Hexosaminidase variants and compositions

Abstract

The present invention relates to novel hexosaminidase variants that exhibit modifications compared to parent hexosaminidases that have been found to provide improved stability, such as improved detergent stability.The present invention also relates to detergent compositions that include the hexosaminidase variants, as well as methods of using and producing the hexosaminidase variants, and isolated hexosaminidase sequences, expression vectors, and host cells that code for the variants.The variants of the present invention are suitable for use in cleaning processes and detergent compositions.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to novel hexosaminidase (dispersin) variants, compositions including detergent compositions comprising the variants, polynucleotides encoding the variants, methods of making the variants, methods of using the variants, and uses of the variants and detergent compositions for cleaning. [Background technology]

[0003] Various enzymes, such as proteases and amylases, have long been used in detergent compositions to remove protein- and starch-related stains, respectively.However, most stains, for example, in laundry, are complex mixtures of various organic matter, which may be difficult to completely remove using known enzymes.For example, complex organic stains, such as biosoils from human skin, for example dead skin cells, sweat and sebum, contain various organic materials, such as proteins, starches, oils, and polysaccharides.As a result, to effectively remove stains, such as laundry, requires various enzyme activities.

[0004] Detergent compositions comprising an enzyme with hexosaminidase activity (dispersin) and hexosaminidase enzyme variants have already been disclosed, for example, in WO 2017 / 186943 and WO 2020 / 207944. However, detergent compositions containing dispersin are not yet commercially available, and furthermore, various detergent compositions and washing conditions may present challenges that not all known dispersins can address.

[0005] In general, enzymes such as dispersin need to be stable and effective in a given detergent composition and under given washing conditions to be useful in cleaning processes such as laundry. For example, washing conditions in North America and certain other parts of the world are generally different from washing conditions in Europe and the like. In Europe, washing machines are typically front-loading and use relatively small amounts of water in combination with relatively long wash cycles and often high temperatures, while in North America and other parts, washing machines are generally top-loading and use large amounts of water, relatively short wash cycles and often low temperatures. These conditions in North America and other parts, including low to medium detergent concentrations in the wash water, can make it difficult for enzyme detergents to provide effective soil removal in highly diluted wash water with short wash cycles and often relatively low temperatures. Detergent compositions may also be formulated differently, for example, in North America compared to Europe, to address washing conditions and other local factors such as water hardness.

[0006] The present invention provides hexosaminidase enzyme variants that have been found to be particularly suitable for use under, for example, typical North American washing conditions and in detergent compositions commonly used in, for example, North America. Summary of the Invention [Means for solving the problem]

[0007] The present invention relates to variants of the hexosaminidase polypeptide of SEQ ID NO: 1, which, compared to SEQ ID NO: 1, have Substitutions at one or more positions, preferably 2, 3 or 4 positions, corresponding to positions 163, 227, 252 and 309 of SEQ ID NO:1; and Substitutions at one or more positions corresponding to positions 106, 111, 120, 124, 127, 150, 170, 171, 178, 199, 208, 254, 255, and 278 of SEQ ID NO:1 Includes; The present invention relates to variants having hexosaminidase activity and having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0008] The invention also relates to compositions, such as detergent compositions, comprising the hexosaminidase variants disclosed herein, as well as isolated polynucleotides encoding the variants, nucleic acid constructs, vectors and host cells comprising the polynucleotides, and methods of producing the variants. The invention further relates to methods of using the hexosaminidase variants and compositions for cleaning, such as for laundry. [Brief description of the drawings]

[0009] [Figure 1] 1 is an alignment of the polypeptides of SEQ ID NOs: 1, 2, 3 and 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Sequence Listing Overview SEQ ID NO:1 Mature hexosaminidase polypeptide obtained from Terribacillus saccharophilus SEQ ID NO:2 Hexosaminidase, variant of SEQ ID NO:1 SEQ ID NO:3 Hexosaminidase, variant of SEQ ID NO:1 SEQ ID NO:4 Hexosaminidase, variant of SEQ ID NO:1

[0011] definition In accordance with this Detailed Description, the following definitions apply: Please note that the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0012] Unless otherwise defined or clearly indicated by 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.

[0013] Hexosaminidase: The term "hexosaminidase" includes "dispersin" and the abbreviation "Dsp", which refers to EC 3.2.1., a polypeptide with hexosaminidase activity that catalyzes the hydrolysis of β-1,6-glycosidic bonds in N-acetyl-glucosamine polymers found, for example, in biofilms. The term hexosaminidase includes polypeptides with N-acetylglucosaminidase activity and β-N-acetylglucosaminidase activity. The term "polypeptide with hexosaminidase activity" may be used interchangeably with the term "hexosaminidase", and similarly, the term "polypeptide with β-N-acetylglucosaminidase activity" may be used interchangeably with the term "β-N-acetylglucosaminidase". For the purposes of the present invention, hexosaminidase activity is determined according to the procedures described in assay 1 or 2. In a preferred embodiment, the polypeptide with hexosaminidase activity is dispersin. In a preferred embodiment, the polypeptide having hexosaminidase activity is a β-N-acetylglucosaminidase that targets poly-β-1,6-N-acetylglucosamine.

[0014] Expression: The term "expression" includes all steps involved in the production of a variant (such as, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0015] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a variant, the coding sequence being operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to induce transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0016] Extension: The term "extension" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a variant, such that the "extended" variant has hexosaminidase activity.

[0017] Fragment: The term "fragment" refers to a variant in which one or more amino acids are missing from the amino and / or carboxyl terminus of the variant, which fragment has hexosaminidase activity.

[0018] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a variant has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest.

[0019] Improved property: The term "improved property" refers to a feature associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, improved catalytic efficiency, catalytic rate, chemical stability, oxidative stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, and thermostability. In the context of the present invention, improved properties are preferably improved stability, such as improved thermostability or improved storage stability in detergent compositions. Improved properties, such as improved stability, of the hexosaminidase variants of the present invention can be determined as described in the Examples herein.

[0020] Isolated: The term "isolated" refers to a variant, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including but not limited to other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other substance is in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted variants expressed in host cells.

[0021] Mature Polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide following N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0022] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having hexosaminidase activity.

[0023] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0024] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0025] Nucleic Acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to code for a particular amino acid, and the present compositions and methods encompass nucleotide sequences that code for a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in the 5' to 3' direction.

[0026] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner not normally found in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0027] Operably linked: The term "operably linked" means that the specified components are in a relationship (including, but not limited to, juxtaposition) that permits them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0028] Parent or Parent Hexosaminidase: The terms "parent", "parent hexosaminidase" or "parent enzyme" refer to the hexosaminidase that is modified to generate the enzyme variant of the present invention. Thus, the parent is a hexosaminidase polypeptide that has the same amino acid sequence of the variant disclosed herein but does not have the specific modifications, typically substitutions, disclosed herein.

[0029] In certain embodiments, the hexosaminidase parent is a hexosaminidase having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0030] In one embodiment, the parent hexosaminidase is a hexosaminidase having the sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a preferred embodiment, the parent hexosaminidase is the polypeptide of SEQ ID NO: 1.

[0031] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and recombining, of nucleic acid sequences to form constellations that differ from those found in nature. The term recombinant refers to a cell, nucleic acid, variant, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses native genes at levels or under different conditions than those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0032] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity." Sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), version 6.6.0, as the "longest identity" output. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The nobrief option must be specified on the command line so that the Needle program reports the longest identity. The output of Needle labeled "longest identity" is calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment).

[0033] Sequence identity between two polynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) as implemented in the Needle program in the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, see above) version 6.6.0 as the "longest identity" output. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The nobrief option must be specified on the command line to cause the Needle program to report the longest identity. The output of Needle labeled "longest identity" is calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0034] Variant: The term "variant" refers to a polypeptide having hexosaminidase activity and which contains a substitution, insertion (including extension), and / or deletion (e.g., truncation) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, in particular 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0035] Wild-type: The term "wild-type" in reference to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory, or modifications of a wild-type sequence).

[0036] Biofilm: A biofilm is any microbial community in which cells are attached to each other on a surface such as a fabric or dish, or on other hard surfaces. These attached cells are often embedded in a self-generated matrix of extracellular polymeric substances (EPS). Biofilm EPS is generally a polymeric mass composed of extracellular DNA, proteins, and polysaccharides. Biofilms can form on living and non-living surfaces. Microbial cells growing in a biofilm are physiologically different from planktonic cells of the same organism (planktonic cells, in contrast, are single cells that can float or swim in a liquid medium). Bacteria living in a biofilm usually have properties that are very different from planktonic bacteria of the same species, because the dense and protected environment of the film allows them to cooperate and interact in various ways. One advantage of this environment is that it is highly resistant to detergents and antibiotics, because the dense extracellular matrix and outer layer of cells protect the inside of the population. In the laundry, biofilm-producing bacteria can be found, for example, in the following species: Acinetobacter spp., Aeromicrobium spp., Brevundimonas spp., Microbacterium spp., Micrococcus luteus, Pseudomonas spp., Staphylococcus epidermidis, and Stenotrophomonas spp.

[0037] Stability: The term "stability" includes storage stability and stability during use, e.g. during the washing process, and reflects the stability of the hexosaminidase variant according to the invention as a function of time, e.g. to what extent activity is retained when the hexosaminidase variant is kept in dry form or in solution (e.g. in a detergent liquor). Stability is influenced by many factors, e.g. pH, temperature, and the nature of the detergent composition (e.g. amount and type of builder, surfactant, etc.). Hexosaminidase stability may be measured as described in the Examples and may be expressed as melting temperature (Tm) or half-life improvement factor (HIF), e.g. compared to a parent hexosaminidase or a reference hexosaminidase, e.g. the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. The term "improved stability" or "increased stability" as used herein refers to an increase in the stability of a hexosaminidase variant according to the invention, when compared to a parent hexosaminidase or a reference hexosaminidase without substitutions in the variant. A variant hexosaminidase is defined as a variant hexosaminidase that exhibits increased stability in solution compared to the stability of the hexosaminidase and / or compared to the stability of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. Hexosaminidases having SEQ ID NO: 2 and SEQ ID NO: 3 have greater stability (Tm and HIF) than the hexosaminidase of SEQ ID NO: 1, and therefore hexosaminidase variants with increased stability compared to SEQ ID NO: 2 or 3 also have greater stability than SEQ ID NO: 1. The terms "improved stability" and "increased stability" include detergent stability.

[0038] Cleaning performance: The term "cleaning performance" in the context of the present invention refers to the cleaning effect in laundry, preferably the deep cleaning effect, where "deep cleaning" refers to the destruction or removal of biofilm or its components of the hexosaminidase variant of the present invention compared to the hexosaminidase parent or the hexosaminidase having sequence number 1.

[0039] Wash performance can be expressed as the recovery value of the stained swatches. After washing and rinsing, the swatches are laid flat and allowed to air dry overnight at room temperature. All washed swatches are evaluated the day after washing. The light reflectance of the swatches is evaluated using a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. Measurements are taken without UV in the incident light and reflectance at 460 nm is extracted.

[0040] Laundry: The term "laundry" refers to both domestic and commercial laundry and means the process of treating fabrics with a solution containing, for example, the cleaning or detergent composition of the present invention. The laundry process can be carried out, for example, using a domestic or commercial washing machine, or can be carried out by hand.

[0041] Detergent composition: The term "detergent composition" (or "cleaning composition") includes all forms of detergent or cleaning compositions, unless otherwise specified. These include all-purpose or heavy-duty detergents in granular or powder form, especially cleaning detergents; all-purpose detergents in liquid, gel or paste form, especially so-called heavy duty liquid (HDL) types; single unit dose (SUD) compositions such as pods, capsules, tubs, etc., with one or more chambers; liquid detergents for delicate fabrics; hand dishwashing or light-duty dishwashing, especially high foaming types; dishwasher cleaners, including various tablet, granular, liquid, and rinse aid types for home and commercial use; liquid cleaning and disinfecting agents, including antibacterial hand washing types, cleaning bars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; hair shampoos and hair rinses; shower gels, foam baths; metal cleaners; and cleaning aids such as bleach additives and "stain sticks" or pre-treatment types. The terms "detergent composition" and "detergent formulation" are used in reference to mixtures intended for use in a cleaning medium for cleaning soiled objects. In some embodiments, the term is used in reference to laundering fabrics and / or clothing (e.g., "laundry detergent"). In alternative embodiments, the term refers to other detergents, such as those used for cleaning dishes, cutlery, and the like (e.g., "dishwashing detergent"). It is not intended that the present invention be limited to any particular detergent formulation or composition. The term "detergent composition" is not intended to be limited to compositions containing surfactants. In addition to variants according to the present invention, the term is intended to encompass detergents that may contain, for example, surfactants, builders, chelators or chelating agents, bleach systems or bleach components, polymers, fabric softeners, foam boosters, suds suppressors, dyes, fragrances, yellowing inhibitors, optical brighteners, bactericides, fungicides, soil suspension agents, anticorrosive agents, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and dissolving agents.

[0042] Fabric: The term "fabric" includes any textile material. Thus, the term is intended to include garments, as well as fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile materials.

[0043] Fabric: The term "fabric" refers to woven fabrics and staple fibers and filaments suitable for conversion into or use as yarns, woven fabrics, knits, and nonwoven fabrics. The term encompasses yarns made from natural and synthetic (e.g., manufactured) fibers. The term "textile material" is a general term for fibers, yarn intermediates, yarns, fabrics, and products (e.g., clothing and other articles) made from fabrics.

[0044] Non-fabric detergent compositions: The term "non-fabric detergent compositions" includes detergent compositions for surfaces other than fabrics, such as, but not limited to, compositions for hard surface cleaning, such as dishwashing detergent compositions, including manual dishwashing compositions, oral detergent compositions, denture detergent compositions, and personal cleansing compositions. Another group of non-fabric detergent compositions are compositions for medical cleaning, i.e., cleaning medical devices to remove or prevent biofilms, or coating medical devices, such as indwelling medical devices or implants to prevent biofilm formation. The medical device may be, for example, a catheter, a mechanical valve, a cardiac pacemaker, an arteriovenous shunt, a scleral buckle, an artificial joint, a tympanic ventilation tube, a tracheotomy tube, a voice prosthesis, an artificial penis, an artificial urinary sphincter, a synthetic pubovaginal sling, a suture, a bone anchor, a bone screw, an intraocular lens, a contact lens, an intrauterine contraceptive device, an aortofemoral artery graft, a vascular graft, a needle, a luer lock connector, a needleless connector, or a surgical instrument.

[0045] Effective amount of enzyme: The term "effective amount of enzyme" refers to the amount of enzyme required to achieve the enzymatic activity required for a particular application, e.g., a defined detergent composition. Such effective amounts are easily determined by those skilled in the art and are based on many factors, such as the particular enzyme used, the cleaning application, the particular composition of the detergent composition, and whether a liquid or dry (e.g., granular, bar) composition is required. The term "effective amount" of a hexosaminidase variant refers to the amount of the hexosaminidase variant described above that achieves a desired level of enzymatic activity, e.g., in a defined detergent composition.

[0046] Relevant washing conditions: The term "relevant washing conditions" is used herein to indicate the conditions actually used in households of the detergent market segment, in particular washing temperature, time, washing machine, detergent concentration, detergent type and water hardness.

[0047] Washing solution: The term "washing solution" (or "washing water") refers to an aqueous solution comprising a hexosaminidase variant of the present invention. A washing solution is a solution (e.g., found in a washing machine or dishwasher) that contains water and a detergent composition comprising a hexosaminidase variant. The detergent composition may be in any suitable form, e.g., liquid or powder, as described elsewhere herein, before being mixed with water to form the washing solution.

[0048] Water Hardness: As used herein, the term "water hardness" or "hardness" or "dH" or "° dH" refers to the German hardness scale, where one degree is defined as 10 milligrams of calcium oxide per liter of water.

[0049] Adjunct materials: The term "adjunct materials" or "adjunct ingredients" refers to any liquid, solid, or gaseous material selected for the particular type of detergent composition desired and the form of the product (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, or foam composition), which materials are also preferably compatible with the hexosaminidase variant enzyme used in the composition. More detailed information regarding adjunct materials is provided further below.

[0050] Low detergent concentration: The term "low detergent concentration" system includes detergents in which less than about 800 ppm of detergent components are present in the wash water. Asian, e.g. Japanese, detergents are typically considered to be low detergent concentration systems.

[0051] Mid detergent concentration: The term "mid detergent concentration" system includes detergents in which from about 800 ppm to about 2000 ppm of detergent components are present in the wash water. North American detergents are generally considered to be mid detergent concentration systems.

[0052] High Detergent Concentration: The term "high detergent concentration" systems includes detergents in which more than about 2000 ppm of detergent components are present in the wash water. European detergents are generally considered to be high detergent concentration systems.

[0053] Rules for Naming Variants For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid positions in another hexosaminidase. The amino acid sequence of another hexosaminidase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on this alignment, the amino acid position numbers corresponding to the amino acid residues in the polypeptide disclosed in SEQ ID NO: 1 are determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48; 443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS; The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16; 276-277), preferably version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0054] In describing the variants of the present invention, the following nomenclature has been adapted for ease of reference: Accepted IUPAC one-letter or three-letter amino acid abbreviations are employed.

[0055] Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "T226A" (or "Thr226Ala" using the three-letter code). Multiple mutations may be separated by a plus sign ("+"), e.g., "G205R+S411F" represents the substitution of serine (S) with arginine (R) with glycine (G) and phenylalanine (F) at positions 205 and 411, respectively. Alternatively, multiple mutations may be indicated by a space, comma, or plus sign (e.g., "G205R S411F", "G205R,S411F", or "G205R+S411F").

[0056] Deletions. For amino acid deletions, the following nomenclature is used: Original amino acid, position, *. Thus, a deletion of glycine at position 195 would be designated "G195*." Multiple deletions are separated by a plus sign ("+") (e.g., "G195*+S411*").

[0057] Insertions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of a lysine after the glycine at position 195 would be designated as "G195GK." Insertions of multiple amino acids are designated as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of a lysine and an alanine after the glycine at position 195 would be designated as "G195GKA."

[0058] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence is:

[0059] [Table 1]

[0060] Multiple modifications. Variants containing multiple modifications are separated by a plus sign ("+") as described above, e.g., "R170Y+G195E" represents the substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Alternatively, multiple modifications may be separated by spaces or commas as described above.

[0061] Various modifications. When various modifications can be introduced at a position, the various modifications can be separated by a comma or a slash, for example, "R170Y,E" or "R170Y / E" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Y167G,A+R170G,A" or "Y167G / A+R170G / A" designates the following variants: "Y167G+R170G", "Y167G+R170A", "Y167A+R170G", and "Y167A+R170A".

[0062] The present invention relates to variants of the hexosaminidase polypeptide of SEQ ID NO: 1, which, compared to SEQ ID NO: 1, have Substitutions at one or more positions, preferably 2, 3 or 4 positions, corresponding to positions 163, 227, 252 and 309 of SEQ ID NO:1; and Substitutions at one or more positions corresponding to positions 106, 111, 120, 124, 127, 150, 170, 171, 178, 199, 208, 254, 255, and 278 of SEQ ID NO:1 Includes; The present invention relates to variants having hexosaminidase activity and having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0063] variant In one embodiment, the variant of the invention has, compared to SEQ ID NO:1: One or more, e.g., two, three or four, of the indicated amino acid residues at the following positions: P at position 163, T at position 227, P at position 252, and E at position 309; and One or more of the indicated amino acid residues at the following positions: M or N at position 106, R at position 111, V at position 120, I, K or L at position 124, P at position 127, N at position 150, G at position 170, F or H at position 171, K at position 178, W at position 199, W at position 208, I at position 254, D at position 255, and V at position 278. Includes; Position numbers are based on SEQ ID NO:1.

[0064] Thus, the hexosaminidase variant comprises two, three or four substitutions selected from the group consisting of S163P, N227T, N252P and K309E compared to SEQ ID NO:1; and at least one substitution selected from the group consisting of V106M, V106N, D111R, T120V, Y124I, Y124K, Y124L, R127P, E150N, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D, and I278V. may include.

[0065] In a preferred embodiment, the hexosaminidase variant may further comprise at least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of said substitutions, compared to SEQ ID NO: 1. In such an embodiment, the variant may further comprise one or more substitutions selected from the group consisting of Q3I, A49W and N59E, preferably two or all three of said substitutions; or one or more substitutions selected from the group consisting of Q3F, V140I, Q215K and N267T, preferably two, three or all four of said substitutions, compared to SEQ ID NO: 1.

[0066] In one embodiment, the hexosaminidase variant comprises at least one substitution selected from the group consisting of substitutions S163P, N227T, N252P and K309E; and H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of said substitutions compared to SEQ ID NO:1.

[0067] In a preferred embodiment, the hexosaminidase variant of the invention comprises at least one substitution selected from the group consisting of V106M / N, D111R, T120V, Y124I / K / L, R127P, E150N, L170G, D171F / H, Q178K, S199W, S208W, T255D, N267T, I278V and K308E compared to SEQ ID NO: 1. Preferably, the variant comprises at least two of said substitutions, such as at least three of said substitutions. One such variant is a variant of SEQ ID NO:2 comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, Y124I / K / L, R127P, E150N, L170G, D171F / H, Q178K, S199W, S208W, T255D, N267T, I278V and K308E, such as at least two or at least three of said substitutions. Another such variant is a variant of SEQ ID NO:3 comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, Y124I / K / L, R127P, E150N, L170G, D171F / H, Q178K, S199W, S208W, T255D, N267T, I278V and K308E, such as at least two or at least three of said substitutions.

[0068] In a more preferred embodiment, the hexosaminidase variant of the invention comprises at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V compared to SEQ ID NO: 1. Preferably, the variant comprises at least two of said substitutions, such as at least three of said substitutions. One such variant is a variant of SEQ ID NO: 2 comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V, such as at least two or at least three of said substitutions. Another such variant is a variant of SEQ ID NO:3 comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V, such as at least two or at least three of said substitutions.

[0069] More preferably, the hexosaminidase variant of the present invention comprises at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V compared to SEQ ID NO:1, such as at least two of said substitutions, such as at least three of said substitutions.

[0070] In a particularly preferred embodiment, the hexosaminidase variant of the invention comprises at least one substitution selected from the group consisting of D111R, T120V and E150N compared to SEQ ID NO: 1. Thus, the variant may comprise the substitutions D111R+T120V, D111R+E150N or T120V+E150N, or all three substitutions D111R+T120V+E150N. In these embodiments, the variants may further comprise at least one substitution selected from the group consisting of V106M, V106N, Y124I, Y124K, Y124L, R127P, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V, with preferred substitutions including those selected from the group consisting of V106M / N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V.

[0071] In one embodiment the hexosaminidase variant comprises the substitution D111R, together with at least one substitution selected from T120V and E150N, and optionally at least one additional substitution selected from the group consisting of V106M, V106N, Y124I, Y124K, Y124L, R127P, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V, compared to SEQ ID NO:1.

[0072] In one embodiment the hexosaminidase variant comprises the substitution T120V, together with at least one substitution selected from D111RV and E150N, and optionally at least one additional substitution selected from the group consisting of V106M, V106N, Y124I, Y124K, Y124L, R127P, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V, compared to SEQ ID NO:1.

[0073] In one embodiment the hexosaminidase variant comprises the substitution E150N, together with at least one substitution selected from D111R and T120V, and optionally at least one additional substitution selected from the group consisting of V106M, V106N, Y124I, Y124K, Y124L, R127P, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V, compared to SEQ ID NO:1.

[0074] In some embodiments, the hexosaminidase variant is selected from the group consisting of V106M, V106N, Y124I, Y124K, Y124L, R127P, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V compared to SEQ ID NO:1; for example, the variant comprises at least one substitution selected from the group consisting of V106M / N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V.

[0075] In some embodiments, the hexosaminidase variant has, compared to SEQ ID NO:1, a) two, three, or preferably four substitutions selected from the group consisting of S163P, N227T, N252P, and K309E; b) at least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q, and K312Q. replacements, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions; and c) at least one substitution selected from the group consisting of V106M, V106N, D111R, T120V, Y124I, Y124K, Y124L, R127P, E150N, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V.

[0076] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of the substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution V106M, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of D111R, T120V, E150N, D171F and I278V.

[0077] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of the substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution V106N, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of D111R, T120V, E150N, D171F and I278V.

[0078] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution D111R, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, T120V, E150N, D171F and I278V.

[0079] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution T120V, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, E150N, D171F and I278V.

[0080] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution Y124I, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0081] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution Y124K, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0082] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution Y124L, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0083] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution R127P, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0084] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution E150N, compared to SEQ ID NO: 1. Such variants may further comprise one or more additional substitutions, for example selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, D171F and I278V.

[0085] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution L170G, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0086] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution D171F, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N and I278V.

[0087] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution D171H, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N and I278V.

[0088] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution Q178K, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0089] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution S199W, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0090] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution S208W, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0091] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution F254I, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0092] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution T255D, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V.

[0093] In one embodiment, the hexosaminidase variant comprises a) two, three or preferably four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; b) at least one substitution, preferably at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, more preferably all of said substitutions, selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q; and c) the substitution I278V, compared to SEQ ID NO: 1. Such variants may further comprise, for example, one or more additional substitutions selected from the group consisting of V106M / N, D111R, T120V, E150N and D171F.

[0094] Examples of specific hexosaminidase variants of the invention include variants which comprise at least one of the following substitutions or sets of substitutions compared to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, preferably compared to SEQ ID NO:2: D111R D111R+T120V D111R+T120V+E150N ·D111R+T120V+E150N+D171F+I278V D111R+T120V+T255D ·D111R+T120V+Y124I ·D111R+T120V+Y124K+Q178K ·D111R+T120V+Y124L+Q178K ·D111R+Y124K+D171F ·D171F+S208W+N267T ·D171F+T255D ·D171H ·I278V ·K308E ·N267T ·R127P ·T120V+D171F ·T120V+D171F+N267T ·T120V+S208W+N267T ·T120V+T255D ·T120V+T255D+N267T ·T120V+Y124K+Q178K ·V106M+D111R+T120V+D171F ·V106M+Q178K+T255D ·V106M+T120V+D171F+Q178K ·V106M+T120V+Y124I+D171F ·V106M+T120V+Y124I+Q178K ·V106M+T120V+Y124K+D171F ·V106M+T120V+Y124L+Q178K ·V106N+D111R+T120V+E150N ·V106N+S208W+N267T ·V106N+T120V+D171F ·V106N+T120V+D171F+S208W ·V106N+T120V+E150N+D171F+I278V ·V106N+T120V+N267T

[0095] The particular hexosaminidase variants listed above may have at least 80%, preferably at least 85%, more preferably at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO:2 or SEQ ID NO:3, preferably compared to SEQ ID NO:2.

[0096] Other examples of specific hexosaminidase variants of the invention include variants which comprise at least one of the following substitutions or sets of substitutions compared to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, preferably compared to SEQ ID NO:3: · L170G E150N+F254I ·F254I · V106N D111R+T120V D111R+T120V+E150N ·D111R+T120V+E150N+D171F+I278V D111R+T120V+T255D D111R+T120V+Y124I D111R+T120V+Y124K+Q178K D111R+T120V+Y124L+Q178K D111R+Y124K+D171F D171F+S208W+N267T D171F+T255D D171H I278V · K308E N267T R127P · T120V+D171F T120V+D171F+N267T ·T120V+S208W+N267T ·T120V+T255D T120V+T255D+N267T T120V+Y124K+Q178K V106M+D111R+T120V+D171F · V106M+Q178K+T255D V106M+T120V+D171F+Q178K V106M+T120V+Y124I+D171F V106M+T120V+Y124I+Q178K V106M+T120V+Y124K+D171F V106M+T120V+Y124L+Q178K V106N+D111R+T120V+E150N · V106N+S208W+N267T V106N+T120V+D171F V106N+T120V+D171F+S208W ·V106N+T120V+E150N+D171F+I278V V106N+T120V+N267T

[0097] The particular hexosaminidase variants listed above may have at least 80%, preferably at least 85%, more preferably at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity compared to SEQ ID NO:2 or SEQ ID NO:3, preferably compared to SEQ ID NO:3.

[0098] Examples of preferred hexosaminidase variants of the invention include variants that comprise one of the following sets of substitutions compared to SEQ ID NO:1: ·Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124I+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171H+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308E+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+R127P+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+D111R+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+S163P+D171F+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+D111R+T120V+E150N+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+E150N+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V106N+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q

[0099] The hexosaminidase variants disclosed herein have at least 60% sequence identity to the parent hexosaminidase without substitutions in the variant. The variants may, for example, have at least 65%, at least 70%, at least 75% or at least 80% sequence identity to the parent hexosaminidase. The hexosaminidase variants may, for example, have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, such as at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity to the parent hexosaminidase.

[0100] In particular, any of the hexosaminidase variants disclosed herein have at least 60% sequence identity to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. The hexosaminidase variants may, for example, have at least 65%, at least 70%, at least 75% or at least 80% sequence identity to any of SEQ ID NO:1, 2 or 3. The hexosaminidase variants may, for example, have at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, such as at least 96%, at least 97% or at least 98%, but less than 100% sequence identity to any of SEQ ID NO:1, 2 or 3.

[0101] In one aspect, the hexosaminidase variant of the invention has at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, such as at least 96%, at least 97%, at least 98% or at least 99%, but less than 100%, sequence identity to SEQ ID NO:2.

[0102] In another aspect, the hexosaminidase variant of the invention has at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95%, such as at least 96%, at least 97%, at least 98% or at least 99%, but less than 100%, sequence identity to SEQ ID NO:3.

[0103] In one aspect the number of modifications in a variant of the invention compared to SEQ ID NO: 1, 2 or 3 is between 1 and 20, such as between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications.

[0104] The variants described herein may further comprise an extension of one or more amino acids at the N-terminus and / or C-terminus. Alternatively, the variants may further comprise a truncation of one or more amino acids at the N-terminus and / or C-terminus.

[0105] Thus, the present invention includes variants as disclosed herein comprising an extension of one or more amino acids at the N-terminus and / or C-terminus, or one or more truncations at the N-terminus and / or C-terminus, which variants have hexosaminidase activity and have improved detergent stability compared to the polypeptide of SEQ ID NO:1, or compared to the polypeptide of SEQ ID NO:2 or SEQ ID NO:3.

[0106] Variants of the present invention may optionally include other amino acid changes that are 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- 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 net charge or another function, such as polyhistidine tails, antigenic epitopes, or binding domains.

[0107] Examples of conservative substitutions are in the group consisting of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and low molecular weight amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not change the specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0108] Alternatively, the amino acid changes may be of such a nature that the physicochemical properties of the polypeptide are altered, for example, the amino acid changes may result in increased thermostability, altered substrate specificity, altered pH optimum, etc.

[0109] Essential amino acids in a polypeptide can be identified according to techniques 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 hexosaminidase activity to identify amino acid residues essential for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations of 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. Identification of essential amino acids can also be inferred from alignment with related polypeptides and / or from sequence homology and conserved catalytic mechanism with related polypeptides or with polypeptides / proteins (typically with similar three-dimensional structure, function and significant sequence similarity) derived from a common ancestor within a polypeptide or protein family. Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of polypeptides. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold”, Nature 596:583-589.

[0110] The variant may be composed of 300 to 350 amino acids, for example, 310 to 340, 315 to 335, or 320 to 330 amino acids.

[0111] In one embodiment, the variant has improved stability, in particular improved storage stability, i.e. improved stability under storage in a detergent composition, compared to the parent enzyme. As mentioned above, improved stability may be expressed, for example, by a half-life improvement factor (HIF), as described in the Examples herein.

[0112] In one embodiment, the hexosaminidase variant has improved stability, expressed as half-life improvement factor (HIF), compared to the parent hexosaminidase, i.e. the HIF value is greater than 1. Preferably, the variant has a half-life improvement factor compared to the parent of at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9 or at least 2.0, e.g. determined as described in the Examples herein. In preferred embodiments, the variant may have a half-life improvement factor compared to the parent of at least 2.5, at least 3.0, at least 3.5 or at least 4.0.

[0113] The hexosaminidase variants of the present invention are preferably isolated, and more preferably purified, using standard protein purification methods known in the art.

[0114] Generating variants The present invention also relates to a method for obtaining a variant having hexosaminidase activity, comprising the steps of: (a) introducing into a parent hexosaminidase substitutions at one or more positions, preferably two, three or four positions, corresponding to positions 163, 227, 252 and 309 of SEQ ID NO:1; and substitutions at one or more positions corresponding to positions 106, 111, 120, 124, 127, 150, 170, 171, 178, 199, 208, 254, 255 and 278 of SEQ ID NO:1; (b) recovering variants; Including, The variant relates to a method wherein the variant has hexosaminidase activity and has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0115] Preferably, the method comprises the step of introducing into the parent hexosaminidase (i) two, three or four substitutions selected from the group consisting of S163P, N227T, N252P and K309E; and (ii) at least one substitution selected from the group consisting of V106M, V106N, D111R, T120V, Y124I, Y124K, Y124L, R127P, E150N, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D and I278V, compared to SEQ ID NO:1. Preferred substitutions to be introduced into the parent hexosaminidase in (ii) include V106M / N, D111R, T120V, E150N, D171F and I278V, for example at least two of said substitutions. Particularly preferred substitutions include one, two or three of D111R, T120V and E150N.

[0116] The method preferably further comprises introducing into the parent hexosaminidase at least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of said substitutions, compared to SEQ ID NO: 1. The method more preferably comprises introducing into the parent hexosaminidase one or more substitutions selected from the group consisting of Q3I, A49W and N59E, preferably two or all three of said substitutions; or one or more substitutions selected from the group consisting of Q3F, V140I, Q215K and N267T, preferably two, three or all four of said substitutions, compared to SEQ ID NO: 1.

[0117] In other embodiments, the method comprises: (a) introducing into a parent hexosaminidase any of the sets of substitutions listed above under the heading "Variant", where the position numbers are based on the numbering of SEQ ID NO:1, and where the variant has at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, and where the variant has hexosaminidase activity; and (b) recovering the variant.

[0118] Preferably, the variants obtained by the methods disclosed herein have at least 80%, preferably at least 85%, more preferably at least 90%, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 98% sequence identity compared to SEQ ID NO:2 or SEQ ID NO:3.

[0119] Variants can be prepared using any mutagenesis technique known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, DNA shuffling, and the like.

[0120] Polynucleotides The present invention also relates to polynucleotides encoding the variants of the invention.

[0121] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0122] In certain aspects, the polynucleotide is isolated.

[0123] In another embodiment, the polynucleotide is purified.

[0124] Nucleic Acid Constructs The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant of the present invention, operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Examples of control sequences that may be used include promoters, terminators, mRNA stabilizers, leader sequences, polyadenylation sequences, signal peptides, propeptides, regulatory sequences, and transcription factors, all of which are well known in the art.

[0125] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0126] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding the variant of the invention, a promoter, and transcriptional and translational termination signals. Various nucleotides and control sequences can be linked together to generate a recombinant expression vector that contains one or more convenient restriction sites, allowing for the insertion or substitution of a polynucleotide encoding the variant at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide, or a nucleic acid construct containing the polynucleotide, into a vector suitable for expression. In creating an expression vector, the coding sequence is placed in the vector such that the coding sequence is operably linked to suitable control sequences for expression.

[0127] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA techniques and can result in expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed circular plasmid. Expression vectors suitable for recombinant expression are well known in the art, as are methods for introducing the vector into a host cell.

[0128] host cell 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 the variants of the present invention.

[0129] The construct or vector containing the polynucleotide is introduced into a host cell, whereby the construct or vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The choice of host cell will depend largely on the gene encoding the variant and its source. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or at least two copies, for example, three, four, five or more copies.

[0130] The host cell can be any microbial cell, e.g., a prokaryotic or fungal cell, useful for the recombinant production of the polypeptides of the invention.

[0131] Prokaryotic host cells can be any gram-positive or gram-negative bacteria, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0132] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus The cell may be any Bacillus cell, including cells of Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0133] The fungal host cell may be a yeast cell or a filamentous fungal cell.

[0134] Filamentous fungal host cells include, for example, those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, and Myceliophthora. , Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma, or Fusarium cell, in a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0135] In one embodiment, the host cell is isolated, preferably purified.

[0136] Generation method The present invention also relates to a method of producing a variant of the invention, comprising: (a) culturing a recombinant host cell of the invention under conditions conducive to the production of the variant; and optionally (b) recovering the variant.

[0137] The host cells are cultured in a nutrient medium suitable for the production of the variants, using methods known in the art. For example, the cells can be cultured by shake flask culture or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter in a suitable medium and under conditions that allow expression and / or isolation of the variants. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., in the catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0138] Variants can be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzyme assays to determine the relative or specific activity of the variant.

[0139] The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one embodiment, the whole fermentation broth is recovered. In another embodiment, the cell-free fermentation broth containing the polypeptide is recovered.

[0140] The variants may be purified by various procedures known in the art to obtain substantially pure variants and / or 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).

[0141] In an alternative embodiment, the variant is not recovered from the medium.

[0142] composition The present invention further relates to a cleaning composition comprising at least one hexosaminidase variant according to the invention and at least one cleaning auxiliary ingredient. The cleaning composition may be used, for example, to improve deep cleaning effect, i.e. destruction or removal effect of biofilm or its components, on items such as fabrics, for example, to prevent and / or reduce the stickiness of items, to pre-treat stains on items, to prevent and / or reduce redeposition of soils during the wash cycle, to prevent and / or reduce adhesion of soils to items, to maintain or improve the whiteness of items, and / or to prevent and / or reduce malodour of items. The hexosaminidase variants of the present invention are useful in various types of detergent compositions, such as powder and liquid cleaning compositions, and also, for example, in single unit dose compositions.

[0143] The compositions may contain one or more cleaning adjunct ingredients selected from the group consisting of surfactants, builders, flocculating aids, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, mud stain removal / anti-redeposition agents, brighteners, suds suppressors, dyes, fragrances, structural elastomers, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, defoamers, dispersants, processing aids, and / or pigments.

[0144] Cleaning compositions typically contain surfactants, and usually other cleaning adjunct ingredients such as builders or mud / soil removal / anti-redeposition agents.

[0145] The cleaning adjunct ingredient may be one or more enzymes other than hexosaminidase. The one or more enzymes may be selected from the group consisting of, for example, protease, amylase, lipase, cutinase, cellulase, DNase, endoglucanase, xyloglucanase, pectinase, pectin lyase, xanthanase, peroxidase, haloperoxygenase, catalase and mannanase. Specific enzymes suitable for the detergent composition of the present invention are described below.

[0146] The cleaning composition can be formulated into any suitable form, such as a bar, a homogenous tablet, a tablet with two or more layers, a pouch with one or more compartments, a regular or compressed powder, a granule, a paste, a gel, or a regular, compressed or concentrated solution.Thus, the cleaning composition can be, for example, a liquid detergent, or a powder or granular detergent, optionally in a "concentrated" or "compressed" form.It can also be in the form of a single unit dose composition.

[0147] The amount of hexosaminidase in the cleaning composition may vary depending on factors such as the degree of concentration or compactness of the composition and the desired concentration of hexosaminidase in the cleaning solution. Hexosaminidase is usually included in the cleaning composition in an amount of up to about 10,000 ppm, typically up to about 5000 ppm or up to about 2000 ppm. Hexosaminidase may, for example, be included in the cleaning composition at a level of 1 ppm to 10,000 ppm, such as 10 ppm to 5000 ppm, 20 ppm to 2000 ppm, 50 ppm to 1000 ppm, 80 ppm to 600 ppm, or 100 ppm to 500 ppm. The unit "ppm" in this context is intended to refer to mg / l for enzyme added to liquid compositions (e.g., liquids, gels, etc.) and mg / kg for enzyme added to solid compositions (e.g., powders, granules, tablets, etc.).

[0148] In some embodiments, the detergent composition is a liquid or powder laundry detergent, e.g., suitable for cleaning at high temperatures and / or pH, e.g., 40° C. or higher and / or pH 8 or higher. In some embodiments, the detergent composition is a liquid or powder laundry detergent, e.g., suitable for cleaning at low temperatures and / or pH, e.g., 20° C. or lower and / or pH 6 or lower. The detergent may also be formulated as a unit dose detergent and / or compact detergent, optionally with minimal or no water. The detergent may also be a dishwashing detergent. Such laundry and dishwashing detergents may be phosphate-free.

[0149] Surfactants The surfactant may be selected from nonionic, anionic, and / or amphoteric surfactants, preferably anionic or nonionic surfactants, although amphoteric surfactants may also be used. In general, bleach-stable surfactants are preferred. Preferred anionic surfactants include sulfate surfactants, particularly alkyl ether sulfates, especially C9-C15 alcohol ether sulfates, C12-C15 primary alcohol ethoxylates, C8-C16 sulfate esters, and C10-C14 sulfate esters, such as monododecyl sulfate esters. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefinsulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfofatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and mono-esters of sulfosuccinic acid, or salts of fatty acids (soaps), and combinations thereof.

[0150] The anionic surfactants are preferably added to the detergent in the form of salts, in which suitable cations are alkali metal ions such as sodium, potassium, lithium and ammonium salts, for example (2-hydroxyethyl)ammonium, bis(2-hydroxyethyl)ammonium and tris(2-hydroxyethyl)ammonium salts. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APEs), nonylphenol ethoxylates (NPEs), alkyl polyglycosides (APGs), alkoxylated amines, fatty acid monoethanolamides (FAMs), fatty acid diethanolamides (FADAs), ethoxylated fatty acid monoethanolamides (EFAMs), propoxylated fatty acid monoethanolamides (PFAMs), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GAs or fatty acid glucamides, FAGAs), as well as products available under the trade names SPAN and TWEEN®, and combinations thereof. Commercially available non-ionic surfactants include Plurafac™, Lutensol™, and Pluronic® from BASF, the Dehypon™ series from Cognis, and the Genapol™ series from Clariant.

[0151] builder The builders are preferably selected from among phosphates, sodium citrate builders, sodium carbonate, sodium silicate, sodium aluminosilicate (zeolites). Suitable builders include alkali metal or ammonium phosphates, polyphosphates, phosphonates, polyphosphates, carbonates, bicarbonates, borates, citrates, and polycarboxylates. Citrate builders, such as citric acid and its soluble salts (especially the sodium salt), are polycarboxylate builders. Citrates can be used in combination with zeolites, silicates such as BRITESIL types, and / or layered silicate builders. The builders are preferably added in an amount of about 0-65% by weight, for example about 5% to about 50% by weight. In laundry detergents, the builder level is typically about 40-65% by weight, especially about 50-65% by weight, especially 20-50% by weight. The builders and / or cobuilders may be chelating agents, in particular forming water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in cleaning detergents can be utilized.Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., Hoechst's SKS-6), and (carboxymethyl)inulin (CMI), and combinations thereof.Further non-limiting examples of builders include citrates, chelators such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl or alkenyl succinates.Additional examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine-N,N-diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid, N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-monoacetic acid (ASMA), and aspartic acid-N,N-monoacetic acid (ASMA). diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranil Phosphonates suitable for use herein include 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentaerythritol phosphate (DTP), ... Examples of suitable detergent co-builders include takis(methylene phosphonic acid) (DTMPA or DTPMPA or DTPMP), nitrilotris(methylene phosphonic acid) (ATMP or NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), and hexamethylenediaminetetrakis(methylene phosphonic acid) (HDTMP). The composition may also contain 0 to 50% by weight, for example about 5% to about 30%, of a detergent co-builder. The detergent composition may contain a co-builder alone or in combination with a builder, for example a zeolite builder.Non-limiting examples of co-builders include homopolymers of polyacrylates or their copolymers, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA) or polyaspartic acid. Further exemplary builders and / or co-builders are described, for example, in WO 2009 / 102854 and US Pat. No. 5,977,053. In some embodiments, the builder is a non-phosphorus builder, such as citric acid and / or methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and / or salts thereof. In addition, in the case where the preferred builder comprises citrate and / or methylglycine-N,N-diacetic acid (MGDA) and / or glutamic acid-N,N-diacetic acid (GLDA) and / or salts thereof, the liquid composition may be phosphate-free.

[0152] bleaching ingredients The cleaning composition may contain 0 to 30% by weight of a bleaching system, such as from about 1% to about 20%. Any bleaching system containing ingredients known in the art for use in cleaning detergents may be utilized. Suitable bleaching system ingredients include a source of hydrogen peroxide; a source of peracid; and a bleach catalyst or bleach booster.

[0153] Sources of hydrogen peroxide: Suitable sources of hydrogen peroxide are inorganic persalts, including sodium percarbonate and sodium perborate (usually the monohydrate or tetrahydrate), as well as alkali metal salts such as hydrogen peroxide-urea (1 / 1).

[0154] Source of peracid: Peracid can be (a) directly incorporated as preformed peracid, or (b) formed in situ in the wash liquor from hydrogen peroxide and a bleach activator (perhydrolysis), or (c) formed in situ in the wash liquor from hydrogen peroxide and a perhydrolase and a suitable substrate for the latter, such as an ester.

[0155] a) Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids such as peroxybenzoic acid and its ring-substituted derivatives, peroxy-α-naphthoic acid, peroxyphthalic acid, peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthalimidoperoxyhexanoic acid (PAP)], and o-carboxybenzamidoperoxycaproic acid; aliphatic and aromatic diperoxydicarboxylic acids such as diperoxydodecanedioic acid, diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassic acid, 2-decyldiperoxybutanedioic acid, and diperoxyphthalic acid, diperoxyisophthalic acid, and diperoxyterephthalic acid; perimidic acids; peroxymonosulfuric acid; peroxydisulfuric acid; peroxyphosphoric acid; peroxysilicic acid; and mixtures of the above compounds. It will be appreciated that the peracids mentioned may in some cases best be added as a suitable salt, for example an alkali metal salt (eg Oxone®) or an alkaline earth metal salt.

[0156] b) Suitable bleach activators include those belonging to the classes of esters, amides, imides, nitriles or anhydrides, and salts thereof, as appropriate. Suitable examples include tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), sodium 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), sodium 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoic acid (DOBA), sodium 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of bleach activators of interest is disclosed in EP 624154, of which acetyl triethyl citrate (ATC) is particularly preferred. ATC or short chain triglycerides like triacetin have the advantage of being environmentally friendly. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are effective bleach activators. Finally, ATC is multifunctional because the citrate released during the perhydrolysis reaction can function as a builder.

[0157] Bleaching catalysts and accelerators: The bleaching system may also include bleaching catalysts or accelerators.Some non-limiting examples of bleaching catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts, and manganese triazacyclononane (MnTACN) catalysts;particularly preferred are 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3-TACN) or 1,2,4,7-tetramethyl-1,4,7-triazacyclononane (Me4-TACN), especially complexes of manganese with Me3-TACN, such as: The dinuclear manganese complexes are [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2 and [2,2',2"-nitrilotris(ethane-1,2-diylazanilylidene-κN-methanilylidene)triphenolato-κ3O]manganese(III). The bleach catalyst can also be other metal compounds, such as iron or cobalt complexes. Other suitable bleach catalysts are acylhydrazone catalysts, such as those disclosed in U.S. Patent Application Publication No. 2014 / 0323381.

[0158] In some embodiments where a source of peracid is included, the source is of the formula: [ka] (iii) and mixtures thereof, wherein each R1 is independently a branched alkyl group containing 9 to 24 carbons or a linear alkyl group containing 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing 9 to 18 carbons or a linear alkyl group containing 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl, and isopentadecyl, may be used.

[0159] Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, EP 1 867708 (vitamin K), and WO 2007 / 087242. Suitable photobleaches can include, for example, sulfonated zinc or aluminum phthalocyanines.

[0160] The selection of detergent composition may include, in the case of fabric care, the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is performed, and the consideration of detergent product formulation. The ingredients mentioned below are categorized by general headings according to functionality, but this should not be interpreted as limiting. Because, as will be understood by those skilled in the art, ingredients including the exemplary non-limiting ingredients shown below may also include additional functionality.

[0161] Hydrotropes The detergent composition may contain 0-10% by weight, such as 0-5% by weight, such as about 0.5-5%, or about 3%-5% of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0162] polymer The detergent composition may contain 0-10% by weight of polymer, e.g., 0.5-5%, 2-5%, 0.5-2%, or 0.2-1%. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as described above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning, and / or antifoam properties. Some polymers may have two or more of the above properties and / or two or more of the motifs listed below. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, polyaspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxides and polypropylene oxides (PEO-PPO), and diquaternium ethoxysulfate.Other exemplary polymers are disclosed, for example, in WO 2006 / 130575.Also contemplated are salts of the above-mentioned polymers.

[0163] Fabric color toning agent The detergent composition of the present invention may also include a fabric hueing agent, such as a dye or pigment, which, when incorporated into the detergent composition, can be deposited on the fabric when the fabric contacts the washing liquor containing the detergent composition, thereby changing the color shade of the fabric by absorbing / reflecting visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents absorb at least a portion of the visible light spectrum, thus changing the color shade of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes classified as Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red in the Color Index (CI) classification, or mixtures thereof, as described in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276, and EP 1876226 (incorporated herein by reference). The detergent composition preferably comprises from about 0.00003% to about 0.2%, from about 0.00008% to about 0.05%, or from about 0.0001% to about 0.04% by weight of the fabric hueing agent. The composition may contain from 0.0001% to about 0.2% by weight of a fabric hueing agent, which may be preferred, particularly when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0164] enzyme The detergent composition may comprise one or more additional enzymes such as proteases, lipases, cutinases, amylases, DNases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, hexosaminidases, oxidases such as laccases, and / or peroxidases.

[0165] In general, the properties of the enzyme selected should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.) and the enzyme should be included in an effective amount.

[0166] Cellulase Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered variants are also included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as the fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and WO 89 / 09259.

[0167] Particularly suitable cellulases are alkaline or neutral cellulases, which have advantages in terms of color protection.Examples of such cellulases include those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940.Other examples include cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, U.S. Patent No. 5,457,046, U.S. Patent No. 5,686,593, U.S. Patent No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and WO 99 / 001544.

[0168] Other cellulases include endo-beta-1,4-glucanase enzymes having a sequence that is at least 97% identical to amino acid sequence positions 1 to 773 of SEQ ID NO:2 of WO 2002 / 099091, or family 44 xyloglucanases, which have a sequence that is at least 60% identical to amino acid sequence positions 40 to 559 of SEQ ID NO:2 of WO 2001 / 062903.

[0169] Commercially available cellulases include Celluzyme™ and Carezyme™ (Novozymes A / S), Carezyme Premium™ (Novozymes A / S), Celluclean™ (Novozymes A / S), Celluclean Classic™ (Novozymes A / S), Cellusoft™ (Novozymes A / S), Whitezyme™ (Novozymes A / S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0170] Proteases Suitable proteases may be of any origin, but preferably of bacterial or fungal origin, and may be in the form of protein engineered or chemically modified variants. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease may be, for example, from the S1 family, such as trypsin, or the S8 family, such as subtilisin. The metalloprotease may be, for example, thermolysin, for example from the M4 family, or another metalloprotease, for example from the M5, M7, or M35 family.

[0171] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subdivisions: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family.

[0172] Although proteases suitable for detergent use can be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases are commonly obtained from bacteria, particularly Bacillus. Examples of Bacillus species from which subtilases are derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168, as well as, for example, protease PD138 (described in WO 93 / 18140). Other useful proteases include, for example, those described in WO 01 / 16285 and WO 02 / 16547.

[0173] Examples of trypsin-like proteases include the Fusarium proteases described in WO 94 / 25583 and WO 2005 / 040372, and the chymotrypsin proteases from Cellumonas, described in WO 2005 / 052161 and WO 2005 / 052146.

[0174] Examples of metalloproteases include the neutral metalloproteases described in WO 2007 / 044993, such as those derived from Bacillus amyloliquefaciens, and the metalloproteases described in WO 2015 / 158723 and WO 2016 / 075078.

[0175] Examples of useful proteases include those described in WO 89 / 06279, WO 92 / 19729, WO 96 / 34946, WO 98 / 20115, WO 98 / 20116, WO 99 / 11768, WO 01 / 44452, WO 03 / 006602, WO 04 / 006602, WO 05 / 006602, WO 06 / 006602, WO 07 / 006602, WO 08 / 006602, WO 09 / 006602, WO 10 / 006602, WO 11 / 006602, WO 12 / 006602, WO 13 / 006602, WO 14 / 006602, WO 15 / 006602, WO 16 / 006602, WO 17 / 006602, WO 18 / 006602, WO 19 ... and protease variants described in WO 2004 / 003186, WO 2004 / 041979, WO 2007 / 006305, WO 2011 / 036263, WO 2014 / 207227, WO 2016 / 087617, and WO 2016 / 174234. Preferred protease variants are, for example: S3T, V4I, S9R, S9E, A15T, S24G, S24R, K27R, N42R, S55P, G59E, G59D, N60D, N60E, V66A, N74D, S85R, A96S, S97G, S97D, S97A, S97SD, S99E, S99D, S99G, S99M, S99N, S99R, S99H, S101A, V102I, V102Y, V102N, S104A, G116V, G116R, H118D, H118N, A120S, S1 26L, P127Q, S128A, S154D, A156E, G157D, G157P, S158E, Y161A, R164S, Q176E, N179E, S182E, Q185N, A188P, G189E, V193M, N198D, V199I, Q200L, Y203W, S206G, L211Q, L211D, N212D, N212S, M216S, A226V, K229L, Q230H, Q239R, N246K, S253D, N255W, N255D, N255E, L256E, L256D and R269H, wherein the position numbers correspond to the positions of the Bacillus lentus protease shown in SEQ ID NO: 1 of WO 2016 / 001449.A protease variant having one or more of these mutations is preferably a variant of Bacillus lentus protease (Savinase®, also known as subtilisin 309) as set forth in SEQ ID NO: 1 of WO 2016 / 001449, or a variant of Bacillus amyloliquefaciens protease (BPN') as set forth in SEQ ID NO: 2 of WO 2016 / 001449. Such a protease variant preferably has at least 80% sequence identity to SEQ ID NO: 1 or to SEQ ID NO: 2 of WO 2016 / 001449.

[0176] Another protease of interest is the alkaline protease from Bacillus lentus DSM 5483, described, for example, in WO 91 / 02792, and variants thereof described, for example, in WO 92 / 21760, WO 95 / 23221, EP 1921147, EP 1921148, and WO 2016 / 096711.

[0177] Alternatively, the protease may be a variant of the TY145 protease having SEQ ID NO:1 of WO 2004 / 067737, for example a variant comprising substitutions at one or more positions corresponding to positions 27, 109, 111, 171, 173, 174, 175, 180, 182, 184, 198, 199 and 297 of SEQ ID NO:1 of WO 2004 / 067737, said protease variant having at least 75% but less than 100% sequence identity to SEQ ID NO:1 of WO 2004 / 067737. TY145 variants of interest are described, for example, in WO 2015 / 014790, WO 2015 / 014803, WO 2015 / 014804, WO 2016 / 097350, WO 2016 / 097352, WO 2016 / 097357, and WO 2016 / 097354.

[0178] Suitable commercially available protease enzymes include Alcalase®, Duralase®, Durazym®, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Blaze Evity® 25 ... Products sold under the trade names Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In, Progress® Key and Progress® Excel (Novozymes A / S), Maxatase™, Maxacal™, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, FN4 exTM, Excellase®, Excellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz® P100, Preferenz® P300, Purafect Prime, Preferenz P110™, Effectenz P1000™, Purafect®, Effectenz P1050™, Purafect® Ox, Effectenz™ P2000, Purafast™, Properase®, Opticlean™, and Optimase® (Danisco / DuPont), BLAP (sequence shown in Figure 29 of U.S. Pat. No. 5,352,604) and variants thereof (Henkel AG), and KAP (Bacillus alkalophilus) from Kao. Examples of such antibacterial agents include those sold under the trade name Bacillus subtilisin.

[0179] Lipase and cutinase Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include those from the genus Thermomyces, such as T. lanuginosus (formerly Humicola lanuginosa), as described in EP 258068 and EP 305216. lanuginosa), cutinases from Humicola, e.g. H. insolens (WO 96 / 13580), Pseudomonas (some of which have now been renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconinensis (P.Lipases from strains of Pseudomonas wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), cutinases from Magnaporthe grisea (WO 10 / 107560), cutinases from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipases from Thermobifida fusca (WO 11 / 084412), Geobacillus stearothermophilus lipase (WO 11 / 084417), Bacillus subtilis cutinases (U.S. Pat. No. 5,389,536), and the like. Examples include lipases from S. subtilis (WO 11 / 084599), as well as lipases from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0180] Other examples include lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0181] Preferred commercially available lipase products include Lipolase™, Lipex™; Lipolex™, and Lipoclean™ (Novozymes A / S), Lumafast (originally manufactured by Genencor), and Lipomax (originally manufactured by Gist-Brocades).

[0182] Further examples include lipases, sometimes referred to as acyltransferases or perhydrolases, such as acyltransferases with homology to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782), perhydrolases from the CE 7 family (WO 09 / 67279), and variants of M. smegmatis perhydrolase, in particular the S54V variant used in the product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0183] amylase Suitable amylases that may be used with the hexosaminidases of the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin, including chemically modified or protein engineered variants. Amylases include, for example, alpha-amylases obtained from special strains of Bacillus, such as Bacillus licheniformis, as described in more detail in GB Patent No. 1,296,839.

[0184] Suitable amylases include amylases having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 1. Preferred variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

[0185] Different suitable amylases include the amylase having SEQ ID NO: 6 in WO 02 / 010355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 is one having deletions at positions 181 and 182 and a substitution at position 193.

[0186] Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof with 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those with substitutions, deletions or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase obtained from B. amyloliquefaciens as set forth in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 comprises the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It has the following characteristics.

[0187] Further suitable amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions at positions R181 and G182, or H183 and G184.

[0188] Additional amylases that may be used are those having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2 or SEQ ID NO:7 of WO 96 / 023873 or variants thereof having 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:7 are those having substitutions, deletions or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476, using the numbering of SEQ ID NO:2 of WO 96 / 023873. More preferred variants are those having deletions at two positions selected from positions 181, 182, 183 and 184, such as positions 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 have a deletion of positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0189] Other amylases that may be used include amylases having SEQ ID NO: 2 in WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 in WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 include those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0190] Further suitable amylases include those having SEQ ID NO: 2 of WO 09 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 include those having C-terminal truncations and / or substitutions, deletions or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 include substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO:2 include substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0191] Other suitable amylases are the alpha-amylases having SEQ ID NO:12 in WO 01 / 66712, or variants having at least 90% sequence identity to SEQ ID NO:12. Preferred amylase variants include those that have substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, N195F, R320K, and R458K, as well as variants having additional substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, and most preferred are variants having additional substitutions at all of these positions.

[0192] Other examples include amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087.

[0193] Commercially available amylases include Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, and BAN™ (from Novozymes A / S), as well as Rapidase™, Purastar™ / Effectenz™, Powerase, and Preferenz S100 (from Genencor International Inc. / DuPont).

[0194] DNase The term "DNase" refers to a polypeptide having DNase (deoxyribonuclease) activity that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thus degrading DNA. DNase polypeptides have been found to be useful in deep cleaning of microbial biofilms, which may be present on surfaces such as fabrics or dishes or other hard surfaces and consist of extracellular DNA, proteins, and a matrix of extracellular polymeric substances (EPS) composed of polysaccharides.

[0195] The DNase polypeptide is typically a microbial enzyme, preferably of fungal or bacterial origin, or a genetically engineered variant of a microbial DNase.

[0196] Suitable bacterial DNases may be obtained, for example, from species of the genus Bacillus and related genera (see Patel and Gupta, Int. J. Syst. Evol. Microbiol. 2020;70:406-438 (proposing six new genera of the family Bacillaceae from species previously classified as belonging to the genus Bacillus)), such as from the genera Bacillus, Cytobacillus, Metabacillus, Alkalihalobacillus, Rossellomorea, or Mesobacillus. Examples of species from which DNase can be obtained include Bacillus licheniformis, Bacillus subtilis, Sutcliffiella horikoshii, Cytobacillus horneckiae, Metabacillus indicus, Alkalihalobacillus algicola, Rossellomorea vietnamensis, Alkalihalobacillus hwajinpoensis, Metabacillus indicus, Mesobacillus campisalis, and others. Preferred bacterial DNases include those obtained from Metabacillus indicus (formerly known as Bacillus cibi) and variants thereof.

[0197] DNases can also be obtained from fungal species. Examples of preferred fungal DNases are those obtained from the genus Aspergillus, e.g., Aspergillus oryzae, Trichoderma, e.g., Trichoderma harzianum, Vibressa, e.g., Vibressea flavovirens, Morchella, e.g., Morchella costata, and Rhizoctonia, e.g., Rhizoctonia solani, and variants thereof. Preferred fungal DNases include those obtained from Aspergillus oryzae and variants thereof.

[0198] Suitable DNases, DNase variants, and their use in detergent compositions are described, for example, in WO 2014 / 087011, WO 2015 / 155350, WO 2015 / 155351, WO 2017 / 060475, WO 2017 / 060493, WO 2017 / 060505, WO 2017 / 064269, WO 201 These are described in WO 8 / 011277, WO 2018 / 177203, WO 2018 / 177936, WO 2018 / 177938, WO 2019 / 081724, WO 2019 / 081721, WO 2021 / 130167, WO 2022 / 194668 and WO 2022 / 194673.

[0199] Peroxidase / Oxidase The peroxidase may be any fragment exhibiting peroxidase activity that may be included by or derived from the enzyme classification EC 1.11.1.7 described by the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (IUBMB). Suitable peroxidases include those of plant, bacterial, or fungal origin. Chemically modified or protein engineered variants are included. Examples of useful peroxidases include peroxidases from Coprinopsis, e.g. C. cinerea (EP 179,486), and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Peroxidases may also include haloperoxidase enzymes, such as chloroperoxidase, bromoperoxidase, and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (EC 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions. In one embodiment, the haloperoxidase is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium peroxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method, a vanadate-containing haloperoxidase is combined with a source of chloride ions.Haloperoxidases have been isolated from many different fungi, particularly from the fungal group of dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis. Haloperoxidases have also been isolated from bacteria, such as Pseudomonas, e.g., P. pyrrocinia, and Streptomyces, e.g., S. aureofaciens. In a preferred embodiment, the haloperoxidase is derived from a Curvularia species, in particular Curvularia verruculosa or Curvularia inaequalis, e.g. C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or Drechslera hartlebii as described in WO 01 / 79459, Dendrophiella salina as described in WO 01 / 79458, salina, Phaeotrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium species as described in WO 01 / 79460.

[0200] Oxidases include any laccase enzyme encompassed by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom that exhibits laccase activity, or a compound exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0201] Preferred laccase enzymes include those of bacterial origin. The enzymes may be obtained from plants, bacteria or fungi (including filamentous fungi and yeasts).

[0202] Suitable examples of fungal origin include Bacillus, Neurospora, e.g. N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, For example, T. villosa and T. versicolor, Rhizoctonia species such as R. solani, Coprinopsis species such as C. cinerea, C. comatus, C. friesii, and C. plicata. atilis, Psathyrella such as P. condoelleana, Panaeolus such as P. papilionaceus, Myceliophthora such as M. thermophila, Schytalidium such as S. thermophilum, Polyporus such as P. pinsitus, Phlebia such as P. radiata (WO 92 / 01046), or Coriolus such as C. hirsutus (JP 2238885 A).

[0203] Suitable examples of bacterial origin include laccase inducible from strains of Bacillus.

[0204] Laccases obtained from the genus Coprinopsis or Myceliophthora; laccases obtained from Coprinopsis cinerea as disclosed in WO 97 / 08325; or laccases obtained from Myceliophthora thermophila as disclosed in WO 95 / 33836 are preferred.

[0205] Detergent enzymes can be included in detergent compositions by adding separate additives containing one or more enzymes or by adding combined additives containing these enzymes. The detergent additives of the present invention, i.e., separate additives or combined additives, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, non-shattering granules, liquids, stabilized liquids, and slurries.

[0206] Non-shattering granules can be produced, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycols, PEGs) with an average molecular weight of 1000-20000; ethoxylated nonylphenols with 16-50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12-20 carbon atoms and there are 15-80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed techniques are given in GB 1483591. Liquid enzyme preparations may be stabilized, for example, by adding polyols, such as propylene glycol, sugars or sugar alcohols, lactic acid, or boric acid, according to established methods. The protected enzyme may be prepared according to the method disclosed in EP238216.

[0207] Dispersants The detergent composition of the present invention can also contain a dispersant. The powder detergent can include a dispersant. Suitable water-soluble organic materials include homo- or copolymeric acids or their salts, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by not more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.

[0208] Dye transfer inhibitor The cleaning compositions of the present invention may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, and polyvinylimidazoles, or mixtures thereof. When present in the subject compositions, the dye transfer inhibitors may be present at a level of about 0.0001% to about 10%, about 0.01% to about 5%, or even about 0.1% to about 3% by weight of the composition.

[0209] Optical brightener The detergent composition may also preferably contain additional ingredients that can adjust the color tone of the article being cleaned, such as fluorescent or optical brighteners. When present, the brightener is preferably at a level of about 0.01% to about 0.5%. Any fluorescent brightener suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used fluorescent brighteners belong to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyl-distyryl derivatives. Examples of fluorescent whitening agents of the diaminostilbene-sulfonic acid derivative type are: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydro) Examples of suitable optical brighteners include sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate, sodium salts of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and sodium salts of 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred optical brighteners are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)-disulfonate. A preferred optical brightener is also commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is the disodium salt of 4,4'-bis-(sulfostyryl)-biphenyl, also known as disodium distyrylbiphenyl disulfonate.Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins.

[0210] Suitable fluorescent brightener levels include lower levels of about 0.01, 0.05, about 0.1, or about 0.2% by weight, to upper levels of 0.5 or 0.75% by weight.

[0211] Dirt Release Polymer The detergent composition may also include one or more soil release polymers that aid in removing soil from fabrics, such as cotton and polyester fabrics, and in removing hydrophobic soil from polyester fabrics. The soil release polymer may be, for example, a nonionic or anionic terephthalate-based polymer, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Chapter 7, Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc. Another type of soil release polymer is an amphiphilic alkoxylated grease cleaning polymer that includes a core structure and a plurality of alkoxylate groups attached to the core structure. The core structure may include a polyalkyleneimine structure or a polyalkanolamine structure, as detailed in WO 2009 / 087523 (herein incorporated by reference). Additionally, random graft copolymers are suitable soil release polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (incorporated herein by reference). Other soil release polymers are substituted polysaccharide structures, especially substituted cellulosic structures, such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003 / 040279 (both of which are incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

[0212] Anti-redeposition agent The detergent compositions of the present invention may also include one or more anti-redeposition agents, such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulosic polymers described above under soil release polymers may also function as anti-redeposition agents.

[0213] Rheology Modifiers The detergent compositions of the present invention may also contain one or more rheology modifiers, structurants, or thickeners different from the viscosity reducing agent. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers that impart shear-thinning properties to the aqueous liquid matrix of the liquid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted by methods known in the art, for example as shown in EP 2169040.

[0214] Other suitable adjunct materials include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, disinfectants, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, suds suppressors, solvents, and liquid detergent structurants and / or elasticizing agents.

[0215] Other materials Also, any detergent ingredient known in the art can be used in the cleaning composition of the present invention.Other optional detergent ingredients include anticorrosive agents, shrinkage inhibitors, soil redeposition inhibitors, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric softeners including clay, fillers / processing aids, optical brighteners / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil suspension agents, softeners, foam suppressors, tan inhibitors, and wicking agents, all of which are known in the art and used in detergents.The selection of such ingredients is well within the skill of the person skilled in the art.

[0216] Formulation of detergent products The detergent composition may be in any convenient form, such as a bar, a homogenous tablet, a tablet having two or more layers, a plain or compressed powder, a granule, a paste, a gel, or a plain, compressed or concentrated liquid. Other detergent formulation forms include single unit dose forms, such as layered forms and pouches.

[0217] The pouch can be configured as a single compartment or multiple compartments. It can be of any form, shape, and material suitable for holding the composition without releasing it from the pouch, for example, prior to contact with water. The pouch is made of a water-soluble film that encompasses the contents and can be divided into compartments. The preferred film is a polymeric material, preferably a polymer that can be formed into a film or sheet. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film is, for example, at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. The membrane can also be of a mixed composition including a hydrolytically degradable and water-soluble polymer mixture, such as polylactide and polyvinyl alcohol, and a plasticizer, such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch can contain a solid laundry cleaning composition or some components and / or a liquid cleaning composition or some components separated by a water-soluble film. The compartment for the liquid components can be different in composition from the compartment containing the solids (see, for example, US Patent Application Publication No. 2009 / 0011970A1).

[0218] The detergent ingredients can be physically separated from each other by compartments in a water-soluble pouch or in different layers of a tablet to avoid negative storage interactions between the ingredients, and the different dissolution profiles of each of the compartments can result in delayed dissolution of selected ingredients in the wash solution.

[0219] Non-unit dose liquid or gel detergents may be water-soluble, typically containing at least 20% by weight and up to 95% water, e.g., up to about 70%, up to about 65%, up to about 55%, up to about 45%, or up to about 35% water. Concentrated liquid detergents may have a lower water content, e.g., up to about 30% or up to about 20%, e.g., in the range of about 1% to about 20%, such as about 2% to about 15%. Other types of liquids may be included in the aqueous liquid or gel, including, but not limited to, alkanols, amines, diols, ethers, and polyols. Water-soluble liquid or gel detergents may contain 0 to 30% organic solvents. Alternatively, the liquid or gel detergent may be non-aqueous.

[0220] The liquid detergent compositions can be formulated to have a moderate pH of about 6 to about 10, such as about pH 7, about pH 8, or about pH 9, or can be formulated to have a higher pH of about 10 to about 12, such as about pH 10, about pH 11, or about pH 12.

[0221] Unless otherwise indicated, the term "liquid" as used herein should be understood to encompass any type of liquid detergent composition, e.g., concentrated liquids, gels, or the liquid or gel portion of, e.g., a pouch having one or more compartments.

[0222] Granular detergent formulations Enzymes in the form of granules containing an enzyme-containing core and optionally one or more coatings are commonly used in granular (powder) detergents. Various methods of preparing the cores are well known in the art, including, for example, a) spray drying a liquid enzyme-containing solution, b) producing a layered product in which the enzyme is coated as a layer surrounding a preformed inert core particle, for example using a fluid bed apparatus, c) absorbing the enzyme onto and / or into the preformed core, d) extruding the enzyme-containing paste, e) suspending the enzyme-containing powder in molten wax to yield a micronized, pelletized product, f) mixer granulation by adding an enzyme-containing liquid to a dry powder composition of the granulation ingredients, g) size reduction of the enzyme-containing core by grinding or crushing larger particles, pellets, etc., and h) fluid bed granulation. The enzyme-containing cores may be dried, for example using a fluid bed dryer or other known methods for drying granules in the feed or enzyme industry, to yield a moisture content of typically 0.1-10% water by weight.

[0223] The enzyme-containing core is sometimes provided with a coating to improve storage stability and / or reduce dust formation. One type of coating often used for enzyme granules for detergents is a salt coating, typically a coating of an inorganic salt, which can be applied as a solution of the salt, for example, using a fluidized bed. Other coating materials that can be used include, for example, polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC), and polyvinyl alcohol (PVA). The granules can contain more than one coating, for example, a salt coating followed by an additional coating of a material such as PEG, MHPC, or PVA.

[0224] For further details regarding enzyme granules and their production, see WO 2013 / 007594, and for example WO 2009 / 092699, EP 1 705 241, EP 1 382 668, WO 2007 / 001262, U.S. Pat. No. 6,472,364, WO 2004 / 074419, and WO 2009 / 102854.

[0225] Formulation of Enzymes in Cogranules Hexosaminidases can be formulated as granules, for example as composite granules combining one or more enzymes, where each enzyme is present in more granules to ensure a more uniform distribution of the enzymes within the detergent. This also reduces physical separation of the various enzymes due to different particle sizes. A method for producing multi-enzyme composite granules for the detergent industry is disclosed in IP.com disclosure IPCOM000200739D.

[0226] Another example of formulating an enzyme with a composite granule is disclosed in WO 2013 / 188331, which relates to a detergent composition comprising: (a) a multi-enzyme composite granule; (b) less than 10 wt. of zeolite (on anhydrous basis); and (c) less than 10 wt. of phosphate (on anhydrous basis), wherein the enzyme complex granule comprises 10-98 wt. % of a hygroscopic component, and the composition additionally comprises 20-80 wt. % of a detergent hygroscopic component. WO 2013 / 188331 also relates to a method for treating and / or cleaning a surface, preferably a textile surface, comprising the steps of (i) contacting the surface with a detergent composition in an aqueous cleaning liquor as claimed and described herein, and (ii) rinsing and / or drying the surface.

[0227] Liquid formulations The present invention also relates to a liquid composition comprising the hexosaminidase variant of the present invention. The composition may contain an enzyme stabilizer (examples 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).

[0228] In some embodiments, fillers or carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts such as sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water, or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain about 5% to about 90% of such materials.

[0229] In one aspect, the liquid formulation comprises 20-80% by weight of polyol. In one embodiment, the liquid formulation comprises 0.001-2% by weight of a preservative.

[0230] In another embodiment, the present invention provides (A) 0.001 to 25% by weight of the variant of the invention; (B) 20 to 80% by weight of a polyol; (C) an optional preservative, from 0.001 to 2% by weight; and (D)Water The present invention relates to a liquid formulation comprising:

[0231] In another embodiment, the present invention provides (A) 0.001 to 25% by weight of the variant of the invention; (B) 0.001 to 2% by weight of a preservative; (C) optionally 20 to 80 weight percent of a polyol; and (D)Water The present invention relates to a liquid formulation comprising:

[0232] In another embodiment, the liquid formulation comprises one or more compounding agents, such as compounding agents 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, acetates, and phosphates, preferably 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 glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0233] In another embodiment, the liquid formulation comprises 20-80% polyol (i.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, where 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., total amount of polyols), 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).

[0234] 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% by weight of the preservative, e.g., 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% by weight of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% by weight of the preservative, 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate, or any combination thereof.

[0235] In another embodiment, the liquid formulation further comprises one or more additional enzymes, for example as described above.

[0236] Use of hexosaminidase variants The hexosaminidase variants of the invention are suitable for use in cleaning processes, such as laundry or hard surface cleaning, particularly in laundry. Thus, one aspect of the invention relates to a method for laundering an item, the item being a fabric, the method comprising: a) exposing the item to a cleaning solution comprising a hexosaminidase variant of the present invention; b) completing at least one cleaning cycle; and, optionally c) Rinsing the items Includes.

[0237] The pH of the liquid washing solution is typically in the range of about 5.5 to about 10, more typically in the range of about 7 to about 9, such as about 7 to about 8.5 or about 7 to about 8.

[0238] The cleaning liquid may have a temperature in the range of 5°C to 95°C, or in the range of 10°C to 80°C, or in the range of 10°C to 70°C, or in the range of 10°C to 60°C, or in the range of 10°C to 50°C, or in the range of 15°C to 40°C, or in the range of 20°C to 30°C.

[0239] The concentration of the hexosaminidase variant enzyme in the wash solution is typically in the range of 0.0001 mg / l to 10 mg / l, 0.0002 mg / l to 10 mg / l, 0.001 mg / l to 10 mg / l, 0.002 mg / l to 10 mg / l, 0.01 mg / l to 10 mg / l, 0.02 mg / l to 10 mg / l, 0.1 mg / l to 10 mg / l, 0.2 mg / l to 10 mg / l or 0.2 mg / l to 5 mg / l of enzyme protein.

[0240] In one embodiment, the hexosaminidase variant or detergent composition comprising the variant may be used to clean hard surfaces, which may be, for example, surfaces such as dishes, table tops, walls or floors, or the interior surfaces of machines such as washing machines or dishwashers.

[0241] When items such as T-shirts or sportswear are used, they are exposed to bacteria from the user's body and from the remaining environment in which they are used. This can cause malodor in the item even after it has been washed. Therefore, the present invention also relates to a method for removing or reducing malodor on fabrics. Malodor can be caused by bacteria that produce unpleasant-smelling compounds. One example of such an unpleasant-smelling compound is E-2-nonenal. Malodor can be present on freshly washed fabrics that are still wet, and malodor can be present on freshly washed and dried fabrics. Malodor can also be present on fabrics that have been stored for some time after washing. Therefore, the present invention also relates to the use of the hexosaminidase variants of the present invention for reducing or removing malodor such as E-2-nonenal from wet or dry fabrics.

[0242] In one embodiment, the hexosaminidase variants of the present invention have improved malodour removal properties compared to a parent or reference hexosaminidase, such as SEQ ID NO:1 (wherein malodour is measured as described in Example 9 of WO 2017 / 186943).

[0243] The invention is further defined by the following numbered paragraphs: 1. A variant of the hexosaminidase polypeptide of SEQ ID NO: 1, which has, compared to SEQ ID NO: 1, Substitutions at one or more positions, preferably 2, 3 or 4 positions, corresponding to positions 163, 227, 252 and 309 of SEQ ID NO:1; and Substitutions at one or more positions corresponding to positions 106, 111, 120, 124, 127, 150, 170, 171, 178, 199, 208, 254, 255, and 278 of SEQ ID NO:1 Includes; A variant having hexosaminidase activity and having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 2. Contains two, three or four substitutions selected from the group consisting of S163P, N227T, N252P and K309E compared to SEQ ID NO:1; 2. The hexosaminidase variant of paragraph 1, further comprising at least one substitution selected from the group consisting of V106M, V106N, D111R, T120V, Y124I, Y124K, Y124L, R127P, E150N, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D, and I278V. 3. The hexosaminidase variant of paragraph 1 or 2, further comprising at least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of said substitutions compared to SEQ ID NO:1. 4. The hexosaminidase variant of paragraph 3, further comprising one or more substitutions selected from the group consisting of Q3I, A49W and N59E, preferably two or all three of said substitutions, compared to SEQ ID NO:1; or one or more substitutions selected from the group consisting of Q3F, V140I, Q215K and N267T, preferably two, three or all four of said substitutions. 5. Compared to SEQ ID NO:1, S163P, N227T, N252P, and K309E substitutions; and At least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q and K312Q, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or all of said substitutions. 5. The hexosaminidase variant of any of paragraphs 1 to 4, comprising: 6. The hexosaminidase variant of any of paragraphs 1 to 5, comprising at least one substitution, such as at least two of the substitutions, selected from the group consisting of V106M / N, D111R, T120V, Y124I / K / L, R127P, E150N, L170G, D171F / H, Q178K, S199W, S208W, T255D, N267T, I278V and K308E compared to SEQ ID NO:1; and having at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. 7. The hexosaminidase variant of any of paragraphs 1 to 6, comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, L170G, D171F, Q178K, S199W, S208W, T255D and I278V, such as at least two of said substitutions, compared to SEQ ID NO:1; and having at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. 8. The hexosaminidase variant of any of paragraphs 1 to 7, comprising at least one substitution selected from the group consisting of V106M / N, D111R, T120V, E150N, D171F and I278V, such as at least two of said substitutions, compared to SEQ ID NO:1; and having at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. 9. The hexosaminidase variant of any of paragraphs 1 to 8, comprising at least one substitution selected from the group consisting of D111R, T120V and E150N; and having at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. 10. The hexosaminidase variant of paragraph 9, comprising the substitutions D111R+T120V, D111R+E150N, T120V+E150N or all three substitutions D111R+T120V+E150N. 11. The following substitution or set of substitutions compared to SEQ ID NO:2: D111R D111R+T120V D111R+T120V+E150N ·D111R+T120V+E150N+D171F+I278V D111R+T120V+T255D D111R+T120V+Y124I D111R+T120V+Y124K+Q178K D111R+T120V+Y124L+Q178K ·D111R+Y124K+D171FD171F+S208W+N267T D171F+T255D D171H I278V · K308E N267T R127P · T120V+D171F T120V+D171F+N267T ·T120V+S208W+N267T ·T120V+T255D T120V+T255D+N267T T120V+Y124K+Q178K V106M+D111R+T120V+D171F · V106M+Q178K+T255D V106M+T120V+D171F+Q178K V106M+T120V+Y124I+D171F V106M+T120V+Y124I+Q178K V106M+T120V+Y124K+D171F V106M+T120V+Y124L+Q178K V106N+D111R+T120V+E150N · V106N+S208W+N267T V106N+T120V+D171F V106N+T120V+D171F+S208W ·V106N+T120V+E150N+D171F+I278V V106N+T120V+N267T 11. The hexosaminidase variant of any of paragraphs 1-10, comprising at least one of: 12. The following substitution or set of substitutions compared to SEQ ID NO:3: · L170G E150N+F254I ·F254I · V106N D111R+T120V D111R+T120V+E150N ·D111R+T120V+E150N+D171F+I278V D111R+T120V+T255D D111R+T120V+Y124I D111R+T120V+Y124K+Q178K D111R+T120V+Y124L+Q178K D111R+Y124K+D171F D171F+S208W+N267T D171F+T255D D171H I278V · K308E N267T R127P · T120V+D171F T120V+D171F+N267T ·T120V+S208W+N267T ·T120V+T255D T120V+T255D+N267T T120V+Y124K+Q178K V106M+D111R+T120V+D171F · V106M+Q178K+T255D V106M+T120V+D171F+Q178K V106M+T120V+Y124I+D171F V106M+T120V+Y124I+Q178K V106M+T120V+Y124K+D171F V106M+T120V+Y124L+Q178K V106N+D111R+T120V+E150N · V106N+S208W+N267T V106N+T120V+D171F V106N+T120V+D171F+S208W ·V106N+T120V+E150N+D171F+I278V V106N+T120V+N267T 11. The hexosaminidase variant of any of paragraphs 1-10, comprising at least one of: 13. The following substitution or set of substitutions compared to SEQ ID NO:1: ·Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+S186R+S225G+N227 T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+D171F+S186R+S225G+N227 T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124I+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+D111R+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+D171H+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308E+K309E+K312Q ·Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+R127P+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+D111R+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+S163P+D171F+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106M+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+D111R+T120V+E150N+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+E150N+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V106N+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G23 5W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E23 2D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+ G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ·Q3F+H15Y+V140I+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G23 5W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q 13. The hexosaminidase variant of any of paragraphs 1-12, including one of: 14. A hexosaminidase variant of any of paragraphs 1 to 13, having at least 80%, at least 85%, at least 90% or at least 95% sequence identity (but less than 100% sequence identity) to SEQ ID NO:2 or SEQ ID NO:3. 15. The hexosaminidase variant of any of paragraphs 1 to 14, having improved stability compared to a parent polypeptide having the same amino acid sequence as the polypeptide of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, or a variant without the substitution. 16. The hexosaminidase variant of paragraph 15, wherein the improved stability is selected from the group consisting of detergent stability and thermal stability. 17. A detergent composition comprising the hexosaminidase variant of any of paragraphs 1 to 16 and at least one detergent adjunct ingredient. 18. Use of a hexosaminidase variant according to any of paragraphs 1 to 16 or a detergent composition according to paragraph 17 in a cleaning process, such as laundry or hard surface cleaning, for example dishwashing. 19. A method of cleaning an item, comprising exposing the item to a cleaning solution comprising the hexosaminidase variant of any of paragraphs 1 to 16, or to a detergent composition of paragraph 17. 20. The method of paragraph 19, wherein the item is a fabric or hard surface, such as dishware, or a surface such as a tabletop, wall or floor, or the interior surface of a machine, such as a washer or dishwasher. 21. A method for washing fabric, comprising: a) exposing a fabric to a wash liquor comprising a hexosaminidase variant according to any of paragraphs 1 to 16 or a detergent composition according to paragraph 17; b) completing at least one cleaning cycle; and, optionally c) Rinsing the items The method includes: 22. The following: (a) a core comprising any one of the hexosaminidase variants of paragraphs 1 to 17, and optionally (b) A coating consisting of one or more layers surrounding the core. Granules containing 23. A liquid composition comprising the hexosaminidase variant of any one of paragraphs 1 to 17, and an enzyme stabilizer, for example a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, for example an aromatic boric acid ester, or a phenylboronic acid derivative, such as 4-formylphenylboronic acid. 24. A polynucleotide encoding a hexosaminidase variant according to any one of paragraphs 1 to 17, a nucleic acid construct or expression vector comprising the polynucleotide, or a recombinant host cell transformed with the polynucleotide. 25. A method for producing a hexosaminidase variant, comprising: a) culturing the recombinant host cell of paragraph 24 under conditions suitable for expression of the variant; and b) Recovering variants The method includes:

[0244] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0245] Materials and Methods Hexosaminidase activity assay: Hexosaminidase activity can be determined, for example, using one of the following assays.

[0246] Assay 1 The hexosaminidase activity of the hexosaminidase polypeptide can be determined using 4-nitrophenyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich) as a substrate. Enzyme reactions are carried out in triplicate in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) with the following conditions: 50 mM 2-(N-morpholino)ethanesulfonic acid pH 6 buffer, 1.5 mg / ml 4-nitrophenyl N-acetyl-β-D-glucosaminide, and 20 μg / ml purified enzyme sample in a total reaction volume of 100 μl. A blank sample without enzyme is run in parallel. Reactions are carried out at 37° C. in a Thermomixer comfort (Eppendorf). After 10 min of incubation, 5 μl of 1 M NaOH is added to each reaction mixture to stop the enzymatic reaction. The formation of 4-nitrophenolate ions released by enzymatic hydrolysis of the 4-nitrophenyl N-acetyl-β-D-glucosaminide substrate was estimated by reading the absorbance at 405 nm using a POLARstar Omega plate reader (BMG LABTECH). The increase in absorbance over the blank (difference in absorbance at 405 nm between sample and blank) indicates hexosaminidase activity.

[0247] Assay 2 The hexosaminidase activity of the hexosaminidase polypeptide can be determined using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich) as a substrate. Enzyme reactions are run in triplicate in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) with the following conditions: 20 mM 3-morpholinopropane-1-sulfonic acid pH 7 buffer, 5 mM 4-methylumbelliferyl N-acetyl-β-D-glucosaminide, and 20 nM purified enzyme sample in a total reaction volume of 200 μl. A blank sample without enzyme is run in parallel. The reaction is carried out at an ambient temperature of 20-25 °C. The reaction kinetics is followed immediately after mixing the enzyme and substrate using a SpectraMax M2e plate reader. The excitation wavelength is set at 368 nm and fluorescence emission readings are taken at 448 nm. The reaction is followed for 30 min at 60 s intervals. The increase in fluorescence signal is used to infer the formation of the liberated 4-methylumbelliferyl ion due to enzymatic hydrolysis of the 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate.

[0248] Results show the average initial reaction velocity measured as relative fluorescence units per minute (RFU / min) using an excitation of 368 nm and a fluorescence emission of 448 nm for each reaction performed in triplicate. Increased values ​​above the blank (ΔRFU / min) indicate hexosaminidase activity.

[0249] Growth and Expression Hexosaminidase polypeptides were expressed in Bacillus host cells grown in standard 96-well microtiter plates (200 μl broth / well). The broth used for growth and expression was Cal18-2 supplemented with 6 μg / ml chloramphenicol (Ostergaard et al., 2010, Identification and characterization of a bacterial glutamic peptidase, BMC Biochem. 11:47). Microtiter plates were grown for 3 days at 30° C. with shaking at 225 rpm. After growth, plates were centrifuged and supernatants were stressed and assayed for stability as described below. All steps related to growth, stress, and assays were performed in microtiter plates in 96-well or 384-well format.

[0250] Determination of variant stability in supernatants in liquid detergent The supernatant was diluted with concentrated 80% (v / v) Persil® ProClean® Power Liquid 2in1 detergent (Henkel). After mixing, the sample was divided into two parts. One part was incubated at room temperature (21° C., non-stressed sample) for the selected stress time, and the second part was incubated in a PCR machine at a selected high stress temperature for the selected stress time ("stressed sample"). The applied stress conditions can be found in the individual examples below. After incubation, the samples were diluted 8-fold with dilution buffer (100 mM Tris-HCl, 0.01% (v / v) Triton® X-100, pH 8.0) and the activity of non-stressed and stressed samples was determined by transferring 5 μl of sample to a 384-well microtiter plate containing 35 μl of assay solution (45 mM citrate buffer pH 5.0 supplemented with 1.0 mg / ml p-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma Aldrich). After 9 h of incubation at room temperature, the reaction was stopped by adding 40 μl of stop solution (0.4 M Na2CO3) and the absorbance at 405 nm (A405) was read. For each sample, the residual activity (RA) was calculated as follows: RA=A405(stressed sample) / A405(non-stressed sample).

[0251] After correcting all A405 measurements with the signal of blank samples (no hexosaminidase), RA values ​​were calculated. RA values ​​were used to calculate the half-life of variants and backbones (SEQ ID NO:2, SEQ ID NO:3, or reference hexosaminidase as shown in the individual examples): Half-life (min) = 60 (min) x Ln (0.5) / Ln (RA) for each variant and backbone. For each variant, the half-life improvement factor (HIF) is calculated as follows: HIF = Half-life (variant) / Half-life (backbone). The HIF of the backbone in this setup is 1 by definition, so variants with improved stability have a HIF > 1.

[0252] Purified variant Growth, expression and purification For purification, the hexosaminidase polypeptide was expressed in Bacillus host cells and grown in 500 ml shake flasks each containing 100 ml of PS-1 medium. The shake flasks were grown for 4 days at 30° C. with shaking at 270 rpm. The culture broth was centrifuged (26,000×g, 20 min) and the supernatant was carefully decanted from the sediment. The supernatant was filtered through a Nalgene 0.2 μm filtration unit to remove the remains of the Bacillus host cells. The 0.2 μm filtrate was transferred onto a G25 Sephadex column (GE Healthcare) in 20 mM MES / NaOH, pH 6.0. The G25 transferred solution was applied to a SOURCE Q column (GE Healthcare) equilibrated with 20 mM MES / NaOH, pH 6.0. After extensive washing of the column with equilibration buffer, the hexosaminidase was eluted with a linear NaCl gradient (0 → 1.0 M NaCl) in the same buffer over 5 column volumes. Fractions were collected during elution and the collected fractions were analyzed by SDS-PAGE. Fractions that showed only one band by Coomassie staining were pooled as purified preparations and used for further experiments.

[0253] Determining the stability (half-life) of purified variants The stability, expressed as half-life, of the mature polypeptides purified as described above was determined by incubating 300 μL, 5 μM samples of each variant in 96-well flat-bottom polystyrene microtiter plates (Thermo Scientific) at 37° C. for 7 days under the following conditions: 20% by volume EPPS (100 mM, pH 8.3), 0.01% by weight Triton® X-100 and 80% by volume Persil® ProClean® Power Liquid 2in1 detergent (= “stress plate”). On days 1, 3 and 7, 20 μL aliquots from the stress plate were analyzed for residual activity using 4-methylumbelliferyl N-acetyl-D-glucosaminide (Sigma-Aldrich) as substrate: 20 μL sample from the stress plate and 100 μL substrate in citrate buffer (45 mM, pH 5.3, 0.01 wt% Brij® L23) were mixed to a concentration of 208 nM sample in a solution containing 6 mM substrate. The reaction mixture was heated at 45° C. for 90 min in a PCR incubator and the reaction was stopped by the addition of 120 μL Na2CO3 (0.6 M, pH 10.3). The absorbance was measured at 405 nm in a standard plate spectrophotometer (SpectraMax 5e).

[0254] The half-life of the stressed samples was estimated by linearization of the sample activity decay data (log(activity) vs. time) followed by linear regression to obtain the sample half-life (t 1 / 2 ) was determined. The half-life improvement factor (HIF) was calculated as the ratio between the half-life of the hexosaminidase variant and the half-life of the reference hexosaminidase: HIF=t 1 / 2 (variant) / t 1 / 2 (reference)

[0255] Example 1 - Stability of hexosaminidase variants The detergent stability of the hexosaminidase variants of the invention in the supernatant was determined as half-life compared to the half-life of the hexosaminidase of SEQ ID NO: 3 using the method described above. The stress conditions in this example were 210 minutes at 62° C. in 80% Persil® ProClean® Power Liquid 2in1 detergent. The results are expressed in Table 1 below as half-life enhancement factor HIF compared to the half-life of SEQ ID NO: 3.

[0256] [Table 2]

[0257] Example 2 - Stability of hexosaminidase variants The detergent stability of the hexosaminidase variants of the invention in the supernatant was determined as half-life compared to the half-life of the hexosaminidase of SEQ ID NO: 2 using the method described above, the stress conditions being a temperature of 57° C. and an incubation time of 360 minutes. The results are presented in Table 2 below as half-life enhancement factor HIF compared to the half-life of the hexosaminidase of SEQ ID NO: 2.

[0258] [Table 3]

[0259] [Table 4]

[0260] [Table 5]

[0261] Example 3 - Stability of hexosaminidase variants The detergent stability of the hexosaminidase variants of the invention in the supernatant was determined as half-life compared to that of SEQ ID NO: 4, a variant of SEQ ID NO: 1 with the substitutions S163P+Q215K+N227T+N252P+F276A+K308E+K309E+K312E, using the method described above. The stress conditions were a temperature of 62°C and an incubation time of 150 minutes. The variants tested in this example are variants of SEQ ID NO: 3 with one or more substitutions as shown in Table 3 below, and the results are expressed as half-life improvement factors HIF compared to the half-life of the reference hexosaminidase of SEQ ID NO: 4.

[0262] [Table 6]

[0263] Example 4 - Stability of purified hexosaminidase variants The detergent stability of the hexosaminidase variants of the invention in purified form was determined as half-life compared to the half-life of the hexosaminidase of SEQ ID NO: 3 using the method described above (7 days at 37°C in 80% by volume Persil® ProClean® Power Liquid 2in1 detergent). The results are expressed as half-life improvement factor HIF compared to the half-life of SEQ ID NO: 3 in Table 4 below.

[0264] [Table 7]

[0265] Example 5: Thermostability of purified hexosaminidase polypeptides The thermal stability of the wild-type hexosaminidase from Terribacillus saccharophilus (SEQ ID NO: 1) and its three variants (SEQ ID NO: 2, 3 and 4) was determined by nDSF using the method described above in a model liquid detergent with the following composition:

[0266] [Table 8]

[0267] The results, expressed as melting temperatures (Tm) at detergent concentrations of 0, 2, 50 and 250 g / L, are provided in Table 6 below, where "-" indicates that the wild-type hexosaminidase of SEQ ID NO:1 was unstable and, as a result, a Tm value could not be determined at higher detergent concentrations.

[0268] [Table 9]

[0269] The present invention described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended as illustrations of some embodiments of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure, including definitions, will control.

Claims

1. A variant of the hexosaminidase polypeptide of SEQ ID NO: 1, compared to SEQ ID NO: 1, Replacement at one or more positions, preferably two, three, or four positions, corresponding to positions 163, 227, 252, and 309 of sequence number 1; and Replacement at one or more positions corresponding to positions 106, 111, 120, 124, 127, 150, 170, 171, 178, 199, 208, 254, 255, and 278 of sequence number 1. Including; A variant having hexosaminidase activity and having at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity (but less than 100% sequence identity) to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

2. Compared to Sequence ID No. 1, it includes two, three, or four substitutions selected from the group consisting of S163P, N227T, N252P, and K309E; The hexosaminidase variant according to claim 1, further comprising at least one substitution selected from the group consisting of D111R, T120V, V106M, V106N, Y124I, Y124K, Y124L, R127P, E150N, L170G, D171F, D171H, Q178K, S199W, S208W, F254I, T255D, and I278V

3. Compared to Sequence ID No. 1, further comprising at least one substitution selected from the group consisting of H15Y, S186R, S225G, E232D, G235W, N260Q, H272V, S279D, Y281P, K308Q, and K312Q, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the above substitutions; The hexosaminidase variant according to claim 2, further comprising, at the option of choice, one or more substitutions selected from the group consisting of Q3I, A49W, and N59E, preferably two or all three of the substitutions, compared to sequence number 1; or one or more substitutions selected from the group consisting of Q3F, V140I, Q215K, and N267T, preferably two, three, or all four of the substitutions.

4. A hexosaminidase variant according to claim 1, comprising, with respect to sequence number 1, at least one substitution selected from the group consisting of D111R, T120V, V106M / N, E150N, D171F, and I278V, for example, at least two of the above substitutions; preferably comprising at least one substitution selected from the group consisting of D111R, T120V, and E150N; and having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity (but less than 100%) with respect to sequence number 2 or sequence number 3.

5. Compared to Sequence ID No. 2, the following substitutions or substitution sets: ・D111R ・D111R+T120V ・D111R+T120V+E150N ・D111R+T120V+E150N+D171F+I278V ・D111R+T120V+T255D ・D111R+T120V+Y124I ・D111R+T120V+Y124K+Q178K ・D111R+T120V+Y124L+Q178K ・D111R+Y124K+D171FD171F+S208W+N267T ・D171F+T255D ・D171H I278V ・K308E ・N267T ・R127P ・T120V+D171F ・T120V+D171F+N267T ・T120V+S208W+N267T ・T120V+T255D ・T120V+T255D+N267T ・T120V+Y124K+Q178K ・V106M+D111R+T120V+D171F ・V106M+Q178K+T255D ・V106M+T120V+D171F+Q178K ・V106M+T120V+Y124I+D171F ・V106M+T120V+Y124I+Q178K ・V106M+T120V+Y124K+D171F ・V106M+T120V+Y124L+Q178K ・V106N+D111R+T120V+E150N ・V106N+S208W+N267T ・V106N+T120V+D171F ・V106N+T120V+D171F+S208W ・V106N+T120V+E150N+D171F+I278V ・V106N+T120V+N267T A hexosaminidase variant according to claim 1, comprising at least one of the following.

6. Compared to Sequence ID No. 3, the following substitutions or substitution sets: L170G ・E150N+F254I F254I ・V106N ・D111R+T120V ・D111R+T120V+E150N ・D111R+T120V+E150N+D171F+I278V ・D111R+T120V+T255D ・D111R+T120V+Y124I ・D111R+T120V+Y124K+Q178K ・D111R+T120V+Y124L+Q178K ・D111R+Y124K+D171F ・D171F+S208W+N267T ・D171F+T255D ・D171H I278V ・K308E ・N267T ・R127P ・T120V+D171F ・T120V+D171F+N267T ・T120V+S208W+N267T ・T120V+T255D ・T120V+T255D+N267T ・T120V+Y124K+Q178K ・V106M+D111R+T120V+D171F ・V106M+Q178K+T255D ・V106M+T120V+D171F+Q178K ・V106M+T120V+Y124I+D171F ・V106M+T120V+Y124I+Q178K ・V106M+T120V+Y124K+D171F ・V106M+T120V+Y124L+Q178K ・V106N+D111R+T120V+E150N ・V106N+S208W+N267T ・V106N+T120V+D171F ・V106N+T120V+D171F+S208W ・V106N+T120V+E150N+D171F+I278V ・V106N+T120V+N267T A hexosaminidase variant according to claim 1, comprising at least one of the following.

7. Compared to SEQ ID NO: 1, the following substitutions or substitution sets: ・Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+S186R+S225G+N227 T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+E150N+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+Y124I+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+D111R+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+D171H+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308E+K309E+K312Q ・Q3I+H15Y+A49W+N59E+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+R127P+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+T120V+Y124K+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+D111R+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+T255D+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+T120V+S163P+D171F+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+T120V+Y124I+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+T120V+Y124K+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106M+T120V+Y124L+S163P+Q178K+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+D111R+T120V+E150N+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+S163P+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+D171F+S186R+S208W+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+T120V+E150N+S163P+D171F+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3I+H15Y+A49W+N59E+V106N+T120V+S163P+S186R+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+F254I+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V106N+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+D111R+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+E150N+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+L170G+S186R+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+G235W+N252P+N260Q+N267T+H272V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+L170G+S186R+S199W+Q215K+S225G+N227T+E232D+ G235W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q ・Q3F+H15Y+V140I+S163P+S186R+S199W+Q215K+S225G+N227T+E232D+G23 5W+N252P+N260Q+N267T+H272V+I278V+S279D+Y281P+K308Q+K309E+K312Q A hexosaminidase variant according to claim 1, comprising one of the above.

8. The hexosaminidase variant according to claim 1, having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity (but less than 100% sequence identity) with respect to sequence number 2 or sequence number 3.

9. The hexosaminidase variant according to claim 1, which has improved stability compared to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or the parent polypeptide having the same amino acid sequence as the variant without substitution.

10. A detergent composition comprising a hexosaminidase variant according to any one of claims 1 to 9 and at least one detergent auxiliary component.

11. Use of the detergent composition according to claim 10 in a cleaning process such as washing clothes or cleaning hard surfaces, for example, washing dishes.

12. A method for cleaning an item, comprising exposing the item to the detergent composition described in claim 10.

13. This is a method for washing fabrics. a) Exposing the fabric to the detergent composition described in claim 10; b) to complete at least one cleaning cycle; and optionally c) Rinse the items mentioned above. A method that includes this.

14. A polynucleotide encoding a hexosaminidase variant according to any one of claims 1 to 9, a nucleic acid construct or expression vector containing the polynucleotide, or a recombinant host cell transformed with the polynucleotide.

15. A method for generating hexosaminidase variants, a) culturing the recombinant host cells described in claim 14 under conditions suitable for the expression of the variant; and b) Recovering the variant. A method that includes this.