 2 Buffer 0.073 0.070 0 0.1% PAA 1.103 1.043 920 EZ-7/Triacetin/H ₂ O ₂ 1.575 1.533 1350

The data in Table 18 confirms that the targeted enzyme-ester-peroxide composition is effective in whitening teeth in a 1-step process. The data in Table 19 demonstrates that peracetic acid is formed by the action of a combination of the EZ-7 enzyme, triacetin and H ₂ O ₂ .

EXAMPLE 14

PRODUCTION OF PERACETIC ACID USING CE-7 PERHYDROLASES WITH RESPECT TO A SERIES OF ETER-BASED SUBSTRATES

The purpose of this example was to demonstrate that a wide range of CE-7 perhydrolases, including an enamel-targeting perhydrolase, catalyze the formation of peracetic acid from a wide range of esters under specific oral conditions.

Thermotoga maritima perhydrolase variants were cloned, recombinantly expressed and purified in a manner similar to that described in Example 4. Sequence changes for these perhydrolase variants are shown in Table 5.

Various ester substrates were tested against at least two or more of the perhydrolase variants shown in Table 5. With the exception of three esters that were specially synthesized, all other esters were purchased from Sigma-Aldrich (St. Missouri), TCI America (Portland, Oregon), Alpha Aesar (Ward Hill, Massachusetts), or Tessendlo Company (Phoenix, Arizona).

For testing different enzymes against an ester substrate, it was convenient to run up to 4 reactions simultaneously, staging the onset of each reaction at 0.5-1 minute intervals. Each individual reaction was carried out in a glass vessel (height: 76 mm; outer diameter: 33 mm; inner diameter: 24 mm) equipped with a magnetic stirrer. The four reaction vessels were fastened together and stored at constant temperature in a 1 liter jacketed stainless steel beaker (KGW IsoTherm, no. TSS-G 1000W) with circulating water controlled by a Thermo NesLab recirculating thermostat (model no. RTE-7 Digital One). Buffer (6 ml, 100 mM sodium phosphate buffer, pH 7.2) was added to each reaction vessel and allowed to equilibrate to 37°C. The ester substrate of interest was added until a concentration of 100 mM was reached. The reaction was initiated by the simultaneous addition of 40 ppm. enzyme and 60 mm H ₂ O ₂ (37 μl, 30% hydrogen peroxide solution). To monitor the production of peracetic acid, 80 μl samples were taken from each reaction at regular intervals from 1 to 15 minutes. The sample was immediately quenched in a microcentrifuge filter tube (NanoSep 30K VWR cat. no. 82031-354) containing a volume and concentration of phosphoric acid solution, which was sufficient to stop the enzymatic reaction (by lowering the pH by 2-3) and to dilute the sample to suitable HPLC analyses. The acid-quenched samples were centrifuged for 5 minutes to remove any particles. After filtration, the Karst reaction and HPLC analyzes for peracetic acid were immediately run on each set of samples using the method previously described in US Pat. No. 7,829,315 to DiCosimo et al . The maximum peracetic acid produced with each ester substrate is shown in Table 20.

Table 20
Maximum peracetic acid obtained under neutral conditions. ¹ Peracetic acid (PAA) (ppm) Ester based substrate
(CAS No.) Without
enzyme C277S
(SEQ ID NO: 424) C277T
(SEQ ID NO: 437) C277T/R296P
(SEQ ID NO: 476) wild type
(SEQ ID NO: 16) 1-thio-β-D-glucose-2,3,4,6-tetraacetate
(19879-84-6) 10 0 171 147 156 1,5-pentanediol diacetate
(542-59-6) eleven 335 484 364 435 Diethylene glycol diacetate
(628-68-2) 17 469 653 530 424 Sorbitolhexaacetate
(7208-47-1) eleven 712 705 616 381 Sucrose octaacetate
(126-14-7) 20 215 493 501 304 4-acetoxybenzoic acid
(2345-34-8) 299 604 426 485 398 Vanillin acetate
(881-68-5) 251 660 500 405 564 propylene glycol methyl ether acetate
(108-65-5) 22 173 197 181 177 2-acetamido-2-deoxy-3,4,6-triacetyl-1-chloride-α-D-glucopyranose
(3068-34-6) 78 863 899 876 548 5-acetoxymethyl-2-furaldehyde
(10551-58-3) 67 1185 1125 1136 778 Ethylene glycol diacetate
(111-55-7) 51 1007 1059 1020 794 propylene glycol diacetate
(623-84-7) 17 1128 1245 1202 780 Diacetin
(25395-31-7) 20 1013 1132 1087 1160 α-D-glucose pentaacetate
(604-68-2) 406 2091 2542 1678 1188 β-D-glucose pentaacetate
(604-69-3) 1124 2178 2531 1716 1158 1,2,3,5-tetra-O-acetyl-ribofuranose
(13035-61-5) 2246 3397 3472 3270 2828 1,2,3,4-tetra-O-acetyl-ribopyranose
(4049-34-7) 2264 3525 3361 3503 2278 Tri-O-acetylglucal
(2873-29-2) 62 1047 1319 1034 729 Triacetin (102-76-1) 51 1977 2311 2291 937 β-D-galactose pentaacetate
(4163-60-4) 176 2539 2546 1947 764 2-acetamido-2-deoxy-1,3,4,6-tetraacetyl-β-D-glucopyranose
(7772-79-4) 2574 3374 3808 3790 2687 β-D-xylofuranose tetraacetate
(CV Chem) ^a 1373 3354 3481 3158 2384 3,4-diacetoxy-1-butene
(18085-02-4) 53 2281 2285 2478 1692 1,2,3,4-tetraacetate β-D-glucopyranose
(13100-46-4) 2102 2309 2225 2235 2129 2,3,4,6-tetraacetyl-β-D-glucopyranose
(10343-06-3) ^b 1500 2820 3072 N/A N/A Triacetin β-methylxyloside
(18531-01-6) ^b 412 3856 3635 N/A N/A 1,3,4,6-tetra-O-acetyl-mannopyranose
(18968-05-3) 1803 3593 3436 N/A 2361 α-D-mannopyranose pentaacetate
(4163-65-9) 3659 4017 4212 3752 4023 TABLE NOTES: ¹ [ester], 100 mM; [H ₂ O ₂ ], 60 mM; [perhydrolase enzyme], 40 ppm with 95 mM phosphate, pH (7.2); ^a custom synthesis with CV-Chem (CiVenti Chem, item no. CV-3146; stock no. 121-RM-134); ^b Synthesized from Robertson et al ., (1934) J. Chem. Soc ., 824-9. N/A = not tested.

These ester substrates were tested for the production of peracetic acid with a targeted variant of C277S perhydrolase, EZ-7, under the same reaction conditions used to generate data for Table 20. Production of peracetic acid with propylene glycol diacetate or sucrose octaacetate with perhydrolase the EZ-7 system is shown in Table 21. The data shown demonstrates that alternative esters are effective substrates for targeted perhydrolase variants as described in Example 5.

Table 21
Maximum peracetic acid produced with alternative substrates using targeted or untargeted CE-7 perhydrolase Peracetic acid (ppm) Enzyme ID propylene glycol diacetate Sucrose octaacetate EZ-1 2165 2768 EZ-7 2109 2070

EXAMPLE 15

TEETH CLARIFICATION USING PERHYDROLASE CE-7 WITH RESPECT TO A SERIES OF ETER BASED SUBSTRATES

The purpose of this example was to demonstrate the efficacy of tooth whitening with CE-7 perhydrolase, which catalyzes the formation of peracetic acid from four esters under appropriate oral conditions.

For this example, the C277S (EZ-1) variant of Thermotoga maritima perhydrolase was cloned, recombinantly expressed, and purified as in Example 4. All substrates were purchased from Sigma-Aldrich (St. Louis, MO) except triacetin (Tessendlo Company (Phoenix, Arizona). )).

The substrates triacetin (TA), α-D-glucose pentaacetate (GPA), sucrose octaacetate (SOC), and propylene glycol diacetate (PGDA) were used to demonstrate enzyme-mediated tooth whitening with EZ-1 (C277S; SEQ ID NO: 424). The optimal conditions used to prepare peracetic acid with each substrate were derived from the studies in Example 14 and are listed below as follows:

40 ppm EZ-1, 100 mM substrate, X mM H ₂ O ₂ , 95 mM phosphate, pH 7.2, where X varies depending on the identity of the substrate; 100 mM H ₂ O ₂ (TA), 360 mM H ₂ O ₂ (PGDA and SOC), 60 mM H ₂ O ₂ (GPA).

Bovine enamel incisors were prepared as described in Example 1. Stained enamel blocks were placed in a 24-well plate with the dentin side facing down and hydrated overnight in phosphate buffer, pH 7.2. Prior to treatment, the color was measured for each tooth using a Konica-Minolta 2600d spectrophotometer and the whiteness index was calculated as described in Example 1.

Each enzyme solution was prepared in a 1.5 ml microcentrifuge tube (Eppendorf, no. 2243102-1) and 1 ml of the solution was immediately transferred to a 24-well plate containing pre-hydrated teeth. Control samples consisted of 100 mM phosphate buffer, pH 7.2, and 9% H ₂ O ₂ in 100 mM citrate/phosphate buffer, pH 5.3. The teeth were incubated at room temperature for 30 minutes, removed, washed with 100 mM phosphate, pH 7.2, and measured for color. The teeth were placed back into the 24-well plate while fresh enzyme solutions were prepared and added to each well. This method was repeated for a total of 3 bleaching treatments; 30 minutes for processing (Table 22).

Table 22
Color measurements of coffee-tea-dyed bovine enamel exposed to 40 ppm. EZ-1, 100 mM substrate, various amounts of H ₂ O ₂ ¹ , 95 mM phosphate, pH 7.2 Sample Whiteness index ΔWI Processing 0 Treatment 1 Processing 2 Processing 3 Buffer ^a -190.4 -183.3 -181.1 -179.0 11.4 9% H ₂ O ₂ ^a -167.6 -129.6 -117.6 -115.2 52.4 TA -189.9 -108.7 -86.7 -86.1 103.8 PGDA -181.4 -108.0 -80.9 -80.1 101.3 SOC -172.8 -135.2 -135.9 -128.8 44.0 GPA -225.2 -128.7 -105.3 -95.9 129.3 Table Notes: ¹ H ₂ O ₂ concentrations vary with substrate and are as follows: 100 mM H ₂ O ₂ (TA), 360 mM H ₂ O ₂ (PGDA and SOC), 60 mM H ₂ O ₂ (GPA). ^a Control samples did not contain the enzyme.

As can be seen from Table 22, all samples of peracetic acid, enzymatically obtained with TA, PGDA, SOC and GPA, show a significant change in whiteness index, as indicated by a shift towards more positive values with each subsequent treatment. The change in whiteness index for the buffer control is within ΔWI of 11.4. These treatments also coincide with a visual examination of the teeth after each 30-minute treatment. These data demonstrate that peracetic acid, enzymatically produced using a number of different substrates with EZ-1, is effective in whitening tea-stained teeth in cattle.

EXAMPLE 16

EFFICACY OF TEETH CLARENING WITH PERHYDROLYTIC ENZYMES IN A TWO-STAGE APPLICATION

The purpose of this example was to demonstrate the efficacy of tooth whitening with perhydrolase systems in a two-stage application.

Bovine enamel incisors were prepared as described in Example 1. Stained enamel blocks were immersed in a 48-well plate to protect the dentinal posterior from exposure to the solution. A solution of 10 µm of each enzyme in 10 mM phosphate buffer, pH 7.2 was prepared and 500 µl of the solution was incubated with each enamel pad for 60 min. The enzyme solution was removed and washed three times each well with an additional 500 μl of buffer. Enamel blocks were placed in an unused well for the bleaching procedure. A final concentration of 40 mM triacetin, 100 mM H ₂ O ₂ in 50 mM phosphate buffer pH 7.2 was re-prepared and 500 μl of the solution was added to each enamel block. After 1 hour, the enamel blocks were removed and the color measured, and then each was returned to the solution in the well plate. Tracked the whiteness index for each sample. The results are listed in Table 23.

Table 23
Color measurements of tea-coffee-stained bovine enamel exposed to various perhydrolases and triacetin/H ₂ O ₂ in 2 stages Enzyme Whiteness index ∆WI 0 min 5 minutes 15 minutes 30 min 45 min 60 min Enzyme absent -108.2 -102.7 -104.0 -107.3 -98.3 -96.7 11.4 EZ-1 -152.7 -126.2 -138.9 -144.8 -107.3 -96.1 56.6 EZ-2 -154.5 -134.6 -97.3 -57.2 -35.8 -25.3 129.1 EZ-3 -159.0 -147.4 -161.5 -117.3 -104.7 -83.8 75.2 EZ-4 -158.9 -143.6 -149.5 -129.4 -106.5 -89.5 69.4 EZ-5 -135.8 -129.8 -97.4 -65.1 -49.9 -42.7 93.2

This example demonstrates that surface-bound perhydrolase can be used to whiten teeth by catalyzing the formation of peracetic acid at the enamel surface. The performance of the clarification in this example correlates with the observed retention of each enzyme on the hydroxyapatite. Untargeted EZ-1 (C277S; SEQ ID NO: 424) shows poor retention on hydroxyapatite and enamel, and hence low potential to perform suitable whitening in a 2-step process. The addition of an effective targeting sequence to retain the enzyme on the enamel makes it possible to obtain peracetic acid in a 2-step application.

Example 17

EFFICACY OF TEETH CLARENING WITH PERHYDROLYTIC ENZYMES IN A TWO-STAGE APPLICATION

The purpose of this example was to demonstrate the efficacy of tooth whitening in targeted and untargeted perhydrolase systems in a two-step application.

Bovine enamel incisors were prepared as described in Example 1. Stained enamel blocks were dipped into a well filled with Silly ^Putty® (Crayola LLC, Easton, PA) 24-well plate to protect the dentinal posterior from exposure to the solution. A solution was prepared with 20 μM of each enzyme in 10 mM phosphate buffer, pH 7.2, and 500 μl of the enzyme solution was added to each enamel block and incubated for 10 min at 37°C. The enamel block was then rinsed three times each time with an additional 500 µl of buffer. The enamel blocks were transferred and immersed in an unused silicone polymer well for the whitening procedure. A solution (200 μl) containing 100 mM phosphate buffer, 100 mM triacetin, 100 mM H ₂ O ₂ , pH 7.2 was added to the enamel and incubated for 10 min at 37°C. The enamel block was removed from the well with tweezers, then rinsed and stored in the well filled with 1.5 ml of water for color measurements. The 2-step method was repeated 5 times (total 50 min). The color measurements of coffee-tea-stained bovine enamel after the 2-step method were shown in Table 24. Each sample data point represents 2 repeats of independent enamel blocks.

To measure the level of peracetic acid formed in the system, the ABTS method was used, 90 μl of reaction mixtures were taken into a new well containing 10 μl of stop buffer (1.33 M H ₃ PO ₄ ). Samples were diluted 1:100 with 100 mM phosphate buffer and added to the ABTS developing reagent as mentioned in Example 8. The results are shown in Table 25. Each sample data point represents 2 repeats of independent enamel blocks.

Table 24
Color measurements of tea-coffee-stained bovine enamel exposed to various perhydrolases and triacetin/H ₂ O ₂ in 2 stages Stage 1 Stage 2 Whiteness index ∆WI Before Rd1 Rd2 Rd3 Rd4 Rd5 Buffer Buffer -122.9 -117.9 -116.3 -126.7 -118.4 -128.6 -5.7 Buffer Triacetin/H ₂ O ₂ -130.9 -150.4 -136.9 -134.4 -145.4 -141.9 -11.1 EZ1 Triacetin/H ₂ O ₂ -140.0 -129.5 -129.5 -129.2 -125.1 -122.1 17.9 EZ7 Triacetin/H ₂ O ₂ -120.8 -106.7 -96.0 -88.7 -85.7 -76.6 44.2

Table 25
The level of peracetic acid formation after each cycle of the bleaching process Stage 1 Stage 2 PAA (ppm) formed after each bleaching cycle Rd1 Rd2 Rd3 Rd4 Rd5 Buffer Buffer 1.2 0.5 8.7 1.3 2.0 Buffer Triacetin/H ₂ O ₂ 31.0 25.8 16.0 45.5 35.9 EZ1 Triacetin/H ₂ O ₂ 33.2 41.0 30.2 39.3 47.1 EZ7 Triacetin/H ₂ O ₂ 383.5 496.7 716.5 998.5 1224.5

This example demonstrates that surface-bound perhydrolase can be used to whiten teeth by catalyzing the formation of peracetic acid at the enamel surface. The lightening performance in this bovine enamel example correlates with the observed retention of each enzyme on the hydroxyapatite. Untargeted EZ-1 (C277S) exhibits poor retention on hydroxyapatite and enamel and therefore low potential to perform suitable whitening in a 2-step process. The addition of an effective targeting sequence to retain the enzyme on the enamel makes it possible to obtain peracetic acid in a 2-step application.

Claims (68)

1. A method of oral care, including: 1) providing a set of reaction components that contains: a) at least one substrate selected from the group consisting of: i) esters with the structure [X] _m R ₅ , where X=ester group with the formula R ₆ C(O)O, R ₆₌ C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ester bonds for R ₆ =C2-C7; R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group, where R ₅ optionally contains one or more ester bonds; m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And where these esters have a solubility in water of at least 5 ppm at 25°C; ii) glycerides with the structure

, where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O); iii) one or more esters of the formula

, where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl , alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )-O) _n H, and n is 1-10; And iv) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides; b) a peroxy oxygen source and c) an enzyme catalyst having perhydrolytic activity of SEQ ID NO: 425-430, 438-459, 461-464, 466, 467 or 479; And 2) combining the reaction components (1) under suitable reaction conditions, whereby at least one peracid is enzymatically obtained; And 3) contacting the oral surface with at least one peracid, whereby the oral surface receives a peracid-based benefit selected from the group consisting of brightening, teeth whitening, decontamination, discoloration, deodorization, biofilm reduction or elimination, or their combinations. 2. The method according to p. 1, where the reaction components are combined in the oral cavity. 3. The method of claim. 1, where the reaction components are combined outside the oral cavity before bringing into contact with the surface of the oral cavity. 4. The method of claim 1, wherein the enzyme catalyst having perhydrolytic activity is present in the oral cavity until the peracid benefit agent is obtained. 5. An oral care product comprising: 1) an enzyme catalyst having perhydrolytic activity, with SEQ ID NO: 425-430, 438-459, 461-464, 466, 467 or 479; 2) at least one substrate selected from the group consisting of: a) esters with the structure [X] _m R ₅ , where X=ester group with the formula R ₆ C(O)O, R ₆ =C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ether linkages for R ₆ =C2-C7; R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds; m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And where these esters have a solubility in water of at least 5 ppm at 25°C; b) glycerides with the structure

, where R ₁ = C1-C7 straight-chain or branched-chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O); c) one or more esters with the formula

, where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl , alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )-O) _n H, and n is 1-10; And d) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides; 3) a source of peroxy group oxygen and 4) a carrier medium acceptable to the mouth. 6. The oral care product of claim 5, wherein the enzyme catalyst binds to tooth enamel or binds to dental plaque. 7. The oral care product of claim 5 or 6, wherein the oral care product is in the form of a powder, paste, gel, liquid, ointment, tablet, rinse, or any combination thereof. 8. The oral care product of claim 5, wherein the oral care product is toothpaste, tooth cream, tooth gel, tooth powder, mouthwash, breath freshener, strip or floss. 9. The oral care product of claim 5, wherein the oral care product is in the form of a tooth whitening strip or mouthguard. 10. The oral care product of claim 5, wherein the enzyme catalyst remains separated from the substrate, the peroxy oxygen source, or both the substrate and the peroxy oxygen source prior to use of the oral care product. 11. The use of a composition for the formation of peracid containing: 1) an enzyme catalyst having perhydrolytic activity, with SEQ ID NO: 425-430, 438-459, 461-464, 466, 467 or 479; And 2) at least one substrate selected from the group consisting of: a) esters with the structure [X] _m R ₅ , where X=ester group with the formula R ₆ C(O)O, R ₆₌ C1-C7 linear, branched or cyclic hydrocarbyl fragment, optionally substituted with hydroxyl groups or C1-C4 alkoxy groups, where R ₆ optionally contains one or more ester bonds for R ₆ =C2-C7; R ₅ =C1-C6 linear, branched or cyclic hydrocarbyl moiety, or a five-membered cyclic heteroaromatic moiety, or a six-membered cyclic aromatic or heteroaromatic moiety, optionally substituted with hydroxyl groups; where each carbon atom in R ₅ individually contains no more than one hydroxyl group or no more than one ester group or carboxylic acid group; where R ₅ optionally contains one or more ether bonds; m is an integer ranging from 1 to the number of carbon atoms in R ₅ ; And where these esters have a solubility in water of at least 5 ppm at 25°C; b) glycerides with the structure

, where R ₁ =C1-C7 straight chain or branched alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₃ and R ₄ individually are H or R ₁ C(O); c) one or more esters with the formula

, where R ₁ is C1-C7 straight chain or branched chain alkyl optionally substituted with hydroxyl or C1-C4 alkoxy, and R ₂ is C1-C10 straight chain or branched chain alkyl, alkenyl, alkynyl, aryl, alkylaryl , alkylheteroaryl, heteroaryl, (CH ₂ CH ₂ O) _n or (CH ₂ CH(CH ₃ )-O) _n H, and n is 1-10; And d) acetylated saccharides selected from the group consisting of acetylated monosaccharides, acetylated disaccharides and acetylated polysaccharides; And 3) a source of oxygen of the peroxy group; moreover, when mixing (1), (2) and (3) peracid is formed; for the treatment or prevention of dental caries, gingivitis, oral candidiasis or periodontitis.