Varianases and oral care compositions comprising varianases

Abstract

The present invention relates to polypeptides having endo-1, 3-alpha-glucanase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, host cells comprising the polynucleotides, methods of producing the polypeptides, and oral care compositions comprising the polypeptides.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Technical Field

[0003] The present invention relates to polypeptides having endo-1,3-α-glucanase activity (i.e., mutanase), polynucleotides encoding these polypeptides, nucleic acid constructs, vectors, host cells comprising these polynucleotides, methods for producing these polypeptides, and oral care compositions comprising these polypeptides. Background Art

[0004] Biofilms are communities of bacteria found on solid surfaces in many different environments, including those in the oral cavity. Oral biofilm, or dental plaque, contains many bacteria that are associated with oral health problems such as bad breath, demineralization, caries, cavities, potential tooth loss, and gum disease (gingivitis and periodontitis).

[0005] The formation of oral biofilm occurs in three stages, respectively referred to as stagnant phase, growth phase, and stable state. In the stagnant phase, glycoproteins from saliva combine with oral surfaces (e.g., teeth) and form a structure called biofilm, which serves as an attachment site for bacteria. In the growth phase, coagulation occurs, i.e., the second bacterial colonizer attaches to the first bacterial colonizer, causing an increase in biofilm diversity and biofilm growth and maturation. In the stable state, biofilm growth slows down and eventually stops. This stage-based formation cycle causes biofilm to exist in several continuous levels, which makes the physical wear of biofilm more difficult.

[0006] Within the biofilm, the resident bacterial cells are distributed in an extracellular polymer matrix that is primarily composed of water, proteins, exopolysaccharides, lipopolysaccharides, lipids, surfactants, and extracellular DNA, with exopolysaccharides accounting for the majority of the biofilm dry weight (HC Flemming and J. Wingender (2010), Nat. Rev. Microbiol. [Nature Review of Microorganisms] 8, 623-633). Exopolysaccharides are major homopolymers of glucose and fructose, including 1,3-α-D-glucan, 1,4-α-D-glucan, 1,6-α-D-glucan, and 2,6-β-D-fructan. These polysaccharides are synthesized from ingested sucrose by glucosyltransferases and fructosyltransferases secreted by oral bacteria (e.g., Streptococcus spp., Lactobacillus spp., and Actinomyces spp.). Mutans and dextran are glucans that are particularly important in the formation of dental plaque. Mutans have a highly branched structure with a main chain consisting of glucose molecules linked to 1,3-α-glycosidic bonds and 1,6-α-glycosidic bonds in the side chains. Dextran is also a high molecular weight polymer of glucose containing multiple consecutive 1,6-α-bonds in the main chain and side chains, starting with a 1,3-α-bond (M. Pleszczynska et al. (2016), Biotechnol. Appl. Biochem. [Applied Biochemistry and Biotechnology] 64(3), 337-346).

[0007] Because of the increased resistance to antimicrobial agents and the mechanical properties of biofilms, many current oral care products are quite ineffective in addressing biofilm formation and alleviating related oral health problems. The primary focus of biofilm removal is mechanical abrasion. However, the multi-layered nature of biofilms makes mechanical abrasion difficult, and mechanical abrasion is further compromised because mechanical removal of biofilms (e.g., by brushing) enlarges and deepens the area where biofilms attach and spread in the oral cavity, potentially increasing rather than decreasing the severity of the problem.

[0008] Given the important role of biofilm in oral diseases, there is a need in the art for oral care compositions that can effectively target oral biofilm. To this end, enzymatic oral care solutions comprising mutanase (ie, 1,3-α-glucanase) and / or dextranase are of particular interest. WO 1997 / 38669 (Novozymes) describes oral care compositions comprising Trichoderma harzianum mutanase and Paecilomyces lilacinum dextranase, WO 1998 / 57653 (Novozymes) provides oral care compositions comprising Paecilomyces lilacinum dextranase and Bacillus pullulanase, U.S. Pat. No. 4,353,981 (Guggenheim et al.) describes the use of Trichoderma harzianum CBS243.71 mutanase, Penicillium funiculum NRRL 1768 mutanase and Penicillium lilacinum NRRL 896 mutanase for removing dental plaque, U.S. Pat. No. 10,472,616 (Genofocus) describes Trichoderma harzianum GF101 mutanase and its use in degrading bacterial biofilms, and WO 1998 / 57653 (Novozymes) provides oral care compositions comprising Paecilomyces lilacinum dextranase and Bacillus pullulanase, U.S. Pat. No. 4,353,981 (Guggenheim et al.) describes the use of Trichoderma harzianum CBS243.71 mutanase, Penicillium funiculum NRRL 1768 mutanase and Penicillium lilacinum NRRL 896 mutanase for removing dental plaque. 2020 / 099490 (Novozymes) describes oral care compositions comprising a Trichoderma harzianum mutanase and a microbial DNase. However, there remains a need for additional and improved oral care compositions that are more effective in degrading oral biofilms.

[0009] It is an object of the present invention to provide a mutanase with improved thermostability and an oral care composition comprising the mutanase. Another object of the present invention is to provide a mutanase and an oral care composition comprising the mutanase, the mutanase providing improved biofilm prevention. Another object of the present invention is to provide a mutanase and an oral care composition comprising the mutanase, the mutanase having improved thermostability and providing comparable or even improved biofilm prevention at the same time. Another object of the present invention is to provide a mutanase and an oral care composition comprising the mutanase, the mutanase showing enzyme activity at pH 5-10, preferably pH 6-9, most preferably pH 7-8. Another object of the present invention is to provide a mutanase and an oral care composition comprising the mutanase, the mutanase having improved thermostability and showing enzyme activity at pH 5-10, preferably pH 6-9, most preferably pH 7-8 at the same time. Another object of the present invention is to provide a mutanase and an oral care composition comprising the same, which mutanase simultaneously has improved thermostability, exhibits enzyme activity at pH 5-10, preferably pH 6-9, most preferably pH 7-8, and provides comparable or even improved biofilm prevention. Summary of the invention

[0010] The present invention provides polypeptides having endo-1,3-α-glucanase activity (ie, mutanase) that have improved thermostability, provide comparable or improved biofilm prevention, and exhibit enzyme activity at typical oral pH levels. The present invention also provides oral care compositions comprising the polypeptides.

[0011] In a first aspect, the present invention is an oral care composition comprising a mutanase selected from the group consisting of:

[0012] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 3;

[0013] (b) a polypeptide having at least 90% sequence identity to SEQ ID NO:6;

[0014] (c) a polypeptide having at least 65% sequence identity to SEQ ID NO:9;

[0015] (d) a polypeptide having at least 65% sequence identity to SEQ ID NO: 12;

[0016] (e) a polypeptide having at least 60% sequence identity to SEQ ID NO: 15;

[0017] (f) a polypeptide having at least 60% sequence identity to SEQ ID NO: 18;

[0018] (g) a polypeptide having at least 60% sequence identity to SEQ ID NO: 21;

[0019] (h) a polypeptide having at least 60% sequence identity to SEQ ID NO: 24;

[0020] (i) a polypeptide having at least 60% sequence identity to SEQ ID NO: 27;

[0021] (j) a polypeptide having at least 60% sequence identity to SEQ ID NO: 30; and

[0022] (k) a polypeptide having at least 60% sequence identity to SEQ ID NO: 33;

[0023] wherein the polypeptide has endo-1,3-alpha-glucanase activity, and wherein the oral care composition further comprises at least one oral care ingredient.

[0024] The present invention also relates to the use of the oral care composition of the present invention as a medicament, methods of using the oral care composition of the present invention, and kits comprising the oral care composition of the present invention.

[0025] In a second aspect, the present invention relates to a polypeptide having endo-1,3-α-glucanase activity, the polypeptide being selected from the group consisting of:

[0026] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 3;

[0027] (b) a polypeptide having at least 90% sequence identity to SEQ ID NO:6;

[0028] (c) a polypeptide having at least 65% sequence identity to SEQ ID NO:9;

[0029] (d) a polypeptide having at least 65% sequence identity to SEQ ID NO: 12;

[0030] (e) a polypeptide having at least 60% sequence identity to SEQ ID NO: 15;

[0031] (f) a polypeptide having at least 60% sequence identity to SEQ ID NO: 18;

[0032] (g) a polypeptide having at least 60% sequence identity to SEQ ID NO: 21;

[0033] (h) a polypeptide having at least 60% sequence identity to SEQ ID NO: 24;

[0034] (i) a polypeptide having at least 60% sequence identity to SEQ ID NO: 27;

[0035] (j) a polypeptide having at least 60% sequence identity to SEQ ID NO: 30; and

[0036] (k) a polypeptide having at least 60% sequence identity to SEQ ID NO:33.

[0037] The present invention also relates to polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs, expression vectors, host cells comprising these polynucleotides; and methods for producing these polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1Shown is an example of thermal stability data generated using a nanoDSF instrument. A sub-graph is an example of data (ratio of fluorescence emission at 350nm to 330nm) of SEQ ID NO:3 obtained in triplicate as a function of temperature. B sub-graph shows the first derivative of the raw data in sub-graph A. The peak maximum in the first derivative graph corresponds to the midpoint of the thermal unfolding transition, referred to as Tm. In this example, for SEQ ID NO:3, the Tm at pH 6 corresponds to 69.6°C, and is highly reproducible within three replicates.

[0039] Figure 2 The effect of pH on the enzymatic activity of selected mutanases (SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 24 and SEQ ID NO: 36) is shown. Endo-1,3-α-glucanase activity was monitored at different pH values ​​ranging from 4 to 10 using standard reaction conditions (50°C for 10 minutes). Each data point represents the average of three measurements, and the error bars represent the standard deviation.

[0040] definition

[0041] In light of this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] Unless defined otherwise or clearly 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 belongs.

[0043] Mutanase: The term "mutanase" means a polypeptide having endo-1,3-α-glucanase activity (EC 3.2.1.59) that catalyzes the hydrolytic cleavage of 1,3-α-glycosidic bonds found, for example, in mutans. The terms "mutanase" and "1,3-α-glucanase" and the expression "polypeptide having endo-1,3-α-glucanase activity" are used interchangeably throughout the application. For the purposes of the present invention, mutanase activity can be determined according to the procedure described in Example 3 below.

[0044] Biofilm prevention: The term "biofilm prevention" means the ability of a polypeptide to reduce the amount of biofilm grown under defined conditions. For the purposes of the present invention, biofilm prevention can be determined according to Example 6 herein.

[0045] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA, which is processed through a series of steps (including splicing) and then appears as a mature, spliced ​​mRNA.

[0046] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are usually determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0047] Control sequences: The term "control sequence" means a nucleic acid sequence that is involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from a different gene) to a polynucleotide encoding a polypeptide, and is native or heterologous to each other. Such control sequences include, but are not limited to, leader polyadenylation sequences, prepropeptides, 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 can be provided with a plurality of linkers for the purpose of introducing specific restriction sites that are conducive to connecting the control sequences to the coding region of the polynucleotide encoding the polypeptide.

[0048] Expression: The term "expression" means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0049] Expression vector: "Expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a polypeptide, the coding sequence being operably linked to a suitable control sequence capable of affecting the expression of the DNA in a suitable host. Such control sequences may include a promoter that affects transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and a sequence that controls the termination of transcription and translation.

[0050] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the "extended" polypeptide has endo-1,3-alpha-glucanase activity.

[0051] Fragment: The term "fragment" means a polypeptide having one or more amino acids missing from the amino and / or carboxyl terminus of a mature polypeptide, wherein the fragment has endo-1,3-alpha-glucanase activity.

[0052] Fusion polypeptide: The term "fusion polypeptide" is a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include connecting the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which fusion polypeptides are produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779). The fusion polypeptide may further comprise a cleavage site between the two polypeptides. When the fusion protein is secreted, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 1998, J. Biotechnol. 76:245-251; 95, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0053] Heterologous: With respect to a host cell, the term "heterologous" means that the polypeptide or nucleic acid is not naturally present in the host cell. With respect to a polypeptide or nucleic acid, the term "heterologous" means that the control sequence (e.g., the promoter of the polypeptide or nucleic acid) is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0054] Host strain or host cell: "Host strain" or "host cell" refers to an organism into which an expression vector, bacteriophage, virus or other DNA construct (including a polynucleotide encoding a polypeptide of interest (e.g., amylase)) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0055] Introduced: In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," as known in the art.

[0056] 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.). Thus, the isolated polypeptide, nucleic acid, cell or other material is in a form that does not exist in nature. An isolated polypeptide includes but is not limited to a culture medium containing a secretory polypeptide expressed in a host cell.

[0057] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form after N-terminal and / or C-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is SEQ ID NO:3. In one aspect, the mature polypeptide is SEQ ID NO:6. In one aspect, the mature polypeptide is SEQ ID NO:9. In one aspect, the mature polypeptide is SEQ ID NO:12. In one aspect, the mature polypeptide is SEQ ID NO:15. In one aspect, the mature polypeptide is SEQ ID NO:18. In one aspect, the mature polypeptide is SEQ ID NO:21. In one aspect, the mature polypeptide is SEQ ID NO:24. In one aspect, the mature polypeptide is SEQ ID NO:27. In one aspect, the mature polypeptide is SEQ ID NO:30. In one aspect, the mature polypeptide is SEQ ID NO:33.

[0058] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having endo-1,3-α-glucanase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 1389 of SEQ ID NO: 1, or a cDNA sequence thereof. In one aspect, the mature polypeptide coding sequence is nucleotides 73 to 2063 of SEQ ID NO: 4, or a cDNA sequence thereof. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 2102 of SEQ ID NO: 7, or a cDNA sequence thereof. In one aspect, the mature polypeptide coding sequence is nucleotides 61 to 2111 of SEQ ID NO: 10, or a cDNA sequence thereof. In one aspect, the mature polypeptide coding sequence is nucleotides 124 to 3352 of SEQ ID NO: 13. In one aspect, the mature polypeptide coding sequence is nucleotides 76 to 2391 of SEQ ID NO: 16. In one aspect, the mature polypeptide coding sequence is nucleotides 79 to 2226 of SEQ ID NO: 19. In one aspect, the mature polypeptide coding sequence is nucleotides 79 to 2310 of SEQ ID NO: 22. In one aspect, the mature polypeptide coding sequence is nucleotides 115 to 2184 of SEQ ID NO: 25. In one aspect, the mature polypeptide coding sequence is nucleotides 133 to 2028 of SEQ ID NO: 28. In one aspect, the mature polypeptide coding sequence is nucleotides 115 to 2436 of SEQ ID NO: 31.

[0059] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0060] 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 can 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 present compositions and methods encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0061] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not originally found in nature to contain a segment of nucleic acid or is synthetic and comprises one or more control sequences operably linked to the nucleic acid sequence.

[0062] Operably linked: The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0063] Purified: The term "purified" means a nucleic acid, polypeptide, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a culture medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, 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., by weight or by mole). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other specified material or component in a composition at a relative or absolute concentration higher than that of the starting composition.

[0064] In one aspect, the term "purified" as used herein refers to a polypeptide or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a polypeptide that is substantially free of insoluble components (especially insoluble components) from the natural organism from which it is obtained. In one aspect, the polypeptide is separated from some soluble components of the organism and culture medium from which it is recovered. The polypeptide can be purified (i.e., isolated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0065] Accordingly, the polypeptide can be purified so that only a small amount of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" may refer to the removal of other components present in the cell of the polypeptide source, particularly other proteins and most particularly other enzymes. The polypeptide may be "substantially pure", i.e., free of other components from the organism that produces it (e.g., a host organism for recombinantly producing the polypeptide). On the one hand, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. On the one hand, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, "substantially pure polypeptide" may refer to a polypeptide preparation containing 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 the polypeptide and other polypeptide materials associated with it naturally or recombinantly.

[0066] Thus, it is preferred that a substantially pure polypeptide 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, based on the weight of the total polypeptide material present in the preparation. The polypeptides of the invention are preferably in substantially pure form (i.e., the preparation is substantially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the polypeptide using well-known recombinant methods or using classical purification methods.

[0067] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cleavage and rejoining) of a nucleic acid sequence to form a sequence population that is different from that found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0068] Recovery: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, such as recovering the polypeptide from a whole fermentation broth or from a cell-free fermentation broth by harvesting the polypeptide crystals, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in box filters, drum filtration, drum filtration, rotary vacuum drum filtration, candle filters, horizontal leaf filters or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, cross-flow, dynamic cross-flow or ultrafiltration in dead-end operation)) or by centrifugation (using a horizontal centrifuge, a disk stack centrifuge, a hyrdo cyclone or the like) or by precipitating the polypeptide and using a related solid-liquid separation method to harvest the polypeptide from a broth medium by using size fractionation. Recovery encompasses the isolation and / or purification of the polypeptide.

[0069] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0070] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferred 6.6.0 version or later). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order to make the Needle program report the longest identity, the -nobrief option must be specified in the command line. The output of the "longest identity" of the Needle mark is calculated as follows:

[0071] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0072] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of "longest identity" using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), which is implemented by the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferred 6.6.0 version or updated version). The parameter used is gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order to make the Needle program report the longest identity, it is necessary to specify the non-simplified (nobrief) option in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0073] (number of identical deoxyribonucleotides × 100) / (length of alignment – ​​total number of gaps in alignment)

[0074] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0075] Subsequence: The term "subsequence" means a polynucleotide having one or more nucleotides deleted from the 5' end and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having endo-1,3-α-glucanase activity.

[0076] Thermal stability: The term "thermostability" means the stability of a polypeptide to heat and can be defined by the thermal unfolding transition midpoint (Tm) of the polypeptide. For the purposes of the present invention, thermal stability can be determined according to Example 4 (thermostability of a polypeptide alone) or Example 5 (thermostability of a polypeptide in the presence of an oral care ingredient) below as thermal stability defined by the thermal unfolding transition midpoint (Tm). In the context of the present invention, the term "comparable thermal stability" means that the thermal stability of a polypeptide is comparable to the thermal stability (Tm) of another polypeptide. m In the context of the present invention, the term "improved thermostability" means that the thermostability of a polypeptide is improved by more than 5%, such as 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or even higher, compared to the thermostability of another polypeptide.

[0077] Variant: The term "variant" means a polypeptide having endo-1,3-α-glucanase activity, comprising an artificial mutation (i.e., substitution, insertion (including extension) and / or deletion (e.g., truncation)) at one or more positions. Substitution means replacing the amino acid occupying a certain position with a different amino acid; deletion means removing the amino acid occupying a certain position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0078] Wild-type: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature. In contrast, 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 a wild-type sequence).

[0079] Sequence review

[0080] SEQ ID NO: 1 is a DNA encoding a mutanase obtained from Humicola insolens.

[0081] SEQ ID NO: 2 is the complete Humicola insolens polysaccharidase (including the native signal peptide).

[0082] SEQ ID NO: 3 is the mature Humicola insolens mutans glycanase.

[0083] SEQ ID NO: 4 is a DNA sequence encoding a mutanase obtained from Trichoderma reesei.

[0084] SEQ ID NO: 5 is the complete Trichoderma reesei mutanoglycanase (including the native signal peptide).

[0085] SEQ ID NO: 6 is the mature Trichoderma reesei mutanoglycanase.

[0086] SEQ ID NO: 7 is a DNA sequence encoding a mutanase obtained from Talaromyces bacillisporus.

[0087] SEQ ID NO: 8 is the complete Talaromyces baculoides mutanase (including the native signal peptide).

[0088] SEQ ID NO:9 is the mature Talaromyces baculoides mutansase.

[0089] SEQ ID NO: 10 is a DNA sequence encoding a mutanase obtained from Talaromyces apiculatus.

[0090] SEQ ID NO: 11 is the complete T. acutus mutanase (including the native signal peptide).

[0091] SEQ ID NO: 12 is the mature T. acutus mutansase.

[0092] SEQ ID NO: 13 is a DNA sequence encoding a mutanase obtained from Streptomyces yanii.

[0093] SEQ ID NO: 14 is the complete Streptomyces yankeii mutanase (including the native signal peptide).

[0094] SEQ ID NO: 15 is the mature S. yankeii mutaglycanase.

[0095] SEQ ID NO: 16 is a DNA sequence encoding a mutanase obtained from Streptomyces alni.

[0096] SEQ ID NO: 17 is the complete S. alnus mutanase (including the native signal peptide).

[0097] SEQ ID NO: 18 is the mature Streptomyces alnus mutanase.

[0098] SEQ ID NO: 19 is a DNA sequence encoding a mutanase obtained from Oribacterium sinus.

[0099] SEQ ID NO: 20 is the complete S. sinusostomatida mutansase (including the native signal peptide).

[0100] SEQ ID NO:21 is the mature S. sinusostomatida mutansase.

[0101] SEQ ID NO: 22 is a DNA sequence encoding a mutanase obtained from Streptomyces galbus.

[0102] SEQ ID NO: 23 is the complete Streptomyces flavonoids mutanase (including the native signal peptide).

[0103] SEQ ID NO:24 is the mature Streptomyces flavonoids mutase.

[0104] SEQ ID NO: 25 is a DNA sequence encoding a mutanase obtained from Streptomyces glomeratus.

[0105] SEQ ID NO:26 is the complete S. glomerans mutanase (including the native signal peptide).

[0106] SEQ ID NO:27 is the mature S. glomerata mutanase.

[0107] SEQ ID NO:28 is the DNA sequence encoding the mutanase obtained from Tetrasphaera sp. 0185.

[0108] SEQ ID NO:29 is the complete Tetragenus sp. 0185 mutanase (including the native signal peptide).

[0109] SEQ ID NO:30 is the mature Tetragenus sp. 0185 mutanase.

[0110] SEQ ID NO: 31 is a DNA sequence encoding a mutanase obtained from Kitasatospora phosalacinea.

[0111] SEQ ID NO: 32 is the complete mitoflocustine Kitasatospora mutanase (including the native signal peptide).

[0112] SEQ ID NO:33 is the mature mitoflocustine Kitasatospora mutanoglycanase.

[0113] SEQ ID NO: 34 is the Bacillus clausii signal peptide (BcSP).

[0114] SEQ ID NO:35 is HHHHHHPR His tag.

[0115] SEQ ID NO:36 is the mature Trichoderma harzianum variant glycanase. DETAILED DESCRIPTION

[0116] Polypeptide having endo-1,3-α-glucanase activity

[0117] The present invention relates to a polypeptide having endo-1,3-α-glucanase activity.

[0118] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0119] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 2;

[0120] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 3;

[0121] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 2;

[0122] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence;

[0123] (e) a polypeptide derived from SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 2, or SEQ ID NO: 3 by substitution, deletion or addition of one or more amino acids;

[0124] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0125] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0126] The polypeptide has endo-1,3-α-glucanase activity.

[0127] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:2 or the mature polypeptide of SEQ ID NO:2.

[0128] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:3.

[0129] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof.

[0130] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:3.

[0131] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0132] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO: 1, or its cDNA sequence.

[0133] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 87 and 142 to 1389 of SEQ ID NO:1, in particular nucleotides 61 to 87 and 142 to 1389 of SEQ ID NO:1.

[0134] In another aspect, the polypeptide is derived from SEQ ID NO:2 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:2 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:3 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:3 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:3 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0135] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0136] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0137] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0138] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0139] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0140] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0141] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0142] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0143] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0144] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0145] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0146] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0147] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0148] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0149] The polypeptide may be a fusion polypeptide.

[0150] In one aspect, the polypeptide is isolated.

[0151] In another aspect, the polypeptide is purified.

[0152] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0153] (a) a polypeptide having at least 90% sequence identity to SEQ ID NO:5;

[0154] (b) a polypeptide having at least 90% sequence identity to SEQ ID NO:6;

[0155] (c) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 5;

[0156] (d) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 4 or its cDNA sequence;

[0157] (e) derived from SEQ ID NO:5, the mature polypeptide of SEQ ID NO:5, or the polypeptide of SEQ ID NO:6 by substitution, deletion or addition of one or more amino acids;

[0158] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0159] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0160] The polypeptide has endo-1,3-α-glucanase activity.

[0161] In one aspect, the polypeptide has at least 90%, e.g., 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 to SEQ ID NO:5 or the mature polypeptide of SEQ ID NO:5.

[0162] In another aspect, the polypeptide has at least 90%, e.g., 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 to SEQ ID NO:6.

[0163] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 5 or the mature polypeptide thereof.

[0164] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:6.

[0165] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0166] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 90%, e.g., 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 to the mature polypeptide coding sequence of SEQ ID NO:4, or its cDNA sequence.

[0167] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 150, 219 to 635, 705 to 1825 and 1883 to 2036 of SEQ ID NO:4, in particular nucleotides 73 to 150, 219 to 635, 705 to 1825 and 1883 to 2036 of SEQ ID NO:4.

[0168] In another aspect, the polypeptide is derived from SEQ ID NO:5 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:5 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:6 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:6 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:6 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0169] In one aspect, the polypeptide is a variant of SEQ ID NO: 6 having endo-1,3-α-glucanase activity and comprising the following amino acid residues: Thr at position 31 and Met at position 323, wherein the variant has at least 90%, e.g., 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%, but not 100% sequence identity to SEQ ID NO: 6, and wherein the position numbering is based on the numbering of SEQ ID NO: 6. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 6 with Thr at position 31 and Met at position 323.

[0170] For the purposes of the present invention, the following nomenclature is used to describe amino acid substitutions: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 323 by methionine is represented as "Thr323Met" or "T323M".

[0171] Thus, in another aspect, the polypeptide is a variant of SEQ ID NO: 6 having endo-1,3-α-glucanase activity and comprising substitutions X31T (e.g., I31T) and X323M (e.g., T323M), wherein the variant has at least 90%, e.g., 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%, but not 100% sequence identity to SEQ ID NO: 6, and wherein the position numbering is based on the numbering of SEQ ID NO: 6. In one embodiment, the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 6 having I31T and T323M substitutions.

[0172] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0173] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0174] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0175] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0176] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0177] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0178] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0179] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0180] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0181] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0182] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0183] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0184] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0185] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0186] The polypeptide may be a fusion polypeptide.

[0187] In one aspect, the polypeptide is isolated.

[0188] In another aspect, the polypeptide is purified.

[0189] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0190] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 8;

[0191] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO:9;

[0192] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 8;

[0193] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 7 or its cDNA sequence;

[0194] (e) derived from SEQ ID NO:8, the mature polypeptide of SEQ ID NO:8, or the polypeptide of SEQ ID NO:9 by substitution, deletion or addition of one or more amino acids;

[0195] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0196] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0197] The polypeptide has endo-1,3-α-glucanase activity.

[0198] In one aspect, the polypeptide has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:8 or the mature polypeptide of SEQ ID NO:8.

[0199] In another aspect, the polypeptide has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:9.

[0200] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof.

[0201] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:9.

[0202] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0203] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO:7, or its cDNA sequence.

[0204] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 129, 196 to 727, 782 to 1007, 1063 to 1520 and 1582 to 2102 of SEQ ID NO: 7, in particular nucleotides 61 to 129, 196 to 727, 782 to 1007, 1063 to 1520 and 1582 to 2102 of SEQ ID NO: 7.

[0205] In another aspect, the polypeptide is derived from SEQ ID NO:8 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:8 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:9 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:9 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:9 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0206] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0207] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0208] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0209] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0210] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0211] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0212] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0213] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0214] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0215] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0216] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0217] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0218] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0219] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0220] The polypeptide may be a fusion polypeptide.

[0221] In one aspect, the polypeptide is isolated.

[0222] In another aspect, the polypeptide is purified.

[0223] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0224] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 11;

[0225] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 12;

[0226] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 11;

[0227] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 10 or its cDNA sequence;

[0228] (e) derived from SEQ ID NO: 11, the mature polypeptide of SEQ ID NO: 11, or the polypeptide of SEQ ID NO: 12 by substitution, deletion or addition of one or more amino acids;

[0229] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0230] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0231] The polypeptide has endo-1,3-α-glucanase activity.

[0232] In one aspect, the polypeptide has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 11 or the mature polypeptide of SEQ ID NO: 11.

[0233] In another aspect, the polypeptide has at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:12.

[0234] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 11 or the mature polypeptide thereof.

[0235] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:12.

[0236] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0237] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO: 10, or its cDNA sequence.

[0238] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 129, 190 to 721, 770 to 995, 1051 to 1511 and 1573 to 2111 of SEQ ID NO: 10, in particular nucleotides 61 to 129, 196 to 727, 782 to 1007, 1063 to 1520 and 1582 to 2102 of SEQ ID NO: 10.

[0239] In another aspect, the polypeptide is derived from SEQ ID NO: 11 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 11 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO: 12 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 12 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 9 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0240] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0241] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0242] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0243] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0244] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0245] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0246] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0247] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0248] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0249] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0250] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0251] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0252] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0253] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0254] The polypeptide may be a fusion polypeptide.

[0255] In one aspect, the polypeptide is isolated.

[0256] In another aspect, the polypeptide is purified.

[0257] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0258] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 14;

[0259] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 15;

[0260] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 14;

[0261] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13;

[0262] (e) derived from SEQ ID NO: 14, the mature polypeptide of SEQ ID NO: 14, or the polypeptide of SEQ ID NO: 15 by substitution, deletion or addition of one or more amino acids;

[0263] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0264] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0265] The polypeptide has endo-1,3-α-glucanase activity.

[0266] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 14 or the mature polypeptide of SEQ ID NO: 14.

[0267] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:15.

[0268] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 14 or the mature polypeptide thereof.

[0269] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:15.

[0270] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0271] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO: 13.

[0272] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 3352 of SEQ ID NO:13, in particular nucleotides 124 to 3352 of SEQ ID NO:13.

[0273] In another aspect, the polypeptide is derived from SEQ ID NO: 14 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 14 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO: 3 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 15 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 15 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 may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0274] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0275] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0276] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0277] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0278] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0279] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0280] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0281] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0282] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0283] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0284] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0285] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0286] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0287] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0288] The polypeptide may be a fusion polypeptide.

[0289] In one aspect, the polypeptide is isolated.

[0290] In another aspect, the polypeptide is purified.

[0291] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0292] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 17;

[0293] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 18;

[0294] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 17;

[0295] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 16;

[0296] (e) derived from SEQ ID NO: 17, the mature polypeptide of SEQ ID NO: 17, or the polypeptide of SEQ ID NO: 18 by substitution, deletion or addition of one or more amino acids;

[0297] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0298] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0299] The polypeptide has endo-1,3-α-glucanase activity.

[0300] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 17 or the mature polypeptide of SEQ ID NO: 17.

[0301] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:18.

[0302] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 17 or the mature polypeptide thereof.

[0303] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:18.

[0304] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0305] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO: 16.

[0306] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2391 of SEQ ID NO:16, in particular nucleotides 76 to 2391 of SEQ ID NO:16.

[0307] In another aspect, the polypeptide is derived from SEQ ID NO: 17 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO: 17 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO: 18 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO: 18 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO: 18 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0308] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0309] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0310] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0311] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0312] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0313] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0314] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0315] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0316] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0317] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0318] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0319] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0320] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0321] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0322] The polypeptide may be a fusion polypeptide.

[0323] In one aspect, the polypeptide is isolated.

[0324] In another aspect, the polypeptide is purified.

[0325] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0326] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 20;

[0327] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 21;

[0328] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 20;

[0329] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 19;

[0330] (e) derived from SEQ ID NO:20, the mature polypeptide of SEQ ID NO:20, or the polypeptide of SEQ ID NO:21 by substitution, deletion or addition of one or more amino acids;

[0331] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0332] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0333] The polypeptide has endo-1,3-α-glucanase activity.

[0334] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 20 or the mature polypeptide of SEQ ID NO: 20.

[0335] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:21.

[0336] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 20 or the mature polypeptide thereof.

[0337] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:21.

[0338] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0339] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO: 19.

[0340] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2226 of SEQ ID NO:19, in particular nucleotides 79 to 2226 of SEQ ID NO:19.

[0341] In another aspect, the polypeptide is derived from SEQ ID NO:20 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:20 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:21 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:21 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:21 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0342] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0343] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0344] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0345] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0346] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0347] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0348] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0349] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0350] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0351] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0352] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0353] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0354] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0355] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0356] The polypeptide may be a fusion polypeptide.

[0357] In one aspect, the polypeptide is isolated.

[0358] In another aspect, the polypeptide is purified.

[0359] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0360] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 23;

[0361] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 24;

[0362] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 23;

[0363] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 22;

[0364] (e) derived from SEQ ID NO:23, the mature polypeptide of SEQ ID NO:23, or the polypeptide of SEQ ID NO:24 by substitution, deletion or addition of one or more amino acids;

[0365] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0366] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0367] The polypeptide has endo-1,3-α-glucanase activity.

[0368] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:23 or the mature polypeptide of SEQ ID NO:23.

[0369] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:24.

[0370] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 23 or the mature polypeptide thereof.

[0371] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:24.

[0372] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0373] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO:22.

[0374] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2310 of SEQ ID NO:22, in particular nucleotides 79 to 2310 of SEQ ID NO:22.

[0375] In another aspect, the polypeptide is derived from SEQ ID NO:23 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:23 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:24 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:24 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:24 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0376] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0377] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0378] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0379] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0380] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0381] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0382] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0383] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0384] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0385] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0386] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0387] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0388] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0389] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0390] The polypeptide may be a fusion polypeptide.

[0391] In one aspect, the polypeptide is isolated.

[0392] In another aspect, the polypeptide is purified.

[0393] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0394] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 26;

[0395] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 27;

[0396] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 26;

[0397] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 25;

[0398] (e) derived from SEQ ID NO:26, the mature polypeptide of SEQ ID NO:26, or the polypeptide of SEQ ID NO:27 by substitution, deletion or addition of one or more amino acids;

[0399] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0400] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0401] The polypeptide has endo-1,3-α-glucanase activity.

[0402] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:26 or the mature polypeptide of SEQ ID NO:26.

[0403] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:27.

[0404] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 29 or the mature polypeptide thereof.

[0405] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:27.

[0406] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0407] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO:25.

[0408] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2184 of SEQ ID NO:25, in particular nucleotides 115 to 2184 of SEQ ID NO:25.

[0409] In another aspect, the polypeptide is derived from SEQ ID NO:26 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:26 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:27 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:27 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:27 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0410] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0411] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0412] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0413] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0414] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0415] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0416] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0417] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0418] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0419] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0420] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0421] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0422] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0423] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0424] The polypeptide may be a fusion polypeptide.

[0425] In one aspect, the polypeptide is isolated.

[0426] In another aspect, the polypeptide is purified.

[0427] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0428] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 29;

[0429] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO:30;

[0430] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO: 29;

[0431] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 28;

[0432] (e) derived from SEQ ID NO:29, the mature polypeptide of SEQ ID NO:29, or the polypeptide of SEQ ID NO:30 by substitution, deletion or addition of one or more amino acids;

[0433] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0434] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0435] The polypeptide has endo-1,3-α-glucanase activity.

[0436] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 29 or the mature polypeptide of SEQ ID NO: 29.

[0437] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:30.

[0438] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 29 or the mature polypeptide thereof.

[0439] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:30.

[0440] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0441] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO:28.

[0442] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2028 of SEQ ID NO:28, in particular nucleotides 133 to 2028 of SEQ ID NO:28.

[0443] In another aspect, the polypeptide is derived from SEQ ID NO:29 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:29 by substitution, deletion or addition of one or several amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:30 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:30 comprising substitutions, deletions and / or insertions at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:30 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0444] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0445] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0446] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0447] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0448] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0449] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0450] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0451] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0452] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0453] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0454] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0455] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0456] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0457] In one embodiment, the polypeptide exhibits endo-1,3-α-glucanase activity at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8. In a preferred embodiment, the polypeptide has improved endo-1,3-α-glucanase activity compared to SEQ ID NO: 36 at pH 5-10, preferably at pH 6-9, and most preferably at pH 7-8 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 5 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 6 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-α-glucanase activity at pH 7 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 8 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 9 compared to SEQ ID NO: 36 when determined according to Example 7 herein. In one embodiment, the polypeptide has improved endo-1,3-alpha-glucanase activity at pH 10 compared to SEQ ID NO: 36 when determined according to Example 7 herein.

[0458] The polypeptide may be a fusion polypeptide.

[0459] In one aspect, the polypeptide is isolated.

[0460] In another aspect, the polypeptide is purified.

[0461] In one aspect, the present invention relates to polypeptides having endo-1,3-α-glucanase activity selected from the group consisting of:

[0462] (a) a polypeptide having at least 60% sequence identity to SEQ ID NO:32;

[0463] (b) a polypeptide having at least 60% sequence identity to SEQ ID NO: 33;

[0464] (c) a polypeptide having at least 60% sequence identity to the mature polypeptide of SEQ ID NO:32;

[0465] (d) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 31;

[0466] (e) derived from SEQ ID NO:32, the mature polypeptide of SEQ ID NO:32, or the polypeptide of SEQ ID NO:33 by substitution, deletion or addition of one or more amino acids;

[0467] (f) a polypeptide derived from the polypeptide of (a), (b), (c), (d) or (e), wherein the N-terminus and / or the C-terminus has been extended by the addition of one or more amino acids; and

[0468] (g) a fragment of the polypeptide of (a), (b), (c), (d) or (e);

[0469] The polypeptide has endo-1,3-α-glucanase activity.

[0470] In one aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:32 or the mature polypeptide of SEQ ID NO:32.

[0471] In another aspect, the polypeptide has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:33.

[0472] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32 or the mature polypeptide thereof.

[0473] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:33.

[0474] The polypeptide may have an N-terminal and / or C-terminal extension of one or more amino acids, for example 1-5 amino acids.

[0475] In some embodiments, the polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to the mature polypeptide coding sequence of SEQ ID NO:31.

[0476] The polynucleotide encoding the polypeptide preferably comprises, consists essentially of, or consists of nucleotides 1 to 2436 of SEQ ID NO:31, in particular nucleotides 115 to 2436 of SEQ ID NO:31.

[0477] In another aspect, the polypeptide is derived from SEQ ID NO:32 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from the mature polypeptide of SEQ ID NO:32 by substitution, deletion or addition of one or more amino acids. In another aspect, the polypeptide is derived from SEQ ID NO:33 by substitution, deletion or addition of one or more amino acids. In some embodiments, the polypeptide is a variant of SEQ ID NO:33 comprising substitution, deletion and / or insertion at one or more positions. In one aspect, the number of amino acid substitutions, deletions and / or insertions introduced into the polypeptide of SEQ ID NO:33 is up to 15, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0478] In one embodiment, the polypeptide has improved thermostability compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 26 at about pH 3 to about pH 9, more preferably at about pH 4 to about pH 8, most preferably at about pH 5 to about pH 7. Preferably, the polypeptide has improved thermostability compared to SEQ ID NO: 36 at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, when determined according to Example 4 herein.

[0479] In one embodiment, the polypeptide has comparable or improved thermal stability compared to SEQ ID NO: 36 in the presence of at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten oral care ingredients, wherein the oral care ingredient is selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol. Preferably, when determined according to Example 5 herein, at about pH 5 and / or about pH 6, most preferably at pH 5 or pH 6, in the presence of at least one oral care ingredient, the polypeptide has improved thermal stability compared to SEQ ID NO: 36.

[0480] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of benzoate (e.g., sodium benzoate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% benzoate, more preferably 0.05%-2.5% benzoate, even more preferably 0.1%-1% benzoate, most preferably 0.1%-0.5% benzoate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM benzoate, more preferably 5-50 mM benzoate, most preferably 10-35 mM benzoate.

[0481] In one embodiment, the polypeptide has improved thermostability at pH 5 and / or pH 6 in the presence of EDTA when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the mutanase has improved thermostability in the presence of 0.1-10 mM EDTA, more preferably 0.5-5 mM EDTA, most preferably 1 mM EDTA.

[0482] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of ethanol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-20% ethanol, more preferably 1%-10% ethanol, even more preferably 2.5%-7.5% ethanol, most preferably 5% ethanol. Preferably, the polypeptide has improved thermal stability in the presence of 1-100000mM ethanol, more preferably 100-10000mM ethanol, most preferably 1000mM ethanol.

[0483] In one embodiment, in the presence of glycerol, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1%-50% glycerol, more preferably 5%-40% glycerol, most preferably 10%-30% glycerol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM glycerol, more preferably 500-5000 mM glycerol, even more preferably 750-4000 mM glycerol, most preferably 1000-3250 mM glycerol.

[0484] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride or stannous fluoride) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-5000ppm fluoride, more preferably 500-2500ppm fluoride, most preferably 1000-1500ppm fluoride. Preferably, the polypeptide has improved thermal stability in the presence of 1-100mM fluoride, more preferably 5-75mM fluoride, even more preferably 10-50mM fluoride, most preferably 20-40mM fluoride.

[0485] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of a peroxide (e.g., hydrogen peroxide) when determined according to Example 5 herein compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM peroxide, more preferably 50-750 mM peroxide, most preferably 100-500 mM peroxide.

[0486] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 in the presence of mannitol, when determined according to Example 5 herein, compared to SEQ ID NO: 36. Preferably, the polypeptide has improved thermal stability in the presence of 1-1000 mM mannitol, more preferably 150-750 mM mannitol, most preferably 250-550 mM mannitol.

[0487] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of phosphate (e.g., sodium phosphate or potassium phosphate), when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 1-50 mM phosphate, more preferably 2.5-25 mM phosphate, even more preferably 5-10 mM phosphate.

[0488] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbate (e.g., sodium sorbate, potassium sorbate or calcium sorbate) when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.01%-5% sorbate, more preferably 0.05%-2.5% sorbate, even more preferably 0.1%-1% sorbate, most preferably 0.1%-0.5% sorbate. Preferably, the polypeptide has improved thermal stability in the presence of 1-100 mM sorbate, more preferably 5-75 mM sorbate, even more preferably 7.5-50 mM sorbate, most preferably 10-35 mM sorbate.

[0489] In one embodiment, the polypeptide has improved thermal stability at pH 5 and / or pH 6 compared to SEQ ID NO: 36 in the presence of sorbitol when determined according to Example 5 herein. Preferably, the polypeptide has improved thermal stability in the presence of 0.1%-70% sorbitol, more preferably 1%-60% sorbitol, even more preferably 5%-50% sorbitol, most preferably 10%-40% sorbitol. Preferably, the polypeptide has improved thermal stability in the presence of 100-10000 mM sorbitol, more preferably 250-5000 mM sorbitol, even more preferably 500-2500 mM sorbitol, most preferably 550-2200 mM sorbitol.

[0490] In one embodiment, the polypeptide provides comparable or improved, preferably improved biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein. In one embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, about 10 ppm and / or about 60 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 1 ppm, preferably 1 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 10 ppm, preferably 10 ppm. In a preferred embodiment, the polypeptide provides comparable or improved biofilm prevention at about 60 ppm, preferably 60 ppm. In preferred embodiments, the polypeptide provides comparable or improved biofilm prevention compared to SEQ ID NO: 36 at at least 1 ppm, e.g., at least 2 ppm, at least 3 ppm, at least 4 ppm, 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 40 ppm, at least 50 ppm, at least 60 ppm, at least 70 ppm, at least 80 ppm, at least 90 ppm, at least 100 ppm or more when determined according to Example 6 herein.

[0491] The polypeptide may be a fusion polypeptide.

[0492] In one aspect, the polypeptide is isolated.

[0493] In another aspect, the polypeptide is purified.

[0494] Essential amino acids in any polypeptide of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for endo-1,3-α-glucanase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides, and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins from a common ancestor (typically having similar three-dimensional structures, functions, and significant sequence similarity) within a family of polypeptides or proteins. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596: 583-589.

[0495] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0496] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0497] Sources of polypeptides having endo-1,3-α-glucanase activity

[0498] The polypeptides having endo-1,3-α-glucanase activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present 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 the source or by a strain into which the polynucleotide of the present invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted outside the cell.

[0499] In one aspect, the polypeptide is a polypeptide obtained from Humicola insolens.

[0500] In one aspect, the polypeptide is a polypeptide obtained from Trichoderma reesei.

[0501] In one aspect, the polypeptide is a polypeptide obtained from Talaromyces baculosporus.

[0502] In one aspect, the polypeptide is a polypeptide obtained from Talaromyces acutus.

[0503] In one aspect, the polypeptide is a polypeptide obtained from Streptomyces yankeii.

[0504] In one aspect, the polypeptide is a polypeptide obtained from Streptomyces alnus.

[0505] In one aspect, the polypeptide is a polypeptide obtained from Bacillus sinusostomatus.

[0506] In one aspect, the polypeptide is a polypeptide obtained from Streptomyces fulvum.

[0507] In one aspect, the polypeptide is a polypeptide obtained from Streptomyces globulus.

[0508] In one aspect, the polypeptide is a polypeptide obtained from Tetragenus sp. 0185.

[0509] In one aspect, the polypeptide is a polypeptide obtained from Bacillus kitasatosporium.

[0510] It should be understood that for the aforementioned species, the present invention encompasses the perfect and imperfect stages and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0511] The above-mentioned probe can be used from other sources, including microorganisms separated from nature (e.g., soil, compost, water, etc.) or DNA samples directly obtained from natural materials (e.g., soil, compost, water, etc.) to identify and obtain polypeptides. The technology for directly separating microorganisms and DNA from natural habitats is well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or the DNA sample mixed. Once the polynucleotide encoding the polypeptide has been detected with a probe, the polynucleotide can be separated or cloned by utilizing technology known to those of ordinary skill in the art (see, for example, Davis et al., 2012, Basic Methods in Molecular Biology [Basic Methods of Molecular Biology], Elsevier [Elsevier]).

[0512] Polynucleotide

[0513] The present invention also relates to polynucleotides encoding the polypeptides of the present invention, as described herein.

[0514] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA or a combination thereof. The polynucleotide can be cloned from a strain of Humicola, Trichoderma, Talaromyces, Streptomyces, Oribacterium, Tetrasphaera, Kitasatosporium or a related organism, and thus, for example, can be a polynucleotide sequence encoding a variant of a polypeptide of the present invention.

[0515] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Humicola cell, preferably a Humicola insolens cell.

[0516] In one embodiment, a polynucleotide encoding a polypeptide of the present invention is isolated from a Trichoderma cell, preferably a Trichoderma reesei cell.

[0517] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Talaromyces cell, preferably a Talaromyces bacillus cell or a Talaromyces acutus cell.

[0518] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Streptomyces cell, preferably a Streptomyces yankeii cell, a Streptomyces alnus cell, a Streptomyces flavos cell or a Streptomyces globulus cell.

[0519] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from an Oralbacterium cell, preferably an Oralbacterium sinusostomatum cell.

[0520] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Tetragenus cell, preferably a Tetragenus sp. 0185 cell.

[0521] In one embodiment, the polynucleotide encoding the polypeptide of the present invention is isolated from a Kitasatospora cell, preferably a phycotoxins Kitasatospora cell.

[0522] These polynucleotides can also be constructed by introducing nucleotide substitutions that do not result in changes in the amino acid sequence of the polypeptide, but correspond to the codon usage of the host organism intended for producing 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 2:95-107.

[0523] In one aspect, the polynucleotide is isolated.

[0524] In another aspect, the polynucleotide is purified.

[0525] Nucleic acid construct

[0526] The present invention also relates to a nucleic acid construct comprising a polynucleotide of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0527] Polynucleotides can be manipulated in a variety of ways to provide expression of a polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0528] Promoter

[0529] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0530] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, supra, and Song et al., 2016, PLOS One 11(7):e0158447.

[0531] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and Schmoll et al., 2014, “Trichoderma: Biology and Applications”. 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0532] For expression in yeast hosts, examples of useful promoters are described by 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 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). 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0533] Terminator

[0534] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell can be used in the present invention.

[0535] Preferred terminators for bacterial host cells can be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0536] Preferred terminators for filamentous fungal host cells are obtainable from Aspergillus or Trichoderma species, such as from the genes for Aspergillus niger glucoamylase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as those described in Mukherjee et al., 2013, “Trichoderma: Biology and Applications” and in Schmoll et al., 2014. 2016. “Gene Expression Systems in Fungi: Advancements and Applications.” Fungal Biology.

[0537] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0538] mRNA Stabilizer

[0539] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0540] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0541] Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0542] Preamble sequence

[0543] The control sequence may also be a leader sequence, i.e., an untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell may be used.

[0544] Suitable leader sequences for bacterial host cells are provided by Hambraeus et al., 2000, Microbiology 146(12):3051-3059 and Kaberdin and 2006, FEMS Microbiol. Rev. [FEMS Microbiology Review] 30(6): 967-979 description.

[0545] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0546] Suitable leaders for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0547] polyadenylation sequence

[0548] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide which, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0549] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0550] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0551] Signal peptide

[0552] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the polypeptide and directs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the encoded polypeptide in the translation open reading frame. Alternatively, the 5' end of the coding sequence can contain a signal peptide coding sequence that is heterologous to the coding sequence. In the case 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 can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. Any signal peptide coding sequence that directs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0553] Effective signal peptide coding sequences for bacterial host cells are those obtained from genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Other signal peptides are described by Freudl, 2018, Microbial Cell Factories 17:52.

[0554] An effective signal peptide coding sequence for a filamentous fungal host cell is a signal peptide coding sequence obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0555] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0556] Propeptide

[0557] The control sequence can also be a propeptide coding sequence encoding a propeptide at the N-terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are generally inactive and can be converted into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the genes of the following items: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease and Saccharomyces cerevisiae α-factor.

[0558] In the case where both the signal peptide sequence and the propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence. Additionally or alternatively, when both the signal peptide sequence and the propeptide sequence are present, the polypeptide may comprise only a portion of the signal peptide sequence and / or only a portion of the propeptide sequence. Alternatively, the final or separated polypeptide may comprise a mixture of a mature polypeptide and a polypeptide comprising a partial or full-length propeptide sequence and / or a signal peptide sequence.

[0559] Adjustment sequence

[0560] It may also be desirable to add regulatory sequences that regulate the expression of polypeptides associated with host cell growth. Examples of regulatory sequences are regulatory sequences that cause gene expression to open or close in response to chemical or physical stimuli (including the presence of regulatory compounds). Regulatory sequences in prokaryotic systems include lac, tac, and trp operon systems. In yeast, ADH2 systems or GAL1 systems may be used. In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter and Trichoderma reesei cellobiohydrolase II promoter may 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.

[0561] Transcription Factors

[0562] The control sequence can also be a transcription factor, i.e. a polynucleotide encoding a polynucleotide-specific DNA binding polypeptide, which controls the transcription rate of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can play a role 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 comprising at least one DNA binding domain, which is usually attached to a specific DNA sequence adjacent to a genetic element regulated by a transcription factor. Transcription factors can directly (i.e., activate the transcription of a gene encoding a protein of interest by binding to its promoter) or indirectly (i.e., such as by binding to a promoter of another transcription factor (which regulates the transcription of a gene encoding a protein of interest) to activate the transcription of another transcription factor) regulate the expression of a protein 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 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0563] Expression vector

[0564] The present invention further relates to a recombinant expression vector comprising a polynucleotide of the present invention, a promoter and a transcription and translation termination signal. A plurality of nucleotides and control sequences can be linked together to produce a recombinant expression vector, and the recombinant expression vector can include one or more convenient restriction sites to allow the insertion or replacement of the polynucleotide encoding a polypeptide at such a site. Alternatively, the polynucleotide can be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing an expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to the suitable control sequence for expression.

[0565] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0566] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicates with the chromosome into which it has been integrated when it is introduced into the host cell. Moreover, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0567] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0568] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0569] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0570] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0571] More than one copy of polynucleotide of the present invention can be inserted into host cell to improve the production of polypeptide.For example, 2 or 3 or 4 or 5 or more copies are inserted into host cell.The copy number of the increase of polynucleotide can be obtained by integrating at least one other copy of sequence into host cell genome or by including amplifiable selectable marker gene together with polynucleotide, wherein the cell containing amplified copy of selectable marker gene and thus other copy of polynucleotide can be selected by cultivating cell in the presence of suitable selective agent.

[0572] Host cells

[0573] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0574] The construct or vector comprising the polynucleotide is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described earlier. The selection of the host cell will depend to a large extent on the gene encoding the polypeptide and its source. The polypeptide can be native or heterologous to the recombinant host cell. In addition, at least one of the one or more control sequences can be heterologous to the polynucleotide encoding the polypeptide. The recombinant host cell can contain a single copy or at least two copies of the polynucleotide of the present invention, such as three, four, five or more copies.

[0575] The host cell may be any microbial cell, such as a prokaryotic cell or a fungal cell, that can be used to recombinantly produce a polypeptide of the present invention.

[0576] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0577] The bacterial host cell can be any Bacillus cell, including but not limited to alkalophilic Bacillus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cell.

[0578] For the purposes of the present invention, the Bacillus genus / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0579] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equi, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0580] The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0581] 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, transduction, wherein DNA is introduced as linearized or circular polynucleotides. Those skilled in the art will be able to easily determine the appropriate method for introducing DNA into a given prokaryotic cell according to, for example, the 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] 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods [Microbiological Methods Magazine] 64: 391-397 and Donald et al., 2013, J. Bacteriol. [Bacteriology Magazine] 195 (11): 2612-2620.

[0582] The host cell may be a fungal cell. "Fungi" as used herein includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitotic spore fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0583] Fungal cells can be transformed by processes involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, gene gun methods, and shock wave-mediated transformation (reviewed in Li et al., 2017, Microbial Cell Factories [Microbial Cell Factories] 16: 168) and EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474; Christensen et al., 1988, Bio / Technology [Biotechnology] 6: 1419-1422 and Lubertozzi and Keasling, 2009, Biotechn. Advances [Biotechnological Progress] 27: 53-75. However, any method known in the art for introducing DNA into fungal host cells may be used, and the DNA may be introduced as a linearized or circular polynucleotide.

[0584] The fungal host cell can be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). For the purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0585] The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In preferred embodiments, the yeast host cell is a Pichia or Komagataella cell, such as a Pichia pastoris cell (Komagataella phaffii).

[0586] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphylum of Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The characteristics of filamentous fungi are generally mycelial walls composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is carried out by hyphae extension, and carbon catabolism is obligate aerobic. In contrast, the vegetative growth of yeast (such as Saccharomyces cerevisiae) is carried out by budding of unicellular thallus, and carbon catabolism can be fermentative.

[0587] The filamentous fungal host cell can be Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, In some embodiments, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In other preferred embodiments, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium. In other preferred embodiments, the filamentous fungal host cell is a cell of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum.

[0588] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinosa, Ceriporiopsis spp. keratinophilum), Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicalum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi), Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium elderberrysambucinum), Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosus, Rhizomucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0589] In one aspect, the host cell is isolated.

[0590] In another aspect, the host cell is purified.

[0591] Generation method

[0592] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell under conditions conducive for production of the polypeptide, the cell producing the polypeptide in its wild-type form; and optionally (b) recovering the polypeptide.

[0593] In one aspect, the cell is a Humicola cell, preferably a Humicola insolens cell.

[0594] In one aspect, the cell is a Trichoderma cell, preferably a Trichoderma reesei cell.

[0595] In one aspect, the cell is a Tallum cell, preferably a Tallum baccata cell or a Tallum acutum cell.

[0596] In one aspect, the cell is a Streptomyces cell, preferably a Streptomyces yankeii cell, a Streptomyces alnus cell, a Streptomyces fulvus cell or a Streptomyces globulus cell.

[0597] In one aspect, the cell is an Oralbacter cell, preferably an Oralbacter cell.

[0598] In one aspect, the cell is a Tetragenus cell, preferably a Tetragenus sp. 0185 cell.

[0599] In one aspect, the cell is a Kitasatospora cell, preferably a phytomycin Kitasatospora cell.

[0600] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally (b) recovering the polypeptide.

[0601] Host cells are cultivated in a nutrient medium suitable for producing polypeptides using methods known in the art. For example, cells can be cultured in suitable medium and under conditions allowing expression and / or separation of polypeptides by shaking flask culture or in a laboratory or industrial fermentor on a small scale or in large scale fermentation (including continuous, batch, fed-batch or solid-state and / or microcarrier-based fermentation). Suitable medium can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0602] The polypeptide can be detected using methods known in the art that are specific for the polypeptide, including but not limited to assays using specific antibodies, enzyme product formation, disappearance of enzyme substrate, or determining the relative or specific activity of the polypeptide.

[0603] The polypeptide can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the whole fermentation broth containing the polypeptide is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0604] The polypeptides can be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science; 80(1):6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129:3-10).

[0605] In alternative aspects, the polypeptide is not recovered.

[0606] Mutanase Granules

[0607] The present invention also relates to enzyme granules / particles comprising the polypeptide of the present invention.In an embodiment, the granule comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0608] The diameter of the core (measured as an equivalent spherical diameter (average particle size based on volume)) may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm. The core diameter measured as an equivalent spherical diameter may be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0609] In embodiments, the core comprises a polypeptide having endo-1,3-alpha-glucanase activity of the invention.

[0610] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0611] The core may include a binder such as a synthetic polymer, wax, fat or carbohydrate.

[0612] The core may include, typically as a homogenous blend, a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposition catalyst, and / or an acidic buffer component.

[0613] The core may comprise an inert particle into which the polypeptide is adsorbed, or applied (eg by fluid bed coating) to the surface of the inert particle.

[0614] The diameter of the core may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm.

[0615] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling or for coloring the granules. Optional coatings may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA).

[0616] The coating may be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10% or at least 15%) by weight of the core. The amount may be up to 100%, 70%, 50%, 40% or 30%.

[0617] The coating is preferably at least 0.1 μm thick, in particular at least 0.5 μm, at least 1 μm or at least 5 μm thick.In some embodiments, the coating has a thickness of less than 100 μm, such as less than 60 μm or less than 40 μm.

[0618] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating with few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0619] The coating may further contain other materials as known in the art, for example fillers, anti-tack agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0620] The salt coating may comprise at least 60% by weight salt, 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% by weight.

[0621] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, for example 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.

[0622] The salt may be added from a salt solution (wherein the salt is completely dissolved), or from a salt suspension (wherein the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm).

[0623] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, particularly having a solubility of at least 0.1 g in 100 g water at 20°C, preferably at least 0.5 g / 100 g water, such as at least 1 g / 100 g water, such as at least 5 g / 100 g water.

[0624] The salt can be an inorganic salt, for example a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or a salt of a simple organic acid (less than 10 carbon atoms, for example 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions or metal ions of the first transition series, for example sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chlorine, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonate, 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 citrates, malonates or acetates may be used.

[0625] The salt in the coating may have a constant humidity of 60% or more, in particular 70% or more, 80% or more or 85% or more at 20°C, or it may be another hydrate form (e.g., anhydrous) of such a salt. The salt coating may be as described in WO00 / 01793 or WO 2006 / 034710.

[0626] A specific example of a suitable salt is NaCl (CH 20 ℃=76%)、Na 2 CO 3 (CH 20 ℃=92%)、NaNO 3 (CH 20 ℃=73%)、Na 2 HPO 4 (CH 20 ℃=95%)、Na 3 PO 4 (CH 25 ℃=92%)、NH 4 Cl(CH 20 ℃=79.5%)、(NH 4 ) 2 HPO 4 (CH 20 ℃=93,0%)、NH 4 H 2 PO 4 (CH 20 ℃=93.1%)、(NH 4 ) 2 SO 4 (CH 20℃=81.1%)、KCl(CH 20 ℃=85%)、K 2 HPO 4 (CH 20 ℃=92%)、KH 2 PO 4( CH 20 ℃=96.5%)、KNO 3 (CH 20 ℃=93.5%)、Na 2 SO 4 (CH 20 ℃=93%)、K 2 SO 4 (CH 20 ℃=98%)、KHSO 4 (CH 20 °C = 86%), MgSO 4 (CH 20 ℃=90%)、ZnSO 4 (CH 20 ℃=90%) and sodium citrate (CH 25 ℃=86%). Other examples include NaH 2 PO 4 NH 4 )H 2 PO 4 ,CuSO 4 Mg(NO 3 ) 2 and magnesium acetate.

[0627] The salt may be in anhydrous form or it may be a hydrated salt, ie a crystalline salt hydrate having one or more bound waters of crystallization, as described, for example, in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na 2 SO 4 ), anhydrous magnesium sulfate (MgSO 4 ), magnesium sulfate heptahydrate (MgSO 4 7H 2 O), zinc sulfate heptahydrate (ZnSO 4 7H 2 O), disodium hydrogen phosphate heptahydrate (Na 2 HPO 4 7H 2 O), magnesium nitrate hexahydrate (Mg(NO 3 ) 2 (6H 2 O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0628] Preferably, the salt is used as a salt solution, for example using a fluidized bed.

[0629] The coating material can be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 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 by fluidized bed technology are given in GB 1483591.

[0630] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with various coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0631] The core may be prepared by granulating a blend of ingredients, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction, drum granulation and / or high shear granulation.

[0632] Methods for preparing the cores 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:

[0633] (a) Spray-dried products, wherein a liquid polypeptide-containing solution is atomized in a spray-drying tower to form small droplets which dry as they descend along the drying tower to form a polypeptide-containing particulate material. Very small particles can be produced in this way (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0634] (b) layered products, wherein the polypeptide is coated in layers around preformed inert core particles, wherein a polypeptide solution is usually atomized in a fluidized bed apparatus, in which the preformed core particles are fluidized and the polypeptide solution is attached to the core particles and dried until the dry polypeptide layer is left on the surface of the core particles. If a useful core particle with a desired size can be found, particles with a desired size can be obtained in this way. This type of product is described in, for example, WO 97 / 23606.

[0635] (c) Absorbed core particles, wherein rather than coating the polypeptide as a layer 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.

[0636] (d) Extruded or pelletized products, in which the polypeptide-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, and the pellets are subsequently dried. Such particles are usually of considerable size, since the material in which the extrusion openings are formed (usually a flat plate with drilled holes) limits the permissible pressure drop across the extrusion openings. Furthermore, when small openings are used, very high extrusion pressures increase the heat generation in the polypeptide paste, which is detrimental to the polypeptides (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0637] (e) Spray granulated products, in which a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed (e.g. by a rotating disk atomizer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; 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. US 4,016,040 and US 4,713,245 describe this technique.

[0638] (f) mixer granulation product, wherein a polypeptide-containing liquid is added to a dry powder composition of a conventional granulation component. Liquid and powder are mixed in a suitable ratio, and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will begin to adhere and condense, and the particles will accumulate to form particles comprising polypeptides. Such methods are described in US4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In a particular aspect of this process, various high shear mixers can be used as granulators. The particles consisting of polypeptide, filler and adhesive etc. are mixed with cellulose fibers to strengthen the particles, thereby producing so-called T-particles. Strengthened particles are more solid and release less enzyme dust.

[0639] (g) Particle size reduction, wherein the core is produced by milling or crushing larger particles, pellets, platelets, briquettes, etc. containing the polypeptide. The desired core particle fraction is obtained by sieving the milled 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.

[0640] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an air stream and spraying a liquid onto the fluidized particles through a nozzle. The particles hit by the sprayed droplets are wetted and become sticky. The sticky particles collide with other particles and attach to them to form granules.

[0641] (i) The cores may be subjected to drying, for example in a fluid bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by the skilled person. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some polypeptides, it is important that the core comprising the polypeptide contains a small amount of water before coating with salt. If a water-sensitive polypeptide is coated with salt before the excess water is removed, the excess water will be trapped in the core and may negatively affect the activity of the polypeptide. After drying, the cores preferably contain 0.1-10% w / w water.

[0642] Non-dusting granules may be produced, 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.

[0643] The granule may further comprise one or more other enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases and transferases. One or more other enzymes are preferably selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, ferulic acid esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or its any combination. Then, every kind of enzyme will be present in more granules, ensuring a more uniform distribution of enzyme, and also reducing the physical separation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granules are disclosed in ip.com disclosure IPCOM000200739D.

[0644] Another example of formulating polypeptides by using co-particles is disclosed in WO 2013 / 188331.

[0645] The present invention also relates to protected polypeptides prepared according to the method disclosed in EP 238216.

[0646] Liquid preparations

[0647] The present invention also relates to a liquid composition comprising the polypeptide of the present invention. The composition may comprise an enzyme stabilizer (examples of the enzyme stabilizer 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 boric acid esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid).

[0648] In certain 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 talcum, 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 certain embodiments, these compositions contain about 5% to about 90% of such materials.

[0649] In one aspect, the liquid formulation comprises 20%-80% w / w polyol.In one embodiment, the liquid formulation comprises 0.001%-2% w / w preservative.

[0650] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0651] (A) 0.001%-25% w / w of the polypeptide having endo-1,3-α-glucanase activity of the present invention;

[0652] (B) 20%-80% w / w polyol;

[0653] (C) optionally 0.001%-2% w / w preservative; and

[0654] (D) Water.

[0655] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0656] (A) 0.001%-25% w / w of the polypeptide having endo-1,3-α-glucanase activity of the present invention;

[0657] (B) 0.001%-2% w / w preservative;

[0658] (C) optionally 20%-80% w / w polyol; and

[0659] (D) Water.

[0660] In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent 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 a formulating agent 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-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having 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.

[0661] In another 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. In one embodiment, the liquid formulation comprises 20%-80% 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, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having 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).

[0662] 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 comprises 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w preservative, 0.05%-1% w / w preservative 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.

[0663] In another embodiment, the liquid formulation further comprises one or more additional enzymes, e.g., hydrolases, isomerases, ligases, lyases, oxidoreductases and transferases. The one or more additional enzymes are preferably selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl 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.

[0664] Signal peptide

[0665] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO:2.

[0666] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 24 of SEQ ID NO:5.

[0667] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO:8.

[0668] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO:11.

[0669] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 41 of SEQ ID NO:14.

[0670] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO:17.

[0671] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 26 of SEQ ID NO:20.

[0672] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 26 of SEQ ID NO:23.

[0673] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 38 of SEQ ID NO:26.

[0674] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 24 of SEQ ID NO:29.

[0675] In one aspect, the present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 38 of SEQ ID NO:32.

[0676] These polynucleotides may further comprise a gene encoding a protein, which is operably linked to the signal peptide. The protein is preferably heterologous to the signal peptide. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 60 of SEQ ID NO:1. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 72 of SEQ ID NO:4. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 60 of SEQ ID NO:7. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 60 of SEQ ID NO:10. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 123 of SEQ ID NO:13. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 75 of SEQ ID NO:16. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 78 of SEQ ID NO:19. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 78 of SEQ ID NO:22. On the one hand, the polynucleotide encoding the signal peptide is nucleotides 1 to 114 of SEQ ID NO:25. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 132 of SEQ ID NO: 28. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 114 of SEQ ID NO:31.

[0677] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.

[0678] The present invention also relates to methods of producing a protein, comprising: (a) culturing a recombinant host cell comprising such a polynucleotide; and optionally (b) recovering the protein.

[0679] The protein may be native or heterologous to the host cell. The term "protein" herein does not refer to a specific length of the encoded product, and therefore encompasses peptides, oligopeptides and polypeptides. The term "protein" also encompasses two or more polypeptides that combine to form the encoded product. Proteins also include hybrid polypeptides and fusion polypeptides.

[0680] Preferably, the protein is a hormone, an enzyme, a receptor or a portion thereof, an antibody or a portion thereof, or a reporter gene. For example, the protein can be a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, such as α-galactosidase, α-glucosidase, an aminopeptidase, an amylase, β-galactosidase, β-glucosidase, β-xylosidase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glycosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, a glucoamylase, an invertase, a laccase, a lipase, a mannosidase, a mutanase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase.

[0681] The gene may be obtained from any prokaryotic, eukaryotic or other source.

[0682] In one aspect, the present invention relates to a polynucleotide encoding a signal peptide selected from the group consisting of:

[0683] (a) a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 2,

[0684] (b) a signal peptide comprising or consisting of amino acids 1 to 24 of SEQ ID NO: 5,

[0685] (c) a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 8,

[0686] (d) a signal peptide comprising or consisting of amino acids 1 to 20 of SEQ ID NO: 11,

[0687] (e) a signal peptide comprising or consisting of amino acids 1 to 41 of SEQ ID NO: 14,

[0688] (f) a signal peptide comprising or consisting of amino acids 1 to 25 of SEQ ID NO: 17,

[0689] (g) a signal peptide comprising or consisting of amino acids 1 to 26 of SEQ ID NO: 20,

[0690] (h) a signal peptide comprising or consisting of amino acids 1 to 26 of SEQ ID NO: 23,

[0691] (i) a signal peptide comprising or consisting of amino acids 1 to 38 of SEQ ID NO: 26,

[0692] (j) a signal peptide comprising or consisting of amino acids 1 to 24 of SEQ ID NO: 29, and

[0693] (k) a signal peptide comprising or consisting of amino acids 1 to 38 of SEQ ID NO: 32;

[0694] The polynucleotide encoding the signal peptide is operably linked to a polynucleotide encoding a polypeptide heterologous to the signal peptide.

[0695] The present invention also relates to a nucleic acid construct or an expression vector comprising a polynucleotide encoding a signal peptide of the present invention, which is operably linked to a polynucleotide encoding a polypeptide heterologous to the signal peptide. The present invention also relates to a recombinant host cell comprising the nucleic acid construct or expression vector. The present invention also relates to a method for producing a polypeptide, comprising culturing the recombinant host cell under conditions conducive to the production of the protein, and optionally recovering the polypeptide.

[0696] Oral Care Ingredients and Forms

[0697] In one aspect, the present invention also relates to an enzymatic oral care composition comprising a polypeptide of the present invention and at least one oral care ingredient.

[0698] Thus, in one aspect, the invention relates to an oral care composition comprising a mutanase selected from the group consisting of:

[0699] (a) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:3;

[0700] (b) a polypeptide having at least 90%, e.g., 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 to SEQ ID NO: 6;

[0701] (c) a polypeptide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:9;

[0702] (d) a polypeptide having at least 65%, e.g., at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 12;

[0703] (e) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 15;

[0704] (f) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO: 18;

[0705] (g) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:21;

[0706] (h) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:24;

[0707] (i) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:27;

[0708] (j) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:30; and

[0709] (k) a polypeptide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID NO:33;

[0710] wherein the polypeptide has endo-1,3-alpha-glucanase activity, and wherein the oral care composition further comprises at least one oral care ingredient.

[0711] Preferably, the mutanase is selected from the group consisting of:

[0712] (a) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 3;

[0713] (b) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:6;

[0714] (c) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:9;

[0715] (d) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 12;

[0716] (e) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 15;

[0717] (f) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 18;

[0718] (g) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:21;

[0719] (h) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:24;

[0720] (i) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:27;

[0721] (j) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:30; and

[0722] (k) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:33;

[0723] The at least one oral care ingredient may vary depending on the type of oral care composition and the characteristics and / or activities desired of the oral care composition.For purposes of the present invention, the terms "ingredient" and "component" are used interchangeably when referring to oral care compositions.

[0724] In a preferred embodiment, the at least one oral care ingredient is selected from the group consisting of abrasives, humectants, solvents, thickeners, binding agents, buffers, foaming agents, foam regulators, sweetening agents, softeners, plasticizers, flavoring agents, colorants, therapeutic agents, antimicrobial agents, tartar control agents, fluoride ion sources, preservatives, detergents, surfactants, colorants, buffers, softeners, plasticizers, whitening agents, bleaching agents, gum base components, and bulking agents.

[0725] The oral care composition of the present invention can be an internal oral care composition, such as toothpaste or toothpaste tablets, dental cream, mouthwash or mouthwash tablets, mouth rinses, lozenges, pastilles, chewing gum, sweets, candies, etc., which is designed to remove biofilms in the oral cavity, such as biofilms residing on teeth, on oral soft tissues, and on dentures residing in the oral cavity.

[0726] In one aspect, the oral care composition is a toothpaste.

[0727] In one aspect, the oral care composition is a mouthwash, a mouthwash tablet or a mouthrinse, preferably a mouthwash.

[0728] In one aspect, the oral care composition is a lozenge.

[0729] In one aspect, the oral care composition is chewing gum.

[0730] The oral care composition of the present invention may also be an external oral care composition, such as a denture cleaning liquid, denture cleaning tablet, denture cleaning powder, etc., which is designed to remove biofilm from a denture that has been removed from the oral cavity for cleaning.

[0731] In preferred embodiments, the oral care composition is an internal oral care composition and the at least one oral care ingredient is selected from the group consisting of abrasives, humectants, solvents, thickeners, binders, buffers, foaming agents, foam regulators, sweeteners, softeners, plasticizers, flavoring agents, colorants, therapeutic agents, antimicrobial agents, tartar control agents, fluoride ion sources, preservatives, detergents, surfactants, colorants, buffers, softeners, plasticizers, whitening agents, bleaching agents, gum base components, and expanders.

[0732] Although the oral care ingredients referred to herein are categorized by general headings according to functionality, this is not to be construed as limiting, as the ingredients may contain additional functionality as will be appreciated by the skilled artisan.

[0733] Toothpaste, tooth cream, mouthwash and mouth rinse

[0734] The internal oral care composition of the present invention is in the form of toothpaste or toothpaste tablets, tooth cream, mouthwash or mouthwash tablets, and mouth rinses, and these internal oral care compositions may include oral care ingredients selected from the following categories:

[0735]

[0736]

[0737] toothpaste

[0738] Toothpastes and tooth creams / tooth gels typically include abrasives, solvents, humectants, detergents / surfactants, thickeners and binders, buffers, flavoring agents, sweeteners, fluoride ion sources, therapeutic agents, enzymes, colorants, and preservatives.

[0739] In a preferred embodiment, the present invention relates to oral care compositions in the form of toothpaste or tooth cream, which oral care compositions comprise the mutanase of the present invention and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0740]

[0741] The oral care composition of the present invention may be a toothpaste comprising the following ingredients (in % by weight of the final toothpaste composition):

[0742] Abrasive: 10% to 70%

[0743] Wetting agent: 0% to 80%

[0744] Thickener: 0.1% to 20%

[0745] Binder: 0.01% to 10%

[0746] Sweeteners: 0.1% to 5%

[0747] Foaming agent: 0% to 15%

[0748] Mutanase: 0.01% to 10%

[0749] Mouthwash

[0750] Mouthwashes and mouth rinses (including plaque removing solutions) of the invention typically comprise a mutanase of the invention, a carrier fluid, a detergent / surfactant, a buffer, a flavoring agent, a humectant, a sweetener, a therapeutic agent, a fluoride ion source, a colorant, and a preservative.

[0751] In a preferred embodiment, the present invention relates to oral care compositions in the form of mouthwashes or mouth rinses, comprising the mutanase of the present invention and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from the following ingredients:

[0752]

[0753]

[0754] The oral care composition of the present invention may be a mouthwash comprising the following ingredients (in % by weight of the final mouthwash composition):

[0755] Water: 0% to 70%

[0756] Ethanol: 0% to 20%

[0757] Wetting agent: 0% to 20%

[0758] Surfactants: 0% to 2%

[0759] Mutanase: 0.01% to 10%

[0760] Other ingredients: 0% to 2% (eg, flavors, sweeteners, fluoride ion sources).

[0761] Mouthwash compositions may be buffered with a suitable buffer (eg, sodium citrate or sodium phosphate) in the pH range of 6-7.5.

[0762] Relevant oral care ingredients suitable for use in toothpastes, tooth creams, mouthwashes, and mouth rinses are described in further detail below. A skilled artisan will be able to vary the oral care ingredients depending on the type of oral care composition and the desired characteristics and / or activity of a particular oral care composition. An oral care composition need not necessarily contain all of the ingredients mentioned.

[0763] Abrasives

[0764] Abrasive and polishing materials can be incorporated into the oral care composition of the present invention. According to the present invention, the abrasive and polishing materials include aluminum oxide and its hydrates (e.g., α-aluminum trihydrate), magnesium trisilicate, magnesium carbonate, kaolin, aluminosilicates (e.g., calcined aluminum silicate and aluminum silicate), calcium carbonate, zirconium silicate, bentonite, silicon dioxide, 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 xerogel, hydrogel and aerogel, etc.

[0765] Also suitable as abrasives are calcium pyrophosphate, water-insoluble alkali metaphosphates, polymetaphosphates, dicalcium phosphate and / or its dihydrate, dicalcium orthophosphate, tricalcium phosphate, particulate hydroxyapatite, etc. It is also possible to use mixtures of these substances.

[0766] Various types of silica dental abrasives are preferred because of their unique benefits of excellent tooth cleaning and polishing performance without excessive abrasion of tooth enamel or dentin, and their good compatibility with other possible ingredients such as metal ions and fluoride.

[0767] Depending on the oral care composition, the abrasive product may be present at 0 to 70%, preferably 1 to 70% by weight.

[0768] For toothpaste, abrasive material levels typically range from 10% to 70% by weight of the final toothpaste product.

[0769] Wetting agent

[0770] Humectants are used to prevent water from escaping from, for example, toothpaste and to prevent toothpaste from becoming hard when exposed to air. Some humectants also impart a desirable sweet taste to toothpaste and mouthwash compositions. Humectants suitable for use in oral care compositions according to the present invention include the following compounds and mixtures thereof: glycerol, 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 the like.

[0771] The humectant is typically present at 0 to 80%, preferably 5 to 70% by weight.

[0772] Thickener / Binder

[0773] Suitable thickeners and / or binders include silicon dioxide, starch, tragacanth gum, xanthan gum, karaya gum, carrageenan (an extract of Irish moss), gum arabic, alginates, pectin, cellulose derivatives (e.g., hydroxyethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, and hydroxyethylpropylcellulose), 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.

[0774] Thickeners may be present in toothpastes, tooth creams and gels and mouthwashes in amounts ranging from 0.1% to 20% by weight of the final product and in adhesives ranging from 0.01% to 10% by weight of the final product.

[0775] Foaming agents and foaming regulators

[0776] As foaming agents, soaps, anionic, cationic, nonionic, amphoteric and / or zwitterionic surfactants may be used alone or in combination. These may be present at a level of 0% to 15%, preferably 0.1% to 13%, more preferably 0.25% to 10% by weight of the final product. The surfactant is only suitable to the extent that it does not inactivate the enzymes and other components contained in the oral care composition. Useful surfactants include anionic, nonionic, and amphoteric compounds, preferably anionic compounds.

[0777] 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 atom acids) with lower aliphatic amino acids, such as sodium N-methyl-N-palmitoyl taurine, sodium N-lauroyl sarcosinate, sodium N-myristoyl sarcosinate, and sodium N-palmitoyl sarcosinate; salts of esters of such fatty acids with isotopic acids or with glycerol monosulfate; such as the sodium salt of the monosulfate monoglyceride of hydrogenated coconut oil fatty acid; olefin sulfonates, such as olefin sulfonates or 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.

[0778] The cation of salt can be sodium, potassium or monoethanolamine, diethanolamine or triethanolamine.Nonionic surfactants include sucrose / fatty acid ester, maltose / fatty acid ester, maltitol / fatty acid ester, malttriol / fatty acid ester, malt tetraol / fatty acid ester, maltpentaol / fatty acid ester, malthexaol / fatty acid ester, mahoheptaitol / fatty acid ester, sorbitan / fatty acid ester, lactose / fatty acid ester, lactinose / fatty acid ester, polyoxyethylene / polyoxypropylene copolymer, polyoxyethylene alkyl ether, polyoxyethylene / fatty acid ester, fatty acid alkanolamide, polyoxyethylene sorbitan / fatty acid ester, polyoxyethylene / hydrogenated castor oil, and polyglycerol / fatty acid ester.

[0779] Most preferred are sodium lauryl sulfate, sodium dodecylbenzene sulfonate and sodium lauryl sarcosinate.

[0780] Preferred foam modulators include polyethylene glycols.

[0781] The foaming agent and foaming regulator may be present in an amount of 0 to 15%, preferably 0.01 to 10% by weight.

[0782] Sweeteners

[0783] Suitable sweeteners include, but are not limited to, saccharin and its water-soluble salts, dextrose, sucrose, lactose, maltose, levulose, aspartame, cyclamate, D-tryptophan, dihydrochalcone, acesulphame, stevioside, levaudioside, glycyrrhizin, pellartine, thaumatin, p-methoxycinnamaldehyde, hydrogenated starch hydrolysates, xylitol, sorbitol, erythritol, mannitol, and mixtures thereof.

[0784] The sweetener may be present in an amount of 0.001% to 60% by weight, preferably 0.01% to 50% by weight.

[0785] Flavoring

[0786] Flavoring agents are usually present in small amounts, such as 0.01% to about 5%, especially 0.1% to 5%, by weight. Flavors that can be used in the present 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, oxanonane, α-ionone, marjoram, lemon, orange, cranberry, propenyl ethyl guaiacol, cinnamon, vanillin, ethyl vanillin, piperonal, 4-cis-heptenal, diacetyl, methyl p-tert-butyl acetate, carvone, cineole, menthone, cinnamaldehyde, limonene, ocimene, n-decanol, citronellol, α-terpineol, methyl acetate, citronellyl acetate, methyl eugenol, linalool, thymol, rosemary oil, allspice oil, diatom oil, eucalyptus oil, and mixtures thereof.

[0787] Coolants may also be part of the flavoring system or added separately to the composition. Preferred coolants in the compositions of the present invention are p-menthane carboxyamide-based agents 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.

[0788] Brightener / bleach

[0789] Brighteners / bleaches include H 2 O 2 And can be added in an amount of less than 5%, preferably 0.05% to 4%, calculated based on the weight of the final composition.

[0790] Other bleaching components that may be included in the present invention include peroxydiphosphates, urea, peroxides, metal peroxides (e.g., calcium peroxide, sodium peroxide, strontium peroxide, magnesium peroxide), hypochlorites (e.g., sodium hypochlorite), and salts of perborates, persilicates, perphosphates and percarbonates (e.g., sodium perborate, potassium persilicate and sodium percarbonate). Peroxide compounds may be stabilized by the addition of triphenylmethane dyes, chelating agents or antioxidants (e.g., butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT)).

[0791] Solvents

[0792] The solvent is typically added to the composition of the present invention in an amount sufficient to render the composition flowable, for example, in the case of a toothpaste, tooth cream or tooth gel, or in an amount sufficient to dissolve the other components of the composition, for example, in the case of a mouthwash or mouth rinse.

[0793] Suitable solvents include water, ethanol and water / ethanol mixtures, which may be present in amounts ranging from 0.1% to 70%.

[0794] Antimicrobial Agents

[0795] The present invention also includes water-soluble antimicrobial agents such as chlorhexidine, triclosan, digluconates, hexetidine, Alexidine, quaternary ammonium antimicrobial compounds; and may also include water-soluble sources of certain metal ions such as zinc, copper, silver and stannous (e.g., zinc, cupric and stannous chlorides, and silver nitrate).

[0796] Sparingly soluble zinc salts such as zinc citrate, C14-alkyl zinc maleate, zinc benzoate, zinc hexanoate, zinc carbonate may also be included in the compositions of the present invention to prolong the antimicrobial efficacy of the zinc ions due to the slow dissolution of these zinc salts in saliva.

[0797] The antimicrobial agent may be present in an amount of 0% to 50% by weight, preferably 0.01% to 40% by weight, most preferably 0.1% to 30% by weight.

[0798] Tartar control agents

[0799] The compositions of the present invention may contain a tartar control agent, such as an inorganic phosphorus tartar control agent including any pyrophosphate salt, such as disodium pyrophosphate, dipotassium pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, and mixtures thereof.

[0800] Organic phosphorus compounds useful as tartar control agents include polyphosphonates such as disodium ethane-1-hydroxy-1,1-diphosphate (EHDP), methane diphosphonic acid, and 2-phosphonobutane-1,2,4-tricarboxylic acid.

[0801] Tartar control agents may be present in an amount of 0 to 10% by weight, preferably 0.1 to 5% by weight.

[0802] preservative

[0803] Suitable preservatives include sodium benzoate, potassium sorbate, parabens, methylparaben, ethylparaben, propylparaben, citric acid, calcium citrate, and mixtures thereof.

[0804] The preservative may be present in an amount of 0 to 40% by weight, preferably 0.01 to 30% by weight.

[0805] Fluoride ion source

[0806] The compositions of the present invention may also contain ingredients useful 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, acidic fluorophosphates, ammonium bifluoride, titanium tetrafluoride, and amine fluorides.

[0807] Particularly preferred are sodium fluoride and sodium monofluorophosphate.

[0808] The fluoride ion source may be present in an amount of 0 to 20% by weight, preferably 0.01 to 15% by weight, most preferably 0.1 to 10% by weight.

[0809] 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.

[0810] Colorants

[0811] Colorants or pigments suitable for use in the oral care compositions of the present invention include nontoxic, water-insoluble inorganic pigments such as titanium dioxide and chromium oxide green, ultramarine blue and pink, and iron oxide, and water-insoluble dye lakes prepared by extending calcium or aluminum salts of FD&C dyes on aluminum oxide, such as FD&C Green No. 1 Lake, FD&C Blue No. 2 Lake, FD&C Red No. 30 Lake, FD&C Yellow No. 16 Lake, and FD&C Yellow No. 10 Lake.

[0812] A preferred sunscreen is titanium dioxide.

[0813] The colorant may be present in an amount of 0 to 20% by weight, preferably 0.01 to 15% by weight, most preferably 0.1 to 10% by weight.

[0814] Buffer

[0815] The oral care compositions of the present invention may also include a buffering agent, ie, a pH adjusting agent, such as alkali metal hydroxides, carbonates, sesquicarbonates, borates, silicates, phosphates, imidazoles, and mixtures thereof.

[0816] Specific buffering agents 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 of these.

[0817] The buffer may be present in an amount of 0 to 10% by weight, preferably 0.01 to 5% by weight.

[0818] chewing gum

[0819] When the oral composition according to the invention is a chewing gum, it may be any known type of chewing gum, such as optionally coated chewing gum tablets, and sticks or chewing gum provided in any desired shape in response to the intended use. The chewing gum preparation may be of any quality, including bubble gum quality.

[0820] In preferred embodiments, the present invention is directed to enzymatic oral care compositions in the form of chewing gum, comprising the mutanase of the present invention and at least one oral care ingredient, wherein the at least one oral care ingredient is selected from elastomers, softeners, plasticizers, emulsifiers, waxes, colorants, sweeteners, flavoring agents, bulking agents, and thickeners.

[0821] Gum base ingredients

[0822] Traditionally, chewing gum is considered to consist of a water-insoluble or matrix portion and a water-soluble portion containing flavorings, sweeteners and coloring agents. The gum base portion of chewing gum is the chewing material that imparts chewing characteristics to the final product. It defines the release profile of flavorings and sweeteners and plays a significant role in chewing gum products. Flavorings, sweeteners and coloring agents can be considered to provide the sensory appeal of chewing gum. There is no restriction on the chewing gum base used in the chewing gum preparation according to the present invention. Conventional chewing gum base can be obtained from, for example, Dansk Tyggegummi Fabrik A / S, LA. Dreyfus or Cafasa Gum SIA are generally suitable, but specially manufactured formulations can also be used. The formulation depends on the type of desired chewing gum or the desired structure type. Suitable raw materials for gum base include materials according to the U.S. chewing gum base regulations - Title 21 of the Code of Federal Regulations Section 172,615 and according to other national and international lists (or positive lists), and include elastomers, resins, waxes, polyvinyl acetates, oils, fats, emulsifiers, fillers and antioxidants.

[0823] The gum base typically comprises 15 to 90% by weight, preferably 30 to 40% by weight, more preferably 5 to 25% by weight of the final product.

[0824] Elastomers provide chewiness, elasticity or rebound for the matrix, and control the bubbles and flavor release in the final chewing gum. They can be any water-insoluble polymer known in the art. They include styrene butadiene copolymer (SBR) and non-SBR types, including both natural and synthetic. The example of natural elastomers includes but is not limited to rubber (e.g. rubber latex (natural rubber)) and guayule, and gum (e.g. chicle, jelutong, balata, gutta percha, lechi capsi, sorva, crown gum, nispero, rosidinha, perillo, niger gutta, tunu, gutta kay, pendare, leche de vaca, chiquibul, crown gum, etc.), and mixtures thereof. Examples of synthetic elastomers include, but are not limited to, polyisobutylene, isobutylene-isoprene copolymers (butyl rubber), polyethylene, polybutadiene, styrene-butadiene copolymers, polyisoprene, and the like, and mixtures thereof.

[0825] The amount of elastomer (rubber) used in the gum base composition will vary widely depending on factors such as the type of gum base used (viscous or regular, bubble or standard), the consistency of the desired gum base composition, and other components used in the composition to prepare the final chewing gum product. Typically, 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, based on the total weight of the gum base composition.

[0826] Elastomer solvents help soften or plasticize the elastomer component. In this way, they provide expansion properties to the chew.

[0827] Elastomer solvents include, but are not limited to, natural rosin esters and synthetic derivatives such as terpenes. Examples of elastomer solvents suitable for use herein include tall oil rosin esters; partially hydrogenated wood and gum rosins; glycerol esters of wood and gum rosins, partially hydrogenated glycerol esters of wood / gum rosins, partially dimerized glycerol esters of wood and gum rosins, polymerized glycerol esters of wood and gum rosins, and glycerol esters of tall oil rosins; deodorized glycerol esters of wood rosins; pentaerythritol esters of wood and gum rosins; partially hydrogenated wood and gum rosins; methyl esters of partially hydrogenated wood rosins; methyl esters, glycerol esters, and pentaerythritol esters of rosins and modified rosins (e.g., hydrogenated, dimerized, and polymerized rosins); terpene resins (e.g., polymers of α-pinene or β-pinene), terpene olefin resins; polyterpenes; and the like and mixtures thereof. The elastomer solvent may be used in the gum base composition in an amount of about 2% to about 40%, preferably about 7% to about 15%, by weight of the gum base composition.

[0828] Polyvinyl acetates provide extensibility or elasticity to the gum base. They also affect chewing expansion, softness and gas bubbles, hydrophilicity and flavor release.

[0829] The amount of polyvinyl acetate of different molecular weights present in the gum base composition should effectively provide the desired chewing characteristics of the finished chewing gum, such as integrity, softness, chewing expansion, film-forming characteristics, hydrophilic characteristics and flavor release. The total amount of polyvinyl acetate for gum base composition is usually based on the total gum base composition by weight about 45% to about 92%. Vinyl polymer can have a molecular weight of about 2000Da to about 95,000Da.

[0830] 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 55,000 Da to about 95,000 Da, but can be as high as 500,000 Da.

[0831] Waxes, fats and oils plasticize the elastomer mixture and improve the elasticity of the gum base. Waxes can provide soft or hard chews, affect flavor release and provide expansibility and smoothness to the gum base. Fats and oils provide soft chews. Fats, oils and waxes can be used alone or in combination, or the gum base can be a wax-free gum base.

[0832] When wax is used, it can be of mineral, animal, vegetable or synthetic origin. Non-limiting examples of mineral waxes include petroleum waxes (e.g. paraffin wax and microcrystalline wax), animal waxes (including beeswax), vegetable waxes (including carnauba wax, candelilla wax, rice wax, esparto wax, flax wax and sugarcane wax), synthetic waxes (including those produced by the Fischer-Tropsch synthesis) and mixtures thereof.

[0833] Suitable oils and fats that can be used 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 or esters and salts (such as sodium stearate and potassium stearate). When used, these ingredients are generally present in an amount 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.

[0834] Preferred as softeners are hydrogenated vegetable oils, and include soybean oil and cottonseed oil, which can be used alone or in combination. These softeners provide good texture and soft chewing characteristics for the gum base composition. These softeners are usually used in an amount of about 5% to about 14% by weight of the gum base composition.

[0835] Emulsifiers help to disperse the immiscible components of the gum base composition into a single stable system. They provide hydrophilic characteristics for the gum base and help plasticizing resins and polyvinyl acetate. They also affect the softness of the matrix and the bubble property of the substrate. Typical emulsifiers include acetylated monoglycerides, glyceryl monostearate, lecithin, fatty acid monoglycerides, diglycerides, propylene glycol monostearate, lecithin, triacetin, glyceryl triacetate, etc. and mixtures thereof.

[0836] Preferred emulsifiers are glyceryl monostearate and acetylated monoglycerides. These are used as plasticizers. Emulsifiers can be used in an amount of about 2% to about 15% by weight of the gum base composition, and are preferably used in an amount of about 7% to about 11% by weight of the gum base composition.

[0837] Fat, oil, wax, emulsifier and some sugar swelling agent are combined together and are called softener usually.Due to the low molecular weight of these compositions, softener can penetrate the basic structure of gum base, make it have plasticity and less viscosity.Above-mentioned useful plasticizer and softener comprise lanolin, palmitic acid, oleic acid, stearic acid, sodium stearate, potassium stearate, triacetin, glyceryl lecithin, glyceryl monostearate, propylene glycol monostearate, acetylated monoglyceride, glycerine, completely unsaturated vegetable oil (for example non-hydrogenated cottonseed oil, hydrogenated vegetable oil, petroleum wax, sorbitan monostearate, tallow etc.) and its mixture, and also comprise high fructose corn syrup, corn syrup, sorbitol solution, hydrogenated starch hydrolysate etc. and its mixture.

[0838] The amount of softener present should be an effective amount to provide the desired chewing swellability and softness for the finished chewing gum.When used as softeners, these materials are generally used in gum base compositions in an amount up to about 25% by weight of the gum base composition, preferably about 1% to about 17%.

[0839] The gum base may further contain a surfactant. 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 be effective to provide the desired softness for the finished chewing gum. Typically, based on the gross weight of the gum base, the surfactant is used in the matrix in an amount of about 0.5% to about 3.0% by weight.

[0840] Gum base composition of the present invention can also include the filler of effective amount, it is sometimes called swelling agent.These materials increase hardness and swelling degree and influence the texture and the flavor release of chewing gum.Useful filler comprises organic and inorganic compound (mineral adjuvant), for example calcium carbonate, magnesium carbonate, heavy calcium carbonate, magnesium silicate, calcium phosphate, cellulose polymer, clay, aluminum oxide, aluminum hydroxide, aluminum silicate, talc, tricalcium phosphate, dicalcium phosphate etc. and its mixture.These fillers or adjuvant can be used in gum base composition with various amounts.The amount that filler exists should be that finished product chewing gum effectively provides desired flavor release and integrity.Typically, filler can be used in gum base composition with the amount of about 1% to about 40% by gum base composition weight and preferably about 5% to about 20%.

[0841] The gum base may also include an antioxidant to provide improved stability, reduce any oily taste and provide a longer shelf life. Typical non-limiting examples of antioxidants are butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate. Mixtures thereof may also be used.

[0842] Other glue ingredients

[0843] The remaining ingredients in the chewing gum composition are conventional and typically comprise from 10% to 85% by weight of the final product.

[0844] Examples are sweeteners, softeners, colorants, bulking agents, thickeners, and flavoring agents of the types and amounts conventionally used in chewing gum.

[0845] Suitable flavoring agents are those spices known to the technician, such as natural and artificial flavoring agents. These flavoring agents can 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. Non-restrictive representative flavoring oils include spearmint oil, cinnamon oil, wintergreen oil (methyl salicylate), peppermint oil, clove oil, bay oil, anise oil, eucalyptus oil, thyme oil, cedar leaf oil, nutmeg oil, allspice oil, sage oil, mace oil, bitter almond oil, and cassia oil. Other useful flavoring agents are artificial, natural and synthetic fruit flavors (such as vanilla and citrus oil, including lemon, orange, lime, grapefruit) and fruit essences (including apple, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, etc.). These flavoring agents can be used in liquid or solid form, and can be used alone or in combination. Commonly used flavors include mints, such as peppermint, menthol, artificial vanilla, cinnamon derivatives, and various fruit flavors, which can be used alone or in combination.

[0846] Other useful flavoring agents include aldehydes and esters, for example, cinnamyl acetate, cinnamaldehyde, diethyl acetal of citrate, dihydrocarvacetate, eugenyl formate, p-methylanisole, etc. Generally, any flavoring agent or food additive can be used.

[0847] Additional examples of aldehyde flavoring agents include, but are not limited to, acetaldehyde (apple), benzaldehyde (cherry, almond), anisaldehyde (licorice, anise), cinnamaldehyde (cinnamon), citral, i.e., α-citral (lemon, lime), neral, i.e., β-citral (lemon, lime), decanal (orange, lemon), ethyl vanillin (vanilla, cream), heliotropin, i.e., piperonal (vanilla, cream), vanillin (vanilla, cream), α-amylcinnamaldehyde (spicy fruity flavors), butyraldehyde (butter, cheese), valeraldehyde (butter, cheese), vanilla, Calendula (various types), decanal (citrus fruits), aldehyde C-8 (citrus fruits), aldehyde C-9 (citrus fruits), aldehyde C-12 (citrus fruits), 2-ethylbutyraldehyde (berry fruits), hexenal i.e. trans-2-hexenal (berry fruits), tolualdehyde (cherries, almonds), veratraldehyde (vanilla), 2,6-dimethyl-5-heptaldehyde i.e. melonal (melon), 2,6-dimethyloctanal (green fruits) and 2-dodecanal (citrus, mandarin), cherries, grapes, strawberry shortcake, and mixtures thereof.

[0848] The amount of flavoring agent used herein is generally influenced by factors such as the type of final chewing gum composition, the individual flavor, the gum used, and the desired flavor intensity. Therefore, the amount of flavoring agent can be varied to obtain the desired result in the final product, and such variations are within the ability of those skilled in the art without undue experimentation. In chewing gum compositions, flavoring agents are generally present in an amount of about 0.02% to about 5% by weight of the chewing gum composition.

[0849] The chewing gum composition generally includes a bulking agent. These carders may be water-soluble and include, but are not limited to, bulking agents selected from the group consisting of monosaccharides, disaccharides, polysaccharides, sugar alcohols, and mixtures thereof; sorbitol, xylitol, maltitol, mannitol, isomalt (a racemic mixture of α-D-pyranosyl-1,6-mannitol and α-D-pyranosyl-1,6-sorbitol, marketed by Suddeutsche Zucker under the trade name Palatinit TM Manufacturing), glycerin, aspartame, Glycerin, galactitol, acesulfame potassium, saccharin and its salts, cyclamates 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 disaccharides; minerals such as calcium carbonate, talc, titanium dioxide, dicalcium phosphate, cellulose, etc., and mixtures thereof. The bulking agent can be used in an amount of up to about 60% by weight of the chewing gum composition, preferably in an amount of about 25% to about 60%.

[0850] The chewing gum composition may also include a high intensity sweetener (sweetener). The sweetness intensity of a high intensity sweetener is substantially greater than the sweetness intensity of sucrose. Examples of suitable strong sweeteners include:

[0851] a) water-soluble, naturally occurring intense sweeteners such as dihydrochalcone, monellin, stevioside, glycyrrhizin, flavanone alcohols, and L-aminodicarboxylic acids, aminoalkanoic acid ester amides, such as those disclosed in U.S. Pat. No. 4,619,834, and mixtures thereof;

[0852] b) water-soluble artificial sweeteners, including soluble saccharin salts (e.g., saccharin sodium or saccharin calcium salts), cyclamate, sodium, ammonium or calcium salts of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, potassium salt of 3,4-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide (acesulfame potassium), the free acid form of saccharin, etc., and mixtures thereof;

[0853] c) dipeptide-based sweeteners, including sweeteners derived from L-aspartic acid (e.g., 1-aspartyl-L-phenylalanine methyl ester (aspartame) and materials described in U.S. Pat. No. 3,492,131), L-α-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alaninamide hydrate (alitame), L-aspartyl-L-phenylglycerol and methyl ester of L-aspartyl-L-2,5-dihydrophenyl-glycine, L-aspartyl-2,5-dihydro-L-phenylalanine, L-aspartyl-L-(1-cyclohexene)-alanine, and the like, and mixtures thereof;

[0854] d) water-soluble intense sweeteners derived from naturally occurring water-soluble sweeteners, for example chlorinated derivatives of ordinary sugar (sucrose), for example chlorodeoxysugar derivatives (e.g. derivatives of chlorodeoxysucrose or chlorodeoxygalactosucrose), known for example in ; 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-fructofuranoside, or 4,1',6'- trichloro-4,1',6'-trideoxygalactosucrose; 4,6-dichloro-4,6-dideoxy-α-D-galactopyranosyl-6-chloro-6-deoxy-β-D-fructofuranoside, 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'-tetrideoxygalactosucrose; and 4,6,1',6'-tetrideoxy-sucrose, and mixtures thereof;

[0855] E) protein-based intensive sweeteners, such as Thaumaoccous daniclii (thaumatin I and II). The amount of sweetener used in the chewing gum composition will vary with the sweetener selected for a particular chewing gum. Therefore, for any given sweetener, a sufficient amount of sweetener is used to provide the desired sweetness level. Based on the gross weight of the chewing gum composition, the above-mentioned carbohydrate sweeteners and sugar alcohols are typically used in an amount of about 1% to about 70%, preferably about 40% to about 50%, by weight. Based on the gross weight of the chewing gum composition, the above-mentioned intensive sweeteners are typically used in an amount of up to about 1%, preferably about 0.05% to about 0.4%, by weight.

[0856] The coloring agents useful in the present invention are used in an amount effective to produce the desired color. These coloring agents include pigments, which can be incorporated in an amount up to about 6% by weight of the chewing gum composition. A preferred pigment, namely titanium dioxide, can be incorporated in an amount up to about 2%, preferably less than about 1%, by weight of the chewing gum composition. Coloring agents can also include natural food pigments and dyes suitable for food, drug and cosmetic applications. These coloring agents are known as FD&C. dyes and lakes. Acceptable materials for the aforementioned uses are preferably water-soluble. Illustrative non-limiting examples include indigo dyes known as FD&C. Blue No. 2, which is the disodium salt of 5,5-indigo disulfonic acid. Similarly, the dye known as FD&C. Green No. 1 contains triphenylmethane dyes and is the monosodium salt of 4-[4-(N-ethyl-N-p-thiobenzylamino)diphenylmethylene]-[1-(N-ethyl-N-p-thiobenzyl)-δ-2,5-cyclohexadienimine].

[0857] Examples of thickening agents include methylcellulose, alginates, carrageenan, xanthan gum, gelatin, carob, tragacanth and locust bean gums, emulsifiers such as lecithin and glyceryl monostearate, acidulants such as malic acid, adipic acid, citric acid, tartaric acid, fumaric acid, and mixtures thereof.

[0858] The plasticizers, softeners, emulsifiers, waxes and antioxidants discussed above as suitable for use in the gum base may also be used in the chewing gum composition.

[0859] Active chewing gum ingredients

[0860] The oral care composition of the present invention in the form of chewing gum may also contain various active ingredients such as antimicrobial agents, Zn salts, fluoride, and urea.

[0861] Furthermore, if necessary, the oral composition according to the present invention may include any other active ingredients such as anticaries agents, anticalculus agents, antiplaque agents, antiperiodontal disease agents, antifungal agents, antismoking agents, anticold agents, antigingivitis agents, etc.

[0862] The antimicrobial agent used in the composition can be any of a variety of cationic antimicrobial agents, such as quaternary ammonium compounds (e.g., cetylpyridinium chloride) and substituted guanidines (e.g., chlorhexidine and the corresponding compound Alexidine). Mixtures of cationic antimicrobial agents can also be used in the present invention.

[0863] 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, tetradecylpyridinium chloride, tetradecylethylpyridinium chloride, dodecyldimethyl(2-phenoxyethyl)ammonium ...

Claims

1. An oral care composition comprising a mutanase selected from the group consisting of: (a) a polypeptide having at least 60% sequence identity to SEQ ID NO: 3; (b) a polypeptide having at least 90% sequence identity to SEQ ID NO:6; (c) a polypeptide having at least 65% sequence identity to SEQ ID NO:9; (d) a polypeptide having at least 65% sequence identity to SEQ ID NO: 12; (e) a polypeptide having at least 60% sequence identity to SEQ ID NO: 15; (f) a polypeptide having at least 60% sequence identity to SEQ ID NO: 18; (g) a polypeptide having at least 60% sequence identity to SEQ ID NO: 21; (h) a polypeptide having at least 60% sequence identity to SEQ ID NO: 24; (i) a polypeptide having at least 60% sequence identity to SEQ ID NO: 27; (j) a polypeptide having at least 60% sequence identity to SEQ ID NO: 30; and (k) a polypeptide having at least 60% sequence identity to SEQ ID NO: 33; wherein the polypeptide has endo-1,3-alpha-glucanase activity, and wherein the oral care composition further comprises at least one oral care ingredient.

2. The oral care composition according to claim 1, wherein the mutanase is selected from the group consisting of: (a) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 3; (b) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:6; (c) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:9; (d) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 12; (e) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 15; (f) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO: 18; (g) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:21; (h) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:24; (i) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:27; (j) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:30; and (k) a polypeptide comprising, consisting essentially of, or consisting of SEQ ID NO:

33.

3. An oral care composition according to any one of claims 1-2, wherein the mutanase has improved thermostability compared to SEQ ID NO: 36; preferably when measured at pH 5 and / or pH 6 according to Example 4 herein, the mutanase has improved thermostability compared to SEQ ID NO: 36; preferably at pH 5 or pH 6.

4. An oral care composition according to any one of claims 1 to 3, wherein the mutanase has improved thermostability compared to SEQ ID NO: 36 in the presence of at least one oral care ingredient selected from the group consisting of: benzoate (e.g., sodium benzoate), EDTA, ethanol, glycerol, fluoride (e.g., sodium fluoride, sodium monofluorophosphate, calcium fluoride, or stannous fluoride), hydrogen peroxide, mannitol, phosphate (e.g., sodium phosphate or potassium phosphate), sorbate (e.g., potassium sorbate) and sorbitol; preferably the mutanase has improved thermostability compared to SEQ ID NO: 36 when measured at pH 5 and / or pH 6 according to Example 5 herein; preferably at pH 5 or pH 6.

5. An oral care composition according to any one of claims 1 to 4, wherein the mutanase provides comparable or improved, preferably improved, biofilm prevention compared to SEQ ID NO: 36 when determined according to Example 6 herein; preferably at about 1 ppm, about 10 ppm and / or about 60 ppm, the mutanase provides comparable or improved biofilm prevention.

6. An oral care composition according to any one of claims 1 to 5 for use as a medicament.

7. An oral care composition according to any one of claims 1 to 5, for use in treating oral diseases; preferably for use in treating periodontal disease (eg gingivitis) and / or dental caries.

8. A method for preventing and / or removing oral biofilm, the method comprising contacting the oral biofilm with an oral care composition according to any one of claims 1 to 5.

9. A set box, the set box comprising: (1) The oral care composition according to any one of claims 1 to 5; and (2) Instructions for use.

10. A polypeptide having endo-1,3-α-glucanase activity, the polypeptide being selected from the group consisting of: a. A polypeptide having at least 60% sequence identity with SEQ ID NO: 3; b. A polypeptide having at least 90% sequence identity with SEQ ID NO: 6; c. A polypeptide having at least 65% sequence identity to SEQ ID NO: 9; d. A polypeptide having at least 65% sequence identity with SEQ ID NO: 12; e. A polypeptide having at least 60% sequence identity with SEQ ID NO: 15; f. A polypeptide having at least 60% sequence identity with SEQ ID NO: 18; g. A polypeptide having at least 60% sequence identity with SEQ ID NO: 21; h. A polypeptide having at least 60% sequence identity with SEQ ID NO: 24; i. A polypeptide having at least 60% sequence identity with SEQ ID NO: 27; j. A polypeptide having at least 60% sequence identity with SEQ ID NO: 30; and k. A polypeptide having at least 60% sequence identity to SEQ ID NO:

33.

11. A polypeptide according to claim 10, which has improved thermal stability compared to SEQ ID NO: 36; preferably when measured at pH 5 and / or pH 6 according to Example 4 herein, compared to SEQ ID NO: 36, the polypeptide has improved thermal stability; preferably at pH 5 or pH 6.

12. A polynucleotide encoding the polypeptide according to any one of claims 10-11.

13. A nucleic acid construct or expression vector comprising the polynucleotide according to claim 12, operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

14. A recombinant host cell comprising the nucleic acid construct or expression vector according to claim 13.

15. A method for producing a polypeptide having endo-1,3-α-glucanase activity, the method comprising culturing the recombinant host cell according to claim 14 under conditions conducive for production of the polypeptide.

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