Oral care composition comprising a DNase

Abstract

The present invention relates to an oral care composition comprising a DNase, uses of the composition, uses of the composition in the treatment of oral diseases, methods of treatment comprising administering the composition to a human subject, methods of preventing or removing oral biofilm comprising contacting an oral biofilm with the composition, methods for reducing the risk of oral biofilm formation, and kits comprising the composition.

Description

[0001] References to sequence lists This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Technical Field

[0002] This invention relates to an oral care composition comprising a DNA enzyme, the use of the composition in medicine, the use of the composition in treating oral diseases, a treatment method including administering the composition to a human subject, a method for preventing or removing oral biofilms by contacting the composition with the oral biofilm, a method for reducing the risk of oral biofilm formation, and a kit containing the composition. Background Technology

[0003] Biofilms are bacterial communities found on solid surfaces in many different environments, including the oral cavity. Oral biofilms, or dental plaque, contain many bacteria associated with oral health problems such as bad breath, demineralization, tooth decay, cavities, potential tooth loss, and gum disease (gingivitis and periodontitis).

[0004] Oral biofilm formation occurs in three phases, known as the retardation phase, the growth phase, and the stationary phase. During retardation, glycoproteins from saliva bind to the oral surface (e.g., teeth) and form a structure called a biofilm, which serves as an attachment site for bacteria. During the growth phase, co-aggregation occurs, where a second bacterial colonist attaches to the first, leading to increased biofilm diversity and growth and maturation. In the stationary phase, biofilm growth slows and eventually ceases. This phase-based formation cycle results in biofilms existing in several consecutive layers, making physical wear and tear on the biofilm more difficult.

[0005] Within biofilms, resident bacterial cells are distributed within an extracellular polymeric matrix primarily composed of water, proteins, exopolysaccharides, lipopolysaccharides, lipids, surfactants, and extracellular DNA (eDNA). eDNA is a crucial component of the extracellular polymeric matrix and plays a vital role in biofilm formation and stability, as well as the antimicrobial properties of the bacteria it encapsulates. eDNA in biofilms is known to influence initial attachment and adhesion to surfaces, as well as subsequent accumulation, and it also has a stabilizing effect on biofilms as it coats the biofilm surface. Furthermore, eDNA derived from lysed bacteria may contain genes conferring resistance to antimicrobial agents. In cases where such DNA fragments transfer within the biofilm and integrate into the chromosome of living bacterial cells, this can lead to novel phenotypes with improved antimicrobial resistance.

[0006] Due to increased resistance to antimicrobial agents and the mechanical properties of biofilms, many current oral care products are highly ineffective in addressing biofilm formation and alleviating related oral health problems. The focus of biofilm removal is on mechanical abrasion. However, the multilayered nature of biofilms makes mechanical abrasion difficult, and because mechanical removal of biofilms (e.g., by brushing) expands and deepens the area where biofilms adhere and spread in the mouth, it potentially increases rather than reduces the severity of the problem, thus further impacting mechanical abrasion.

[0007] Given the important role of biofilms in oral diseases, there is a need in the art for oral care compositions that can provide improved prevention and / or removal of oral biofilms. In particular, there is a need for agents capable of effectively targeting the eDNA components of oral biofilms.

[0008] WO 2020 / 099491 relates to oral care compositions containing NUC1 / NUC2 type DNA enzymes and methods for the prevention and removal of biofilms. Summary of the Invention

[0009] The inventors of this invention have identified certain microbial DNases that are highly effective in degrading several types of DNA secondary structures. As illustrated in the examples of this application, the DNases of this invention are capable of degrading B-DNA (the conventional right-handed double helix structure of genomic DNA), Z-DNA (a naturally occurring, limited, alternative left-handed double helix structure), and G-quadruplex DNA (G4-DNA; a helical structure of guanidine-rich DNA sequences that can exhibit different topologies through Hoogsteen base pairing). The broad substrate specificity of the DNases of this invention is associated with improved biofilm prevention / removal, particularly for oral biofilms containing B-DNA, Z-DNA, and G4-DNA as part of the eDNA component. Furthermore, the DNases of this invention are highly stable in the presence of various oral care ingredients, making them well-suited for oral care applications.

[0010] In a first aspect, the present invention relates to an oral care composition comprising a DNase selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0011] In a second aspect, the present invention relates to an oral care composition of the first aspect for use in the treatment of oral diseases.

[0012] In a third aspect, the present invention relates to the oral care composition of the first aspect for use as a medicine.

[0013] In a fourth aspect, the present invention relates to methods for preventing and / or removing oral biofilms, the methods comprising contacting the oral biofilm with an oral care composition of the first aspect.

[0014] In a fifth aspect, the present invention relates to polypeptides having DNase activity selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0015] In another aspect, the present invention also relates to a polynucleotide encoding a polypeptide of the fifth aspect, a nucleic acid construct or expression vector comprising said polynucleotide, and a recombinant host cell comprising said nucleic acid construct or expression vector. The present invention also relates to a method for producing a polypeptide having DNase activity.

[0016] definition Based on this detailed description, the following definitions apply. Note that unless otherwise explicitly stated in the context, the singular forms “a / an” and “the” include plural indicators.

[0017] Unless otherwise defined or explicitly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0018] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks intron sequences that can be present in the corresponding genomic DNA. The initial primary RNA transcript is the precursor of mRNA, which is processed through a series of steps (including splicing) to become mature, spliced ​​mRNA.

[0019] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined by an open reading frame (ORF), which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0020] Control Sequences: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of polynucleotides in a particular organism, either in vivo or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding a polypeptide, and is native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction sites that facilitate the linking of control sequences to the coding regions of polynucleotides encoding polypeptides.

[0021] Dentures: The term “dentures” is intended to encompass dentures themselves as well as orthodontic appliances, clear aligners, fixation devices, etc.

[0022] DNase: The term "DNase" refers to a polypeptide having deoxyribonuclease (DNase) (EC 3.1.21 or EC 3.1.22) activity, which catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. The terms "DNase" and "polypeptide having deoxyribonuclease activity" are used interchangeably throughout this application. For the purposes of this invention, DNase activity can be determined according to DNase activity assay I or DNase activity assay II as described in the examples below.

[0023] Expression: The term “expression” refers to any step involved in polypeptide production, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0024] Expression vector: An expression vector is a linear or circular DNA construct containing a DNA sequence encoding a polypeptide, with the coding sequence operatively linked to a suitable control sequence that can influence the expression of the DNA in a suitable host. Such control sequences may include promoters that affect transcription, optional operon sequences that control transcription, sequences encoding suitable ribosome binding sites on mRNA, enhancers, and sequences that control the termination of transcription and translation.

[0025] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has DNase activity.

[0026] Fragment: The term "fragment" refers to a polypeptide that has one or more amino acids missing from the amino and / or carboxyl termini of a mature polypeptide, wherein the fragment has DNase activity.

[0027] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one of the polypeptides of the present invention is fused to the N-terminus and / or C-terminus. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide with a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences of the polypeptides such that they conform to reading frames, and that the expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intronomer technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, ). EMBO J. [Journal of the European Society for Molecular Biology] 12: 2575-2583; Dawson et al., 1994. Science [Science] 266: 776-779). Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003. J. Ind. Microbiol. Biotechnol. [Journal of Industrial Microbiology and Biotechnology] 3: 568-576; Svetina et al., 2000, J. Biotechnol [Journal of Biotechnology] 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63:3488-3493; Ward et al., 1995. Biotechnology [Biotechnology] 13:498-503; and Contreras et al., 1991, Biotechnology [Biotechnology] 9:378-381; Eaton et al., 1986, Biochemistry [Biochemistry] 25:505-512; Collins-Racie et al., 1995, Biotechnology [Biotechnology] 13: 982-987; Carter et al., 1989, Proteins : Structure, Function, and Genetics [Proteins: Structure, Function, and Genetics] 6:240-248; and Stevens, 2003. Drug Discovery World [Drug Discovery World] 4:35-48.

[0028] Heterogeneous: For host cells, the term "heterogeneous" means that the polypeptide or nucleic acid is not naturally present in the host cell. For polypeptides or nucleic acids, the term "heterogeneous" means that the control sequence (e.g., the promoter of the polypeptide or nucleic acid) is not naturally associated with that polypeptide or nucleic acid; that is, the control sequence comes from a gene other than the gene encoding the mature polypeptide.

[0029] Host strain or host cell: A “host strain” or “host cell” refers to an organism in which an expression vector, bacteriophage, virus, or other DNA construct (including a polynucleotide encoding a polypeptide of interest, such as an amylase) has been introduced. An exemplary host strain is a microbial cell (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term “host cell” includes protoplasts produced by cells.

[0030] Introduction: In the context of inserting a nucleic acid sequence into a cell, the term “introduction” means “transfection,” “conversion,” or “transduction,” as is known in the art.

[0031] Isolated: The term "isolated" means a polypeptide, nucleic acid, cell, or other specific material or component that has been separated from at least one other material or component (including, but not limited to, other proteins, nucleic acids, cells, etc.). Therefore, the isolated polypeptide, nucleic acid, cell, or other material exists in a form not found in nature. Isolated polypeptides include, but are not limited to, culture media containing secreted polypeptides expressed in host cells.

[0032] Mature peptide: The term “mature peptide” refers to a peptide that has been processed at its N-terminus and / or C-terminus (e.g., removal of the signal peptide) to be in its mature form.

[0033] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to the polynucleotide that encodes a mature polypeptide with DNase activity.

[0034] Natural: The term "natural" refers to nucleic acids or polypeptides that are naturally present in host cells.

[0035] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding polypeptides. Nucleic acids can be single-stranded or double-stranded and may include chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention cover nucleotide sequences encoding specific amino acid sequences. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0036] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a way that does not originally exist in nature to contain a segment of nucleic acid or is synthesized and contains one or more control sequences that are operatively linked to the nucleic acid sequence.

[0037] Operationally linked: The term "operationally linked" means that specified components are in a relationship that allows them to function in the intended manner (including, but not limited to, juxtaposition). For example, a regulatory sequence is operationally linked to a coding sequence such that the expression of the coding sequence is under the control of the regulatory sequence.

[0038] Parent: The term "parent" or "parental polypeptide" refers to an enzyme that has been altered to produce an enzyme variant. In one respect, a parent is a parental DNAse that has been altered to produce a DNAse variant.

[0039] Purified: The term "purified" means nucleic acids, peptides, or cells that are substantially free of other components, as determined by analytical techniques well known in the art (e.g., in electrophoretic gels, chromatographic eluates, and / or media subjected to density gradient centrifugation, where purified peptides or nucleic acids form discrete bands). Purified nucleic acids or peptides are at least about 50% pure, and typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, or more pure (e.g., weight percentage or molar percentage). In a relevant sense, a composition is enriched with the molecule when the concentration of the molecule increases significantly after the application of purification or enrichment techniques. The term “enrichment” refers to the presence of compounds, peptides, cells, nucleic acids, amino acids, or other specified materials or components in a composition at a relative or absolute concentration higher than that of the starting composition.

[0040] In one respect, the term "purified," as used herein, means that the polypeptide or cell is substantially free of components (especially insoluble components) from the producing organism. In other respects, the term "purified" means that the polypeptide is substantially free of insoluble components (especially insoluble components) from the natural organism from which it was obtained. In one respect, the polypeptide is separated from some soluble components of the organism from which it was recovered and the culture medium. The polypeptide can be purified (i.e., separated) by one or more of the following unit operation methods: filtration, precipitation, or chromatography.

[0041] Accordingly, the polypeptide can be purified such that only small amounts of other proteins, particularly other polypeptides, are present. The term "purified" as used herein can mean the removal of other components, particularly other proteins and most particularly other enzymes, present in the cells from which the polypeptide originates. The polypeptide can be "substantially pure," meaning it is free from other components from the organism that produced it (e.g., the host organism used to recombinantly produce the polypeptide). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the formulation. In another aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the formulation. As used herein, "substantially pure polypeptide" can mean a polypeptide formulation containing, by weight, at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other polypeptide material naturally or recombinantly associated with the polypeptide.

[0042] Therefore, it is preferred that the substantially pure polypeptide, based on the weight of the total polypeptide material present in the formulation, is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure. The polypeptides of the present invention are preferably in a substantially pure form (i.e., the formulation is substantially free of other polypeptide materials associated with its natural or recombinant form). For example, this can be achieved by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0043] Recombination: The term "recombination," used in its conventional sense, refers to the manipulation (e.g., cutting and rejoining) of nucleic acid sequences to form a sequence group different from that found in nature. The term recombination refers to cells, nucleic acids, polypeptides, or vectors that have been modified from their natural state. Thus, for example, recombinant cells express genes not found in their natural (non-recombinant) forms, or express natural genes at different levels or under different conditions compared to those found in nature. The term "recombination" is synonymous with "genetically modified" and "transgenic."

[0044] Recovery: The term "recovery" refers to the removal of peptides from at least one fermentation broth component selected from a list of cells, nucleic acids, or other specified materials, for example, by methods such as: harvesting peptides by peptide crystallization, by filtration (e.g., deep filtration (using filter aids or packed filter media, cloth filtration in a box filter, rotary drum filtration, drum filtration, rotary vacuum drum filtration, candle filter, horizontal leaf filter, or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, modular filtration, candle filtration, microfiltration, crossflow, dynamic crossflow, or dead-end operation ultrafiltration)), or by centrifugation (using a horizontal centrifuge, disc stack centrifuge, hydrocyclone, or the like), or by precipitation of peptides and using relevant solid-liquid separation methods to harvest peptides from broth media by particle size fractionation. Recovery encompasses the separation and / or purification of peptides.

[0045] Sequence identity: The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0046] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48: 443-453) is used to determine the sequence identity between two amino acid sequences as the output of "longest identity". This algorithm is implemented in the Niedel program using the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. For the Niedel program to report the longest identity, the non-brief (-nobrief) option must be specified in the command line. The Niedel-marked "longest identity" output is calculated as follows: (identical residues × 100) / (alignment length - total number of vacancies in the alignment) For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two polynucleotide sequences as the output of "longest identity," as implemented by the Niedle program in the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 6.6.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. For the Niedle program to report the longest identity, the non-simplified option must be specified in the command line. The Niedle-marked "longest identity" output is calculated as follows: (Identical deoxyribonucleotides × 100) / (Alignment length – Total number of vacancies in the alignment) Signal peptide: A signal peptide is an amino acid sequence attached to the N-terminal portion of a polypeptide that promotes the secretion of the polypeptide outside the cell. The mature form of the extracellular polypeptide lacks the signal peptide, which is cleaved during the secretion process.

[0047] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more nucleotides are deleted from the 5' and / or 3' end of the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment with DNase activity.

[0048] Variant: The term "variant" refers to a DNase that contains artificial mutations (i.e., substitutions, insertions (including extensions), and / or deletions (e.g., truncations)) at one or more positions compared to the parental DNase. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, especially 1 amino acid) adjacent to and immediately following an amino acid occupying a position.

[0049] Wild-type: When referring to an amino acid or nucleic acid sequence, the term "wild-type" means that the amino acid or nucleic acid sequence is a naturally occurring or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences). Attached Figure Description

[0050] Figure 1An example of thermal stability data generated using a nanoDSF instrument is shown, as described in Example 4. Subplot A is an example of data from SEQ ID NO:1 obtained in triplicate as a function of temperature (ratio of fluorescence emission at 350 nm to 330 nm). Subplot B shows the first derivative of the raw data from Subplot A. The peak maximum in the first derivative plot corresponds to the midpoint of the pyrolysis fold transition, termed Tm. In this example, Tm at pH 6 corresponds to 52.9 °C and is highly reproducible across three replicates.

[0051] Sequence Overview

[0052] SEQ ID NO:1 is a species obtained from the genus Bacillus ( Bacillus Mature DNAase sp)-62490.

[0053] SEQ ID NO:2 is derived from Sackievirus 1,000 (Sackievirus 1,000). Bacillus horikoshii Mature DNA enzymes.

[0054] SEQ ID NO:3 is obtained from *Salmonella floccosum* ( Halalkalibacter akibai Mature DNA enzymes.

[0055] SEQ ID NO:4 is Bacillus clausti ( Bacillus clausii Signal peptide.

[0056] SEQ ID NO:5 is a His tag.

[0057] SEQ ID NO:6 is the G4 DNA model substrate (G4_1).

[0058] SEQ ID NO:7 is the G4 DNA model substrate (G4_2).

[0059] SEQ ID NO:8 is the Z-DNA model substrate (ds Z-DNA_1).

[0060] SEQ ID NO:9 is the Z-DNA model substrate (ds Z-DNA_2).

[0061] SEQ ID NO:10 is a B-DNA model substrate (ds B-DNA_1).

[0062] SEQ ID NO:11 is a B-DNA model substrate (ds B-DNA_2).

[0063] SEQ ID NO:12 is a nucleic acid construct used to express SEQ ID NO:1.

[0064] SEQ ID NO:13 is a nucleic acid construct used to express SEQ ID NO:2.

[0065] SEQ ID NO:14 is a nucleic acid construct used to express SEQ ID NO:3. Detailed Implementation

[0066] The inventors of this invention have identified certain microbial DNases that are highly effective in degrading several types of DNA secondary structures. As illustrated in the examples of this application, the DNases of this invention are capable of degrading B-DNA (the conventional right-handed double helix structure of genomic DNA), Z-DNA (a naturally occurring, limited, alternative left-handed double helix structure), and G-quadruplex DNA (G4-DNA; a helical structure of guanidine-rich DNA sequences that can present different topologies through Hussant base pairing). The broad substrate specificity of the DNases of this invention is associated with improved biofilm prevention / removal, particularly for oral biofilms containing B-DNA, Z-DNA, and G4-DNA as part of the eDNA component. Furthermore, the DNases of this invention are highly stable in the presence of various oral care ingredients, making them well-suited for oral care applications.

[0067] Polypeptides with DNA enzyme activity In one respect, DNA enzymes are selected from the following group: (a) A polypeptide having DNase activity and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1; (b) A polypeptide encoded by a polynucleotide having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:12; (c) A polypeptide derived from SEQ ID NO:1 by substitution, deletion or addition of one or more amino acids; (d) A polypeptide derived from (a), (b), or (c); wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids; and (e) A fragment of a polypeptide of (a), (b), (c) or (d); wherein the polypeptide has DNase activity.

[0068] In a preferred embodiment, the DNase has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1.

[0069] In a preferred embodiment, the DNA enzyme comprises SEQ ID NO:1, consists substantially of SEQ ID NO:1, or consists of SEQ ID NO:1.

[0070] DNA enzymes can have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1-5 amino acids).

[0071] On the other hand, DNases are derived from SEQ ID NO:1 through the substitution, deletion or addition of one or more amino acids.

[0072] In some embodiments, the DNase is a variant of the parental DNase (preferably SEQ ID NO:1) that includes substitutions, deletions, and / or insertions at one or more sites. In one aspect, the DNase is a variant of SEQ ID NO:1, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:1 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine fragment, an antigenic epitope, or a binding module.

[0073] In one respect, DNA enzymes are selected from the following group: (a) A polypeptide having DNase activity and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2; (b) A polypeptide encoded by a polynucleotide having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:13; (c) A polypeptide derived from SEQ ID NO:2 by substitution, deletion or addition of one or more amino acids; (d) A polypeptide derived from (a), (b), or (c); wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids; and (e) A fragment of a polypeptide of (a), (b), (c) or (d); wherein the polypeptide has DNase activity.

[0074] In a preferred embodiment, the DNase has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2.

[0075] In a preferred embodiment, the DNA enzyme comprises SEQ ID NO:2, is substantially composed of SEQ ID NO:2, or is composed of SEQ ID NO:2.

[0076] DNA enzymes can have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1-5 amino acids).

[0077] On the other hand, DNases are derived from SEQ ID NO:2 through the substitution, deletion or addition of one or more amino acids.

[0078] In some embodiments, the DNase is a variant of the parental DNase (preferably SEQ ID NO:2) that includes substitutions, deletions, and / or insertions at one or more sites. In one aspect, the DNase is a variant of SEQ ID NO:2, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:2 is up to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine fragment, an antigenic epitope, or a binding module.

[0079] In one respect, DNA enzymes are selected from the following group: (a) A polypeptide having DNase activity and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3; (b) A polypeptide encoded by a polynucleotide having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide encoding sequence of SEQ ID NO:14; (c) A polypeptide derived from SEQ ID NO:3 by substitution, deletion or addition of one or more amino acids; (d) A polypeptide derived from (a), (b), or (c); wherein the N-terminus and / or C-terminus have been extended by adding one or more amino acids; and (e) A fragment of a polypeptide of (a), (b), (c) or (d); wherein the polypeptide has DNase activity.

[0080] In a preferred embodiment, the DNAse has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3.

[0081] In a preferred embodiment, the DNA enzyme comprises SEQ ID NO:3, is substantially composed of SEQ ID NO:3, or is composed of SEQ ID NO:3.

[0082] DNA enzymes can have an N-terminus and / or C-terminus extension of one or more amino acids (e.g., 1-5 amino acids).

[0083] On the other hand, DNases are derived from SEQ ID NO:3 through the substitution, deletion or addition of one or more amino acids.

[0084] In some embodiments, the DNase is a variant of the parental DNase (preferably SEQ ID NO:3) that includes substitutions, deletions, and / or insertions at one or more sites. In one aspect, the DNase is a variant of SEQ ID NO:3, and the number of amino acid substitutions, deletions, and / or insertions introduced into the polypeptide of SEQ ID NO:3 is up to 15, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1-30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, such as a polyhistidine fragment, an antigenic epitope, or a binding module.

[0085] Procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989), can be used. Science [Science] 244:1081-1085) to identify essential amino acids in polypeptides. In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the DNase activity of the resulting molecule is tested to identify the amino acid residues critical to the molecule's activity. See also Hilton et al., 1996. J. Biol. Chem.[Journal of Biochemistry] 271: 4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutagenesis of the amino acids at the presumed contact sites. See, for example, de Vos et al., 1992. Science [Science] 255:306-312; Smith et al., 1992. J. Mol. Biol. [Journal of Molecular Biology] 224: 899-904; Wlodaver et al., 1992. FEBS Lett. [FEC Biochemical Society Letters] 309: 59-64. The identity of essential amino acids can also be inferred from alignment with related peptides, and / or from sequence homology and conserved catalytic mechanisms with related peptides or peptide / protein families from a common ancestor (typically possessing similar three-dimensional structures, functions, and significant sequence similarities). Alternatively or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of peptides. See, for example, Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”. Nature [Nature] 596:583-589.

[0086] Using known mutagenesis, recombination, and / or tampering methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested. Such screening procedures include those developed by Reidhaar-Olson and Sauer, 1988. Science [Science] 241: 53-57; Bowie and Sauer, 1989. Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 86:2152-2156; WO 95 / 17413; or those disclosed in WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry [Biochemistry] 30:10832-10837; US 5,223,409; WO 92 / 06204) and regional directed mutagenesis (Derbyshire et al., 1986, Gene [Gene] 46:145; Ner et al., 1988, DNA 7:127).

[0087] Mutagenesis / recombination methods can be combined with high-throughput, automated screening methods to detect the activity of clonal, mutagenic peptides expressed by host cells (Ness et al., 1999). Nature Biotechnology [Nature Biotechnology] 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the peptide.

[0088] The DNase of the present invention prevents the formation of oral biofilm. Preferably, the DNase has an improved effect on the prevention of oral biofilm. In the embodiments, the DNase prevents the formation of oral biofilm by at least 5%, for example 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%. For the purposes of the present invention, oral biofilm prevention can be determined, for example, according to Example 4 below.

[0089] The DNase of the present invention reduces the risk of oral biofilm formation. Preferably, the DNase reduces the risk of oral biofilm formation by at least 5%, for example 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100%.

[0090] The DNase of the present invention can also remove oral biofilm. Preferably, the DNase has an improved effect on the removal of oral biofilm. In the embodiments, the DNase removes at least 5%, for example 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the oral biofilm.

[0091] The DNase of the present invention is highly stable in formulations and / or forms suitable for oral care, particularly in formulations or forms such as toothpaste, mouthwash, lozenges, mints, chewing gum, and candy. High stability, for example, equivalent or improved stability, can be equivalent or improved physical and / or chemical stability. When the DNase is co-formulated and / or co-administered with another agent, preferably in co-formulation, equivalent or improved chemical stability can occur, i.e., equivalent or improved stability in the presence of the other agent (e.g., another enzyme, active ingredient, excipient, or solvent).

[0092] In a preferred aspect, the DNase has comparable or improved thermal stability. In the context of this invention, the term "comparable thermal stability" means that, in the presence of a particular oral care ingredient or component (or alternatively, as co-formulated with a particular oral care ingredient or component), the thermal stability of the DNase is within + / - 5% of the thermal stability of the same DNase alone (i.e., in the absence of said oral care ingredient). In the context of this invention, the term "improved thermal stability" means that, compared to the thermal stability of the same DNase alone (i.e., in the absence of said oral care ingredient), the thermal stability of the DNase is improved by at least 5%, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or even higher. For the purposes of this invention, thermal stability may be determined according to Example 4 below and defined by the pyrolysis fold transition midpoint (Tm).

[0093] In one embodiment, the DNase has comparable or improved thermal stability in the presence of at least one of the following groups, for example, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or all of the oral care ingredients: benzoate (preferably sodium benzoate), EDTA, ethanol, fluoride (preferably sodium fluoride), glycerol, hydrogen peroxide, mannitol, phosphate (preferably sodium phosphate), SDS, sorbate (preferably potassium sorbate), and sorbitol.

[0094] In a preferred embodiment, the DNase exhibits comparable or improved thermal stability at pH 4-8, for example, at pH 4, 5, 6, 7, or 8. Preferably, the DNase exhibits comparable or improved thermal stability at pH 5-7, more preferably at pH 5-6, and most preferably at pH 5 and / or pH 6.

[0095] In one embodiment, the oral care composition contains arginine, and the DNase exhibits comparable or improved thermal stability in the presence of arginine. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-200 mM arginine, more preferably 5-150 mM arginine, even more preferably 10-100 mM arginine, and most preferably 30-90 mM arginine.

[0096] In one embodiment, the oral care composition comprises a benzoate (e.g., sodium benzoate), and the DNase exhibits comparable or improved thermal stability in the presence of the benzoate (e.g., sodium benzoate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.01%-5% benzoate (e.g., sodium benzoate), more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, and most preferably 0.1%-0.5% benzoate. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0097] In one embodiment, the oral care composition comprises EDTA, and the DNase exhibits comparable or improved thermal stability in the presence of EDTA. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0098] In one embodiment, the oral care composition comprises ethanol, and the DNase exhibits comparable or improved thermal stability in the presence of ethanol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, and most preferably 5% ethanol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100,000 mM ethanol, more preferably 100-10,000 mM ethanol, and most preferably 1,000 mM ethanol.

[0099] In one embodiment, the oral care composition comprises a fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), and the DNase exhibits comparable or improved thermal stability in the presence of the fluoride. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-5000 ppm fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm fluoride, and most preferably 1000-1500 ppm fluoride. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM fluoride (e.g., sodium fluoride), more preferably 5-75 mM fluoride, even more preferably 10-50 mM fluoride, and most preferably 20-40 mM fluoride.

[0100] In one embodiment, the oral care composition comprises glycerol, and the DNase exhibits comparable or improved thermal stability in the presence of glycerol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, and most preferably 10%-30% glycerol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 100-10,000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0101] In one embodiment, the oral care composition comprises mannitol, and the DNase exhibits comparable or improved thermal stability in the presence of mannitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0102] In one embodiment, the oral care composition comprises a phosphate (e.g., sodium phosphate or potassium phosphate), and the DNase exhibits comparable or improved thermal stability in the presence of the phosphate (e.g., sodium phosphate or potassium phosphate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0103] In one embodiment, the oral care composition comprises a sorbate (e.g., sodium sorbate, potassium sorbate, or calcium sorbate), and the DNase exhibits comparable or improved thermal stability in the presence of the sorbate (e.g., sodium sorbate, potassium sorbate, or calcium sorbate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.01%-5% sorbate (e.g., potassium sorbate), more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, and most preferably 0.1%-0.5% sorbate. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM sorbate (e.g., potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0104] In one embodiment, the oral care composition comprises sorbitol, and the DNase exhibits comparable or improved thermal stability in the presence of sorbitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, and most preferably 10%-40% sorbitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 100-10,000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0105] In one aspect, the DNA enzyme of the present invention may be a fusion polypeptide.

[0106] In one aspect, the DNA enzyme of the present invention is isolated.

[0107] In one aspect, the DNAse of the present invention is purified.

[0108] Sources of polypeptides with DNase activity The DNAse of this invention can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from," as used herein in conjunction with a given source, should mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has inserted the polynucleotide of this invention. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0109] In one respect, the polypeptide is obtained from Bacillus species, preferably Bacillus species-62490.

[0110] In one respect, the polypeptides were obtained from Sacchariformis (Sacchariformis spp.) Sutcliffiella (Preferably, *Sacchariformis sackievirus*).

[0111] In one respect, the polypeptide was obtained from the genus *Halophilus* ( Halalkalibacter (Preferably, *Hydrocalcinus aucubin*).

[0112] It should be understood that, for the aforementioned species, this invention covers complete and incomplete stages, as well as other taxonomic equivalents, such as asexual forms, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0113] The probes mentioned above can be used to identify and obtain polypeptides from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the polypeptide has been detected with the probe, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0114] Polynucleotides This invention also relates to polynucleotides encoding the polypeptides of this invention, as described herein. The polynucleotides may be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or combinations thereof.

[0115] In one aspect, the polynucleotide can be cloned from a strain of Bacillus, preferably Bacillus species-62490, or an associated organism, and thus, for example, can be a polynucleotide sequence encoding a variant of the polypeptide of the present invention. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from Bacillus cells, preferably Bacillus species-62490 cells.

[0116] In one aspect, the polynucleotide can be cloned from strains of the genus *Sacchariformis*, preferably *Sacchariformis burmannii*, or related organisms, and thus, for example, can be a polynucleotide sequence encoding a variant of the polypeptide of the present invention. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from *Sacchariformis* cells, preferably *Sacchariformis burmannii* cells.

[0117] In one aspect, the polynucleotide can be cloned from strains of the genus *Haloxybacterium*, preferably *Haloxybacterium flavum*, or related organisms, and thus, for example, can be a polynucleotide sequence encoding a variant of the polypeptide of the present invention. In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from *Haloxybacterium* cells, preferably *Haloxybacterium flavum* cells.

[0118] These polynucleotides can also be constructed by introducing nucleotide substitutions that do not cause a change in the amino acid sequence of the polypeptide but correspond to the codons used by the host organism intended to produce the enzyme, or by introducing nucleotide substitutions that may produce different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991. Protein Expression and Purification [Protein Expression and Purification] 2:95-107.

[0119] On the one hand, polynucleotides are isolated.

[0120] On the other hand, the polynucleotides are purified.

[0121] Nucleic acid constructs The present invention also relates to nucleic acid constructs comprising the polynucleotide of the present invention, wherein the polynucleotide is operatively linked to one or more control sequences, which, under conditions compatible with the control sequences, direct the expression of the coding sequence in a suitable host cell.

[0122] Polynucleotides can be manipulated in various ways to provide polypeptide expression. Depending on the expression vector, manipulating the polynucleotide before insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0123] promoter The control sequence may be a promoter, i.e., a polynucleotide recognized by the host cell for expressing the polypeptide encoding the present invention. The promoter contains a transcriptional control sequence that mediates polypeptide expression. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutant promoters, truncated promoters, and heterozygous promoters, and can be a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0124] Examples of suitable promoters for guiding the transcription of polynucleotides in bacterial host cells in this invention are described in Sambrook et al., 1989, *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Lab, New York; Davis et al., 2012, ibid.; and Song et al., 2016. PLOS One [Public Library of Science General] 11(7): e0158447.

[0125] Examples of suitable promoters for guiding the transcription of polynucleotides in filamentous fungal host cells in this invention are obtained from Aspergillus and Fusarium species. Fusarium ), Rhizopus ( Rhizomucor ) and Trichoderma ( Trichoderma ) Cell promoters, such as those described in Mukherjee et al., 2013, " Trichoderma"Trichoderma: Biology and Applications" and Schmoll and Dattenböck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications". Fungal Biology [Fungal Biology]

[0126] Examples of useful promoters for expression in yeast hosts are described in: Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae) and Schmoll and Dattenböck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”. Fungal Biology [Fungal Biology]

[0127] Termination The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operatively linked to the 3' end of a polynucleotide encoding a polypeptide. Any terminator that is functional in the host cell can be used in this invention.

[0128] Preferred terminators for bacterial host cells can be obtained from the genes of Bacillus clausti alkaline protease (Bc. clausti). aprH ), Bacillus licheniformis ( Bacillus licheniformis α-Amylase ( amyL ) and Escherichia coli ( Escherichia coli ) Ribosomal RNA ( rrnB ).

[0129] Preferred terminators for filamentous fungal host cells can be obtained from species of the genera *Aspergillus* or *Trichoderma*, such as those obtained from *Aspergillus niger*. Aspergillus niger ) glucosyl amylase, Trichoderma reesei ( Trichoderma reeseiGenes for β-glucosidase, Trichoderma reesei cellobiase I, and Trichoderma reesei endoglucanase I, such as those described in the terminator below: Mukherjee et al., 2013, " Trichoderma "Trichoderma: Biology and Applications" and Schmoll and Dattenböck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications". Fungal Biology [Fungal Biology]

[0130] Preferred terminators for yeast host cells can be obtained from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells were proposed by Romanos et al., 1992. Yeast [Yeast] 8:423-488 description.

[0131] mRNA stabilizers Control sequences can also be mRNA stabilizer regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[0132] Examples of suitable mRNA stabilizer regions were obtained from Bacillus thuringiensis (Bt). Bacillus thuringiensis ) cryIIIA Gene (WO 94 / 25612) and Bacillus subtilis ( Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. [Journal of Bacteriology] 177: 3465-3471.

[0133] An example of a stabilizer region in fungal cell mRNA is described in Geisberg et al., 2014. Cell 156(4): 812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11): 1838-1846.

[0134] Leader sequence The control sequence can also be a leader sequence, i.e., an untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of a polynucleotide encoding a polypeptide. Any leader sequence that is functional in the host cell can be used.

[0135] The appropriate leader sequence for bacterial host cells was determined by Hambraeus et al., 2000. Microbiology study] 146(12): 3051-3059 and Kaberdin and Bläsi, 2006, FEMS Microbiol. Rev. [FEMS Microbiology] [Scholarly Review] 30(6): 967-979 Description.

[0136] Preferred leader sequences for filamentous fungal host cells can be obtained from the genes of Aspergillus oryzae (…). Aspergillus oryzae TAKA amylase and Aspergillus nidulans ( Aspergillus nidulans Triose phosphate isomerase.

[0137] Suitable leader sequences for yeast host cells can be obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0138] polyadenylation sequence The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of a polynucleotide that is recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell can be used.

[0139] Preferred polyadenylated sequences for filamentous fungal host cells were obtained from the genes of the following: *Aspergillus nidulans* o-aminobenzoic acid synthase, *Aspergillus niger* glucosidase, *Aspergillus niger* α-glucosidase, *Aspergillus oryzae* TAKA amylase, and *Fusarium oxysporum* (…). Fusarium oxysporum Trypsin-like proteases.

[0140] Useful polyadenylation sequences in yeast host cells were discovered by Guo and Sherman, 1995. Mol. Cellular Biol. [Molecular Cell Biology] 15:5983-5990 (description).

[0141] signal peptide The control sequence can also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and guides the polypeptide into the secretory pathway of the cell. The 5' end of the polynucleotide coding sequence itself may contain a signal peptide coding sequence naturally linked to the coding sequence segment of the polypeptide within the translation read frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is heterologous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a heterologous signal peptide coding sequence may be required. Alternatively, a heterologous signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance polypeptide secretion. Any signal peptide coding sequence that guides the expressed polypeptide into the host cell's secretory pathway can be used.

[0142] The effective signal peptide coding sequence of bacterial host cells is obtained from the signal peptide coding sequences of the following genes: maltose amylase produced by Bacillus NCIB 11837, Bacillus subtilis protease, Bacillus licheniformis β-lactamase, and Bacillus stearothermophilus (…). Bacillus stearothermophilus α-Amylase, thermophilic Bacillus stearothermophilus neutral protease ( nprT , nprS , nprM ) and Bacillus subtilis prsA Other signal peptides were identified by Freudl, 2018. Microbial Cell Factories [Microbial Cell Factory] 17:52 Description.

[0143] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, and *Pseudomonas spp.* Humicola insolens Cellulase, specific humic fungus endoglucanase V, and cottony humic fungus ( Humicola lanuginosa Lipase and Rhizopus oryzae ( Rhizomucor miehei Aspartic proteases, such as those by Xu et al., 2018, Biotechnology Letters The signal peptide described in [Biotechnology Letters] 40:949-955.

[0144] Useful signal peptides in yeast host cells are obtained from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Sequences encoding other useful signal peptides are described above by Romanos et al., 1992.

[0145] Propeptide The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (…). aprE ), Bacillus subtilis neutral protease ( nprT ), thermophilic pyridamus ( Myceliophthora thermophila Laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease and Saccharomyces cerevisiae α-factor.

[0146] When both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide, and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence. Alternatively, when both a signal peptide sequence and a propeptide sequence are present, the polypeptide may contain only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or isolated polypeptide may comprise a mixture of a mature polypeptide and a polypeptide containing partial or full-length propeptide sequences and / or signal peptide sequences.

[0147] Regulatory sequence It is also desirable to add regulatory sequences that regulate the expression of host cell growth-related peptides. Examples of regulatory sequences are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include... lac , tac and trp Operant systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, the *Aspergillus oryzae* glucosylamylase promoter, the *Trichoderma reesei* cellobiose hydrolase I promoter, and the *Trichoderma reesei* cellobiose hydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In fungal systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals.

[0148] Transcription factor Control sequences can also be transcription factors, which are polynucleotides encoding polynucleotide-specific DNA-binding polypeptides that control the rate of transcription of genetic information from DNA to mRNA by binding to specific polynucleotide sequences. Transcription factors can function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by containing at least one DNA-binding domain, which is typically attached to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of proteins of interest directly (i.e., by binding to their promoters to activate the transcription of genes encoding proteins of interest) or indirectly (i.e., by binding to the promoters of other transcription factors that regulate the transcription of genes encoding proteins of interest). Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011. Subcellular Biochemistry [Subcellular Biochemistry] 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. [FEMS Microbiology Reviews] 33(1): 133-151.

[0149] expression carrier The present invention also relates to recombinant expression vectors comprising the polynucleotide of the present invention, a promoter, and transcription and translation termination signals. Multiple nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction sites to allow the insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located in the vector such that the coding sequence is operatively linked to a suitable control sequence for expression.

[0150] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids.

[0151] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity and replicates independently of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids collectively containing the total DNA of the host cell genome to be introduced, or transposons can be used.

[0152] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, or prototrophic auxotrophic traits, etc.

[0153] The vector preferably contains at least one element that allows the vector to integrate into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0154] In order to integrate into the host cell genome, the vector may depend on a polynucleotide sequence encoding a polypeptide or any other element of the vector used for integration into the genome via homologous recombination (such as homologous directed repair (HDR)) or non-homologous recombination (such as non-homologous end joining (NHEJ)).

[0155] For autonomous replication, the vector may further include an origin of replication, which enables the vector to replicate autonomously in the host cell discussed. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0156] More than one copy of the polynucleotide of the present invention can be inserted into host cells to enhance polypeptide production. For example, two, three, four, five, or more copies can be inserted into host cells. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene along with the polynucleotide, wherein cells containing the amplified copy of the selective marker gene and thus additional copies of the polynucleotide can be selected by culturing cells in the presence of a suitable selective reagent.

[0157] host cells The present invention also relates to recombinant host cells containing polynucleotides of the present invention operably linked to one or more control sequences that direct the production of polypeptides of the present invention.

[0158] A construct or vector containing a polynucleotide is introduced into a host cell, such that the construct or vector is maintained as a chromosomal integrase or as a self-replicating extrachromosomal vector, as previously described. The choice of host cell will depend largely on the gene encoding the polypeptide and its origin. The polypeptide can be native or heterologous to the recombinant host cell. Furthermore, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell may contain a single copy or at least two copies, such as three, four, five or more copies, of the polynucleotide of the invention.

[0159] The host cell can be any microbial cell that can be used to recombinantly generate the polypeptides of the present invention, such as prokaryotic cells or fungal cells.

[0160] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus and Clostridium species. Clostridium ), Enterococcus ( Enterococcus ), Bacillus spp. Geobacillus Lactobacillus ( ) Lactobacillus Lactococcus spp. Lactococcu s), Bacillus spp. ( Oceanobacillus Staphylococcus spp. Staphylococcus Streptococcus ( Streptococcus ) and Streptomyces ( Streptomyces Gram-negative bacteria include, but are not limited to, Campylobacter spp. ( Campylobacter ), Escherichia coli, Flavobacterium spp. Flavobacterium ), Fusobacterium genus ( Fusobacterium ), Helicobacter spp. Helicobacter ), Colistinia spp. ( Ilyobacter ), Neisseria spp. Neisseria ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella ) and Ureaplasma genus ( Ureaplasma ).

[0161] The bacterial host cell can be any Bacillus spp. cell, including but not limited to Bacillus alkalophilus (B). Bacillus alkalophilus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus brevis ( Bacillus brevis ), Bacillus circularis ( Bacillus circulans ), Bacillus cladei, Bacillus coagulans ( Bacillus coagulans ), Bacillus sclerosus ( Bacillus firmus ), Bacillus splendens ( Bacillus lautus ), Bacillus tarda ( Bacillus lentus ), Bacillus licheniformis, Bacillus megaterium (Bacillus megaterium ), Bacillus pumilus ( Bacillus pumilus The cells used include *Bacillus stearothermophilus*, *Bacillus subtilis*, and *Bacillus thuringiensis*. In the examples, the *Bacillus* cells are *Bacillus amyloliquefaciens*, *Bacillus licheniformis*, and *Bacillus subtilis* cells.

[0162] In a particularly preferred embodiment, the host cell is a Bacillus subtilis cell.

[0163] In a particularly preferred embodiment, the host cell is a Bacillus licheniformis cell.

[0164] For the purposes of this invention, the class / genus / species of Bacillus should be as Patel and Gupta, 2020. Int. J. Syst.Evol.Microbiol. The definition is as described in [International Journal of Systematic and Evolutionary Microbiology] 70:406-438.

[0165] The bacterial host cell can also be any streptococcal cell, including but not limited to Streptococcus equi (Streptococcus equi). Streptococcus equisimilis Streptococcus pyogenes, Streptococcus lactis ( Streptococcus uberis ) and Streptococcus equi subsp. porcupineus ( Streptococcus equi subsp. Zooepidemicus )cell.

[0166] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces ( Streptomyces achromogenes ), insecticide streptomycin ( Streptomyces avermitilis Streptomyces azureum ( Streptomyces coelicolor ), Streptomyces grayi ( Streptomyces griseus ) and Streptomyces purpureus ( Streptomyces lividans )cell.

[0167] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, and transduction, wherein the DNA is introduced as a linearized or circular polynucleotide. Those skilled in the art will be able to readily determine, for example, a suitable method for introducing DNA into a given prokaryotic cell based on its genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018. BMC Microbiology [BMC Microbiology] 18:56, Burke et al., 2001, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 98: 6289-6294, Choi et al., 2006. J. Microbiol. Methods[Journal of Microbiological Methods] 64:391-397 and Donald et al., 2013, J. Bacteriol. [Journal of Bacteriology] 195(11): 2612-2620.

[0168] The host cell can be a fungal cell. As used herein, “fungi” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al. in the following literature: Ainsworth and Bisby’s Dictionary of The Fungi [Ainsworth and Bysby Dictionary of Fungi], 8th edition, 1995, CAB International, University Press, Cambridge, UK.

[0169] Fungal cells can undergo transformation through processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (e.g., Li et al., 2017). Microbial Cell Factories [Microbial Cell Factory] 16:168 (reviewed) and EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 81: 1470-1474; Christensen et al., 1988, Bio / Technology [Biotechnology] 6:1419-1422 and Lubertozzi and Keasling, 2009. Biotechn.Advances The procedure described in [Advances in Biotechnology] 27:53-75 can be used for transformation. However, any method known in the art for introducing DNA into fungal host cells can be used, and the DNA can be introduced as a linearized or cyclic polynucleotide.

[0170] The fungal host cell can be a yeast cell. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the class Fungi Imperfecti (Blastomycetes). For the purposes of this invention, yeast should be as follows... Biology and Activities of Yeast [Biology and Activity of Yeast] (edited by Skinner, Passmore, and Davenport)Soc. App. Bacteriol. Symposium Series The definition is as described in Proceedings of the Society for Applied Bacteriology, No. 9 (Series 9), 1980.

[0171] Yeast host cells can be Candida genus ( White ), Hansenula genus ( Little Hansen Kluyveromyces ( ) Kluyveromyces Pichia pastoris ( ) Peach ), Yeast ( Saccharomyces ), genus *Fissionyomyces* Schizosaccharomyces ) or Yersinia genus ( Yarrow ) cells, such as Kluyveromyces lactis ( Kluyveromyces lactis ), Kelvin yeast ( Saccharomyces carlsbergensis ), brewing yeast, saccharifying yeast ( Saccharomyces diastaticus ), Douglas yeast ( Saccharomyces douglasii ), Klufer yeast ( Saccharomyces kluyveri ), Nodi yeast ( Saccharomyces norbensis ), oval yeast ( Saccharomyces oviformis ) or Yersinia lipophila ( Yarrowia lipolytica ) cells. In a preferred embodiment, the yeast host cell is a Pichia pastoris or a Pichia pastoris ( Komagataella ) cells, such as Pichia pastoris ( Shepherd's pie ) (Faffia colomata ( Komagataella phaffii ))cell.

[0172] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes the phylum Fungi (…). Eumycota Filamentous fungi are all filamentous forms of the subphylum Oomycetes (as defined by Hawksworth et al., 1995, ibid.). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, yeasts (such as *Saccharomyces cerevisiae*) undergo vegetative growth through budding of single-celled cells, and carbon metabolism can be fermentative.

[0173] The host cell for filamentous fungi can be *Cladosporium* ( ). Acremonium Aspergillus, Bruxelles Aureobasidium ), genus *Citropa* ( Bjerkandera ), genus Pseudocybe Ceriporiopsis ), genus Aureospora ( Chrysosporium ), Coprinus ( Coprinus ), genus *Gynostemma* Coriolus Cryptococcus ( Cryptococcus ), Ustilago aceae ( Filibasidium Fusarium, Humus ( ), Humicola ), Pyreosporium ( Magnaporth Mucor ( ) I am sick. ), genus *Hypericum* Mycelium ), genus *Neotrichum* ( Neocallimastix Neurospora ( Neurospora ), Penicillium genus ( Paecilomyces ), Penicillium ( Penicillium ), genus *Platycodon* Phanerochaete ), *Neuromyces* genus ( Phlebia ), Rumenichthys ( Pyromyces ), Pleurotus ( Oyster mushroom ), genus *Schizophyllum* ( Schizophyllum ), genus *Basilaria* Talaromyces ), genus Thermophilic Ascomycetes ( Thermoscus ), genus *Clostridium* ( Thielavia ), genus *Cyclophorus* ( Tolypocladium ), spp. Trametes The host cells are *Aspergillus*, *Trichoderma*, or *Fusarium* cells. In a preferred embodiment, the filamentous fungal host cells are *Aspergillus*, *Trichoderma*, or *Fusarium* cells. In another preferred embodiment, the host cells are *Aspergillus niger*, *Aspergillus oryzae*, *Trichoderma reesei*, or *Fusarium moniliforme*. Fusarium wilt )cell.

[0174] For example, the host cell for filamentous fungi can be Aspergillus bubomori (… Aspergillus awamori Aspergillus smut () Aspergillus foetidus Aspergillus fumigatus ( ) Aspergillus fumigatus ), Aspergillus japonicum ( Aspergillus Japanese Aspergillus nidus, Aspergillus niger, Aspergillus oryzae, Aspergillus niger ( Bjerkandera scordata ), dried cereus ( Ceriporiopsis aneirina ), Carnegie cereus ( Ceriporiopsis caregiea ), pale yellow cereus ( Ceriporiopsis gilvescens Panochita cereus ( Ceriporiopsis pannocinta ), Circulating cereus ( Ceriporiopsis rivulosa ), *Pseudomonas erythroptera* ( Ceriporiopsis subrufa ), Insectoid cereus ( Ceriporiopsis subvermispora ), Narrow-sided golden spores ( Chrysosporium inops ), Keratoplasmosis ( Chrysosporium keratinophilus ), Lukenowens golden spores ( Chrysosporium from Lucknow ), Fecal spores ( Chrysosporium merdarius ), spores of the genus *Pseudomonas* Chrysosporium pannicola ), Queensland golden spores ( Chrysosporium queenslandicum ), Tropical golden spores ( Chrysosporium tropicum ), Brown spores ( Chrysosporium zonatum ), Grey-covered Ghost Umbrella ( Coprinus cinereus ), scabra ( Coriolus hirsutus ), Fusarium moniliforme ( Fusarium bactridioides ), Cereal Fusarium ( Fusarium cerealis ), Fusarium oxysporum ( Fusarium crookwellense ), Fusarium solani ( Fusarium culmorum Fusarium graminearum ( ), Fusarium graminearum Fusarium graminearum ( ), Fusarium graminearum ( ) Fusarium graminum Fusarium heterosporum ( ), Fusarium heterosporum ), Albizia julibrissin ( Fusarium negundi ), Fusarium oxysporum, Fusarium multibranchii ( Fusarium reticulatum ), pink Fusarium ( Fusarium roseum ), Fusarium elderberry ( Fusarium sambucinum ), Skin-colored Fusarium ( Fusarium sarcochroum Fusarium pseudobryophyte ( ), Fusarium sporotrichioides ), Fusarium sulfonata ( Fusarium sulphureum ), Fusarium ( Fusarium torulosum Fusarium pseudofilariae ( ) Fusarium trichothecioides ), Fusarium moniliforme, specific humic mold, cottony humic mold, *Rhizopus oryzae*, thermophilic *Neurospora crassa* ( Neurospora crassa ), Penicillium purpureum ( Penicillium purpurogenum ), Chloris chrysophagus ( Phanerochaete chrysosporium ), Epireobacterium ( Phlebia radiata ), Erycibe and Pleurotus eryngii ( Pleurotus eryngii Emerson's basket bacteria ( Talaromyces emersonii ), terrestrial closporidum ( Thielavia terrestris ), long-haired cork bacteria ( Trametes villosa ), discoloration thrombus ( Trametes versicolor Trichoderma harzianum ( Trichoderma harzianum Corning Trichoderma ( Trichoderma koningii Trichoderma longifolia ( Trichoderma longibrachiatum ), Trichoderma reesei or Trichoderma viride ( Trichoderma viride )cell.

[0175] In a particularly preferred embodiment, the host cell is an Aspergillus niger cell.

[0176] In a particularly preferred embodiment, the host cell is an Aspergillus oryzae cell.

[0177] On the one hand, the host cell is isolated.

[0178] On the other hand, the host cell is purified.

[0179] Generation method The present invention also relates to methods for producing the polypeptides of the invention, the methods comprising (a) culturing cells under conditions conducive to the production of the polypeptide, the cells producing the polypeptide in their wild-type form; and optionally (b) recovering the polypeptide.

[0180] In one respect, the cells are Bacillus cells, preferably Bacillus species-62490 cells.

[0181] In one respect, the cells are Sackleaf cells, preferably Sackleaf burmannii cells.

[0182] In one respect, the cells are halophilic bacteria, preferably *Haloxybacterium flavum* cells.

[0183] The present invention also relates to methods for generating the polypeptides of the invention, the methods comprising (a) culturing the recombinant host cells of the invention under conditions conducive to the generation of the polypeptide; and optionally (b) recovering the polypeptide.

[0184] In one aspect, the recombinant host cell is a Bacillus cell, preferably a Bacillus subtilis cell or a Bacillus licheniformis cell, most preferably a Bacillus licheniformis cell.

[0185] In one respect, the recombinant host cell is an Aspergillus cell, preferably an Aspergillus niger cell or an Aspergillus oryzae cell.

[0186] The host cells are cultured in a nutrient medium suitable for producing peptides using methods known in the art. For example, cells can be cultured in a suitable medium and under conditions that allow for peptide expression and / or isolation by shake-flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state and / or microcarrier-based fermentation) in a laboratory or industrial fermenter. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the peptide is secreted into the nutrient medium, it can be recovered directly from that medium. If the peptide is not secreted, it can be recovered from cell lysates.

[0187] Peptides can be detected using methods known in the art that are specific to peptides, including but not limited to the use of specific antibodies, enzyme product formation, enzyme substrate disappearance, or determination of the relative or specific activity of the peptide.

[0188] Peptides can be recovered from culture media using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, whole fermentation broth containing peptides is recovered. In another aspect, cell-free fermentation broth containing peptides is recovered.

[0189] Peptides can be purified using a variety of procedures known in the art to obtain substantially pure peptides and / or peptide fragments (see, for example, Wingfield, 2015). Current Protocols in Protein Science [The latest approach in protein science]; 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing [Downstream protein processing], 1129:3-10).

[0190] In terms of alternatives, peptides are not recycled.

[0191] Enzyme granules The present invention also relates to enzyme particles / granules comprising the polypeptides of the present invention. In embodiments, the particles comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0192] The core diameter (measured as equivalent sphere diameter (volume-average particle size)) can be 20–2000 µm, particularly 50–1500 µm, 100–1500 µm, or 250–1200 µm. The core diameter as an equivalent sphere diameter can be determined using laser diffraction methods such as those using the Malvern Mastersizer and / or those described under ISO 13320 (2020).

[0193] The core may include other materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0194] The core may include adhesives such as synthetic polymers, waxes, fats, or carbohydrates.

[0195] The core, typically as a homogeneous blend, may include salts of polyvalent cations, reducing agents, antioxidants, peroxide decomposition catalysts, and / or acidic buffer components.

[0196] The core may contain inert particles into which the polypeptide is adsorbed or applied (e.g., by fluidized bed coating) to the surface of the inert particles.

[0197] The diameter of the core can be 20-2000 µm, especially 50-1500 µm, 100-1500 µm or 250-1200 µm.

[0198] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during handling, or to color the particles. Optional coatings may include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0199] The coating may be applied at a rate of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) of the core weight. This amount may be at most 100%, 70%, 50%, 40%, or 30%.

[0200] The coating is preferably at least 0.1 µm thick, particularly at least 0.5 µm, at least 1 µm, or at least 5 µm thick. In some embodiments, the coating thickness is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0201] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer should be understood as a coating with very few or no pores, such that the core unit has very few or no uncoated areas. This layer or coating should be uniform in thickness.

[0202] The coating may further comprise other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0203] Salt coatings may contain at least 60% salt by weight, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% salt by weight.

[0204] To provide acceptable protection, the salt coating is preferably at least 0.1 µm thick, such as at least 0.5 µm, at least 1 µm, at least 2 µm, at least 4 µm, at least 5 µm, or at least 8 µm. In particular embodiments, the thickness of the salt coating is less than 100 µm, such as less than 60 µm or less than 40 µm.

[0205] Salt can be added from a salt solution (where the salt is completely dissolved) or from a salt suspension (where the fine particles are less than 50 µm, for example less than 10 µm or less than 5 µm).

[0206] Salt coatings may contain a single salt or a mixture of two or more salts. The salts may be water-soluble, particularly having a solubility of at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g / 100 g of water, for example at least 1 g / 100 g of water, or for example at least 5 g / 100 g of water.

[0207] Salts can be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (less than 10 carbon atoms, such as 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions, or first transition metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonic acid, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate, or salts of simple organic acids such as citrate, malonate or acetate can be used.

[0208] The salt in the coating may have a constant humidity of 60% or more, particularly 70%, 80% or more or 85% or more at 20°C, or it may be another hydrated form of such salt (e.g., anhydrous form). Salt coatings may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0209] A specific example of a suitable salt is NaCl (CH4). 20℃ =76%), Na2CO3 (CH 20℃ =92%), NaNO3 (CH 20℃ =73%), Na2HPO4 (CH 20℃ =95%), Na3PO4 (CH 25℃ =92%), NH4Cl (CH 20℃ = 79.5%), (NH4)2HPO4 (CH 20℃ = 93.0%), NH4H2PO4 (CH 20℃ = 93.1%), (NH4)2SO4 (CH 20℃ =81.1%), KCl (CH 20℃ =85%), K2HPO4 (CH 20℃ =92%), KH2PO4 (CH 20℃ =96.5%), KNO3 (CH 20℃ =93.5%), Na2SO4 (CH 20℃ =93%), K2SO4 (CH 20℃ =98%), KHSO4 (CH 20℃ =86%), MgSO4 (CH20℃ =90%), ZnSO4 (CH 20℃ =90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0210] Salts can be in anhydrous form, or they can be hydrated salts, i.e., crystalline salt hydrates with one or more bound water crystals, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na₂SO₄), anhydrous magnesium sulfate (MgSO₄), magnesium sulfate heptahydrate (MgSO₄•7H₂O), zinc sulfate heptahydrate (ZnSO₄•7H₂O), disodium hydrogen phosphate heptahydrate (Na₂HPO₄•7H₂O), magnesium nitrate hexahydrate (Mg(NO₃)₂(6H₂O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0211] Preferably, the salt is used as a salt solution, for example, in a fluidized bed.

[0212] Coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591.

[0213] The particles may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA). Examples of enzyme particles with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0214] The core can be prepared by blends of granulated components, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, granulation, spheronization, particle size reduction, drum granulation, and / or high-shear granulation.

[0215] Methods for preparing the core can be found in *Handbook of Powder Technology*; CE Capes, *Particle Size Enlargement*; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as: (a) Spray-dried products, wherein a liquid polypeptide solution is atomized in a spray drying tower to form small droplets, which are dried as they descend along the drying tower to form polypeptide-containing particulate material. This method can produce very small particles (Michael S. Showell (ed.); Powdered detergents [Powdered Detergents]; Surfactant Science Series; 1998; Volume 71; pp. 140-142; Marcel Dekker.

[0216] (b) Layered products, wherein a polypeptide is coated in layers around a pre-formed inert core particle, wherein the polypeptide-containing solution is typically atomized in a fluidized bed apparatus, in which the pre-formed core particle is fluidized and the polypeptide-containing solution adheres to the core particle and is dried until a dry polypeptide layer remains on the surface of the core particle. If useful core particles of the desired size can be found, particles of the desired size can be obtained in this manner. This type of product is described, for example, in WO 97 / 23606.

[0217] (c) Absorbed core particles, wherein instead of coating the polypeptide in layers around the core, the polypeptide is absorbed onto and / or into the surface of the core. Such a method is described in WO 97 / 39116.

[0218] (d) Extruded or pelletized products, wherein a peptide-containing paste is compressed into pellets or extruded and cut into particles under pressure through small openings, followed by drying of these pellets. Such particles typically have a fairly large size because the material with the extrusion openings (usually a flat plate with perforations) limits the pressure drop allowed through the extrusion openings. Furthermore, when using small openings, the very high extrusion pressure increases the heat generated in the peptide paste, which is detrimental to the peptide (Michael S. Showell (ed.); Powdered detergents [Powdered Detergents]; Surfactant Science Series; 1998; Volume 71; pp. 140-142; Marcel Dekker.

[0219] (e) Spray-granulated products, wherein peptide-containing powder is suspended in molten wax and the suspension is sprayed (e.g., via a rotary sprayer) into a cooling chamber in which the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents [Powdered Detergent]; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker. The resulting product is one in which the polypeptide is uniformly distributed throughout the inert material rather than concentrated on its surface. This technique is described in US 4,016,040 and US 4,713,245.

[0220] (f) A mixer-granulated product in which a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. The liquid and powder are mixed in a suitable ratio, and as the moisture from the liquid is absorbed into the dry powder, the components of the dry powder begin to adhere and aggregate, and the particles accumulate to form polypeptide-containing granules. Such methods are described in US4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440, and WO 90 / 09428. In certain aspects of this process, various high-shear mixers can be used as granulators. Granules consisting of polypeptides, fillers, and binders are mixed with cellulose fibers to reinforce the granules, thereby producing so-called T-granules. The reinforced granules are more robust and release less enzyme dust.

[0221] (g) Particle size reduction, in which a core is generated by grinding or crushing larger particles, pellets, flat sheets, briquettes, etc., containing polypeptides. The desired core particle fraction is obtained by sieving the ground or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0222] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an airflow and spraying liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets become wetted and sticky. The sticky particles collide with and adhere to other particles to form granules.

[0223] (i) These cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying particles in the feed or enzyme industries. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some peptides, it is important that the peptide-containing core contains a small amount of water before salt coating. If water-sensitive peptides are salt-coated before removing excess water, the excess water will be trapped in the core and may negatively affect the activity of the peptide. After drying, these cores preferably contain 0.1-10% w / w water.

[0224] Dust-free particles may be generated, for example, as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.

[0225] The particles may further contain one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. One or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranylase, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof. Each enzyme will then be present in more particles, ensuring a more uniform distribution of the enzymes and reducing the physical separation of different enzymes due to the varying particle sizes. The method for generating multi-enzyme coparticles is disclosed in IP.com disclosure IPCOM000200739D.

[0226] Another example of using coparticles to formulate peptides is disclosed in WO 2013 / 188331.

[0227] The present invention also relates to protected polypeptides prepared according to the method disclosed in EP 238216.

[0228] Liquid preparations The present invention also relates to liquid compositions comprising the polypeptides of the present invention. The compositions may comprise enzyme stabilizers (examples of which include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borate esters, or phenyl boric acid derivatives such as 4-formylphenylboronic acid).

[0229] In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfate, carbonate, and silicate, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, these compositions contain about 5% to about 90% of such materials.

[0230] In one aspect, the liquid formulation contains 20%-80% w / w polyol. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative.

[0231] In another embodiment, the present invention relates to a liquid preparation comprising: (a) 0.001%-25% w / w of the polypeptide of the present invention having deoxyribonuclease activity; (b) 20%-80% w / w polyols; (c) Optionally 0.001%-2% w / w preservative; and (d) Water.

[0232] In another embodiment, the present invention relates to a liquid preparation comprising: (a) 0.001%-25% w / w of the polypeptide of the present invention having deoxyribonuclease activity; (b) 0.001%-2% w / w preservative; (c) Optionally 20%-80% w / w polyols; and (d) Water.

[0233] In another embodiment, the liquid formulation comprises one or more formulations, such as formulations selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate, and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0234] In another embodiment, the liquid formulation comprises 20%-80% polyols (i.e., the total amount of polyols), such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols. In one embodiment, the liquid formulation comprises 20%-80% polyols, such as 25%-75% polyols, 30%-70% polyols, 35%-65% polyols, or 40%-60% polyols, wherein the polyols are selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propanediol or 1,3-propanediol, dipropylene glycol, polyethylene glycol (PEG) with an average molecular weight of less than about 600, and polypropylene glycol (PPG) with an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0235] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation contains 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation contains 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w, 0.05%-1% w / w, or 0.1%-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0236] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetylxylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranoside, cellobiase, cellulase, ferulic acid esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannoside, β-mannoside (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0237] Oral care composition In one aspect, the present invention relates to oral care compositions comprising the DNase of the present invention. The oral care compositions of the present invention can be of any type. Suitable forms of oral care compositions and methods for preparing these forms are well known in the art and are further described herein.

[0238] In one aspect, the oral care composition contains a DNA enzyme selected from the group consisting of: a) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1; b) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2; and c) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3.

[0239] In one embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0240] In a preferred embodiment, the oral care composition comprises a DNase having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1. In a particularly preferred embodiment, the DNase comprises SEQ ID NO:1, consists substantially of SEQ ID NO:1, or consists of SEQ ID NO:1.

[0241] In a preferred embodiment, the oral care composition comprises a DNase having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2. In a particularly preferred embodiment, the DNase comprises SEQ ID NO:2, consists substantially of SEQ ID NO:2, or consists of SEQ ID NO:2.

[0242] In a preferred embodiment, the oral care composition comprises a DNase having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3. In a particularly preferred embodiment, the DNase comprises SEQ ID NO:3, consists substantially of SEQ ID NO:3, or consists of SEQ ID NO:3.

[0243] The oral care composition of the present invention may contain any effective amount or concentration of the DNase of the present invention. In a preferred embodiment, the oral care composition contains from about 1 ppm of DNase to about 500 ppm, preferably from about 1 ppm to about 100 ppm, more preferably from about 5 ppm to about 75 ppm, even more preferably from about 10 ppm to about 60 ppm, and most preferably from 10 ppm to 60 ppm.

[0244] In a preferred embodiment, the oral care composition contains at least 1 ppm, such as at least 5 ppm, at least 10 ppm, at least 15 ppm, at least 20 ppm, at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, at least 50 ppm, at least 55 ppm, at least 60 ppm, at least 65 ppm, at least 70 ppm, at least 75 ppm, at least 80 ppm, at least 85 ppm, at least 90 ppm, at least 95 ppm, at least 100 ppm, or more, of the DNA enzyme of the present invention.

[0245] In a particularly preferred embodiment, the oral care composition contains at least 10 ppm of the DNA enzyme of the present invention. In another particularly preferred embodiment, the oral care composition contains at least 60 ppm of the DNA enzyme of the present invention.

[0246] In one embodiment, the oral care composition is an internal oral care composition, such as toothpaste or toothpaste tablet, dental cream, mouthwash or mouthwash tablet, oral cleaner, lozenge, soft lozenge, chewing gum, sweets, candy, etc., which is designed to remove biofilms in the oral cavity, such as biofilms residing on teeth, oral soft tissues, and dentures residing in the oral cavity.

[0247] In one embodiment, the oral care composition is an external oral care composition, such as denture cleaning solution, denture cleaning tablets, denture cleaning powder, etc., which is designed to remove biofilm from dentures that have been removed from the mouth for cleaning.

[0248] In a preferred embodiment, the oral care composition is toothpaste.

[0249] In a preferred embodiment, the oral care composition is a mouthwash.

[0250] In a preferred embodiment, the oral care composition is an tablet.

[0251] In a preferred embodiment, the oral care composition is chewing gum.

[0252] The oral care composition of the present invention further comprises oral care ingredients that can vary depending on the type of oral care composition. Those skilled in the art can modify the oral care ingredients and their dosage according to the type of oral care composition and the desired characteristics of the oral care composition.

[0253] Although the oral care ingredients mentioned below are classified under a general heading based on their functionality, this is not to be construed as limiting, as ingredients may contain additional functionality as would be understood by a technician.

[0254] In one embodiment, the oral care composition comprises a benzoate (e.g., sodium benzoate), and the DNase exhibits comparable or improved thermal stability in the presence of the benzoate (e.g., sodium benzoate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.01%-5% benzoate (e.g., sodium benzoate), more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, and most preferably 0.1%-0.5% benzoate. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM benzoate (e.g., sodium benzoate), more preferably 5-50 mM benzoate, and most preferably 10-35 mM benzoate.

[0255] In one embodiment, the oral care composition comprises EDTA, and the DNase exhibits comparable or improved thermal stability in the presence of EDTA. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, and most preferably 1 mM EDTA.

[0256] In one embodiment, the oral care composition comprises ethanol, and the DNase exhibits comparable or improved thermal stability in the presence of ethanol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, and most preferably 5% ethanol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100,000 mM ethanol, more preferably 100-10,000 mM ethanol, and most preferably 1,000 mM ethanol.

[0257] In one embodiment, the oral care composition comprises a fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), and the DNase exhibits comparable or improved thermal stability in the presence of the fluoride. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-5000 ppm fluoride (e.g., sodium fluoride), more preferably 500-2500 ppm fluoride, and most preferably 1000-1500 ppm fluoride. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM fluoride (e.g., sodium fluoride), more preferably 5-75 mM fluoride, even more preferably 10-50 mM fluoride, and most preferably 20-40 mM fluoride.

[0258] In one embodiment, the oral care composition comprises glycerol, and the DNase exhibits comparable or improved thermal stability in the presence of glycerol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, and most preferably 10%-30% glycerol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, and most preferably 1000-3250 mM glycerol.

[0259] In one embodiment, the oral care composition comprises a peroxide (e.g., hydrogen peroxide), and the DNase exhibits comparable or improved thermal stability in the presence of the peroxide (e.g., hydrogen peroxide). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, and most preferably 100-500 mM peroxide.

[0260] In one embodiment, the oral care composition comprises mannitol, and the DNase exhibits comparable or improved thermal stability in the presence of mannitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, and most preferably 250-550 mM mannitol.

[0261] In one embodiment, the oral care composition comprises a phosphate (e.g., sodium phosphate or potassium phosphate), and the DNase exhibits comparable or improved thermal stability in the presence of the phosphate (e.g., sodium phosphate or potassium phosphate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-50 mM phosphate (e.g., sodium phosphate), more preferably 2.5-25 mM phosphate, and even more preferably 5-10 mM phosphate.

[0262] In one embodiment, the oral care composition comprises sodium lauryl sulfate (SDS), and the DNase exhibits comparable or improved thermal stability in the presence of SDS. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 10-50 mM SDS, more preferably 15-25 mM SDS, and most preferably 17 mM SDS.

[0263] In one embodiment, the oral care composition comprises a sorbate (e.g., sodium sorbate, potassium sorbate, or calcium sorbate), and the DNase exhibits comparable or improved thermal stability in the presence of the sorbate (e.g., sodium sorbate, potassium sorbate, or calcium sorbate). Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.01%-5% sorbate (e.g., potassium sorbate), more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, and most preferably 0.1%-0.5% sorbate. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 1-100 mM sorbate (e.g., potassium sorbate), more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, and most preferably 10-35 mM sorbate.

[0264] In one embodiment, the oral care composition comprises sorbitol, and the DNase exhibits comparable or improved thermal stability in the presence of sorbitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, and most preferably 10%-40% sorbitol. Preferably, the DNase exhibits comparable or improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, and most preferably 550-2200 mM sorbitol.

[0265] Toothpaste, tooth cream, mouthwash, and oral cleaning agents The internal oral care compositions of the present invention are in the form of toothpaste, tooth cream, mouthwash, and oral cleansing agents, and these internal oral care compositions may include ingredients and / or substances selected from the following categories:

[0266] toothpaste Toothpaste and tooth cream / tooth gel typically include abrasives, solvents, humectants, detergents / surfactants, thickeners and binders, buffers, flavorings, sweeteners, fluoride ions, therapeutic agents, colorants, and preservatives as oral care ingredients.

[0267] In a preferred embodiment, the present invention relates to oral care compositions in the form of toothpaste or tooth cream, which contain the DNA enzyme of the present invention. The oral care composition may contain at least one oral care ingredient selected from:

[0268] The oral care composition of the present invention may be a toothpaste comprising the following ingredients (in weight percent of the final toothpaste composition): Abrasive: 10% to 70% Wetting agent: 0% to 80% Thickener: 0.1% to 20% Binder: 0.01% to 10% Sweetener: 0.1% to 5% Foaming agent: 0% to 15% Enzyme (DNase): 0.01% to 20%

[0269] mouthwash The mouthwashes and oral cleaning agents (including plaque removers) of the present invention typically include a carrier fluid, detergent / surfactant, buffer, flavoring agent, humectant, sweetener, therapeutic agent, fluoride ion source, colorant, and preservative as oral care ingredients.

[0270] In a preferred embodiment, the present invention relates to oral care compositions in the form of mouthwashes or oral cleansers, which contain the DNase of the present invention. The oral care compositions may contain at least one oral care ingredient selected from:

[0271] The oral care composition of the present invention can be a mouthwash containing the following ingredients (in weight percent of the final mouthwash composition): Water: 0% to 70% Ethanol: 0% to 20% Wetting agent: 0% to 20% Surfactants: 0% to 2% Enzyme (DNase): 0.01% to 20% Other ingredients: 0% to 2% (e.g., flavorings, sweeteners, fluoride ion sources).

[0272] Mouthwash compositions can be buffered in a suitable buffer solution (such as sodium citrate or sodium phosphate) in the pH range of 6-7.5.

[0273] The relevant oral care components applicable to toothpaste, tooth cream, mouthwash, and oral rinses are further detailed below. Technicians can modify oral care components depending on the type of oral care composition and the desired characteristics and / or activities of a particular oral care composition. Oral care compositions do not necessarily need to contain all the mentioned ingredients.

[0274] abrasive Abrasive and polishing materials can be incorporated into the oral care compositions of the present invention. According to the present invention, the abrasive and polishing materials include alumina and its hydrates (e.g., α-alumina trihydrate), magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates (e.g., calcined aluminum silicate and aluminum silicate), calcium carbonate, zirconium silicate, bentonite, silica, sodium bicarbonate, and powdered plastics (e.g., polyvinyl chloride), polyamide, polymethyl methacrylate, polystyrene, phenolic resin, melamine-formaldehyde resin, urea-formaldehyde resin, epoxy resin, powdered polyethylene, silica dry gel, hydrogel, and aerogel, etc.

[0275] Other suitable abrasives include calcium pyrophosphate, water-insoluble alkaline metaphosphates, polymetaphosphates, dicalcium phosphate and / or its dihydrates, dicalcium orthophosphate, tricalcium phosphate, and particulate hydroxyapatite. Mixtures of these substances may also be used.

[0276] Various types of silica dental abrasives are preferred because of their unique benefits, such as excellent tooth cleaning and polishing performance without excessive abrasion of tooth enamel or dentin, and their good compatibility with other possible components, such as metal ions and fluoride.

[0277] Depending on the oral care composition, the abrasive product may be present in 0% to 70% by weight, preferably 1% to 70%.

[0278] For toothpaste, the content of abrasive materials typically ranges from 10% to 70% by weight of the final toothpaste product.

[0279] wetting agent Humectants are used to prevent water from escaping from, for example, toothpaste and to prevent toothpaste from hardening when exposed to air. Some humectants also impart a desired sweetness to toothpaste and mouthwash compositions. Humectants suitable for oral care compositions according to the present invention include the following compounds and mixtures thereof: glycerin, polyols, sorbitol, xylitol, maltitol, lactitol, polyoxyethylene, polyethylene glycol (PEG), polypropylene glycol, propylene glycol, 1,3-propanediol, 1,4-butanediol, hydrogenated partially hydrolyzed polysaccharides, coconut oil fatty acids, amides of N-methyl-taurine, and Pluronic®.

[0280] The wetting agent is typically present in an amount of 0% to 80%, preferably 5% to 70% by weight.

[0281] Thickener / Binder Suitable thickeners and / or binders include silica, starch, tragacanth gum, xanthan gum, ebony gum, carrageenan gum (an extract of Irish moss), gum arabic, alginate, pectin, cellulose derivatives (e.g., hydroxyethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, and hydroxyethylpropyl cellulose), polyacrylic acid and its salts, polyvinylpyrrolidone and carboxyvinyl polymers, and inorganic thickeners (e.g., amorphous silica compounds). These agents stabilize the oral care compositions of the present invention.

[0282] Thickeners may be present in toothpaste, tooth cream, gel, and mouthwash in amounts ranging from 0.1% to 20% by weight of the final product, and binders may be present in amounts ranging from 0.01% to 10% by weight of the final product.

[0283] Foaming agents and foaming regulators As foaming agents, soaps, anionic, cationic, nonionic, amphoteric, and / or facultative zwitterionic surfactants can be used alone or in combination. These can be present at levels of 0% to 15%, preferably 0.1% to 13%, and more preferably 0.25% to 10% by weight of the final product. Surfactants are only used to the extent that they do not inactivate enzymes and other components contained in the oral care composition. Useful surfactants include anionic, nonionic, and amphoteric compounds, preferably anionic compounds.

[0284] Examples of suitable surfactants include salts of higher alkyl sulfates, such as sodium lauryl sulfate or other suitable alkyl sulfates having 8 to 18 carbon atoms in the alkyl group; sodium lauryl sulfoacetate, salts of sulfonated monoglycerides of higher fatty acids, such as sodium coconut monoglyceride sulfonate or other suitable sulfonated monoglycerides of fatty acids having 10 to 18 carbon atoms; amide salts of higher fatty acids (e.g., 12 to 16 carbon atoms acids) with lower aliphatic amino acids, such as sodium N-methyl-N-palmitoyl taurate, sodium N-lauroyl sarcosinate, sodium N-myristoyl sarcosinate, and sodium N-palmitoyl sarcosinate; salts of such fatty acids with isotopic acids or with esters of glycerol monosulfates; sodium salts of monosulfate monoglycerides, such as hydrogenated coconut oil fatty acids; olefin sulfonates, such as chain olefin sulfonates or chain olefin sulfonates or mixtures thereof having 12 to 16 carbon atoms in the carbon chain of the molecule; and soaps of higher fatty acids, such as those having 12 to 18 carbon atoms, such as coconut oil fatty acids.

[0285] The cation of the salt can be sodium, potassium, or monoethanolamine, diethanolamine, or triethanolamine. Nonionic surfactants include sucrose / fatty acid esters, maltose / fatty acid esters, maltitol / fatty acid esters, malttriol / fatty acid esters, maltitol / fatty acid esters, maltpentol / fatty acid esters, malthexol / fatty acid esters, mahoheptaitol / fatty acid esters, sorbitan / fatty acid esters, lactose / fatty acid esters, lactinose / fatty acid esters, polyoxyethylene / polyoxypropylene copolymers, polyoxyethylene alkyl ethers, polyoxyethylene / fatty acid esters, fatty acid alkanolamides, polyoxyethylene sorbitan / fatty acid esters, polyoxyethylene / hydrogenated castor oil, and polyglycerol / fatty acid esters.

[0286] The preferred options are sodium lauryl sulfate, sodium dodecylbenzene sulfonate, and sodium lauryl sarcosinate.

[0287] Preferred foaming regulators include polyethylene glycol.

[0288] The foaming agent and foaming regulator may be present in an amount of 0% to 15%, preferably 0.01% to 10% by weight.

[0289] sweeteners Suitable sweeteners include, but are not limited to, saccharin and its water-soluble salts, dextrose, sucrose, lactose, maltose, levulose, aspartame, cyclohexanesulfonate, D-tryptophan, dihydrochalcone, acesulphame, steviol, levaudioside, glycyrrhizin, pellartine, kiwifruit protein, p-methoxycinnamaldehyde, hydrogenated starch hydrolysate, xylitol, sorbitol, erythritol, mannitol, and mixtures thereof.

[0290] The sweetener may be present in an amount of 0.001% to 60% by weight, preferably 0.01% to 50% by weight.

[0291] Flavorings Flavorings are typically present in small amounts, for example, from 0.01% to 5% by weight, particularly from 0.1% to 5%. Flavors that can be used in this invention include, but are not limited to, wintergreen oil, peppermint oil, spearmint oil, clove oil, menthol, anethole, methyl salicylate, eucalyptol, cinnamon, 1-menthyl acetate, sage, eugenol, celery oil, xanone, α-ionone, marjoram, lemon, orange, cranberry, propenyl ethyl guaiacol, cinnamon, vanillin, ethyl vanillin, piperaldehyde, 4-cis-heptenal, diacetyl, methyl p-tert-butylphenylacetate, carvone, eucalyptol, menthone, cinnamaldehyde, limonene, ocimene, n-decanol, citronellol, α-terpineol, methyl acetate, citronellol acetate, methyl eugenol, linalool, thymol, rosemary oil, allspice oil, diatomaceous earth oil, eucalyptus oil, and mixtures thereof.

[0292] The coolant may be added to the composition as part of the flavoring system or separately. Preferred coolants in the compositions of the present invention are p-menthane carboxylamide reagents, such as N-ethyl-p-menthane-3-carboxamide (commercially known as "WS-3"), menthol, 3-1-menthoxy-1,2-propanediol ("TK-10"), menthone glycerol acetal ("MGA"), menthyl lactate, and mixtures thereof.

[0293] Whitening agent / bleaching agent The whitening / bleaching agent includes H2O2 and can be added in an amount of less than 5%, preferably from 0.05% to 4%, based on the weight of the final composition.

[0294] Other bleaching components that may be included in this invention include peroxydiphosphate, urea, peroxides, metal peroxides (e.g., calcium peroxide, sodium peroxide, strontium peroxide, magnesium peroxide), hypochlorites (e.g., sodium hypochlorite), and salts of perborate, persilicate, superphosphate, and percarbonate (e.g., sodium perborate, potassium persilicate, and sodium percarbonate). Peroxide compounds can be stabilized by adding triphenylmethane dyes, chelating agents, or antioxidants (e.g., butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)).

[0295] solvent Solvents are typically added to the compositions of the present invention in an amount sufficient to make the composition flowable in the case of a toothpaste, tooth cream, or gel, or to dissolve other components of the composition in the case of a mouthwash or oral rinse.

[0296] Suitable solvents include water, ethanol, and water / ethanol mixtures, which can be present in amounts from 0.1% to 70%.

[0297] antimicrobial agents The present invention also includes water-soluble antimicrobial agents, such as chlorhexidine, digluconate, hexetine, alexiconine, and quaternary ammonium antimicrobial compounds; and may also include water-soluble sources of certain metal ions, such as zinc, copper, silver, and stannous chloride (e.g., zinc chloride, copper chloride, stannous chloride, and silver nitrate).

[0298] Because of the slow dissolution of these zinc salts in saliva, slightly soluble zinc salts such as zinc citrate, C14-alkyl zinc maleate, zinc benzoate, zinc hexanoate, and zinc carbonate can also be included in the compositions of the present invention to prolong the antimicrobial efficacy of zinc ions.

[0299] The antimicrobial agent can be present in an amount of 0% to 50% by weight, preferably 0.01% to 40% by weight, and most preferably 0.1% to 30% by weight.

[0300] Dental plaque control agent The compositions of the present invention may contain tartar control agents, such as inorganic phosphorus tartar control agents, which include any pyrophosphate, such as disodium pyrophosphate, dipotassium pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, and mixtures thereof.

[0301] Organophosphorus compounds that can be used as tartar control agents include polyphosphonates, such as ethane-1-hydroxy-1,1-diphosphate disodium (EHDP), methane diphosphonic acid, and 2-phosphonobutane-1,2,4-tricarboxylic acid.

[0302] The tartar control agent may be present in an amount of 0% to 10% by weight, preferably 0.1% to 5% by weight.

[0303] preservative Suitable preservatives include sodium benzoate, potassium sorbate, parabens, methylparaben, ethylparaben, propylparaben, citric acid, calcium citrate, and mixtures thereof.

[0304] The preservative may be present in an amount of 0% to 40% by weight, preferably 0.01% to 30% by weight.

[0305] Fluoride ion source The compositions of the present invention may further contain components that can be used as fluoride ion sources. Preferred soluble fluoride ion sources include sodium fluoride, potassium fluoride, stannous fluoride, indium fluoride, sodium monofluorophosphate, sodium hexafluorosilicate, zinc fluoride, lithium fluoride, aluminum fluoride, acid fluorophosphate, ammonium hydrogen fluoride, titanium tetrafluoride, and fluorinated amines.

[0306] The preferred sources of fluoride ions are sodium fluoride and sodium monofluorophosphate.

[0307] The fluoride ion source can be present in an amount of 0% to 20% by weight, preferably 0.01% to 15% by weight, and most preferably 0.1% to 10% by weight.

[0308] In a preferred embodiment, at least one oral care ingredient is a fluoride ion source; preferably, the fluoride ion source is selected from the group consisting of sodium fluoride, calcium fluoride, stannous fluoride, or sodium monofluorophosphate.

[0309] Colorant Colorants or pigments suitable for oral care compositions of the present invention include non-toxic, water-insoluble inorganic pigments such as titanium dioxide and chromium oxide green, ultramarine blue and pink, and iron oxide, as well as water-insoluble dye lakes prepared by extending calcium or aluminum salts of FD&C dyes onto alumina, such as FD&C Green Lake No. 1, FD&C Blue Lake No. 2, FD&C Red Lake No. 30, FD&C Yellow Lake No. 16, and FD&C Yellow Lake No. 10.

[0310] The preferred light-blocking agent is titanium dioxide.

[0311] The colorant may be present in an amount of 0% to 20% by weight, preferably 0.01% to 15% by weight, and most preferably 0.1% to 10% by weight.

[0312] buffer The oral care composition of the present invention may also include a buffer, i.e. a pH adjuster, such as alkali metal hydroxides, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazoles, and mixtures thereof.

[0313] Specific buffers include monosodium phosphate, trisodium phosphate, sodium hydroxide, potassium hydroxide, alkali metal carbonates, sodium carbonate, imidazole, pyrophosphate, sodium citrate, hydrochloric acid, sodium hydroxide, triethanolamine, triethylamine, lactic acid, malic acid, fumaric acid, tartaric acid, phosphoric acid, and mixtures thereof.

[0314] The buffer can be present in an amount of 0% to 10% by weight, preferably 0.01% to 5% by weight.

[0315] chewing gum When the oral composition according to the invention is chewing gum, it can be any known type of chewing gum, such as optionally coated chewing gum sheets, and sticks or chewing gum provided in any desired shape in response to the intended use. Chewing gum articles can have any quality, including bubble gum quality.

[0316] In a preferred embodiment, the present invention relates to oral care compositions in the form of chewing gum, which contain the DNase of the present invention. The oral care compositions may contain at least one oral care ingredient selected from: elastomers, softeners, plasticizers, emulsifiers, waxes, colorants, sweeteners, flavorings, leavening agents, and thickeners.

[0317] Collagen base components Traditionally, chewing gum is considered to consist of a water-insoluble or base portion and a water-soluble portion containing flavorings, sweeteners, and colorings. The gum base portion of chewing gum is the chewing substance that imparts the chewing characteristics of the final product. It defines the release profiles of flavorings and sweeteners and plays a significant role in chewing gum products. Flavorings, sweeteners, and colorings can be considered to contribute to the sensory appeal of chewing gum. There are no limitations on the chewing gum base used in chewing gum products according to the invention. Conventional chewing gum bases can be obtained, for example, from Dansk Tyggegummi Fabrik A / S, LA. Dreyfus or Cafasa Gum SIA are generally suitable, but specially manufactured formulations may also be used. The formulation depends on the desired type of chewing gum or the desired structural type. Suitable raw materials for gum bases include substances in accordance with the U.S. Gum Base Regulations—Title 21, Section 172,615 of the Federal Regulations and other national and international lists (or positive lists), and include elastomers, resins, waxes, polyvinyl acetate, oils, fats, emulsifiers, fillers, and antioxidants.

[0318] The adhesive base typically comprises 15% to 90% by weight of the final product, preferably 30% to 40% by weight, and more preferably 5% to 25% by weight.

[0319] Elastomers provide chewiness, elasticity, or resilience to the matrix and control the release of bubbles and flavor in the final chewing gum. They can be any water-insoluble polymer known in the art. They include styrene-butadiene copolymers (SBR) and non-SBR types, both natural and synthetic. Examples of natural elastomers include, but are not limited to, rubbers (e.g., rubber latex (natural rubber)) and guar gum, as well as gums (e.g., chicle, jelutong, balata, guttapercha, lechi capsi, sorva, crown gum, nispero, rosedinha, perillo, nigergutta, tunu, gutta kay, pendar, leche de vaca, chiquibul, crown gum, etc.), and mixtures thereof. Examples of synthetic elastomers include, but are not limited to, polyisobutylene, isobutylene-isoprene copolymer (butyl rubber), polyethylene, polybutadiene, styrene-butadiene copolymer, polyisoprene, and mixtures thereof.

[0320] The amount of elastomer (rubber) used in a gum base composition will vary considerably depending on various factors, such as the type of gum base used (viscous or conventional, aerated or standard), the desired consistency of the gum base composition, and other components used in the composition to prepare the final chewing gum product. Typically, based on the total weight of the gum base composition, the elastomer is present in the gum base composition in an amount of about 15% to about 60%, preferably about 25% to about 30% by weight.

[0321] Elastomer solvents help soften or plasticize elastomeric components. In this way, they provide swelling properties to chewable substances.

[0322] Elastomer solvents include, but are not limited to, natural rosin esters and synthetic derivatives such as terpenes. Examples of elastomeric solvents suitable for use herein include tall oil rosin esters; partially hydrogenated wood rosin and resin rosin; glycerol esters of wood rosin and resin rosin, partially hydrogenated wood rosin / resin rosin glycerol esters, partially dimerized wood rosin and resin rosin glycerol esters, polymerized wood rosin and resin rosin glycerol esters, and tall oil rosin glycerol esters; deodorized glycerol esters of wood rosin; pentaerythritol esters of wood rosin and resin rosin; partially hydrogenated wood rosin and resin rosin; methyl esters of partially hydrogenated wood rosin; methyl esters, glycerol esters, and pentaerythritol esters of rosin and modified rosin (e.g., hydrogenated, dimerized, and polymerized rosin); terpene resins (e.g., polymers of α-pinene or β-pinene), terpene resins; polyterpenes; and mixtures thereof. The elastomer solvent can be used in the adhesive composition in an amount of about 2% to about 40%, preferably about 7% to about 15%, based on the weight of the adhesive composition.

[0323] Polyvinyl acetate provides extensibility or elasticity to the gum base. They also affect chewing swelling, softness and air bubbles, hydrophilic characteristics, and flavor release.

[0324] The amount of polyvinyl acetate of different molecular weights present in the gum base composition should effectively provide the desired chewing properties of the finished chewing gum, such as integrity, softness, chewing swelling, film-forming characteristics, hydrophilic characteristics, and flavor release. The total amount of polyvinyl acetate used in the gum base composition is typically from about 45% to about 92% by weight of the total gum base composition. The vinyl polymer may have a molecular weight from about 2000 Da to about 95,000 Da.

[0325] Typically, low molecular weight polyvinyl acetate has a weight-average molecular weight of about 2,000 Da to about 14,000 Da. Medium molecular weight polyvinyl acetate typically has a weight-average molecular weight of about 15,000 Da to 55,000 Da. High molecular weight polyvinyl acetate typically has a weight-average molecular weight of about 55,000 Da to about 95,000 Da, but can be as high as 500,000 Da.

[0326] Waxes, fats, and oils plasticize elastomer blends and improve the elasticity of the gum base. Waxes provide a soft or firm chewiness, affect flavor release, and provide swelling and smoothness to the gum base. Fats and oils provide a soft chewiness. Fats, oils, and waxes can be used alone or in combination, or the gum base can be wax-free.

[0327] When waxes are used, they can be of mineral, animal, plant, or synthetic origin. Non-limiting examples of mineral waxes include petroleum waxes (such as paraffin and microcrystalline wax), animal waxes (including beeswax), plant waxes (including carnauba wax, candelilla wax, rice bran wax, fine-stemmed needle wax, linseed wax, and sugarcane wax), synthetic waxes (including those produced by the Fischer-Tropsch synthesis process), and mixtures thereof.

[0328] Suitable oils and fats for use in chewing gum compositions include hydrogenated or partially hydrogenated vegetable or animal fats, such as cottonseed oil, soybean oil, coconut oil, palm kernel oil, tallow, hydrogenated tallow, lard, cocoa butter, lanolin, etc.; fatty acids such as palmitic acid, oleic acid, stearic acid, linoleic acid, lauric acid, myristic acid, caproic acid, caprylic acid, capric acid, decanoic acid, or esters and salts (such as sodium stearate and potassium stearate). When used, these ingredients are typically present in amounts up to about 7% by weight of the chewing gum composition, and preferably up to about 3.5% by weight of the chewing gum composition.

[0329] Hydrogenated vegetable oils, including soybean oil and cottonseed oil, are preferred softeners and can be used alone or in combination. These softeners provide the gum-based composition with a good texture and a soft chewy character. These softeners are typically used in amounts from about 5% to about 14% by weight of the gum-based composition.

[0330] Emulsifiers help disperse the immiscible components of a gum base composition into a single, stable system. They provide hydrophilic properties to the gum base and help plasticize the resin and polyvinyl acetate. They also affect the softness and bubble characteristics of the matrix. Typical emulsifiers include acetylated monoglycerides, glyceryl monostearate, lecithin, fatty acid monoglycerides, diglycerides, propylene glycol monostearate, lecithin, triacetin, triacetic acid glycerides, and mixtures thereof.

[0331] Preferred emulsifiers are glyceryl monostearate and acetylated glyceryl monostearate. These are used as plasticizers. The emulsifier can be used in an amount of about 2% to about 15% by weight of the rubber-based composition, and preferably in an amount of about 7% to about 11% by weight of the rubber-based composition.

[0332] Fats, oils, waxes, emulsifiers, and certain sugar swelling agents are often combined and referred to as softeners. Due to the low molecular weight of these components, softeners can penetrate the basic structure of the gum base, giving it plasticity and less stickiness. The aforementioned useful plasticizers and softeners include lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, glyceryl triacetate, glyceryl lecithin, glyceryl monostearate, propylene glycol monostearate, acetylated monoglycerides, glycerol, fully unsaturated vegetable oils (e.g., non-hydrogenated cottonseed oil, hydrogenated vegetable oil, petroleum wax, sorbitan monostearate, tallow, etc.) and mixtures thereof, and also include high-fructose corn syrup, corn syrup, sorbitol solutions, hydrogenated starch hydrolysates, and mixtures thereof.

[0333] The amount of softener present should be an effective amount to provide the desired chewing expansion and softness of the finished chewing gum. When used as a softener, these materials are typically used in the gum base composition in an amount of up to about 25% by weight of the gum base composition, preferably about 1% to about 17%.

[0334] The gum base may further contain surfactants. Examples of suitable surfactants include polyoxyethylene (20) sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, polyethylene (4) sorbitan monolaurate, polyoxyethylene (20) sorbitan monopalmitate, polyoxyethylene (20) sorbitan monostearate, polyoxyethylene (4) sorbitan monostearate, polyoxyethylene (20) sorbitan tristearate, polyoxyethylene (5) sorbitan monooleate, polyoxyethylene (20) sorbitan trioleate, sorbitan monolaurate, etc. The amount of surfactant present should effectively provide the desired softness of the finished chewing gum. Typically, surfactants are used in the matrix in an amount of about 0.5% to about 3.0% by weight, based on the total weight of the gum base.

[0335] The gum base composition of the present invention may also include an effective amount of filler, sometimes referred to as a swelling agent. These materials increase hardness and swelling and affect the texture and flavor release of the chewing gum. Useful fillers include organic and inorganic compounds (mineral adjuvants), such as calcium carbonate, magnesium carbonate, heavy calcium carbonate, magnesium silicate, calcium phosphate, cellulose polymers, clay, alumina, aluminum hydroxide, aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate, and mixtures thereof. These fillers or adjuvants can be used in the gum base composition in various amounts. The amount of filler present should effectively provide the desired flavor release and integrity of the finished chewing gum. Typically, fillers can be used in the gum base composition in an amount of about 1% to about 40% by weight of the gum base composition, and preferably about 5% to about 20%.

[0336] The gum base may also contain antioxidants to provide improved stability, reduce any oily odor, and provide a longer shelf life. Typical, non-limiting examples of antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and propyl gallate. Mixtures thereof may also be used.

[0337] Other adhesive components The remaining ingredients in the chewing gum composition are conventional and typically comprise 10% to 85% by weight of the final product.

[0338] Examples include sweeteners, softeners, colorants, leavening agents, thickeners, and flavoring agents commonly used in the types and amounts of chewing gum.

[0339] Suitable flavorings are those spices known to those skilled in the art, such as natural and artificial flavorings. These flavorings may be selected from synthetic flavoring oils and flavoring aromatics and / or oils, oleoresins and extracts derived from plants, leaves, flowers, fruits, etc., and combinations thereof. Representative, non-limiting flavoring oils include spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, clove oil, laurel oil, anise oil, eucalyptus oil, thyme oil, cedarwood leaf oil, nutmeg oil, sweet pepper oil, sage oil, mace oil, bitter almond oil, and cinnamon oil. Other useful flavorings are artificial, natural, and synthetic fruit flavorings (such as vanilla and citrus oils, including lemon, orange, lime, and grapefruit) and fruit essences (including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, etc.). These flavorings can be used in liquid or solid form and can be used alone or in combination. Commonly used spices include peppermint, such as peppermint, menthol, artificial vanilla, cinnamon derivatives, and various fruit spices, which can be used alone or in combination.

[0340] Other useful flavoring agents include aldehydes and esters, such as cinnamyl acetate, cinnamaldehyde, diethyl citrate, dihydrocarboxylate, eugenol formate, and p-methyl anisole. Generally, any flavoring agent or food additive can be used.

[0341] Other examples of aldehyde flavoring agents include, but are not limited to, acetaldehyde (apple), benzaldehyde (cherry, almond), anisaldehyde (licorice, anise), cinnamaldehyde (cinnamon), citral (α-citral, lemon, lime), neraldehyde (β-citral, lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, butter), heliotrope (piperaldehyde, vanillin), vanillin (vanilla, butter), α-pentylcinnamaldehyde (spicy fruit flavoring), butyraldehyde (butter, cheese), pentanalaldehyde (butter, cheese), and more. Veratral (various types), decanal (citrus fruits), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrus fruits), aldehyde C-12 (citrus fruits), 2-ethylbutanal (berry fruits), hexenal (trans-2-hexenal, berry fruits), toluenealdehyde (cherries, almonds), veratral (vanilla), 2,6-dimethyl-5-heptanal (melon aldehyde, melon aldehyde), 2,6-dimethyloctanal (green fruits), and 2-dodecanoal (citrus fruits), cherry, grape, strawberry shortbread, and mixtures thereof.

[0342] The amount of flavoring agent used herein is typically influenced by factors such as the type of the final chewing gum composition, the individual flavor profile, the gum used, and the desired flavor intensity. Therefore, the amount of flavoring agent can be varied to achieve the desired results in the final product, and such variations are within the capabilities of those skilled in the art without requiring excessive experimentation. In chewing gum compositions, flavoring agents are typically present in amounts from about 0.02% to about 5% by weight of the chewing gum composition.

[0343] Chewing gum compositions typically include expanding agents. These expanding agents (carders, fillers) can be water-soluble and include, but are not limited to, expanding agents selected from the group consisting of: monosaccharides, disaccharides, polysaccharides, sugar alcohols, and mixtures thereof; sorbitol, xylitol, maltitol, mannitol, isomaltitol (a racemic mixture of α-D-glucopyranosyl-1,6-mannitol and α-D-glucopyranosyl-1,6-sorbitol, marketed by Suddeutsche Zucker under the trade name Palatinit). TM The ingredients include: glycerin, aspartame, Lycasin® glycerin, galactitol, acetylsupan potassium, saccharin and its salts, cyclohexanesulfonate and its salts, neohesperidin dihydrochalcone, glycyrrhizic acid and its salts, thaumantine and sucralose and mixtures thereof, or mixtures thereof with other suitable sweeteners; maltodextrin; hydrogenated starch hydrolysate; hydrogenated hexose; hydrogenated disaccharide; minerals such as calcium carbonate, talc, titanium dioxide, dicalcium phosphate, cellulose, etc., and mixtures thereof. The leavening agent may be used in an amount up to about 60% by weight of the chewing gum composition, preferably about 25% to about 60%.

[0344] Chewing gum compositions may also include high-intensity sweeteners (sweeteners). The sweetness intensity of high-intensity sweeteners is substantially greater than that of sucrose. Examples of suitable high-intensity sweeteners include: a) Water-soluble, naturally occurring potent sweeteners, such as dihydrochalcone, monoline, steviol, glycyrrhizin, flavanone alcohols and L-aminodicarboxylic acid, aminoalkyl ester amides, such as those disclosed in U.S. Patent No. 4,619,834, and mixtures thereof; b) Water-soluble artificial sweeteners, including soluble saccharin salts (e.g., sodium or calcium saccharin), cyclohexanesulfonate, sodium, ammonium, or calcium salts of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, potassium salts of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide (acetylsupan potassium), free acid form of saccharin, and mixtures thereof; c) Dipeptide-based sweeteners, including sweeteners derived from L-aspartic acid (e.g., methyl 1-aspartic-L-phenylalanine (aspartame) and the material described in U.S. Patent No. 3,492,131), L-α-aspartic-N-(2,2,4,4-tetramethyl-3-thiecyclobutyl)-D-propanamide hydrate (alitame), methyl esters of L-aspartic-L-phenylglycerol and L-aspartic-L-2,5-dihydrophenyl-glycine, L-aspartic-2,5-dihydro-L-phenylalanine, L-aspartic-L-(1-cyclohexene)-alanine, and mixtures thereof; d) Water-soluble potent sweeteners derived from naturally occurring water-soluble sweeteners, such as chlorinated derivatives of common sugars (sucrose), such as chlorodeoxy sugar derivatives (e.g., derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose), known for example under the product name Sucralose®; examples of chlorodeoxysucrose and chlorodeoxygalactosucrose derivatives include, but are not limited to: 1-chloro-1'-deoxysucrose; 4-chloro-4-deoxy-α-D-galactopyranosyl-α-D-fructofuranoside, or 4-chloro-4-deoxygalactosucrose; 4-chloro-4-deoxy-α-D-galactopyranosyl-1-chloro-deoxy-β-D-fructofuranoside, or 4,1'-dichloro-4,1'-dideoxygalactosucrose; 1',6'-dichloro-1',6'-dideoxysucrose; 4-chloro-4-deoxy-α- D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl, or 4,1',6'-trichloro-4,1',6'-trideoxygalactosucrose; 4,6-dichloro-4,6-dideoxy-α-D-galactopyranosyl-6-chloro-6-deoxy-β-D-fructofuranosyl, or 4,6,6'-trichloro-4,6,6'-trideoxygalactosucrose; 6,1',6'-trichloro-6,1',6'-trideoxysucrose; 4,6-dichloro-4,6-dideoxy-α-D-galactopyranosyl-1,6-dichloro-1,6-dideoxy-β-D-fructofuranoside, or 4,6,1',6'-tetrachloro-4,6,1',6'-tetradeoxygalactosucrose; and 4,6,1',6'-tetradeoxy-sucrose, mixtures thereof; and e) Protein-based potent sweeteners, such as Thaumaoccous daniclii (Kiwi sweet protein I and II). The amount of sweetener used in the chewing gum composition will vary depending on the sweetener selected for a particular chewing gum. Therefore, for any given sweetener, a sufficient amount of sweetener is used to provide the desired level of sweetness. Based on the total weight of the chewing gum composition, the aforementioned sugar sweeteners and sugar alcohols are typically used in an amount of about 1% to about 70% by weight, preferably about 40% to about 50% by weight. Based on the total weight of the chewing gum composition, the aforementioned strong sweeteners are typically used in an amount of up to about 1% by weight, preferably about 0.05% to about 0.4% by weight.

[0345] The colorants useful in this invention are used in amounts that effectively produce the desired color. These colorants include pigments that can be incorporated in amounts up to about 6% by weight of the chewing gum composition. Preferred pigments, namely titanium dioxide, can be incorporated in amounts up to about 2% by weight of the chewing gum composition, preferably less than about 1%. Colorants may also include natural food pigments and dyes suitable for food, pharmaceutical, and cosmetic applications. These colorants are referred to as FD&C. dyes and lakes. Acceptable materials for the foregoing uses are preferably water-soluble. Illustrative and non-limiting examples include indigo dye known as FD&C. Blue 2, which is a disodium salt of 5,5-indigo disulfonic acid. Similarly, dye known as FD&C. Green 1 comprises a triphenylmethane dye and is a monosodium salt of 4-[4-(N-ethyl-N-p-thiobenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-thiobenzyl)-δ-2,5-cyclohexadieneimine].

[0346] Examples of thickeners include methylcellulose, alginate, carrageenan, xanthan gum, gelatin, carob gum, tragacanth gum and locust bean gum, emulsifiers (such as lecithin and glyceryl monostearate), acidifiers (such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid) and mixtures thereof.

[0347] The plasticizers, softeners, emulsifiers, waxes, and antioxidants discussed above that are suitable for gum bases can also be used in chewing gum compositions.

[0348] Active chewing gum ingredients The oral care composition of the present invention in the form of chewing gum may also contain various active ingredients, such as antimicrobial agents, zinc salts, fluorides, and urea.

[0349] Furthermore, if desired, the oral composition according to the invention may include any other active ingredients, such as anti-caries agents, anti-calculus agents, anti-plaque agents, anti-periodontal agents, antifungal agents, anti-smoking agents, anti-cold agents, anti-gingivitis agents, etc.

[0350] The antimicrobial agent used in the composition can be any of a variety of cationic antimicrobial agents, such as quaternary ammonium compounds (e.g., hexadecyl pyridine chloride) and substituted guanidines (e.g., chlorhexidine and the corresponding compound alexidin). Mixtures of cationic antimicrobial agents can also be used in this invention.

[0351] Antimicrobial quaternary ammonium compounds include those in which one or two substituents on the quaternary nitrogen have a carbon chain length of about 8 to 20, typically 10 to 18 carbon atoms (typically alkyl groups), while the remaining substituents (typically alkyl or benzyl groups) have a smaller number of carbon atoms (e.g., 1 to 7 carbon atoms), typically methyl or ethyl groups. Dodecyltrimethylammonium bromide, tetradecylpyridine chloride, tetradecylethylpyridine chloride, dodecyldimethyl(2-phenoxyethyl)ammonium bromide, benzyldimethylstearylammonium chloride, hexadecylpyridine chloride, quaternized 5-amino-1,3-bis(2-ethyl-hexyl)-5-methylhexahydropyrimidine, and benzyl chloride are typical examples of quaternary ammonium antibacterial agents. Other compounds are bis[4-(R-amino)-1-pyridine]alkanes, as disclosed in U.S. Patent 4,206,215 to Bailey, issued June 3, 1980 (which is incorporated herein by reference). Pyridine compounds are preferred quaternary ammonium compounds.

[0352] Cationic antimicrobial agents are typically used in the compositions of the present invention at levels of about 0.02% to about 1%, preferably about 0.3% to about 0.7%, and most preferably about 0.3% to about 0.5%.

[0353] As readily soluble zinc salts, any physiologically acceptable, readily soluble zinc salt of inorganic or organic acids can be used in principle, which is capable of releasing zinc ions and is approved for its intended use, such as in food, cosmetic, or pharmaceutical products. Non-limiting examples include, for example, zinc citrate, zinc sulfate, zinc lactate, zinc chloride, zinc acetate, and mixtures thereof. Among these salts, zinc acetate is preferred.

[0354] The zinc salt used must be readily soluble to ensure that the amount of zinc ions released in the oral cavity within the appropriate time period is effective for the target purpose.

[0355] Advantageously, the zinc salt is present in the oral composition in an amount of 0.001% to 1.25% by weight. The amount used depends on the form of application and the intended use, and is adjusted such that the amount of zinc ions released is effective for the intended use.

[0356] As a masking salt, at least one salt selected from sodium chloride, ammonium chloride, and physiologically acceptable alkali metals, alkaline earth metals, and / or ammonium carbonate is used.

[0357] Alkali metals, particularly sodium or potassium, and alkaline earth metals, preferably calcium or magnesium. Particularly preferred masking salts are sodium carbonate, potassium carbonate and magnesium carbonate, sodium chloride, ammonium chloride and mixtures thereof.

[0358] Flavor-masking salts are advantageously used in oral compositions in amounts of 0.05% to 6.25% by weight, more preferably 0.25% to 3.50% by weight, for example 0.50% to 2.50% by weight.

[0359] In each case, the amount of masking salt used to mask the taste of zinc can be determined by those skilled in the art and depends on the specific zinc salt in question and the chosen form of application.

[0360] Urea is used in anti-caries products to neutralize acids produced in dental plaque after eating or drinking. In addition to urea, the composition may also contain pharmacologically acceptable substances capable of releasing urea under predominantly oral conditions. Examples include salts and addition compounds between urea and inorganic compounds such as magnesium sulfate, calcium phosphate, and sodium chloride.

[0361] The urea content of the composition according to the invention varies between 0.05% and 80% by weight, preferably between 0.2% and 25% by weight.

[0362] The chewing gum composition can be prepared using standard techniques and equipment known to those skilled in the art. The useful apparatus according to the invention also includes mixing and pulping devices.

[0363] Tablets and soft tablets Tablets are flavored drug dosage forms intended to be inhaled and held in the mouth or throat. They may contain vitamins, antibiotics, antiseptics, local anesthetics, antihistamines, decongestants, corticosteroids, astringents, analgesics, aromatics, soothing agents, or combinations of these ingredients. Tablets can come in various shapes, most commonly flat, round, octagonal, and biconvex. Another type, called bacilli, comes in short rod or cylindrical shapes. Softer types of tablets are called soft tablets, which consist of a drug in a gelatin or glycerin-gelatin matrix or a gum arabic, sucrose, and water matrix (HA Lieberman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (1980), Marcel Dekker, Inc., New York, NY.).

[0364] In a preferred embodiment, the present invention relates to oral care compositions in the form of lozenges or soft lozenges, which contain the DNase of the present invention. The oral care compositions may contain at least one oral care ingredient selected from lubricants, bulking agents, sweeteners, and flavoring agents.

[0365] lubricant Lubricants are used in the manufacture of compressed tablets to facilitate the release of the tablets from the mold in which they are formed. The lubricant used in this invention is a solid material that is non-charged and does not interfere with (e.g., composite) cationic antimicrobial agents. The material should preferably be insoluble in water. One type of suitable material that meets these requirements is a non-toxic hydrocarbon fat or derivative. Examples include hydrogenated tallow and hydrogenated vegetable oil. Polyethylene glycol can also be used as a lubricant as long as it is a solid material; this generally means that the polyethylene glycol has a molecular weight in the range of 4000 Da to 6000 Da. These materials can also be used as fillers, as described below.

[0366] Mixtures of lubricants can also be used in this invention. The lubricant is used at a level of about 0.1% to about 4.0%, preferably about 0.5% to about 2%.

[0367] Tablet Carrier This article uses the term "tablet carrier" to refer to one or more materials that carry the active ingredient (i.e., enzyme) and a lubricant. These materials are also called expanders or fillers. Because carriers are non-cariogenic, they should be free of sucrose and similar materials.

[0368] Acceptable filler materials include mannitol, sorbitol, xylitol, polyethylene glycol, and non-cariogenic dextran. Fillers can be used alone or in combination.

[0369] Mannitol is a naturally occurring sugar alcohol and can be obtained as a fine powder. Its sweetness is only about 50% that of sucrose. However, mannitol's negative heat of solution allows it to impart a pleasant, cooling sensation in the mouth when dissolved in tablet form.

[0370] Sorbitol is a chemical isomer of mannitol and has a similar level of sweetness. Its heat of solution (which is negative) also provides a pleasant, cooling sensation in the mouth. Sorbitol is available as free-flowing granules or as a crystalline powder. Polyethylene glycol (PEG) can also be used in the compositions of this invention. These materials have the general formula HOCH2(CH2OCH2). n PEG is a polymer of ethylene oxide (CH2OH). Using PEG alone is not advantageous, but its use in combination with other fillers is acceptable. The most desirable molecular weights were found to be between 4000 Da and 6000 Da.

[0371] The filler is typically used in the compositions of the present invention at a level of about 85% to about 99.8%, preferably about 90% to about 98%, and most preferably about 94% to about 97%.

[0372] Other tablet components Acceptable tablets can be manufactured using only the active ingredients, lubricants, and filler materials as outlined above. However, to make the tablets more aesthetically acceptable, they typically include materials such as spray-dried or encapsulated flavorings or liquid flavorings adsorbed onto a suitable diluent. Spray-dried or encapsulated flavorings are preferred. Suitable flavorings include peppermint oil, wintergreen oil, sassafras oil, spearmint oil, and clove oil. Sweeteners are also acceptable for use in the compositions of the present invention. Suitable agents include aspartame, acetylsupan, saccharin, dextrose, and levose. Sweeteners and flavorings are typically used in the compositions of the present invention at levels of about 0.1% to about 2%, preferably about 0.25% to about 1.5%.

[0373] It is also acceptable that the amount of water-soluble fluoride compound present in the tablets in solid form is sufficient to provide a fluoride concentration of about 0.0025% to about 5.0% by weight, preferably about 0.005% to about 2.0% by weight, to provide additional anti-caries efficacy. Preferred fluorides are sodium fluoride, stannous fluoride, indium fluoride, and sodium monofluorophosphate. The tablets may also contain various active ingredients, such as antimicrobial agents, zinc salts, fluorides, and urea (see above).

[0374] Sweets and candies In a preferred embodiment, the present invention relates to oral care compositions in the form of sweets or candies, which contain the DNase of the present invention. The oral care compositions may contain at least one oral care ingredient selected from colorants, sweeteners, flavoring agents, and oil modifiers.

[0375] The preparation of confectionery products is historically well-known and has remained largely unchanged over the years. Confectionery products have been classified as either "hard" or "soft" confectionery. The volatile oil modifier of this invention can be incorporated into conventional hard and soft confectionery products by mixing the modifier into them.

[0376] Hard desserts can be processed and formulated using conventional methods. Typically, hard desserts have a matrix consisting of a mixture of sugars and other carbohydrate leavening agents maintained in an amorphous or glassy state. This form is considered to be a solid sugar syrup, which typically contains about 0.5% to about 1.5% water. Such materials typically contain up to about 92% corn syrup, up to about 55% sugar, and about 0.1% to about 5% water by weight of the final composition. The syrup component is typically prepared from fructose-rich corn syrup, but may include other materials. Additional ingredients, such as flavoring agents, sweeteners, acidulants, coloring agents, etc., may also be added.

[0377] Such sweets can be prepared using conventional methods, such as those involving fire cookers, vacuum cookers, and scraper cookers (also known as high-speed atmospheric cookers).

[0378] The fire-cooking apparatus relates to a traditional method for manufacturing confectionery bases. In this method, a desired amount of carbohydrate leavening agent is dissolved in water by heating a reagent in a pot until the leavening agent dissolves. Additional leavening agent can then be added, and cooking continues until the final temperature reaches 145°C to 156°C. The batch is then cooled and processed as a plastic sample lump to incorporate additives such as flavorings, colorings, etc.

[0379] High-speed atmospheric cookers utilize heat exchanger surfaces, which involve spreading a layer of candy on the heat exchange surface, heating the candy to 165°C to 170°C within minutes. The candy is then rapidly cooled to 100°C to 120°C and functions as a plastic-like mass in which additives (such as flavorings, colorings, etc.) can be incorporated.

[0380] In a vacuum cooker, the carbohydrate leavening agent is boiled to 125°C to 132°C under vacuum, and additional water is evaporated without further heating. When cooking is complete, the mass is semi-solid and has a plastic-like consistency. At this point, flavorings, colorings, and other additives are mixed into the mass using conventional mechanical mixing operations.

[0381] During the production of conventional hard confectionery, the optimal mixing time required to evenly combine spices, colorings, and other additives is determined by the time needed to achieve a uniform distribution of the materials. Typically, a mixing time of 4 to 10 minutes has been found to be acceptable.

[0382] Once the candy block has been properly tempered, it can be cut into workable portions or shaped as desired. A variety of forming techniques can be used depending on the desired shape and size of the final product. A general discussion of the composition and preparation of hard confectionery can be found in HA Lieberman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (1980), Marcel Dekker, Inc., NY, New York City.

[0383] According to the present invention, useful equipment includes cooking and mixing apparatus well-known in the field of confectionery manufacturing, and the selection of specific apparatus will be obvious to a person skilled in the art. In contrast, compressed tablet confectionery contains specific materials and forms a structure under pressure.

[0384] These sweets typically contain up to about 95% sugar by weight of the composition, as well as typical tablet excipients, such as binders and lubricants, and flavorings, colorings, etc. Similar to hard sweets, soft sweets can be used in this invention. The preparation of soft sweets (e.g., nougat) involves conventional methods, such as a combination of two main components, namely (1) a high-boiling-point syrup, such as corn syrup, hydrogenated starch hydrolysate, etc., and (2) a relatively lightweight frozen sweet, which is typically prepared from ovalbumin, gelatin, plant proteins (e.g., soy-derived compounds), unsweetened milk-derived compounds (e.g., milk proteins), and mixtures thereof. Frozen sweets are typically relatively light and can have a density range, for example, from about 0.5 to about 0.7 g / cc.

[0385] Flavoring components in desserts are spices that have a associated bitterness or other unpleasant aftertaste. These flavoring components can be selected from natural and synthetic flavoring liquids, such as volatile oils, synthetic flavoring oils, flavoring aromas, and oils, liquids, oleoresins, or extracts derived from plants, leaves, flowers, fruits, stems, and combinations thereof. Non-limiting representative examples of volatile oils include spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, menthol, clove oil, laurel oil, anise oil, eucalyptus oil, thyme oil, cedarwood leaf oil, nutmeg oil, sweet pepper oil, sage oil, nutmeg skin extract, bitter almond oil, and cinnamon oil. In addition, desserts may contain artificial, natural, or synthetic flavorings, including single and mixed fruit flavorings (such as vanilla and citrus oils, including lemon, orange, grape, lime, and grapefruit) and fruit essences (including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, etc.).

[0386] Other useful flavorings include aldehydes and esters, such as benzaldehyde (cherries, almonds), citral (i.e., α-citral (lemons, limes)), neraldehyde (i.e., β-citral (lemons, limes)), decanal (oranges, lemons), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrus fruits), aldehyde C-12 (citrus fruits), tolualdehyde (cherries, almonds), 2,6-dimethyl-octanal (green fruits), and 2-dodecaldehyde (citrus fruits), and mixtures thereof.

[0387] In the use of sweeteners, this invention is considered to include those sweeteners well known in the art, including both natural and artificial sweeteners. Sweeteners may be selected from the following non-limiting list: sugars, such as sucrose, glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof; saccharin and its various salts (e.g., sodium or calcium salts); cyclohexane and its various salts, such as sodium salts; dipeptide sweeteners, such as aspartame, dihydrochalcone compounds, glycyrrhizin; stevia (… Stevia Rebaudiana(Steviol glycosides); chlorinated derivatives of sucrose; flavanone alcohols; hydroxyguaiacol esters; L-aminodicarboxylic acid geminiamine; L-aminodicarboxylic acid aminoolefin ester amides; and sugar alcohols such as sorbitol, sorbitol syrup, mannitol, xylitol, etc. The synthetic sweetener 3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularly its potassium salt (acetylsupan potassium), sodium salt, and calcium salt, was also considered.

[0388] Sweets may also include coloring agents. These coloring agents can be selected from a variety of dyes suitable for food, pharmaceutical, and cosmetic applications, and are referred to as FD&C dyes, etc. Acceptable materials for the aforementioned application spectrum are preferably water-soluble. Illustrative examples include indigo dye known as FD&C Blue No. 2, which is a disodium salt of 5,5'-indodisulfonic acid. Similarly, dye known as FD&C Green No. 1 comprises a triphenylmethane dye and is a monosodium salt of 4-[4-N-ethyl-p-sulfonylbenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-thiobenzyl)-2-5-cyclohexadieneimine]. A complete description of all FD&C and D&C dyes and their corresponding chemical structures can be found in Volume 5 of the Kirk-Othmer Encyclopedia of Chemical Technology.

[0389] Sweets can also include volatile oil modifiers, such as capsicum oleoresin. These oil modifiers are present in quantities that are not detected as separate components in the oral cavity, but they can still alter the sensory perception of volatile oils.

[0390] Oil modifiers are present in amounts ranging from about 1 to about 150 ppm, similar to those used in sweet peppers. Chili peppers can be obtained from small peppers (Capsicum minium), ornamental peppers (Capsicum frutescens), bell peppers (Capsicum annuum), and similar varieties. Commercially, the fruit of the chili pepper is called chilies or peppers. These fruits are known for their strong bite, pungent taste, and distinctive aroma.

[0391] Regarding confectionery compressed tablet formulations, they will contain a tablet granulation matrix and various additives, such as sweeteners and flavorings. The tablet granulation matrix used will vary depending on a number of factors, such as the type of matrix used, the desired crispness, and other components used to manufacture the final product. These confectionery products typically contain up to 95% sugar by weight of the composition.

[0392] Sweetened compressed tablets may additionally include tablet excipients, such as binders or lubricants, as well as flavoring agents, coloring agents, and volatile oils and volatile oil modifiers.

[0393] The variations that can be practiced with regard to these sweets are very extensive and are within the capabilities of those skilled in the art, particularly with respect to the use of additional fillers, flavorings, colorings, etc.

[0394] External oral care composition External oral care preparations (such as denture cleaning solutions, denture cleaning tablets, denture cleaning powders, etc.) may include ingredients and / or substances selected from the following categories:

[0395] In a preferred embodiment, at least one oral care ingredient is selected from the group consisting of: carriers, disinfectants and bleaching agents, detergents, washing agents and surfactants, foaming agents, preservatives, and flavoring agents.

[0396] Alternatively, the oral care composition of the present invention may also be contained in a filament suitable for cleaning teeth (e.g., a filament used as dental floss). Preferably, the oral care composition is coated onto the outside of the filament. Thus, in a preferred embodiment, the present invention relates to a filament comprising an oral care composition containing the DNase of the present invention, wherein the filament is suitable for cleaning teeth.

[0397] use The oral care compositions of the present invention are suitable for use in the treatment of oral diseases, wherein the desired outcome is the prevention or removal of oral biofilms. The compositions of the present invention are particularly suitable for the treatment of periodontal disease and dental caries.

[0398] Periodontal disease, also known as gingivitis, is a group of inflammatory conditions caused by bacterial infection and the subsequent formation of a biofilm on the teeth and surrounding tissues. Periodontal disease can be classified according to severity as follows: gingivitis (including plaque-induced gingivitis), chronic periodontitis, aggressive periodontitis, periodontitis as a manifestation of systemic disease, necrotizing ulcerative gingivitis / periodontitis, periodontal ligament abscess, and combined pulpoperiodontal disease. Depending on the extent of the affected area, periodontal disease can be further considered local or systemic.

[0399] Tooth decay, also known as cavities or caries, is caused by organic acids (such as lactic acid), which are produced by certain biofilm-forming bacteria residing in the oral cavity (including Streptococcus mutans). Streptococcus mutans ) and some Lactobacillus species ( Lactobacillus ) Species) release. Dental caries can be associated with other complications, such as inflammation of the tissues surrounding the teeth, tooth loss, and infection or abscess formation. Dental caries can be classified by location, etiology, rate of progression, and affected hard tissues, for example, according to the GVBlack classification (categories I, II, III, IV, V, and VI).

[0400] In one aspect, the present invention relates to an oral care composition comprising a DNase for use as a medicine, wherein the DNase is selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0401] In a preferred embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0402] In one aspect, the present invention relates to an oral care composition comprising a DNase for use in the treatment of oral diseases, preferably periodontal diseases and / or dental caries, wherein the DNase is selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0403] In a preferred embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0404] In one aspect, the present invention relates to the use of an oral care composition comprising a DNase for the treatment or preventative treatment of a human subject, wherein the DNase is selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0405] In a preferred embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0406] In a preferred embodiment, the oral care composition is applied to the oral cavity of a human subject.

[0407] In one aspect, the present invention relates to a method for preventing or removing oral biofilms, the method comprising contacting the biofilm with an oral care composition comprising a DNase, wherein the DNase is selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0408] In a preferred embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0409] In one embodiment, the oral care composition is an external oral care composition, and the biofilm is located on the target; preferably, the target is a denture. In another embodiment, the target is located inside or outside the oral cavity.

[0410] In one aspect, the present invention relates to a method for preventing or removing dental plaque, the method comprising contacting the dental plaque with an oral care composition comprising a DNase, wherein the DNase is selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0411] In a preferred embodiment, the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0412] Example DNA enzyme activity assay DNA enzyme activity assay I DNase activity can be determined on DNase assay agar (prepared according to the manufacturer's instructions) containing methyl green (BD, Franklin Lakes, NJ, USA). In short, 21 g of agar is dissolved in 500 ml of water and then autoclaved at 121°C for 15 min. The autoclaved agar is then warmed to 48°C in a water bath, and 20 ml of agar is poured into a Piper dish and allowed to solidify overnight at room temperature. On the solidified agar plate, 5 µl of enzyme solution is added, and DNase activity is observed as a colorless area appearing around the spot of enzyme solution.

[0413] DNA enzyme activity assay I DNase activity was determined using the DNase Alert Kit (11-02-01-04, IDT Integrated DNA Technologies) according to the manufacturer's instructions. In short, 95 µl of DNase sample was mixed with 5 µl of substrate in a microtiter plate, and fluorescence was immediately measured using a Clariostar microplate reader (536 nm excitation, 556 nm emission) from BMG Labtech.

[0414] Example 1: Cloning, expression, and purification of the DNAse of the present invention Linear integration vector systems are used for cloning and expressing the DNA enzymes of the present invention (exemplified by SEQ ID NO: 1, 2 and 3). The linear integration construct is a PCR fusion product formed by fusing corresponding coding sequences between two Bacillus subtilis homologous chromosomal regions with a strong promoter and a chloramphenicol resistance marker. This fusion is prepared by SOE PCR (Horton, RM, Hunt, HD, Ho, SN, Pullen, JK and Pease, LR (1989)). Engineered heterozygous genes without restriction enzymes and gene splicing are generated by overlap extension (Gene [Gene] 77:61-68; WO 2003 / 095658). The gene is expressed under the control of a triple promoter system (as described in WO 1999 / 43835), consisting of a Bacillus licheniformis α-amylase gene promoter containing a stable sequence, a Bacillus amyloliquefaciens α-amylase gene promoter (amyQ), and a Bacillus thuringiensis cryIIIA promoter. The gene encoding chloramphenicol acetyltransferase was used as a marker (described, for example, in Diderichsen, B.; Poulsen, GB; Joergensen, ST 1993). Plasmid [Plasmid] "A useful cloning vector for Bacillus subtilis" (30:312). The final gene construct was integrated into the pectin lyase locus via homologous recombination in the Bacillus chromosome.

[0415] The genes encoding the DNases of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were amplified from the genomic DNA of strains of *Bacillus* species-62490, *Sacchariformis Horikoshi*, and *Haloxybacterium falciparum*, respectively. SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 were expressed by nucleic acid constructs provided as SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively, wherein the *Bacillus clausti* signal peptide (MKKPLGKIVASTALLISVAFSSSIASA; SEQ ID NO:4) replaced the native secretion signal of the gene, and the His tag (HHHHHH; SEQ ID NO:5) was directly fused to the C-terminus of the mature polypeptide.

[0416] PCR amplification was performed using gene-specific primers containing overhangs to the two flanking vector fragments. Upstream and downstream vector fragments (described in WO 2003 / 095658 as Bacillus subtilis PL3598-37) were amplified from the genomic DNA of Bacillus subtilis MB1361. The two linear vector fragments and their corresponding gene fragments were assembled into a linear vector construct by SOE-PCR. Aliquots of each PCR product were transformed into Bacillus subtilis strains. Transformants were selected on LB plates supplemented with chloramphenicol (6 µg / ml). For each of the three constructs, recombinant Bacillus subtilis clones containing the integrated expression construct were cultured on a rotary shaker in 500 mL baffled Erlenmeyer flasks containing 100 mL of yeast extract-based medium. The clones were incubated at 26°C for 4 days, and the enzyme-containing supernatant was harvested.

[0417] Prior to purification, the pH of the supernatant was adjusted to pH 8 with 3 M Tris. The pH-adjusted supernatant was incubated for 1 hour and then filtered using a filtration device (Nalgene) equipped with a 0.2 µm filter. The filtered supernatant was applied to a 5 ml HisTrap™ Excel column (GE Healthcare LifeSciences) and pre-equilibrated with 5 column volumes (CV) of 50 mM Tris / HCl at pH 8. Unbound proteins were eluted by washing the column with 8 CV of 50 mM Tris / HCl at pH 8. The DNase of the present invention was then eluted with 50 mM HEPES buffer containing 10 mM imidazole at pH 7, with elution monitored by absorbance at 280 nm. The eluted DNase was desalted on a HiPrep™ 26 / 10 desalting column (General Healthcare Life Sciences) pre-equilibrated with 50 mM HEPES buffer (pH 7) containing 100 mM NaCl at 3 CV, and then eluted from the column at a flow rate of 10 ml / min using the same buffer. Relevant fractions were selected, pooled, and analyzed by SDS-PAGE using 4%–12% Bis-Tris gel (Invitrogen) and 2-(N-morpholino)ethanesulfonic acid (MES) SDS-PAGE run buffer (Invitrogen). The gels were stained with Instant Blue (Novexin) and destained with MilliQ water. The concentration of the purified enzyme was determined by absorbance at 280 nm.

[0418] Example 2: In vitro assays for evaluating substrate specificity The DNA model substrate solution was prepared as follows: The DNA model substrate (see Table 1) was dissolved in G4 buffer (10 mM Tris, 100 mM KCl, pH 7) or B / Z buffer (0.025% chitosan, 25 mM Tris, 6.25 mM CaCl2, 1 mM MgSO4, pH 6), then annealed at 95°C for 5 min, and then gradually cooled to 35°C over 90 min to form the secondary structure.

[0419] Dilute the DNA model substrate solution (1 µM) to 200 nM in G4 buffer or B / Z buffer, and use DNase (SEQ ID NO: 1, 2, and 3) at a concentration of 10 mg / L. Add 40 µL of model substrate and 4 µL of enzyme solution (or water as a negative control) to a sterile microtiter plate, seal with sterile tape, and incubate at 37°C with shaking at 50 rpm for 1 hour.

[0420] After incubation, the tape was removed, and the remaining model substrate was quantified by staining with 1 µM SYTO60 (G4), 0.5% PicoGreen (Z-DNA), or µM TOTO-1 (B-DNA) using a Clario Star microplate reader with the following settings: (i) for G4 studies, excitation at 630–10 nm, emission at 670–10 nm, gain 2500; and (ii) excitation at 488–15 nm, emission at 530–20 nm, gain 1300–1800. Table 2 provides the relative activities of the DNase of the present invention against G4, Z-DNA, and B-DNA model substrates.

[0421] As can be clearly seen from Table 2, the DNAse of the present invention has broad substrate specificity and is capable of degrading B-DNA as well as Z-DNA and G4-DNA.

[0422] Example 3: Multi-species biofilm prevention assay Using Streptococcus mutans UA159, one of the three dental pathogens, and Actinobacillus negriensis ( Actinomyces naeslundii ATCC 12104 and oral streptococci ( Streptococcus oralis Evaluation of the effects of the DNase of the present invention on biofilm prevention in mixed species biofilms of ATCC 35037 (H. Koo et al., Journal of Bacteriology 2010; Ahn KB et al., PLoS ONE, 2018; HMNassar and RL Gregory, Journal of Oral Microbiology, 2017).

[0423] A 96-well microtiter plate (Nunclon Delta surface, Thermo Scientific, catalog number 167008) was filled with 94 µl of Tripticase soybean broth (TSB) + 2% glucose (a bacterial inoculum containing a mixture of Streptococcus mutans UA159, Actinomyces negrinosum ATCC 12104, and Streptococcus oralis ATCC 35037 (1 × 10⁷ CFU / ml)) and 6 µl of enzyme solution in assay buffer (50 mM HEPES, 100 mM NaCl, pH 7) to obtain a final concentration of 60 ppm. As a control, the enzyme solution was replaced with assay buffer. The microtiter plate was then incubated anaerobically at 37°C without shaking for 72 hours in a Thermo Scientific™ Rectangular AnaeroBox™ container (Thermo Scientific AnaeroGen 2.5L, catalog number AN0025A). The enzyme and control samples were evaluated eight times.

[0424] After incubation, airborne bacteria were removed by two gentle washes with 100 µl of 0.9% NaCl solution, and the biofilm was stained with 0.095% crystal violet solution at room temperature for 15 min. The plate was rinsed twice with 100 µL of 0.9% NaCl, and the adhering dye was dissolved in an aqueous solution of 96% ethanol and 0.1% acetic acid. The absorbance at 600 nm was measured using a microplate reader (SpectraMax M3, Molecular Devices).

[0425] For data processing, the resulting absorbance is directly proportional to the degree of residual biofilm after enzyme or control treatment. The results are expressed as the percentage of biofilm prevention and calculated as follows: 100 - ((A600nm enzyme-treated sample) / (A600nm buffer-treated control sample) x 100) Where A600nm refers to the average of eight measurements. As shown in Table 2, the DNase of the present invention significantly prevents the formation of multi-species biofilms containing dental pathogens such as Streptococcus mutans, Streptococcus stomatologicus, and Actinomyces negrius.

[0426] Example 4: Thermal stability measurement Preparation of oral care formulations for thermal stability measurement The thermal stability, or pyrolysis folding midpoint (Tm), of the DNase of the present invention was measured in the presence of several widely used oral care ingredients within concentration ranges commonly found in oral care product formulations and selected commercial oral care products. The Tm parameter is used to assess thermal stability because it is the temperature at which the populations of folded and unfolded protein molecules are equal, and it is a widely accepted parameter used in assessing thermal stability. High-purity and biotechnology-grade reagents were obtained from various suppliers, and stock solutions were freshly prepared using MilliQ water. These formulation chemicals and their stock solutions, as well as the final concentrations used in the Tm measurements, are listed in Table 4.

[0427] The purified protein samples were diluted to a stock concentration of 2 mg / ml, and then further diluted 10-fold in an oral care formulation consisting of separately formulated chemicals, citrate phosphate buffer (McIlvaine buffer), and Milli-Q water, corresponding to a final protein concentration of 0.2 mg / ml. All dilutions were performed using a robotic arm in 384-well small-volume deep-well plates (Greiner Bio-One International) with a final volume of 70 µl, and were used for thermal stability measurements. Tm measurements were performed for each protein within a physiological pH range close to that of the oral cavity using McIlvaine buffer at pH 5.0 and pH 6.0. A 100 ml McIIvaine buffer with pH 5.0 was prepared by mixing 51.50 ml of 0.2 M Na2HPO4+ and 48.50 ml of 0.1 M citric acid, while a McIIvaine buffer with pH 6.0 was prepared by mixing 63.15 ml of 0.2 M Na2HPO4+ and 36.85 ml of 0.1 M citric acid.

[0428] Determination of Tm Thermal stability measurements were performed using a capillary-based nanodiffraction differential fluorescence spectrometer (nanoDSF; Prometheus NT.Plex, NanoTemper Technologies GmbH, Munich, Germany). Standard nanoDSF-grade capillary action sheets (catalog number: PR-AC002) from NanoTemper Technologies were used. Protein samples were loaded into capillaries via capillary action (three copies of each sample). The emission intensity at 330 nm and 350 nm was optimized by varying the LED power on the instrument to ensure sufficient signal strength. Fluorescence signals at 330 nm and 350 nm were continuously monitored as a function of temperature (heating rates for pyrolysis were 3.3 °C / min from 20 °C to 95 °C). Data were analyzed using the manufacturer's PR.StabilityAnalysis_1.1.0.11077 software. This analysis is model-independent and uses only the peak maximum of the first derivative, which corresponds to the approximate midpoint of the pyrolysis transition, defined as Tm (see [link to analysis]). Figure 1 ).

[0429] Reproducibility of thermal stability data Figure 1 An example of thermal stability data generated using a nanoDSF instrument is shown. Subplot A is an example of data from SEQ ID NO:1 obtained in triplicate as a function of temperature (ratio of fluorescence emission at 350 nm to 330 nm). Subplot B shows the first derivative of the raw data from subplot A. The peak maximum in the first derivative plot corresponds to the midpoint of the pyrolysis fold transition, termed Tm. In this example, Tm at pH 6 corresponds to 52.9 °C and is highly reproducible across three replicates.

[0430] Figure 1 The data shown are examples of data types generated using nanoDSF in different formulations for the DNAse of the present invention (SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:1). In all cases, the data show obvious unfolding transitions and clearly distinguishable peaks in the first derivative, and are highly reproducible.

[0431] Thermal stability of DNAases in oral care formulations Tables 5 and 6 show the average thermal stability of the three DNAses of the present invention, measured in triplicate at pH 5.0 and pH 6.0, respectively, in the presence of a range of commonly used oral care ingredients.

[0432] These data clearly show that none of these components have an adverse effect on the thermal stability of DNA enzymes, and that under conditions similar to those in the oral cavity, these enzymes exhibit considerable or even improved stability in the presence of these components, making them suitable for use in oral care products and in the oral cavity.

[0433] The invention is further defined by the following numbered paragraphs:

[0434] 1. An oral care composition comprising a DNase selected from the group consisting of: a) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1; b) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2; and c) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3.

[0435] 2. An oral care composition comprising a DNase selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0436] 3. The oral care composition according to any one of the preceding paragraphs, wherein the DNA enzyme is present in an effective amount; preferably in an amount of about 1 ppm to about 500 ppm; most preferably in an amount of about 50 ppm to about 200 ppm.

[0437] 4. The oral care composition according to any one of paragraphs 1-3, wherein the oral care composition is in the form of an internal oral care composition; preferably in the form of toothpaste or toothpaste tablets, tooth cream, mouthwash or mouthwash tablets, oral cleanser, lozenges, soft lozenges, chewing gum, sweets, or candies.

[0438] 5. The oral care composition according to any one of paragraphs 1-3, wherein the oral care composition is in the form of an external oral care composition; preferably in the form of a denture cleaning solution, denture cleaning tablets, or denture cleaning powder.

[0439] 6. The oral care composition according to any one of paragraphs 1-5, for use as a medicine.

[0440] 7. The oral care composition according to any one of paragraphs 1-5, for use in the treatment of oral diseases; preferably for use in the treatment of periodontal disease (e.g., gingivitis) and / or dental caries.

[0441] 8. Use of the oral care composition according to any one of paragraphs 1-5 for the treatment or preventive treatment of human subjects.

[0442] 9. A method of treating a human subject, the method comprising applying an oral care composition according to any one of paragraphs 1-5; preferably, the oral care composition is applied to the oral cavity of the human subject.

[0443] 10. A method for preventing and / or removing oral biofilms, the method comprising contacting the oral biofilm with an oral care composition according to any one of paragraphs 1-5.

[0444] 11. The method according to paragraph 10, wherein the oral biofilm is located on the target, preferably denture.

[0445] 12. The method described in paragraph 11, wherein the denture is located inside or outside the oral cavity.

[0446] 13. A kit comprising a) an oral care composition according to any one of paragraphs 1-5; and b) instructions for use.

[0447] 14. A polypeptide having DNase activity, the polypeptide being selected from the group consisting of: a) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:1; b) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:2; and c) A polypeptide having at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:3.

[0448] 15. A polypeptide having DNase activity, the polypeptide being selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:3.

[0449] 16. A polynucleotide encoding a polypeptide according to any one of paragraphs 14-15.

[0450] 17. The polynucleotide described in paragraph 16 is purified.

[0451] 18. The polynucleotide described in paragraph 16 is isolated.

[0452] 19. A nucleic acid construct or expression vector comprising a polynucleotide as described in paragraph 16, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0453] 20. A recombinant host cell comprising a nucleic acid construct or expression vector as described in paragraph 19.

[0454] 21. The recombinant host cell according to paragraph 20, wherein the polypeptide is heterologous to the recombinant host cell.

[0455] 22. The recombinant host cell according to paragraph 20 or 21, wherein at least one of the one or more control sequences is heterologous to the polynucleotide encoding the polypeptide.

[0456] 23. The recombinant host cell according to any one of paragraphs 20-22, wherein the recombinant host cell comprises at least two copies, such as three, four, five or more copies, of the polynucleotide described in paragraph 16.

[0457] 24. The recombinant host cell according to any one of paragraphs 20-23, wherein the recombinant host cell is a prokaryotic recombinant host cell, for example, a Bacillus cell, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceae, Bacillus coagulans, Bacillus sclerotiorum, Bacillus brilliance, Bacillus retardans, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, or Bacillus thuringiensis cells.

[0458] 25. The recombinant host cell according to any one of paragraphs 20-23, wherein the recombinant host cell is a yeast recombinant host cell, for example, cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia*, *Saccharomyces*, *Saccharomyces*, or *Yersinia*, such as *Kluyveromyces lactis*, *Kalvatia*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Douglas*, *Kluyveromyces kluyvernsis*, *Nordiya*, *Ovoyces*, or *Yersinia lipolytica* cells.

[0459] 26. The recombinant host cell according to any one of paragraphs 20-23, wherein the recombinant host cell is a filamentous fungal recombinant host cell, such as *Cladosporium*, *Aspergillus*, *Briefomus*, *Cirsium*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Cladosporium*, *Cryptococcus*, *Ustilago*, *Fusarium*, *Pyrophyllus*, *Mucor*, *Hydrophyllus*, *Neurospora*, *Penicillium*, etc. Cells of the genera *Moldae*, *Pleurotus*, *Gastromycetes*, *Rumenichthys*, *Pleurotus*, *Schizophyllum*, *Basilaria*, *Thermophilic Ascomycetes*, *Fusporium*, *Cyclophorus*, *Vallisneria*, or *Trichoderma*, especially *Aspergillus buergerianus*, *Aspergillus scabiosifolius*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus niger*, *Aspergillus oryzae*, *Cyclophorus nidus ... , Insectivorous lacewings, Narrow-sided augenosa, Keratopteric augenosa, Lukenowens augenosa, Coprophytic augenosa, Femtosporum augenosa, Queensland augenosa, Tropical augenosa, Brown augenosa, Coprinus comatus, Trichoderma spp., Fusarium moniliforme ... Spores, skin-colored Fusarium, pseudo-branchial Fusarium, sulfur-colored Fusarium, round Fusarium, pseudo-filamentous Fusarium, patchy Fusarium, specific humic mold, loose cottony humic mold, rice black root mold, thermophilic filamentous mold, rough spores, purple-producing Penicillium, yellow-spore flat fungus, radiating fungus, erythrophorus, Emerson basket fungus, terrestrial closporium, long-haired cork fungus, discoloration cork fungus, Trichoderma harzianum, Corning Trichoderma, long-branched Trichoderma, Trichoderma reesei, or green Trichoderma cells.

[0460] 27. The recombinant host cell according to any one of paragraphs 20-26, wherein the recombinant host cell is isolated.

[0461] 28. The recombinant host cell according to any one of paragraphs 20-26, wherein the recombinant host cell is purified.

[0462] 29. A method for producing a polypeptide according to any one of paragraphs 14-15, the method comprising culturing a recombinant host cell according to any one of paragraphs 20-26 under conditions conducive to the production of the polypeptide.

[0463] 30. The method according to paragraph 29, further comprising recovering the polypeptide.

[0464] 31. A method for producing a polypeptide according to any one of paragraphs 14-15, the method comprising culturing cells under conditions conducive to the production of the polypeptide, the cells producing the polypeptide in their wild-type form.

[0465] 32. The method according to paragraph 31, further comprising recovering the polypeptide.

[0466] 33. A whole culture medium formulation or cell culture composition comprising a polypeptide according to any one of paragraphs 14-15.

Claims

1. An oral care composition comprising a DNase selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

2. The oral care composition according to claim 1, wherein the DNA enzyme is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:

3.

3. The oral care composition according to any one of the preceding claims, wherein the DNase is present in an effective amount; preferably in an amount of about 1 ppm to about 500 ppm; most preferably in an amount of about 50 ppm to about 200 ppm.

4. The oral care composition according to any one of the preceding claims, wherein the oral care composition is in the form of toothpaste or toothpaste tablets, tooth cream, mouthwash or mouthwash tablets, oral cleanser, lozenges, soft lozenges, chewing gum, sweets or candies.

5. The oral care composition according to any one of claims 1-4, for use as a medicine.

6. The oral care composition according to any one of claims 1-4, for use in the treatment of oral diseases; preferably for use in the treatment of periodontal disease and / or dental caries.

7. A method for preventing and / or removing oral biofilms, the method comprising contacting the oral biofilm with an oral care composition according to any one of claims 1-4.

8. A polypeptide having DNase activity, said polypeptide being selected from the group consisting of: a) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:1, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; b) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:2, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity; and c) A polypeptide having DNase activity and having at least 60% sequence identity with SEQ ID NO:3, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

9. The polypeptide of claim 8, wherein the polypeptide is selected from the group consisting of: a) A polypeptide comprising SEQ ID NO:1, substantially consisting of SEQ ID NO:1, or consisting of SEQ ID NO:1; b) A polypeptide comprising SEQ ID NO:2, substantially consisting of SEQ ID NO:2, or consisting of SEQ ID NO:2; and c) A polypeptide comprising SEQ ID NO:3, consisting essentially of SEQ ID NO:3, or consisting of SEQ ID NO:

3.

10. A polynucleotide encoding a polypeptide according to any one of claims 8-9.

11. A nucleic acid construct or expression vector comprising a polynucleotide according to claim 10, the polynucleotide being operatively linked to one or more control sequences that direct the production of the polypeptide in an expression host.

12. A recombinant host cell comprising the nucleic acid construct or expression vector according to claim 11.

13. The recombinant host cell according to claim 12, wherein the recombinant host cell is a Bacillus spp. ( Bacillus ) cells; preferably Bacillus alkalophilus ( Bacillus alkalophilus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus brevis ( Bacillus brevis ), Bacillus circularis ( Bacillus circulans ), Bacillus clausti ( Bacillus clausii Bacillus coagulans ( Bacillus coagulans ), Bacillus sclerosus ( Bacillus firmus ), Bacillus splendens ( Bacillus lautus ), Bacillus tarda ( Bacillus lentus ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus megaterium ( Bacillus megaterium ), Bacillus pumilus ( Bacillus pumilus ), thermophilic steatobacterium ( Bacillus stearothermophilus Bacillus subtilis ( Bacillus subtilis ) or Bacillus thuringiensis ( Bacillus thuringiensis Cells; most preferably Bacillus licheniformis or Bacillus subtilis cells.

14. A method for producing a polypeptide having DNase activity according to any one of claims 8-9, the method comprising: The recombinant host cell according to any one of claims 12-13 is cultured under conditions conducive to the production of the polypeptide, and optionally, the polypeptide is recovered.

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