Mannanase variants and polynucleotides encoding same

Abstract

The present invention relates to mannanase variants with improved stability. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] References to sequence listings

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

[0004] The present invention relates to mannanase variants, polynucleotides encoding these variants, methods of producing these variants, and methods of using these variants. The variants of the present invention are suitable for use in cleaning processes and detergent compositions (such as laundry detergent compositions and dishwashing compositions). In particular, the mannanase variants of the present invention show improved in-detergent stability and / or protease stability. Background Art

[0005] Mannans are polysaccharides with a backbone of β-1,4-linked D-mannopyranosyl residues, which may contain galactose or acetyl substitutions and may have glucose residues in the backbone. The main type of enzyme involved in mannan degradation is endo-1,4-β-mannanase (EC 3.2.1.78), which hydrolyzes internal glycosidic bonds in the mannan backbone.

[0006] Mannans are a type of hemicellulose that make up up to 25% of the dry weight of the wood in softwoods, but are also found in other plant materials, especially various seeds. Mannan-containing guar gum is used as a stabilizer in many food products.

[0007] Therefore, it may be advantageous to use endomannanases in applications where degradation of mannans is desired. Examples where mannanases may be used are in detergents to remove stains containing mannans; in the removal of mannans from cork (Várnai et al., (2011) "Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood", Bioresource tech., 102(19), pp. 9096-9104) and palm kernel cake ( et al., (2010) "Production of ethanol and feed by high dry matter hydrolysis and fermentation of palm kernel press cake", Applied Biochem. Biotech., 161(1-8), pp. 318-332), in the production of bioethanol for use in improving animal feed (Cai et al., (2011), "Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application", J. Biosci. Bioeng., 112(6), pp. 551-557); and in the hydrolysis of coffee extract (Nunes et al., (2006), "Characterization of Galactomannanase Derivatives in Roasted Coffee Beverages”, J. Agricultural Food Chem., 54(9), pp. 3428-3439).

[0008] In the home care industry it is known to use mannanases in eg laundry detergents.In WO 1999 / 064619 an alkaline mannanase is disclosed which exhibits mannanase activity also in the alkaline pH range when applied in cleaning compositions.

[0009] According to CAZy (cazy.org), endo-1,4-β-mannanases have been found in glycoside hydrolase families 5, 26 and 113. In WO 2019 / 068713, WO 2019 / 068715, WO 2021 / 152120 and WO 152123, GH 26 family mannanases exhibiting β-mannanase activity are disclosed. WO 152123 also discloses that SEQ ID NO: 2 has improved stability in the presence of proteases (page 370, second to last line). Summary of the invention

[0010] Stability under conditions relevant to end use (i.e., customer use) is key to the performance and usefulness of enzymes, including mannanases. Mannanases are desirable and industrially applicable for the detergent manufacturing industry, for example, as discussed above. The present invention provides mannanase variants having further improved stability compared to the above-mentioned mannanase variants having SEQ ID NO: 2, in particular, in detergent compositions comprising proteases as disclosed in Example 3, the mannanase has improved stability (i.e., improved half-life).

[0011] The present invention relates to isolated mannanase variants comprising deletions at positions 490 and 491 of SEQ ID NO:2, and optionally one or more deletions or substitutions at positions corresponding to positions 16, 20, 26, 30, 36, 46, 48, 53, 61, 64, 65, 69, 70, 74, 76, 78, 82, 101, 103, 109, 111, 112, 118, 120, 126, 137, 139, 141, 143, 155, 160, 161, 162, 163, 164, 165, 166, 167, 168, 171, 172, 176, 178, 181, 182, 183, 190, 197, 214, 215, 219, 239, 244, 248, 253, 258, 271, 276, 280, 283, 286, 299, 315, 324, 366, 378, 385, 408, 410, 413, 473, 485, 486, and optionally deletions at positions corresponding to the following positions, which positions are SEQ ID NO:2, wherein the variants have at least 60% sequence identity to the polypeptide of SEQ ID NO:3, 4, 5, 6, 7, 22, 23, 24, 25, 26, 27, 28 or 29, and wherein the variants have mannanase activity.

[0012] In a further aspect, the invention relates to a variant of the polypeptide of SEQ ID NO: 2, wherein the amino acids at positions 490 and 491 are deleted, wherein the variant has at least 60% sequence identity with the polypeptide of SEQ ID NO: 3, and wherein the variant has mannanase activity.

[0013] The present invention also relates to compositions comprising the variants as disclosed herein, the use of such compositions in household or industrial cleaning processes, isolated polynucleotides encoding these variants; nucleic acid constructs, vectors and host cells comprising these polynucleotides; and methods of producing these variants, as well as methods of washing using the compositions disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is an alignment of the polypeptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6 and 7.

[0015] Sequence Listing Overview

[0016] SEQ ID NO: 1 is a mature mannanase polypeptide obtained from Paenibacillus illinoisensis

[0017] SEQ ID NO:2 is a mature mannanase polypeptide obtained from Paenibacillus ilinifolia, which is a variant of SEQ ID NO:1

[0018] SEQ ID NO:3 to SEQ ID NO:7, SEQ ID NO:9 to SEQ ID NO:16 and SEQ ID NO:22 to SEQ ID NO:29 are variants of SEQ ID NO:2

[0019] SEQ ID NO: 8 is a GH5 mannanase from Bacillus bogoriensis

[0020] SEQ ID NO: 17 is a protease from Bacillus lentus

[0021] SEQ ID NO: 18 is a mannanase from Bacillus sp.

[0022] SEQ ID NO: 19 is a mannanase from Bacillus clausii

[0023] SEQ ID NO: 20 is a mannanase from Bacillus lentus

[0024] SEQ ID NO: 21 is a mannanase from Paenibacillus sp.

[0025] SEQ ID NO:30 is a xanthan gum lyase from Paenibacillus sp.

[0026] SEQ ID NO:31 is a xanthan endoglucanase from Paenibacillus sp.

[0027] SEQ ID NO:32 is a protease from Bacillus lentus

[0028] definition

[0029] In light of this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0030] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] Mannanase activity: To estimate the mannose yield after substrate hydrolysis, the reducing end assay developed by Lever (1972), Anal. Biochem. 47:273-279 was used. The assay is based on 4-hydroxybenzoic acid hydrazide, which reacts with the reducing end of sugars under alkaline conditions. The product is a strong yellow anion that absorbs at 410 nm.

[0032] The mannanase activity of the variants was determined as described in Example 1.

[0033] Adjunct materials: The term "adjunct materials" or "adjunct ingredients" means any liquid, solid or gaseous material selected for the particular type of detergent composition and product form desired (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, or foam composition), which is also preferably compatible with the mannanase variant enzyme used in the composition. More detailed information on adjunct materials is provided further below.

[0034] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are usually determined by an open reading frame that begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0035] Control sequences: The term "control sequences" means nucleic acid sequences that are involved in regulating the expression of polynucleotides in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) to the polynucleotides encoding the variants, and are native or heterologous to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, prepropeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, these control sequences include promoters and transcription and translation termination signals. For the purpose of introducing specific restriction sites that promote the connection of control sequences to the coding regions of the polynucleotides encoding the variants, the control sequences can be provided with joints.

[0036] Detergent composition: Unless otherwise specified, the term "detergent composition" (or "cleaning composition") includes any form of detergent or cleaning composition. These include general-purpose or heavy-duty detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, gel or paste form, especially the so-called heavy-duty liquid (HDL) type; single unit dose (SUD) compositions with one or more chambers, such as pods, capsules, tablets, etc.; liquid fine fabric detergents; hand dishwashing detergents or light-duty dishwashing detergents, especially those of the high-sudsing type; machine dishwashing detergents, including different tablet, granular, liquid and rinse aid types for domestic and institutional use; liquid cleaners and disinfectants, including antibacterial hand-washing types, cleaning bars, soap bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; shampoos and hair rinses; shower gels, foaming baths; metal cleaners; as well as cleaning aids (such as bleach additives) and "stain-sticks" or pre-treatment types. The terms "detergent composition" and "detergent formulation" are used with respect to mixtures in a washing medium intended for dirt cleaning. In some embodiments, the term is used with respect to washing fabrics and / or clothes (e.g., "laundry detergent"). In alternative embodiments, the term refers to other detergents such as those used to clean dishes, cutlery, etc. (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. It is intended that, except for variants according to the present invention, the term encompasses detergents that may contain: for example, surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foam boosters, foam suppressors, dyes, perfumes, tarnish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0037] Effective amount of enzyme: The term "effective amount of enzyme" refers to the amount of enzyme necessary to achieve the desired enzyme activity in a particular application, such as in a defined detergent composition. Such an effective amount is readily determined by one of ordinary skill in the art and is based on many factors, such as the specific enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granular, stick) composition is desired, etc. The term "effective amount" of a mannanase variant refers to the amount of the aforementioned mannanase variant that achieves the desired level of enzyme activity (e.g., in a defined detergent composition).

[0038] Expression: The term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0039] Expression vector: "Expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which is operably linked to a suitable control sequence capable of affecting expression of the DNA in a suitable host. Such control sequences may include a promoter that affects transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0040] Fabric: The term "fabric" encompasses any textile material. Thus, it is intended that the term encompasses garments, as well as fabrics, yarns, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material.

[0041] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a variant, wherein the "extended" variant has mannanase activity.

[0042] Fragment: The term "fragment" means a variant having one or more amino acids deleted at the amino and / or carboxyl terminus of the variant; wherein the fragment has mannanase activity. In one aspect, the fragment comprises at least amino acids 13 to 489 of SEQ ID NO:5, such as at least amino acids 14 to 489 of SEQ ID NO:5, such as at least amino acids 15 to 489 of SEQ ID NO:5. In another aspect, the fragment comprises at least amino acids 13 to 489 of SEQ ID NO:6, such as at least amino acids 14 to 489 of SEQ ID NO:6, such as at least amino acids 15 to 489 of SEQ ID NO:6. In yet another aspect, the fragment comprises at least amino acids 13 to 489 of SEQ ID NO:7, such as at least amino acids 14 to 489 of SEQ ID NO:7, such as at least amino acids 15 to 489 of SEQ ID NO:7.

[0043] Hard surface cleaning: The term "hard surface cleaning" is defined herein as cleaning hard surfaces, where hard surfaces can include floors, tables, walls, ceilings, etc., as well as surfaces of hard objects such as cars (car washing) and tableware (dishwashing). Dishwashing includes, but is not limited to, cleaning plates, cups, glasses, bowls and cutlery (e.g., spoons, knives, forks), serving utensils, ceramics, plastics, metals, porcelain, glass and acrylics.

[0044] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that can hydrolyze hemicellulosic materials. See, e.g., Shallom and Shoham, Current Opinion In Microbiology, 2003, 6(3):219-228. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, acetylmannan esterases, acetylxylan esterases, arabinanases, arabinofuranosidases, coumaric acid esterases, ferulic acid esterases, galactosidases, glucuronidases, glucuronidases, mannanases, mannosidases, xylanases, and xylosidases. The substrates for these enzymes (hemicellulose) are a heterogeneous group of branched and linear polysaccharides that bind to cellulose microfibrils in plant cell walls via hydrogen bonds, thereby cross-linking them into a robust network. Hemicellulose is also covalently attached to lignin, thereby forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the synergistic action of many enzymes to degrade it completely. The catalytic module of hemicellulase is a glycoside hydrolase (GH) that hydrolyzes glycosidic bonds, or a carbohydrate esterase (CE) that hydrolyzes the ester bonds of acetic acid or ferulic acid side groups. These catalytic modules can be assigned to GH and CE families based on the homology of their primary sequences. Some families (with generally similar folds) can be further grouped into clans, marked with letters (e.g., GH-A). The most detailed and up-to-date classification of these and other carbohydrate-active enzymes is available in the carbohydrate-active enzyme (CAZy) database. Hemicellulolytic enzyme activity can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752 at a suitable temperature (e.g., 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C) and a suitable pH (e.g., 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0).

[0045] Heterologous: With respect to a host cell, the term "heterologous" means that the polypeptide or nucleic acid is not naturally present in the host cell. With respect to a polypeptide or nucleic acid, the term "heterologous" means that the control sequence (e.g., the promoter of the polypeptide or nucleic acid) is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0046] Host strain or host cell: "Host strain" or "host cell" refers to an organism into which an expression vector, bacteriophage, virus or other DNA construct (including a polynucleotide encoding a variant) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0047] Improved properties: The term "improved properties" means a characteristic associated with a variant that is improved relative to a parent. Such improved properties include, but are not limited to, in-detergent stability, thermostability, protease stability, surfactant stability, pH stability.

[0048] Improved wash performance: The term "improved wash performance" can be defined as the improved cleaning effect of the mannanase variants according to the present invention compared to the mannanase having SEQ ID NO: 1 or SEQ ID NO: 2. Wash performance can be expressed as the reflectance value of the stained swatch. After washing and rinsing, the swatches are spread flat and allowed to air dry overnight at room temperature. All washed swatches are evaluated the next day after washing. Light reflectance evaluation of the swatches is performed using a Macbeth Color Eye 7000 reflectance spectrophotometer with a very small aperture. The measurements are performed without UV in the incident light and the reflectance value at 460nm is extracted.

[0049] In-detergent stability: The term "in-detergent stability" or "detergent stability" refers to the stability of a mannanase (whether wild-type, parent or variant) incubated in detergent. For the purposes of the present invention, in-detergent stability can be determined as shown in Example 3.

[0050] Introduced: In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," as known in the art.

[0051] Isolated: The term "isolated" means a variant, nucleic acid, cell or other specified material or component that is separated from at least one other material or component (including but not limited to other proteins, nucleic acids, cells, etc.). Thus, an isolated polypeptide, nucleic acid, cell or other material is in a form that does not exist in nature. An isolated polypeptide includes but is not limited to a culture medium containing a secreted variant expressed in a host cell.

[0052] Laundry: The term "laundry" refers to both domestic and industrial laundry and means the process of treating textiles with a solution containing a cleaning or detergent composition of the present invention. The laundry process can be carried out, for example, using, for example, a domestic or industrial washing machine, or can be carried out manually.

[0053] Mannanase: The term "mannanase" means a polypeptide having mannan endo-1,4-β-mannosidase activity (EC 3.2.1.78) that catalyzes the hydrolysis of 1,4-β-D-mannosidic bonds in mannans, galactomannans, glucomannans. Alternative names for mannan endo-1,4-β-mannosidase are: 1,4-β-D-mannan mannan hydrolase; endo-1,4-β-mannanase; endo-β-1,4-mannanase; β-mannanase B; β-1,4-mannan 4-mannan hydrolase; endo-β-mannanase; and β-D-mannanase.

[0054] According to CAZy (www.cazy.org) mannanases can be found in two groups: glycoside hydrolase family 5 (GH5) and glycoside hydrolase family 26 (GH26). The substrate specificity of the two groups is different, and therefore a combination of GH5 and GH26 may be advantageous.

[0055] Mannanases belonging to the GH5 family are disclosed in WO2018 / 206300 and WO2018 / 206302.

[0056] Mannanases belonging to the GH26 family are disclosed in WO2019 / 068715, WO2021 / 152120 and WO152123.

[0057] For purposes of the present invention, mannanase activity may be determined using the reducing end assay as described in Example 1 herein.

[0058] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form after N-terminal processing and / or C-terminal processing (e.g., removal of a signal peptide). In one aspect, the mature polypeptide is amino acids 1 to 474 of SEQ ID NO:3. In one aspect, the mature polypeptide is amino acids 1 to 474 of SEQ ID NO:4. In one aspect, the mature polypeptide is amino acids 1 to 489 of SEQ ID NO:5. In one aspect, the mature polypeptide is amino acids 1 to 489 of SEQ ID NO:6. In one aspect, the mature polypeptide is amino acids 1 to 489 of SEQ ID NO:7.

[0059] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having mannanase activity.

[0060] Modification: In the context of the polypeptides of the present invention, the term "modification" means changing one or more amino acids within a reference amino acid sequence (i.e., SEQ ID NO: 1 or 2) by substitution with a different amino acid, by insertion of an amino acid, or by deletion (preferably by at least one deletion). The terms "modification," "alteration," and "mutation" can be used interchangeably and constitute the same meaning and purpose.

[0061] Mutant: The term "mutant" means a polynucleotide encoding a variant.

[0062] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0063] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids can be single-stranded or double-stranded, and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0064] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not originally found in nature to contain a segment of nucleic acid or is synthetic and comprises one or more control sequences operably linked to the nucleic acid sequence.

[0065] Operably linked: The term "operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) that allows them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that the expression of the coding sequence is under the control of the regulatory sequence.

[0066] Parent or parent mannanase: The term "parent" or "parent mannanase" means the mannanase to which changes are made to produce the enzyme variants of the present invention. The mannanase having SEQ ID NO: 2 is the parent mannanase.

[0067] Protease stability: The term "protease stability" refers to the stability of a mannanase (whether wild-type, parent or variant) incubated in the presence of a protease, optionally in a detergent. For the purposes of the present invention, protease stability may be determined as shown in Example 4.

[0068] Purified: The term "purified" means a nucleic acid, variant, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., purified variants or nucleic acids may form discrete bands in electrophoretic gels, chromatographic eluates, and / or culture media subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., by weight or by molar percentage). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, variant, cell, nucleic acid, amino acid or other specified material or component in a composition at a relative or absolute concentration higher than that of the starting composition.

[0069] In one aspect, the term "purified" as used herein refers to a variant or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a variant that is substantially free of insoluble components (especially insoluble components) from the natural organism from which it is obtained. In one aspect, the variant is separated from some soluble components of the organism and culture medium from which it is recovered. The variant may be purified (i.e., isolated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0070] Accordingly, variants can be purified so that only a small amount of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" may refer to the removal of other components present in the cell of the polypeptide source, particularly other proteins and most particularly other enzymes. Variants may be "substantially pure", i.e., free of other components from the organism that produces it (e.g., a host organism for recombinantly producing the variant). On the one hand, by the weight of the total polypeptide material present in the preparation, the polypeptide is at least 40% pure. On the one hand, by the weight of the total polypeptide material present in the preparation, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure. As used herein, "substantially pure polypeptide" may refer to a polypeptide preparation containing by weight up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1% and even most preferably up to 0.5% of other polypeptide materials naturally or recombinantly associated with the polypeptide.

[0071] Thus, it is preferred that a substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure, based on the weight of the total polypeptide material present in the preparation. The variants of the invention are preferably in substantially pure form (i.e., the preparation is substantially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0072] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cleavage and rejoining) of a nucleic acid sequence to form a sequence population that is different from that found in nature. The term recombinant refers to a cell, nucleic acid, variant, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0073] Recovery: The term "recover" or "recovery" means removing a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids or other specified materials, e.g., recovering a polypeptide from a whole fermentation broth or from a cell-free fermentation broth by harvesting the polypeptide crystals, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in box filters, drum filtration, drum filtration, rotary vacuum drum filtration, candle filters, horizontal leaf filters or the like, using sheet or pad filtration in a frame or modular device) or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, cross-flow, dynamic cross-flow or ultrafiltration in dead-end operation)) or by centrifugation (using a horizontal centrifuge, a disk stack centrifuge, a hyrdo cyclone or the like) or by precipitating the polypeptide and using a related solid-liquid separation method to harvest the polypeptide from a broth medium by using size fractionation. Recovery encompasses isolation and / or purification of the polypeptide.

[0074] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0075] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48: 443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [Genetics Trend] 16: 276-277) (preferably version 6.6.0 or later). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, a non-simplified (-nobrief) option must be specified in the command line. The output of the "longest identity" of the Needle tag is calculated as follows:

[0076] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0077] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of "longest identity" using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same), which is implemented by the Needle program of EMBOSS software package (EMBOSS:The European Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, the same) (preferred 6.6.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order to make the longest identity reported by the Needle program, the non-simplified (nobrief) option must be specified in the command line. The output of "longest identity" marked by Needle is calculated as follows:

[0078] (number of identical deoxyribonucleotides x 100) / (length of alignment – ​​total number of gaps in the alignment)

[0079] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0080] Subsequence: The term "subsequence" means a polynucleotide having one or more nucleotides deleted from the 5' end and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having mannanase activity.

[0081] Surfactant stability: The term "surfactant stability" refers to the stability of a mannanase (whether wild-type, parent or variant) incubated in the presence of a surfactant, such as a surfactant in a detergent. Exemplary surfactants are those described in detail below, and in particular embodiments, surfactant stability refers to stability in the presence of anionic surfactants such as LAS. For purposes of the present invention, surfactant stability can be determined as shown in the Examples.

[0082] Textiles: The term "textile" means any textile material, including yarn, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile materials, fabrics made from these materials, and products made from fabrics (e.g., garments and other articles). The textile or fabric can be in the form of knitwear, woven fabrics, denim, nonwoven fabrics, felt, yarn, and terry cloth. The textile can be cellulose-based, such as natural cellulose, including cotton, flax / linen, jute, ramie, sisal, or coconut husk fiber; or artificial cellulose (e.g., derived from wood pulp), including viscose / rayon, ramie, cellulose acetate fibers (tricell), lyocell fibers, or blends thereof. The textile or fabric can also be based on non-cellulose, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair and silk; or synthetic polymers, such as nylon, aromatic polyamide, polyester, acrylate, polypropylene and spandex / elastane; or its blends together with blends based on cellulose and based on non-cellulose fibers. Examples of blends are blends of cotton and / or rayon / viscose with one or more accompanying materials, such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aromatic polyamide fibers) and cellulose-containing fibers (e.g., rayon / viscose fibers, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers). Fabrics can be conventional washable clothing, such as household clothing with stains. When the term fabric or clothing is used, it is intended to also include the broad term textiles.

[0083] Thermal stability: The term "thermostability" refers to the stability of a mannanase (whether wild type, parent or variant) incubated in the presence of an elevated temperature. For purposes of the present invention, nDSF (Prometheus, Nanotemper) can be used to determine thermal stability by measuring the melting temperature (Tm) of a variant in the presence of a detergent (e.g., the standard detergent of Table 1). Samples were prepared to a concentration of 10% of the standard detergent of Table 1 and a protein concentration of 200ppm. After simple mixing, the sample was loaded into a capillary and placed in an nDSF sample tray. The sample was run in Tm estimation mode with a temperature range of 25°C-85°C and a heating rate of 1°C / min. After the run was complete, the 350nm signal was used to estimate Tm in the software.

[0084] Variant: The term "variant" means a polypeptide having mannanase activity, comprising substitutions, insertions (including extensions) and / or deletions (e.g., truncations) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0085] Wash liquor: The term "wash liquor" refers to an aqueous solution comprising a mannanase variant of the present invention. A wash liquor is a solution, e.g., in a washing machine or dishwasher, containing water and a detergent composition comprising a mannanase. Before the detergent composition is mixed with water to form the wash liquor, the detergent composition can be in any form as described elsewhere herein, e.g., a liquid or powder.

[0086] Water hardness: As used herein, the term "water hardness" or "degree of hardness" or "dH" or "°dH" refers to the German degrees of hardness. One degree is defined as 10 mg calcium oxide per liter of water.

[0087] Wild-type: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of a wild-type sequence).

[0088] Variant naming conventions

[0089] For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid position in another mannanase. The amino acid sequence of another mannanase is aligned with the polypeptide disclosed in SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 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 software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 6.6.0 or later). The parameters used were gap opening penalty of 10, gap extension penalty of 0.5 and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0090] In describing the variants of the present invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0091] replace .For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 by alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represents that glycine (G) and serine (S) at position 205 and position 411 are replaced by arginine (R) and phenylalanine (F), respectively.

[0092] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position, * Accordingly, the deletion of glycine at position 195 is indicated as "Gly195*" or "G195*". Multiple deletions are separated by plus signs ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0093] insert.For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0094] In such a case, the inserted one or more amino acid residues are numbered by adding lowercase letters to the position number of the amino acid residue preceding the inserted one or more amino acid residues. In the above example, the sequence would therefore be:

[0095] Parents: Variants: 195 195 195a 195b G GKA

[0096] Multiple changes Variants containing multiple alterations are separated by plus signs ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent that arginine at position 170 and glycine at position 195 are replaced by tyrosine and glutamic acid, respectively.

[0097] Different changes . In the case where different changes can be introduced at one position, the different changes are separated by commas, for example, "Arg170Tyr,Glu" represents that the arginine at position 170 is replaced by tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0098] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION

[0099] The present invention relates to isolated mannanase variants comprising deletions at positions 490 and 491 of SEQ ID NO:2, and optionally one or more deletions or substitutions at positions corresponding to positions 16, 20, 26, 30, 36, 46, 48, 53, 61, 64, 65, 69, 70, 74, 76, 78, 82, 101, 103, 109, 111, 112, 118, 120, 126, 137, 139, 141, 143, 155, 160, 161, 162, 163, 164, 165, 166, 167, 168, 171, 172, 176, 178, 181, 182, 183, 190, 197, 214, 215, 219, 239, 244, 248, 253, 258, 271, 276, 280, 283, 286, 299, 315, 324, 366, 378, 385, 408, 410, 413, 473, 485, 486, and optionally deletions at positions corresponding to the following positions, which positions are SEQ ID NO:2, wherein the variants have at least 60% sequence identity to the polypeptide of SEQ ID NO:3, 4, 5, 6, 7, 22, 23, 24, 25, 26, 27, 28 or 29, and wherein the variants have mannanase activity.

[0100] In a further aspect, the invention relates to a variant of the polypeptide of SEQ ID NO:2, wherein the amino acids at positions 490 and 491 are deleted, wherein the variant has mannanase activity, and wherein the variant is a polypeptide of SEQ ID NO:3, a polypeptide of SEQ ID NO:4, a polypeptide of SEQ ID NO:5, a polypeptide of SEQ ID NO:6, a polypeptide of SEQ ID NO:7, a polypeptide of SEQ ID NO:22, a polypeptide of SEQ ID NO:23, a polypeptide of SEQ ID NO:24, a polypeptide of SEQ ID NO:25, a polypeptide of SEQ ID NO:26, a polypeptide of SEQ ID NO:27, a polypeptide of SEQ ID NO:28, or a polypeptide of SEQ ID NO:3. The polypeptide of NO:29 has at least 60%, for example at least 65%, 70%, 75%, 80%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, at least 90.6%, at least 90.7%, at least 90.8%, at least 90.9%, for example at least 91%, at least 91.1%, at least 91.2%, at least 91.3%, at least 91.4%, at least 91.5%, at least 91.6 %, at least 91.7%, at least 91.8%, at least 91.9%, for example at least 92%, at least 92.1%, at least 92.2%, at least 92.3%, at least 92.4%, at least 92.5%, at least 92.6%, at least 92.7%, at least 92.8%, at least 92.9%, for example at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%. 9%, for example at least 94%, at least 94.1%, at least 94.2%, at least 94.3%, at least 94.4%, at least 94.5%, at least 94.6%, at least 94.7%, at least 94.8%, at least 94.9%, for example at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97.1%, at least 97.9%, at least 97.1%, at least 97.2%, at least 97.9%, at least 97.1%, at least 97.2%, at least 97. %, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, for example at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, for example at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, such as at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity or even 100% sequence identity, and wherein the variants have mannanase activity. In particular embodiments, the variants comprise a truncation of one or more amino acids at the N-terminus. .

[0101] Variants

[0102] In the following, unless expressly stated otherwise, the amino acid numbering of the variant refers to the numbering obtained when aligned with SEQ ID: 2.

[0103] In one embodiment, the mannanase variant of the invention comprises deletions W490* and R491* of SEQ ID NO:2.

[0104] In a preferred embodiment, in addition to the amino acid deletions corresponding to the amino acids at positions W490* and R491*, the variant comprises at least 11 but less than 16 amino acid deletions from the N-terminus of the polypeptide with SEQ ID NO: 2. The deletion may comprise 11-15 (e.g., 11, 12, 13, 14, or 15) amino acids from the N-terminus of the polypeptide with SEQ ID NO: 2. In a preferred embodiment, the variant comprises the following deletion: A1*+I2*+G3*+V4*+P5*+G6*+G7*+V8*+A9*+E10*+P11*+H12*+T13*+S14*+Q15* of SEQ ID NO: 2. In particular, when the N-terminus of the polypeptide with SEQ ID NO: 2 is missing 15 amino acids, the variant may further comprise the substitution D16A.

[0105] In another embodiment, in addition to the deletions and substitutions disclosed in the above embodiments, the variant further comprises one or more modifications selected from the group consisting of I30L, D48P, Y155H, T167P, Q215E, H276C, R280K, F286C, G366N and D486E of SEQ ID NO:2.

[0106] In an embodiment, the variant comprises substitutions H276C+R280K+F286C, and optionally one or more substitutions selected from the group consisting of: SEQ ID I30L, D48P, Y155H, T167P, Q215E, G366N and D486E of NO:2, for example, substitutions of D48P+H276C+R280K+F286C, T167P+H276C+R280K+F286C, Q215E+H276C+R280K+F286C, D48P+T167P+H276C+R280K+F286C, D48P+Q215E+H276C+R280K+F286C, T167P+Q215E+H276C+R280K+F286C or D48P+T167P+Q215E+H276C+R280K+F286C.

[0107] In an embodiment, a variant of SEQ ID NO: 2 comprises at least two of the substitutions D48P, T167P and Q215E, e.g., D48P+T167P, and optionally one or more substitutions selected from the group consisting of I30L, Y155H, H276C, R280K, F286CG366N and D486E.

[0108] In an embodiment, a variant of SEQ ID NO: 2 comprises at least two of the substitutions D48P, T167P and Q215E, e.g., D48P+Q215E, and optionally one or more substitutions selected from the group consisting of I30L, Y155H, H276C, R280K, F286CG366N and D486E.

[0109] In an embodiment, a variant of SEQ ID NO: 2 comprises at least two of the substitutions D48P, T167P and Q215E, e.g., T167P+Q215E, and optionally one or more substitutions selected from the group consisting of I30L, Y155H, H276C, R280K, F286CG366N and D486E.

[0110] In an embodiment, a variant of SEQ ID NO: 2 comprises at least two of the substitutions D48P, T167P and Q215E, e.g., D48P+T167P+Q215E, and optionally one or more substitutions selected from the group consisting of I30L, Y155H, H276C, R280K, F286C G366N and D486E.

[0111] In one aspect, the variant comprises a substitution at a position corresponding to a position selected from the group consisting of: I30L, D48P, Y155H, T167P, Q215E, H276C, R280K, F286C, G366N, D486E, I30L+D48P, I30L+Y155H, I30L+T167P, I30L+Q215E, I30L+H276C, I30L+R280K, I30L+F286C, I30L+G366N, I30L+D486E, D48P+Y155H, D48P+T167P, D48P+Q215E, D48P+H276C, D48P+R280K, D48P+ F286C, D48P+G366N, D48P+D486E, Y155H+T167P, Y155H+Q215E, Y155H+H276 C. Y155H+R280K, Y155H+F286C, Y155H+G366N, Y155H+D486E, T167P+Q215E, T167P+H276C, T167P+R280K, T167P+F286C, T167P+G366N, T167P+D486E, Q2 15E+H276C, Q215E+R280K, Q215E+F286C, Q215E+G366N, Q215E+D486E, H276C +R280K, H276C+F286C, H276C+G366N, H276C+D486E, R280K+F286C, R280K+G 366N, R280K+D486E, F286C+G366N, F286C+D486E, G366N+D486E, I30L+D48P +Y155H, I30L+D48P+T167P, I30L+D48P+Q215E, I30L+D48P+H276C, I30L+D4 8P+R280K, I30L+D48P+F286C, I30L+D48P+G366N, I30L+D48P+D486E, I30L+Y 155H+T167P, I30L+Y155H+Q215E, I30L+Y155H+H276C, I30L+Y155H+R280K, I30L+Y155H+F286C, I30L+Y155H+G366N, I30L+Y155H+D486E, I30L+T167P+ Q215E, I30L+T167P+H276C, I30L+T167P+R280K, I30L+T167P+F286C, I30L+ T167P+G366N, I30L+T167P+D486E,, 30L+Q215E+H276C, I30L+Q215E+R280K,I30L+Q215E+F286C、I30L+Q215E+G366N、I30L+Q215E+D486E、I30L+H276C+R280K、I30L+H276C+F286C、I30L+H276C+G366N、I30L+H276C+D486E、I30L+R280K+F286C、I30L+R280K+G366N、I30L+R280K+D486E、I30L+F286C+G366N、I30L+F286C+D486E、I30L+G366N+D486E、D48P+Y155H+T167P、D48P+Y155H+Q215E、D48P+Y155H+H276C、D48P+Y155H+R280K、D48P+Y155H+F286C、D48P+Y155H+G366N、D48P+Y155H+D486E、D48P+T167P+Q215E、D48P+T167P+H276C、D48P+T167P+R280K、D48P+T167P+F286C、D48P+T167P+G366N、D48P+T167P+D486E、D48P+Q215E+H276C、D48P+Q215E+R280K、D48P+Q215E+F286C、D48P+Q215E+G366N、D48P+Q215E+D486E、D48P+H276C+R280K、D48P+H276C+F286C、D48P+H276C+G366N、D48P+H276C+D486E、D48P+R280K+F286C、D48P+R280K+G366N、D48P+R280K+D486E、D48P+F286C+G366N、D48P+F286C+D486E、D48P+G366N+D486E、Y155H+T167P+Q215E、Y155H+T167P+H276C、Y155H+T167P+R280K、Y155H+T167P+F286C、Y155H+T167P+G366N、Y155H+T167P+D486E、Y155H+Q215E+H276C、Y155H+Q215E+R280K、Y155H+Q215E+F286C、Y155H+Q215E+G366N、Y155H+Q215E+D486E、Y155H+H276C+R280K、Y155H+H276C+F286C、Y155H+H276C+G366N、Y155H+H276C+D486E、Y155H+R280K+F286C、Y155H+R280K+G366N、Y155H+R280K+D486E、Y155H+F286C+G366N、Y155H+F286C+D486E、Y155H+G366N+D486E、T167P+Q215E+H276C、T167P+Q215E+R280K、T167P+Q215E+F286C、T167P+Q215E+G366N、T167P+Q215E+D486E、T167P+H276C+R280K、T167P+H276C+F286C、T167P+H276C+G366N、T167P+H276C+D486E、T167P+R280K+F286C、T167P+R280K+G366N、T167P+R280K+D486E、T167P+F286C+G366N、T167P+F286C+D486E、T167P+G366N+D486E、Q215E+H276C+R280K、Q215E+H276C+F286C、Q215E+H276C+G366N、Q215E+H276C+D486E、Q215E+R280K+F286C、Q215E+R280K+G366N、Q215E+R280K+D486E、Q215E+F286C+G366N、Q215E+F286C+D486E、Q215E+G366N+D486E、H276C+R280K+F286C、H276C+R280K+G366N、H276C+R280K+D486E、H276C+F286C+G366N、H276C+F286C+D486E、H276C+G366N+D486E、R280K+F286C+G366N、R280K+F286C+D486E、R280K+G366N+D486E、F286C+G366N+D486E、I30L+D48P+Y155H+T167P、I30L+D48P+Y155H+Q215E、I30L+D48P+Y155H+H276C、I30L+D48P+Y155H+R280K、I30L+D48P+Y155H+F286C、I30L+D48P+Y155H+G366N、I30L+D48P+Y155H+D486E、I30L+D48P+T167P+Q215E、I30L+D48P+T167P+H276C、I30L+D48P+T167P+R280K、I30L+D48P+T167P+F286C、I30L+D48P+T167P+G366N、I30L+D48P+T167P+D486E、I30L+D48P+Q215E+H276C、I30L+D48P+Q215E+R280K、I30L+D48P+Q215E+F286C、I30L+D48P+Q215E+G366N、I30L+D48P+Q215E+D486E、I30L+D48P+H276C+R280K、I30L+D48P+H276C+F286C、I30L+D48P+H276C+G366N、I30L+D48P+H276C+D486E、I30L+D48P+R280K+F286C、I30L+D48P+R280K+G366N、I30L+D48P+R280K+D486E、I30L+D48P+F286C+G366N、I30L+D48P+F286C+D486E、I30L+D48P+G366N+D486E、I30L+Y155H+T167P+Q215E、I30L+Y155H+T167P+H276C、I30L+Y155H+T167P+R280K、I30L+Y155H+T167P+F286C、I30L+Y155H+T167P+G366N、I30L+Y155H+T167P+D486E、I30L+Y155H+Q215E+H276C、I30L+Y155H+Q215E+R280K、I30L+Y155H+Q215E+F286C、I30L+Y155H+Q215E+G366N、I30L+Y155H+Q215E+D486E、I30L+Y155H+H276C+R280K、I30L+Y155H+H276C+F286C、I30L+Y155H+H276C+G366N、I30L+Y155H+H276C+D486E、I30L+Y155H+R280K+F286C、I30L+Y155H+R280K+G366N、I30L+Y155H+R280K+D486E、I30L+Y155H+F286C+G366N、I30L+Y155H+F286C+D486E、I30L+Y155H+G366N+D486E、I30L+T167P+Q215E+H276C、I30L+T167P+Q215E+R280K、I30L+T167P+Q215E+F286C、I30L+T167P+Q215E+G366N、I30L+T167P+Q215E+D486E、I30L+T167P+H276C+R280K、I30L+T167P+H276C+F286C、I30L+T167P+H276C+G366N、I30L+T167P+H276C+D486E、I30L+T167P+R280K+F286C、I30L+T167P+R280K+G366N、I30L+T167P+R280K+D486E、I30L+T167P+F286C+G366N、I30L+T167P+F286C+D486E、I30L+T167P+G366N+D486E、I30L+Q215E+H276C+R280K、I30L+Q215E+H276C+F286C、I30L+Q215E+H276C+G366N、I30L+Q215E+H276C+D486E、I30L+Q215E+R280K+F286C、I30L+Q215E+R280K+G366N、I30L+Q215E+R280K+D486E、I30L+Q215E+F286C+G366N、I30L+Q215E+F286C+D486E、I30L+Q215E+G366N+D486E、I30L+H276C+R280K+F286C、I30L+H276C+R280K+G366N、I30L+H276C+R280K+D486E、I30L+H276C+F286C+G366N、I30L+H276C+F286C+D486E、I30L+H276C+G366N+D486E、I30L+R280K+F286C+G366N、I30L+R280K+F286C+D486E、I30L+R280K+G366N+D486E、I30L+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E、D48P+Y155H+T167P+H276C、D48P+Y155H+T167P+R280K、D48P+Y155H+T167P+F286C、D48P+Y155H+T167P+G366N、D48P+Y155H+T167P+D486E、D48P+Y155H+Q215E+H276C、D48P+Y155H+Q215E+R280K、D48P+Y155H+Q215E+F286C、D48P+Y155H+Q215E+G366N、D48P+Y155H+Q215E+D486E、D48P+Y155H+H276C+R280K、D48P+Y155H+H276C+F286C、D48P+Y155H+H276C+G366N、D48P+Y155H+H276C+D486E、D48P+Y155H+R280K+F286C、D48P+Y155H+R280K+G366N、D48P+Y155H+R280K+D486E、D48P+Y155H+F286C+G366N、D48P+Y155H+F286C+D486E、D48P+Y155H+G366N+D486E、D48P+T167P+Q215E+H276C、D48P+T167P+Q215E+R280K、D48P+T167P+Q215E+F286C、D48P+T167P+Q215E+G366N、D48P+T167P+Q215E+D486E、D48P+T167P+H276C+R280K、D48P+T167P+H276C+F286C、D48P+T167P+H276C+G366N、D48P+T167P+H276C+D486E、D48P+T167P+R280K+F286C、D48P+T167P+R280K+G366N、D48P+T167P+R280K+D486E、D48P+T167P+F286C+G366N、D48P+T167P+F286C+D486E、D48P+T167P+G366N+D486E、D48P+Q215E+H276C+R280K、D48P+Q215E+H276C+F286C、D48P+Q215E+H276C+G366N、D48P+Q215E+H276C+D486E、D48P+Q215E+R280K+F286C、D48P+Q215E+R280K+G366N、D48P+Q215E+R280K+D486E、D48P+Q215E+F286C+G366N、D48P+Q215E+F286C+D486E、D48P+Q215E+G366N+D486E、D48P+H276C+R280K+F286C、D48P+H276C+R280K+G366N、D48P+H276C+R280K+D486E、D48P+H276C+F286C+G366N、D48P+H276C+F286C+D486E、D48P+H276C+G366N+D486E、D48P+R280K+F286C+G366N、D48P+R280K+F286C+D486E、D48P+R280K+G366N+D486E、D48P+F286C+G366N+D486E、Y155H+T167P+Q215E+H276C、Y155H+T167P+Q215E+R280K、Y155H+T167P+Q215E+F286C、Y155H+T167P+Q215E+G366N、Y155H+T167P+Q215E+D486E、Y155H+T167P+H276C+R280K、Y155H+T167P+H276C+F286C、Y155H+T167P+H276C+G366N、Y155H+T167P+H276C+D486E、Y155H+T167P+R280K+F286C、Y155H+T167P+R280K+G366N、Y155H+T167P+R280K+D486E、Y155H+T167P+F286C+G366N、Y155H+T167P+F286C+D486E、Y155H+T167P+G366N+D486E、Y155H+Q215E+H276C+R280K、Y155H+Q215E+H276C+F286C、Y155H+Q215E+H276C+G366N、Y155H+Q215E+H276C+D486E、Y155H+Q215E+R280K+F286C、Y155H+Q215E+R280K+G366N、Y155H+Q215E+R280K+D486E、Y155H+Q215E+F286C+G366N、Y155H+Q215E+F286C+D486E、Y155H+Q215E+G366N+D486E、Y155H+H276C+R280K+F286C、Y155H+H276C+R280K+G366N、Y155H+H276C+R280K+D486E、Y155H+H276C+F286C+G366N、Y155H+H276C+F286C+D486E、Y155H+H276C+G366N+D486E、Y155H+R280K+F286C+G366N、Y155H+R280K+F286C+D486E、Y155H+R280K+G366N+D486E、Y155H+F286C+G366N+D486E、T167P+Q215E+H276C+R280K、T167P+Q215E+H276C+F286C、T167P+Q215E+H276C+G366N、T167P+Q215E+H276C+D486E、T167P+Q215E+R280K+F286C、T167P+Q215E+R280K+G366N、T167P+Q215E+R280K+D486E、T167P+Q215E+F286C+G366N、T167P+Q215E+F286C+D486E、T167P+Q215E+G366N+D486E、T167P+H276C+R280K+F286C、T167P+H276C+R280K+G366N、T167P+H276C+R280K+D486E、T167P+H276C+F286C+G366N、T167P+H276C+F286C+D486E、T167P+H276C+G366N+D486E、T167P+R280K+F286C+G366N、T167P+R280K+F286C+D486E、T167P+R280K+G366N+D486E、T167P+F286C+G366N+D486E、Q215E+H276C+R280K+F286C、Q215E+H276C+R280K+G366N、Q215E+H276C+R280K+D486E、Q215E+H276C+F286C+G366N、Q215E+H276C+F286C+D486E、Q215E+H276C+G366N+D486E、Q215E+R280K+F286C+G366N、Q215E+R280K+F286C+D486E、Q215E+R280K+G366N+D486E、Q215E+F286C+G366N+D486E、H276C+R280K+F286C+G366N、H276C+R280K+F286C+D486E、H276C+R280K+G366N+D486E、H276C+F286C+G366N+D486E、R280K+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E、I30L+D48P+Y155H+T167P+H276C、I30L+D48P+Y155H+T167P+R280K、I30L+D48P+Y155H+T167P+F286C、I30L+D48P+Y155H+T167P+G366N、I30L+D48P+Y155H+T167P+D486E、I30L+D48P+Y155H+Q215E+H276C、I30L+D48P+Y155H+Q215E+R280K、I30L+D48P+Y155H+Q215E+F286C、I30L+D48P+Y155H+Q215E+G366N、I30L+D48P+Y155H+Q215E+D486E、I30L+D48P+Y155H+H276C+R280K、I30L+D48P+Y155H+H276C+F286C、I30L+D48P+Y155H+H276C+G366N、I30L+D48P+Y155H+H276C+D486E、I30L+D48P+Y155H+R280K+F286C、I30L+D48P+Y155H+R280K+G366N、I30L+D48P+Y155H+R280K+D486E、I30L+D48P+Y155H+F286C+G366N、I30L+D48P+Y155H+F286C+D486E、I30L+D48P+Y155H+G366N+D486E、I30L+D48P+T167P+Q215E+H276C、I30L+D48P+T167P+Q215E+R280K、I30L+D48P+T167P+Q215E+F286C、I30L+D48P+T167P+Q215E+G366N、I30L+D48P+T167P+Q215E+D486E、I30L+D48P+T167P+H276C+R280K、I30L+D48P+T167P+H276C+F286C、I30L+D48P+T167P+H276C+G366N、I30L+D48P+T167P+H276C+D486E、I30L+D48P+T167P+R280K+F286C、I30L+D48P+T167P+R280K+G366N、I30L+D48P+T167P+R280K+D486E、I30L+D48P+T167P+F286C+G366N、I30L+D48P+T167P+F286C+D486E、I30L+D48P+T167P+G366N+D486E、30L+D48P+R280K+G366N+D486E,I30L+D48P+F286C+G366N+D486E、I30L+Y155H+T167P+Q215E+H276C、I30L+Y155H+T167P+Q215E+R280K、30L+Y155H+T167P+R280K+D486E、I30L+Y155H+T167P+F286C+G366N、I30L+Y155H+T167P+F286C+D486E、I30L+Y155H+T167P+G366N+D486E、I30L+Y155H+Q215E+H276C+R280K、I30L+Y155H+Q215E+H276C+F286C、I30L+Y155H+Q215E+H276C+G366N、I30L+Y155H+Q215E+H276C+D486E、I30L+Y155H+Q215E+R280K+F286C、I30L+Y155H+Q215E+R280K+G366N、I30L+Y155H+Q215E+R280K+D486E、I30L+Y155H+Q215E+F286C+G366N、I30L+Y155H+Q215E+F286C+D486E、I30L+Y155H+Q215E+G366N+D486E、I30L+Y155H+H276C+R280K+F286C、I30L+Y155H+H276C+R280K+G366N、I30L+Y155H+H276C+R280K+D486E、I30L+Y155H+H276C+F286C+G366N、I30L+Y155H+H276C+F286C+D486E、I30L+Y155H+H276C+G366N+D486E、I30L+Y155H+R280K+F286C+G366N、I30L+Y155H+R280K+F286C+D486E、I30L+Y155H+R280K+G366N+D486E、I30L+Y155H+F286C+G366N+D486E、I30L+T167P+Q215E+H276C+R280K、I30L+T167P+Q215E+H276C+F286C、I30L+T167P+Q215E+H276C+G366N、I30L+T167P+Q215E+H276C+D486E、I30L+T167P+Q215E+R280K+F286C、I30L+T167P+Q215E+R280K+G366N、I30L+T167P+Q215E+R280K+D486E、I30L+T167P+Q215E+F286C+G366N、I30L+T167P+Q215E+F286C+D486E、I30L+T167P+Q215E+G366N+D486E、I30L+T167P+H276C+R280K+F286C、I30L+T167P+H276C+R280K+G366N、I30L+T167P+H276C+R280K+D486E、I30L+T167P+H276C+F286C+G366N、I30L+T167P+H276C+F286C+D486E、I30L+T167P+H276C+G366N+D486E、I30L+T167P+R280K+F286C+G366N、I30L+T167P+R280K+F286C+D486E、I30L+T167P+R280K+G366N+D486E、I30L+T167P+F286C+G366N+D486E、I30L+Q215E+H276C+R280K+F286C、I30L+Q215E+H276C+R280K+G366N、I30L+Q215E+H276C+R280K+D486E、I30L+Q215E+H276C+F286C+G366N、I30L+Q215E+H276C+F286C+D486E、I30L+Q215E+H276C+G366N+D486E、I30L+Q215E+R280K+F286C+G366N、I30L+Q215E+R280K+F286C+D486E、I30L+Q215E+R280K+G366N+D486E、I30L+Q215E+F286C+G366N+D486E、I30L+H276C+R280K+F286C+G366N、I30L+H276C+R280K+F286C+D486E、I30L+H276C+R280K+G366N+D486E、I30L+H276C+F286C+G366N+D486E、I30L+R280K+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E+H276C、D48P+Y155H+T167P+Q215E+R280K、D48P+Y155H+T167P+Q215E+F286C、D48P+Y155H+T167P+Q215E+G366N、D48P+Y155H+T167P+Q215E+D486E、D48P+Y155H+T167P+H276C+R280K、D48P+Y155H+T167P+H276C+F286C、D48P+Y155H+T167P+H276C+G366N、D48P+Y155H+T167P+H276C+D486E、D48P+Y155H+T167P+R280K+F286C、D48P+Y155H+T167P+R280K+G366N、D48P+Y155H+T167P+R280K+D486E、D48P+Y155H+T167P+F286C+G366N、D48P+Y155H+T167P+F286C+D486E、D48P+Y155H+T167P+G366N+D486E、D48P+Y155H+Q215E+H276C+R280K、D48P+Y155H+Q215E+H276C+F286C、D48P+Y155H+Q215E+H276C+G366N、D48P+Y155H+Q215E+H276C+D486E、D48P+Y155H+Q215E+R280K+F286C、D48P+Y155H+Q215E+R280K+G366N、D48P+Y155H+Q215E+R280K+D486E、D48P+Y155H+Q215E+F286C+G366N、D48P+Y155H+Q215E+F286C+D486E、D48P+Y155H+Q215E+G366N+D486E、D48P+Y155H+H276C+R280K+F286C、D48P+Y155H+H276C+R280K+G366N、D48P+Y155H+H276C+R280K+D486E、D48P+Y155H+H276C+F286C+G366N、D48P+Y155H+H276C+F286C+D486E、D48P+Y155H+H276C+G366N+D486E、D48P+Y155H+R280K+F286C+G366N、D48P+Y155H+R280K+F286C+D486E、D48P+Y155H+R280K+G366N+D486E、D48P+Y155H+F286C+G366N+D486E、D48P+T167P+Q215E+H276C+R280K、D48P+T167P+Q215E+H276C+F286C、D48P+T167P+Q215E+H276C+G366N、D48P+T167P+Q215E+H276C+D486E、D48P+T167P+Q215E+R280K+F286C、D48P+T167P+Q215E+R280K+G366N、D48P+T167P+Q215E+R280K+D486E、D48P+T167P+Q215E+F286C+G366N、D48P+T167P+Q215E+F286C+D486E、D48P+T167P+Q215E+G366N+D486E、D48P+T167P+H276C+R280K+F286C、D48P+T167P+H276C+R280K+G366N、D48P+T167P+H276C+R280K+D486E、D48P+T167P+H276C+F286C+G366N、D48P+T167P+H276C+F286C+D486E、D48P+T167P+H276C+G366N+D486E、D48P+T167P+R280K+F286C+G366N、D48P+T167P+R280K+F286C+D486E、D48P+T167P+R280K+G366N+D486E、D48P+T167P+F286C+G366N+D486E、D48P+Q215E+H276C+R280K+F286C、D48P+Q215E+H276C+R280K+G366N、D48P+Q215E+H276C+R280K+D486E、D48P+Q215E+H276C+F286C+G366N、D48P+Q215E+H276C+F286C+D486E、D48P+Q215E+H276C+G366N+D486E、D48P+Q215E+R280K+F286C+G366N、D48P+Q215E+R280K+F286C+D486E、D48P+Q215E+R280K+G366N+D486E、D48P+Q215E+F286C+G366N+D486E、D48P+H276C+R280K+F286C+G366N、D48P+H276C+R280K+F286C+D486E、D48P+H276C+R280K+G366N+D486E、D48P+H276C+F286C+G366N+D486E、D48P+R280K+F286C+G366N+D486E、Y155H+T167P+Q215E+H276C+R280K、Y155H+T167P+Q215E+H276C+F286C、Y155H+T167P+Q215E+H276C+G366N、Y155H+T167P+Q215E+H276C+D486E、Y155H+T167P+Q215E+R280K+F286C、Y155H+T167P+Q215E+R280K+G366N、Y155H+T167P+Q215E+R280K+D486E、Y155H+T167P+Q215E+F286C+G366N、Y155H+T167P+Q215E+F286C+D486E、Y155H+T167P+Q215E+G366N+D486E、Y155H+T167P+H276C+R280K+F286C、Y155H+T167P+H276C+R280K+G366N、Y155H+T167P+H276C+R280K+D486E、Y155H+T167P+H276C+F286C+G366N、Y155H+T167P+H276C+F286C+D486E、Y155H+T167P+H276C+G366N+D486E、Y155H+T167P+R280K+F286C+G366N、Y155H+T167P+R280K+F286C+D486E、Y155H+T167P+R280K+G366N+D486E、Y155H+T167P+F286C+G366N+D486E、Y155H+Q215E+H276C+R280K+F286C、Y155H+Q215E+H276C+R280K+G366N、Y155H+Q215E+H276C+R280K+D486E、Y155H+Q215E+H276C+F286C+G366N、Y155H+Q215E+H276C+F286C+D486E、Y155H+Q215E+H276C+G366N+D486E、Y155H+Q215E+R280K+F286C+G366N、Y155H+Q215E+R280K+F286C+D486E、Y155H+Q215E+R280K+G366N+D486E、Y155H+Q215E+F286C+G366N+D486E、Y155H+H276C+R280K+F286C+G366N、Y155H+H276C+R280K+F286C+D486E、Y155H+H276C+R280K+G366N+D486E、Y155H+H276C+F286C+G366N+D486E、Y155H+R280K+F286C+G366N+D486E、T167P+Q215E+H276C+R280K+F286C、T167P+Q215E+H276C+R280K+G366N、T167P+Q215E+H276C+R280K+D486E、T167P+Q215E+H276C+F286C+G366N、T167P+Q215E+H276C+F286C+D486E、T167P+Q215E+H276C+G366N+D486E、T167P+Q215E+R280K+F286C+G366N、T167P+Q215E+R280K+F286C+D486E、T167P+Q215E+R280K+G366N+D486E、T167P+Q215E+F286C+G366N+D486E、T167P+H276C+R280K+F286C+G366N、T167P+H276C+R280K+F286C+D486E、T167P+H276C+R280K+G366N+D486E、T167P+H276C+F286C+G366N+D486E、T167P+R280K+F286C+G366N+D486E、Q215E+H276C+R280K+F286C+G366N、Q215E+H276C+R280K+F286C+D486E、Q215E+H276C+R280K+G366N+D486E、Q215E+H276C+F286C+G366N+D486E、Q215E+R280K+F286C+G366N+D486E、H276C+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C、I30L+D48P+Y155H+T167P+Q215E+R280K、I30L+D48P+Y155H+T167P+Q215E+F286C、I30L+D48P+Y155H+T167P+Q215E+G366N、I30L+D48P+Y155H+T167P+Q215E+D486E、I30L+D48P+Y155H+T167P+H276C+R280K、I30L+D48P+Y155H+T167P+H276C+F286C、I30L+D48P+Y155H+T167P+H276C+G366N、I30L+D48P+Y155H+T167P+H276C+D486E、I30L+D48P+Y155H+T167P+R280K+F286C、I30L+D48P+Y155H+T167P+R280K+G366N、I30L+D48P+Y155H+T167P+R280K+D486E、I30L+D48P+Y155H+T167P+F286C+G366N、I30L+D48P+Y155H+T167P+F286C+D486E、I30L+D48P+Y155H+T167P+G366N+D486E、I30L+D48P+Y155H+Q215E+H276C+R280K、I30L+D48P+Y155H+Q215E+H276C+F286C、I30L+D48P+Y155H+Q215E+H276C+G366N、I30L+D48P+Y155H+Q215E+H276C+D486E、I30L+D48P+Y155H+Q215E+R280K+F286C、I30L+D48P+Y155H+Q215E+R280K+G366N、I30L+D48P+Y155H+Q215E+R280K+D486E、I30L+D48P+Y155H+Q215E+F286C+G366N、I30L+D48P+Y155H+Q215E+F286C+D486E、I30L+D48P+Y155H+Q215E+G366N+D486E、I30L+D48P+Y155H+H276C+R280K+F286C、I30L+D48P+Y155H+H276C+R280K+G366N、I30L+D48P+Y155H+H276C+R280K+D486E、I30L+D48P+Y155H+H276C+F286C+G366N、I30L+D48P+Y155H+H276C+F286C+D486E、I30L+D48P+Y155H+H276C+G366N+D486E、I30L+D48P+Y155H+R280K+F286C+G366N、I30L+D48P+Y155H+R280K+F286C+D486E、I30L+D48P+Y155H+R280K+G366N+D486E、I30L+D48P+Y155H+F286C+G366N+D486E、I30L+D48P+T167P+Q215E+H276C+R280K、I30L+D48P+T167P+Q215E+H276C+F286C、I30L+D48P+T167P+Q215E+H276C+G366N、I30L+D48P+T167P+Q215E+H276C+D486E、I30L+D48P+T167P+Q215E+R280K+F286C、I30L+D48P+T167P+Q215E+R280K+G366N、I30L+D48P+T167P+Q215E+R280K+D486E、I30L+D48P+T167P+Q215E+F286C+G366N、I30L+D48P+T167P+Q215E+F286C+D486E、I30L+D48P+T167P+Q215E+G366N+D486E、I30L+D48P+T167P+H276C+R280K+F286C、I30L+D48P+T167P+H276C+R280K+G366N、I30L+D48P+T167P+H276C+R280K+D486E、I30L+D48P+T167P+H276C+F286C+G366N、I30L+D48P+T167P+H276C+F286C+D486E、I30L+D48P+T167P+H276C+G366N+D486E、I30L+D48P+T167P+R280K+F286C+G366N、I30L+D48P+T167P+R280K+F286C+D486E、I30L+D48P+T167P+R280K+G366N+D486E、I30L+D48P+T167P+F286C+G366N+D486E、I30L+D48P+Q215E+H276C+R280K+F286C、I30L+D48P+Q215E+H276C+R280K+G366N、I30L+D48P+Q215E+H276C+R280K+D486E、I30L+D48P+Q215E+H276C+F286C+G366N、I30L+D48P+Q215E+H276C+F286C+D486E、I30L+D48P+Q215E+H276C+G366N+D486E、I30L+D48P+Q215E+R280K+F286C+G366N、I30L+D48P+Q215E+R280K+F286C+D486E、I30L+D48P+Q215E+R280K+G366N+D486E、I30L+D48P+Q215E+F286C+G366N+D486E、I30L+D48P+H276C+R280K+F286C+G366N、I30L+D48P+H276C+R280K+F286C+D486E、I30L+D48P+H276C+R280K+G366N+D486E、I30L+D48P+H276C+F286C+G366N+D486E、I30L+D48P+R280K+F286C+G366N+D486E、I30L+Y155H+T167P+Q215E+H276C+R280K、I30L+Y155H+T167P+Q215E+H276C+F286C、I30L+Y155H+T167P+Q215E+H276C+G366N、I30L+Y155H+T167P+Q215E+H276C+D486E、I30L+Y155H+T167P+Q215E+R280K+F286C、I30L+Y155H+T167P+Q215E+R280K+G366N、I30L+Y155H+T167P+Q215E+R280K+D486E、I30L+Y155H+T167P+Q215E+F286C+G366N、I30L+Y155H+T167P+Q215E+F286C+D486E、I30L+Y155H+T167P+Q215E+G366N+D486E、I30L+Y155H+T167P+H276C+R280K+F286C、I30L+Y155H+T167P+H276C+R280K+G366N、I30L+Y155H+T167P+H276C+R280K+D486E、I30L+Y155H+T167P+H276C+F286C+G366N、I30L+Y155H+T167P+H276C+F286C+D486E、I30L+Y155H+T167P+H276C+G366N+D486E、I30L+Y155H+T167P+R280K+F286C+G366N、I30L+Y155H+T167P+R280K+F286C+D486E、I30L+Y155H+T167P+R280K+G366N+D486E、I30L+Y155H+T167P+F286C+G366N+D486E、I30L+Y155H+Q215E+H276C+R280K+F286C、I30L+Y155H+Q215E+H276C+R280K+G366N、I30L+Y155H+Q215E+H276C+R280K+D486E、I30L+Y155H+Q215E+H276C+F286C+G366N、I30L+Y155H+Q215E+H276C+F286C+D486E、I30L+Y155H+Q215E+H276C+G366N+D486E、I30L+Y155H+Q215E+R280K+F286C+G366N、I30L+Y155H+Q215E+R280K+F286C+D486E、I30L+Y155H+Q215E+R280K+G366N+D486E、I30L+Y155H+Q215E+F286C+G366N+D486E、I30L+Y155H+H276C+R280K+F286C+G366N、I30L+Y155H+H276C+R280K+F286C+D486E、I30L+Y155H+H276C+R280K+G366N+D486E、I30L+Y155H+H276C+F286C+G366N+D486E、I30L+Y155H+R280K+F286C+G366N+D486E、I30L+T167P+Q215E+H276C+R280K+F286C、I30L+T167P+Q215E+H276C+R280K+G366N、I30L+T167P+Q215E+H276C+R280K+D486E、I30L+T167P+Q215E+H276C+F286C+G366N、I30L+T167P+Q215E+H276C+F286C+D486E、I30L+T167P+Q215E+H276C+G366N+D486E、I30L+T167P+Q215E+R280K+F286C+G366N、I30L+T167P+Q215E+R280K+F286C+D486E、I30L+T167P+Q215E+R280K+G366N+D486E、I30L+T167P+Q215E+F286C+G366N+D486E、I30L+T167P+H276C+R280K+F286C+G366N、I30L+T167P+H276C+R280K+F286C+D486E、I30L+T167P+H276C+R280K+G366N+D486E、I30L+T167P+H276C+F286C+G366N+D486E、I30L+T167P+R280K+F286C+G366N+D486E、I30L+Q215E+H276C+R280K+F286C+G366N、I30L+Q215E+H276C+R280K+F286C+D486E、I30L+Q215E+H276C+R280K+G366N+D486E、I30L+Q215E+H276C+F286C+G366N+D486E、I30L+Q215E+R280K+F286C+G366N+D486E、I30L+H276C+R280K+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E+H276C+R280K、D48P+Y155H+T167P+Q215E+H276C+F286C、D48P+Y155H+T167P+Q215E+H276C+G366N、D48P+Y155H+T167P+Q215E+H276C+D486E、D48P+Y155H+T167P+Q215E+R280K+F286C、D48P+Y155H+T167P+Q215E+R280K+G366N、D48P+Y155H+T167P+Q215E+R280K+D486E、D48P+Y155H+T167P+Q215E+F286C+G366N、D48P+Y155H+T167P+Q215E+F286C+D486E、D48P+Y155H+T167P+Q215E+G366N+D486E、D48P+Y155H+T167P+H276C+R280K+F286C、D48P+Y155H+T167P+H276C+R280K+G366N、D48P+Y155H+T167P+H276C+R280K+D486E、D48P+Y155H+T167P+H276C+F286C+G366N、D48P+Y155H+T167P+H276C+F286C+D486E、D48P+Y155H+T167P+H276C+G366N+D486E、D48P+Y155H+T167P+R280K+F286C+G366N、D48P+Y155H+T167P+R280K+F286C+D486E、D48P+Y155H+T167P+R280K+G366N+D486E、D48P+Y155H+T167P+F286C+G366N+D486E、D48P+Y155H+Q215E+H276C+R280K+F286C、D48P+Y155H+Q215E+H276C+R280K+G366N、D48P+Y155H+Q215E+H276C+R280K+D486E、D48P+Y155H+Q215E+H276C+F286C+G366N、D48P+Y155H+Q215E+H276C+F286C+D486E、D48P+Y155H+Q215E+H276C+G366N+D486E、D48P+Y155H+Q215E+R280K+F286C+G366N、D48P+Y155H+Q215E+R280K+F286C+D486E、D48P+Y155H+Q215E+R280K+G366N+D486E、D48P+Y155H+Q215E+F286C+G366N+D486E、D48P+Y155H+H276C+R280K+F286C+G366N、D48P+Y155H+H276C+R280K+F286C+D486E、D48P+Y155H+H276C+R280K+G366N+D486E、D48P+Y155H+H276C+F286C+G366N+D486E、D48P+Y155H+R280K+F286C+G366N+D486E、D48P+T167P+Q215E+H276C+R280K+F286C、D48P+T167P+Q215E+H276C+R280K+G366N、D48P+T167P+Q215E+H276C+R280K+D486E、D48P+T167P+Q215E+H276C+F286C+G366N、D48P+T167P+Q215E+H276C+F286C+D486E、D48P+T167P+Q215E+H276C+G366N+D486E、D48P+T167P+Q215E+R280K+F286C+G366N、D48P+T167P+Q215E+R280K+F286C+D486E、D48P+T167P+Q215E+R280K+G366N+D486E、D48P+T167P+Q215E+F286C+G366N+D486E、D48P+T167P+H276C+R280K+F286C+G366N、D48P+T167P+H276C+R280K+F286C+D486E、D48P+T167P+H276C+R280K+G366N+D486E、D48P+T167P+H276C+F286C+G366N+D486E、D48P+T167P+R280K+F286C+G366N+D486E、D48P+Q215E+H276C+R280K+F286C+G366N、D48P+Q215E+H276C+R280K+F286C+D486E、D48P+Q215E+H276C+R280K+G366N+D486E、D48P+Q215E+H276C+F286C+G366N+D486E、D48P+Q215E+R280K+F286C+G366N+D486E、D48P+H276C+R280K+F286C+G366N+D486E、Y155H+T167P+Q215E+H276C+R280K+F286C、Y155H+T167P+Q215E+H276C+R280K+G366N、Y155H+T167P+Q215E+H276C+R280K+D486E、Y155H+T167P+Q215E+H276C+F286C+G366N、Y155H+T167P+Q215E+H276C+F286C+D486E、Y155H+T167P+Q215E+H276C+G366N+D486E、Y155H+T167P+Q215E+R280K+F286C+G366N、Y155H+T167P+Q215E+R280K+F286C+D486E、Y155H+T167P+Q215E+R280K+G366N+D486E、Y155H+T167P+Q215E+F286C+G366N+D486E、Y155H+T167P+H276C+R280K+F286C+G366N、Y155H+T167P+H276C+R280K+F286C+D486E、Y155H+T167P+H276C+R280K+G366N+D486E、Y155H+T167P+H276C+F286C+G366N+D486E、Y155H+T167P+R280K+F286C+G366N+D486E、Y155H+Q215E+H276C+R280K+F286C+G366N、Y155H+Q215E+H276C+R280K+F286C+D486E、Y155H+Q215E+H276C+R280K+G366N+D486E、Y155H+Q215E+H276C+F286C+G366N+D486E、Y155H+Q215E+R280K+F286C+G366N+D486E、Y155H+H276C+R280K+F286C+G366N+D486E、T167P+Q215E+H276C+R280K+F286C+G366N、T167P+Q215E+H276C+R280K+F286C+D486E、T167P+Q215E+H276C+R280K+G366N+D486E、T167P+Q215E+H276C+F286C+G366N+D486E、T167P+Q215E+R280K+F286C+G366N+D486E、T167P+H276C+R280K+F286C+G366N+D486E、Q215E+H276C+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K、I30L+D48P+Y155H+T167P+Q215E+H276C+F286C、I30L+D48P+Y155H+T167P+Q215E+H276C+G366N、I30L+D48P+Y155H+T167P+Q215E+H276C+D486E、I30L+D48P+Y155H+T167P+Q215E+R280K+F286C、I30L+D48P+Y155H+T167P+Q215E+R280K+G366N、I30L+D48P+Y155H+T167P+Q215E+R280K+D486E、I30L+D48P+Y155H+T167P+Q215E+F286C+G366N、I30L+D48P+Y155H+T167P+Q215E+F286C+D486E、I30L+D48P+Y155H+T167P+Q215E+G366N+D486E、I30L+D48P+Y155H+T167P+H276C+R280K+F286C、I30L+D48P+Y155H+T167P+H276C+R280K+G366N、I30L+D48P+Y155H+T167P+H276C+R280K+D486E、I30L+D48P+Y155H+T167P+H276C+F286C+G366N、I30L+D48P+Y155H+T167P+H276C+F286C+D486E、I30L+D48P+Y155H+T167P+H276C+G366N+D486E、I30L+D48P+Y155H+T167P+R280K+F286C+G366N、I30L+D48P+Y155H+T167P+R280K+F286C+D486E、I30L+D48P+Y155H+T167P+R280K+G366N+D486E、I30L+D48P+Y155H+T167P+F286C+G366N+D486E、I30L+D48P+Y155H+Q215E+H276C+R280K+F286C、I30L+D48P+Y155H+Q215E+H276C+R280K+G366N、I30L+D48P+Y155H+Q215E+H276C+R280K+D486E、I30L+D48P+Y155H+Q215E+H276C+F286C+G366N、I30L+D48P+Y155H+Q215E+H276C+F286C+D486E、I30L+D48P+Y155H+Q215E+H276C+G366N+D486E、I30L+D48P+Y155H+Q215E+R280K+F286C+G366N、I30L+D48P+Y155H+Q215E+R280K+F286C+D486E、I30L+D48P+Y155H+Q215E+R280K+G366N+D486E、I30L+D48P+Y155H+Q215E+F286C+G366N+D486E、I30L+D48P+Y155H+H276C+R280K+F286C+G366N、I30L+D48P+Y155H+H276C+R280K+F286C+D486E、I30L+D48P+Y155H+H276C+R280K+G366N+D486E、I30L+D48P+Y155H+H276C+F286C+G366N+D486E、I30L+D48P+Y155H+R280K+F286C+G366N+D486E、I30L+D48P+T167P+Q215E+H276C+R280K+F286C、I30L+D48P+T167P+Q215E+H276C+R280K+G366N、I30L+D48P+T167P+Q215E+H276C+R280K+D486E、I30L+D48P+T167P+Q215E+H276C+F286C+G366N、I30L+D48P+T167P+Q215E+H276C+F286C+D486E、I30L+D48P+T167P+Q215E+H276C+G366N+D486E、I30L+D48P+T167P+Q215E+R280K+F286C+G366N、I30L+D48P+T167P+Q215E+R280K+F286C+D486E、I30L+D48P+T167P+Q215E+R280K+G366N+D486E、I30L+D48P+T167P+Q215E+F286C+G366N+D486E、I30L+D48P+T167P+H276C+R280K+F286C+G366N、I30L+D48P+T167P+H276C+R280K+F286C+D486E、I30L+D48P+T167P+H276C+R280K+G366N+D486E、I30L+D48P+T167P+H276C+F286C+G366N+D486E、I30L+D48P+T167P+R280K+F286C+G366N+D486E、I30L+D48P+Q215E+H276C+R280K+F286C+G366N、I30L+D48P+Q215E+H276C+R280K+F286C+D486E、I30L+D48P+Q215E+H276C+R280K+G366N+D486E、I30L+D48P+Q215E+H276C+F286C+G366N+D486E、I30L+D48P+Q215E+R280K+F286C+G366N+D486E、I30L+D48P+H276C+R280K+F286C+G366N+D486E、I30L+Y155H+T167P+Q215E+H276C+R280K+F286C、I30L+Y155H+T167P+Q215E+H276C+R280K+G366N、I30L+Y155H+T167P+Q215E+H276C+R280K+D486E、I30L+Y155H+T167P+Q215E+H276C+F286C+G366N、I30L+Y155H+T167P+Q215E+H276C+F286C+D486E、I30L+Y155H+T167P+Q215E+H276C+G366N+D486E、I30L+Y155H+T167P+Q215E+R280K+F286C+G366N、I30L+Y155H+T167P+Q215E+R280K+F286C+D486E、I30L+Y155H+T167P+Q215E+R280K+G366N+D486E、I30L+Y155H+T167P+Q215E+F286C+G366N+D486E、I30L+Y155H+T167P+H276C+R280K+F286C+G366N、I30L+Y155H+T167P+H276C+R280K+F286C+D486E、I30L+Y155H+T167P+H276C+R280K+G366N+D486E、I30L+Y155H+T167P+H276C+F286C+G366N+D486E、I30L+Y155H+T167P+R280K+F286C+G366N+D486E、I30L+Y155H+Q215E+H276C+R280K+F286C+G366N、I30L+Y155H+Q215E+H276C+R280K+F286C+D486E、I30L+Y155H+Q215E+H276C+R280K+G366N+D486E、I30L+Y155H+Q215E+H276C+F286C+G366N+D486E、I30L+Y155H+Q215E+R280K+F286C+G366N+D486E、I30L+Y155H+H276C+R280K+F286C+G366N+D486E、I30L+T167P+Q215E+H276C+R280K+F286C+G366N、I30L+T167P+Q215E+H276C+R280K+F286C+D486E、I30L+T167P+Q215E+H276C+R280K+G366N+D486E、I30L+T167P+Q215E+H276C+F286C+G366N+D486E、I30L+T167P+Q215E+R280K+F286C+G366N+D486E、I30L+T167P+H276C+R280K+F286C+G366N+D486E、I30L+Q215E+H276C+R280K+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E+H276C+R280K+F286C、D48P+Y155H+T167P+Q215E+H276C+R280K+G366N、D48P+Y155H+T167P+Q215E+H276C+R280K+D486E、D48P+Y155H+T167P+Q215E+H276C+F286C+G366N、D48P+Y155H+T167P+Q215E+H276C+F286C+D486E、D48P+Y155H+T167P+Q215E+H276C+G366N+D486E、D48P+Y155H+T167P+Q215E+R280K+F286C+G366N、D48P+Y155H+T167P+Q215E+R280K+F286C+D486E、D48P+Y155H+T167P+Q215E+R280K+G366N+D486E、D48P+Y155H+T167P+Q215E+F286C+G366N+D486E、D48P+Y155H+T167P+H276C+R280K+F286C+G366N、D48P+Y155H+T167P+H276C+R280K+F286C+D486E、D48P+Y155H+T167P+H276C+R280K+G366N+D486E、D48P+Y155H+T167P+H276C+F286C+G366N+D486E、D48P+Y155H+T167P+R280K+F286C+G366N+D486E、D48P+Y155H+Q215E+H276C+R280K+F286C+G366N、D48P+Y155H+Q215E+H276C+R280K+F286C+D486E、D48P+Y155H+Q215E+H276C+R280K+G366N+D486E、D48P+Y155H+Q215E+H276C+F286C+G366N+D486E、D48P+Y155H+Q215E+R280K+F286C+G366N+D486E、D48P+Y155H+H276C+R280K+F286C+G366N+D486E、D48P+T167P+Q215E+H276C+R280K+F286C+G366N、D48P+T167P+Q215E+H276C+R280K+F286C+D486E、D48P+T167P+Q215E+H276C+R280K+G366N+D486E、D48P+T167P+Q215E+H276C+F286C+G366N+D486E、D48P+T167P+Q215E+R280K+F286C+G366N+D486E、D48P+T167P+H276C+R280K+F286C+G366N+D486E、D48P+Q215E+H276C+R280K+F286C+G366N+D486E、Y155H+T167P+Q215E+H276C+R280K+F286C+G366N、Y155H+T167P+Q215E+H276C+R280K+F286C+D486E、Y155H+T167P+Q215E+H276C+R280K+G366N+D486E、Y155H+T167P+Q215E+H276C+F286C+G366N+D486E、Y155H+T167P+Q215E+R280K+F286C+G366N+D486E、Y155H+T167P+H276C+R280K+F286C+G366N+D486E、Y155H+Q215E+H276C+R280K+F286C+G366N+D486E、T167P+Q215E+H276C+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+F286C、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+G366N、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+F286C+G366N、I30L+D48P+Y155H+T167P+Q215E+H276C+F286C+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+R280K+F286C+G366N、I30L+D48P+Y155H+T167P+Q215E+R280K+F286C+D486E、I30L+D48P+Y155H+T167P+Q215E+R280K+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+H276C+R280K+F286C+G366N、I30L+D48P+Y155H+T167P+H276C+R280K+F286C+D486E、I30L+D48P+Y155H+T167P+H276C+R280K+G366N+D486E、I30L+D48P+Y155H+T167P+H276C+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+Q215E+H276C+R280K+F286C+G366N、I30L+D48P+Y155H+Q215E+H276C+R280K+F286C+D486E、I30L+D48P+Y155H+Q215E+H276C+R280K+G366N+D486E、I30L+D48P+Y155H+Q215E+H276C+F286C+G366N+D486E、I30L+D48P+Y155H+Q215E+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+H276C+R280K+F286C+G366N+D486E、I30L+D48P+T167P+Q215E+H276C+R280K+F286C+G366N、I30L+D48P+T167P+Q215E+H276C+R280K+F286C+D486E、I30L+D48P+T167P+Q215E+H276C+R280K+G366N+D486E、I30L+D48P+T167P+Q215E+H276C+F286C+G366N+D486E、I30L+D48P+T167P+Q215E+R280K+F286C+G366N+D486E、I30L+D48P+T167P+H276C+R280K+F286C+G366N+D486E、I30L+D48P+Q215E+H276C+R280K+F286C+G366N+D486E、I30L+Y155H+T167P+Q215E+H276C+R280K+F286C+G366N、I30L+Y155H+T167P+Q215E+H276C+R280K+F286C+D486E、I30L+Y155H+T167P+Q215E+H276C+R280K+G366N+D486E、I30L+Y155H+T167P+Q215E+H276C+F286C+G366N+D486E、I30L+Y155H+T167P+Q215E+R280K+F286C+G366N+D486E、I30L+Y155H+T167P+H276C+R280K+F286C+G366N+D486E、I30L+Y155H+Q215E+H276C+R280K+F286C+G366N+D486E、I30L+T167P+Q215E+H276C+R280K+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E+H276C+R280K+F286C+G366N、D48P+Y155H+T167P+Q215E+H276C+R280K+F286C+D486E、D48P+Y155H+T167P+Q215E+H276C+R280K+G366N+D486E、D48P+Y155H+T167P+Q215E+H276C+F286C+G366N+D486E、D48P+Y155H+T167P+Q215E+R280K+F286C+G366N+D486E、D48P+Y155H+T167P+H276C+R280K+F286C+G366N+D486E、D48P+Y155H+Q215E+H276C+R280K+F286C+G366N+D486E、D48P+T167P+Q215E+H276C+R280K+F286C+G366N+D486E、Y155H+T167P+Q215E+H276C+R280K+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+F286C+G366N、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+F286C+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+H276C+F286C+G366N+D486E、I30L+D48P+Y155H+T167P+Q215E+R280K+F286C+G366N+D486E, I30L+D48P+Y155H+T167P+H276C+R280K+F286C+G 366N+D486E, I30L+D48P+Y155H+Q215E+H276C+R280K+F286C+G366N+D486E, I30L+D48P+T167P+Q215E+H276C+R2 80K+F286C+G366N+D486E, I30L+Y155H+T167P+Q215E+H276C+R280K+F286C+G366N+D486E, D48P+Y155H+T167P+Q 215E+H276C+R280K+F286C+G366N+D486E, I30L+D48P+Y155H+T167P+Q215E+H276C+R280K+F286C+G366N+D486E. ,

[0112] In an embodiment, the variant comprises a deletion of the above amino acids corresponding to the amino acids at positions 490 and 491 of SEQ ID NO: 2, optionally substituted D16A and one or more substitutions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 22, 24 or 25 substitutions) selected from the group consisting of G20P, I30L , D48P, A101L, A111P, A118E, S137A, Q143R, Y155H, R160L, E162D, T167P, M171I, N176S, P182R, Q183E, Q215E, V244A, H276C, R280K, F286C, H324K, G366N, D385H, and D486E.

[0113] In an embodiment, a variant of SEQ ID NO: 2 comprises substitutions I30L, D48P, Y155H, T167P, Q215E, H276C, R280K, F286C, G366N, and D486E, and deletions W490* and R491*.

[0114] In an embodiment, a variant of SEQ ID NO:2 comprises substitutions G20P, I30L, D48P, A101L, A111P, A118E, S137A, Q143R, Y155H, R160L, E162D, T167P, M171I, N176S, P182R, Q183E, Q215E, V244A, H276C, R280K, F286C, H324K, G366N, D385H, and D486E, and deletions W490* and R491*.

[0115] In an embodiment, a variant of SEQ ID NO:2 comprises substitutions G20P, I30L, D48P, A101L, A111P, A118E, S137A, Q143R, Y155H, R160L, E162D, T167P, M171I, N176S, P182R, Q183E, Q215E, V244A, H276C, R280K, F286C, H324K, G366N, D385H, and D486E, and deletions W490* and R491* and at least one substitution selected from the group consisting of V161A, R164S, R164P, R164A, R164K, I165G, I165A, I165P, I165T, and I165E.

[0116] In embodiments, the variant is a polypeptide of SEQ ID NO: 3, a polypeptide of SEQ ID NO: 4, a polypeptide of SEQ ID NO: 5, a polypeptide of SEQ ID NO: 6, a polypeptide of SEQ ID NO: 7, a polypeptide of SEQ ID NO: 22, a polypeptide of SEQ ID NO: 23, a polypeptide of SEQ ID NO: 24, a polypeptide of SEQ ID NO: 25, a polypeptide of SEQ ID NO: 26, a polypeptide of SEQ ID NO: 27, a polypeptide of SEQ ID NO: 28, or a polypeptide of SEQ ID NO: 3. The polypeptide of NO:29 has at least 90%, at least 90.1%, at least 90.2%, at least 90.3%, at least 90.4%, at least 90.5%, at least 90.6%, at least 90.7%, at least 90.8%, at least 90.9%, for example at least 91%, at least 91.1%, at least 91.2%, at least 91.3%, at least 91.4%, at least 91.5%, at least 91.6%, at least 91.7%, at least 91.8%, at least 91.9%, for example at least 92%, at least 92.1%, at least 92.2%, at least 92.3 %, at least 92.4%, at least 92.5%, at least 92.6%, at least 92.7%, at least 92.8%, at least 92.9%, for example at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%, for example at least 94%, at least 94.1%, at least 94.2%, at least 94.3%, at least 94.4%, at least 94.5%, at least 94.6%, at least 94.7%, at least 94.8%, at least 94.9%, for example at least 94.1%, at least 94.2%, at least 94.3%, at least 94.4%, at least 94.5%, at least 94.6%, at least 94.7%, at least 94.9%. 8%, at least 94.9%, for example at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, for example at least 97%, at least 97.1%, at least 97.2%, at least 97.3 %, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, for example at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, for example at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.9%.8%, or 99.9% sequence identity or even 100% sequence identity, wherein the variant has mannanase activity.

[0117] In other embodiments, the amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine stretch, antigenic epitope, or binding domain).

[0118] Examples of conservative substitutions are within 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 small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are 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.

[0119] The variants may consist of 474 to 489 amino acids, such as 474 to 485 amino acids, such as 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488 or 489 amino acids.

[0120] In an embodiment, the variant has improved stability in detergent under storage conditions compared to the parent enzyme as described in Example 3.

[0121] The mannanase variants of the present invention are preferably isolated, and more preferably purified, using standard protein purification methods known in the art.

[0122] Preparation of variants

[0123] The present invention also relates to a method for obtaining variants having mannanase activity with substitutions and deletions disclosed herein. In one embodiment, the method comprises: (a) introducing deletions into a parent mannanase at positions 490 and 491, and introducing deletions or substitutions into a parent mannanase at one or more positions corresponding to the following positions, the positions being selected from the group consisting of: SEQ ID 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 26, 30, 36, 46, 48, 53, 61, 64, 65, 69, 70, 74, 76, 78, 82, 101, 103, 109, 111, 112, 118, 120, 126, 137, 139, 141, 143, 155, 160, 161, 162, 163, 164, 165, 1 66, 167, 168, 171, 172, 176, 178, 181, 182, 183, 190, 197, 214, 215, 219, 239, 244, 248, 253, 258, 271, 276, 280, 283, 286, 299, 315, 324, 366, 378, 385, 408, 410, 413, 473, 485 and 486, wherein the variant has mannanase activity; and (b) recovering the variant.

[0124] In particular, the present invention relates to a method for obtaining a variant having mannanase activity as disclosed in the above paragraph variant.

[0125] In another specific embodiment, the present invention relates to a method for obtaining a variant having mannanase activity, wherein the variant is a polypeptide of SEQ ID NO: 3, a polypeptide of SEQ ID NO: 4, a polypeptide of SEQ ID NO: 5, a polypeptide of SEQ ID NO: 6, a polypeptide of SEQ ID NO: 7, a polypeptide of SEQ ID NO: 22, a polypeptide of SEQ ID NO: 23, a polypeptide of SEQ ID NO: 24, a polypeptide of SEQ ID NO: 25, a polypeptide of SEQ ID NO: 26, a polypeptide of SEQ ID NO: 27, a polypeptide of SEQ ID NO: 28, or a polypeptide of SEQ ID NO: 3. The polypeptide of NO:29 has at least 60%, for example at least 65%, 70%, 75%, 80%, 85%, 85.5%, 86%, 86.5%, 87%, 87.5%, 88%, 88.5%, 89%, 89.5%, 90%, 90.5%, at least 90.6%, at least 90.7%, at least 90.8%, at least 90.9%, for example at least 91%, at least 91.1%, at least 91.2%, at least 91.3%, at least 91.4%, at least 91.5%, at least 91.6%, at least 91.7 %, at least 91.8%, at least 91.9%, for example at least 92%, at least 92.1%, at least 92.2%, at least 92.3%, at least 92.4%, at least 92.5%, at least 92.6%, at least 92.7%, at least 92.8%, at least 92.9%, for example at least 93%, at least 93.1%, at least 93.2%, at least 93.3%, at least 93.4%, at least 93.5%, at least 93.6%, at least 93.7%, at least 93.8%, at least 93.9%, for example at least 94%, at least 94. 1%, at least 94.2%, at least 94.3%, at least 94.4%, at least 94.5%, at least 94.6%, at least 94.7%, at least 94.8%, at least 94.9%, for example, at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5 %, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, for example at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, for example at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%.9%, such as at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity or even 100% sequence identity.

[0126] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, and the like.

[0127] Site-directed mutagenesis is a technique that introduces one or more mutations at one or more defined sites in the polynucleotide encoding the parent.

[0128] In vitro site-directed mutagenesis can be achieved by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cutting at a site in a plasmid containing a polynucleotide encoding a parent by a restriction enzyme and then connecting an oligonucleotide containing the mutation to the polynucleotide. Typically, the restriction enzymes that digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to connect to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0129] In vivo site-directed mutagenesis can also be achieved by methods known in the art. See, for example, US 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0130] Any site-directed mutagenesis procedure may be used in the present invention.There are many commercially available kits that can be used to prepare variants.

[0131] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as multiplexed microchip-based techniques described by Tian et al., 2004, Nature 432:1050-1054, and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0132] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0133] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0134] The many aspects of semi-synthetic gene construction are realized by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or reorganization. Semi-synthetic construction typically utilizes the process of synthetic polynucleotide fragments and the combination of PCR technology. Therefore, the restricted area of ​​gene can be synthesized from scratch, and other regions can use site-specific mutagenic primers to increase, and other regions can carry out fallibility PCR or non-fallibility PCR amplification. Then the polynucleotide subsequence can be reorganized.

[0135] Polynucleotide

[0136] The present invention also relates to polynucleotides encoding the variants of the present invention.

[0137] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0138] In one aspect, the polynucleotide is isolated.

[0139] In another aspect, the polynucleotide is purified.

[0140] Nucleic acid construct

[0141] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0142] The polynucleotides can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to its insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0143] Promoter

[0144] The control sequence may be a promoter, i.e., a polynucleotide that is recognized by the host cell to express the polynucleotide encoding the variant of the invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant promoters, truncated promoters, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0145] Examples of suitable promoters for directing transcription of the polynucleotides of the present invention in bacterial host cells are described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevie, and Song et al., 2016, PLOS One 11(7):e0158447.

[0146] Terminator

[0147] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator may be operably linked to the 3'-end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

[0148] Preferred terminators for bacterial host cells can be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0149] mRNA Stabilizer

[0150] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0151] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0152] Examples of mRNA stabilizer regions of fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824 and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0153] Preamble sequence

[0154] The control sequence may also be a leader sequence, i.e., a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence may be operably linked to the 5'-end of the polynucleotide encoding the variant. Any leader sequence that is functional in the host cell may be used.

[0155] Suitable leader sequences for bacterial host cells are provided by Hambraeus et al., 2000, Microbiology 146(12):3051-3059 and Kaberdin and 2006, FEMS Microbiol. Rev. [FEMS Microbiology Review] 30(6): 967-979 description.

[0156] polyadenylation sequence

[0157] The control sequence may also be a polyadenylation sequence, ie, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0158] Signal peptide

[0159] The control sequence can also be a signal peptide coding region, which encodes a signal peptide connected to the N-terminus of the variant, and instructs the variant to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide can inherently contain a signal peptide coding sequence, which is naturally connected to the segment of the coding sequence encoding the variant in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence, so as to enhance the secretion of the variant. However, any signal peptide coding sequence that instructs the variant expressed to enter the secretory pathway of the host cell can be used.

[0160] Propeptide

[0161] The control sequence can also be a propeptide coding sequence encoding a propeptide located at the N-terminal of a variant. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptide is usually inactive and can be converted into an active variant by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the gene of the following items: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease and Saccharomyces cerevisiae α-factor.

[0162] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0163] Adjustment sequence

[0164] It is also desirable to add regulatory sequences that regulate the expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory sequences in prokaryotic systems include lac, tac, and trp operator systems.

[0165] Transcription Factors

[0166] The control sequence can also be a transcription factor, i.e. a polynucleotide encoding a polynucleotide-specific DNA binding polypeptide, which controls the transcription rate of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can play a role alone and / or together with one or more other polypeptides or transcription factors in the complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA binding domain, which is usually attached to a specific DNA sequence adjacent to a genetic element regulated by a transcription factor. Transcription factors can directly (i.e., activate the transcription of a gene encoding a target protein by binding to its promoter) or indirectly (i.e., such as by binding to a promoter of another transcription factor (which regulates the transcription of a gene encoding a target protein) to activate the transcription of another transcription factor) regulate the expression of the target protein. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011, Subcellular Biochemistry 52:7-23 and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0167] Expression vector

[0168] The present invention further relates to a recombinant expression vector comprising a polynucleotide, a promoter and a transcription and translation termination signal for encoding a variant of the present invention. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which can include one or more suitable restriction sites to allow the polynucleotides to be inserted or substituted at such sites for encoding variants. Alternatively, the polynucleotides can be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing an expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to the suitable control sequence for expression.

[0169] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0170] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicates with one or more chromosomes into which it has been integrated when it is introduced into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids can be used, which contain the total DNA to be introduced into the host cell genome together, or a transposon can be used.

[0171] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0172] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0173] For integration into the host cell genome, the vector may rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology directed repair (HDR), or non-homologous recombination, such as non-homologous end joining (NHEJ).

[0174] For autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicon that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0175] More than one copy of polynucleotide of the present invention can be inserted into host cell to improve the production of polypeptide.For example, 2 or 3 or 4 or 5 or more copies are inserted into host cell.The copy number of the increase of polynucleotide can be obtained by integrating at least one other copy of sequence into host cell genome or by including amplifiable selectable marker gene together with the polynucleotide, wherein the cell containing amplified copy of selectable marker gene and other copy of the polynucleotide can be selected by cultivating cell in the presence of suitable selective agent.

[0176] Host cells

[0177] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention operably linked to one or more regulatory sequences that direct the production of a variant of the present invention.

[0178] The construct or vector comprising the polynucleotide is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The selection of the host cell will depend to a large extent on the gene encoding the variant and its source. The recombinant host cell may comprise a single copy or at least two copies, such as three, four, five or more copies of the polynucleotide of the present invention.

[0179] The host cell may be any cell useful for the recombinant production of a variant of the invention, such as a prokaryotic cell or a fungal cell.

[0180] The host cell may be any microbial cell, such as a prokaryotic cell or a fungal cell, that can be used to recombinantly produce a polypeptide of the present invention.

[0181] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0182] The bacterial host cell can be any Bacillus cell, including but 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 stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus subtilis cell.

[0183] For the purposes of the present invention, the Bacillus genus / species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70:406-438.

[0184] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

[0185] The bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0186] Methods for introducing DNA into prokaryotic host cells are well known in the art, and any suitable method can be used, including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, wherein DNA is introduced as linearized or circular polynucleotides. Those skilled in the art will be able to easily determine the appropriate method for introducing DNA into a given prokaryotic cell according to, for example, the genus. Methods for introducing DNA into prokaryotic host cells are described, for example, in Heinze et al., 2018, BMC Microbiology [BMC Microbiology] 18: 56, Burke et al., 2001, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods [Microbiological Methods Magazine] 64: 391-397 and Donald et al., 2013, J. Bacteriol. [Bacteriology Magazine] 195 (11): 2612-2620.

[0187] In one aspect, the host cell is isolated, preferably the host cell is purified.

[0188] Production method

[0189] The present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.

[0190] Host cells are cultivated in a nutrient medium suitable for producing variants using methods known in the art. For example, cells can be cultured by shaking flasks, or in a suitable medium and under conditions allowing variant expression and / or separation, small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to cultivate cells. Suitable culture medium can be obtained from commercial suppliers or can be prepared according to disclosed compositions (for example, in the catalog of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be directly recovered from the culture medium. If the variant is not secreted, it can be recovered from a cell lysate.

[0191] Variants may be detected using methods known in the art that are specific for the variant, including but not limited to enzyme assays using specific antibodies, enzyme product formation, disappearance of enzyme substrate, or determining the relative or specific activity of the variant.

[0192] The variant can be recovered from the culture medium using methods known in the art, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0193] Variants can be purified by a variety of 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).

[0194] In an alternative aspect, the variant is not recovered.

[0195] Mannanase Granules

[0196] The invention also relates to enzyme granules / particles comprising a variant of the invention.In an embodiment, the granule comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0197] The diameter of the core (measured as an equivalent spherical diameter (average particle size based on volume)) may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm. Laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020), may be used to determine the core diameter measured as an equivalent spherical diameter.

[0198] In an embodiment, the core comprises a variant of the invention.

[0199] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0200] The core may include a binder such as a synthetic polymer, wax, fat or carbohydrate.

[0201] The core may include, typically as a homogenous blend, a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposition catalyst, and / or an acidic buffer component.

[0202] The core may comprise an inert particle into which the variant is adsorbed, or applied (eg by fluid bed coating) to a surface.

[0203] The diameter of the core may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm.

[0204] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling or for coloring the granules. The optional coating(s) may include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methylhydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA).

[0205] The coating may be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10% or at least 15%) by weight of the core. The amount may be up to 100%, 70%, 50%, 40% or 30%.

[0206] The coating is preferably at least 0.1 μm thick, in particular at least 0.5 μm, at least 1 μm or at least 5 μm thick.In some embodiments, the coating has a thickness of less than 100 μm, such as less than 60 μm or less than 40 μm.

[0207] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating with few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0208] The coating may further contain other materials as known in the art, for example fillers, anti-tack agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0209] The salt coating may comprise at least 60% by weight salt, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

[0210] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, for example at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm or at least 8 μm. In particular embodiments, the thickness of the salt coating is less than 100 μm, such as less than 60 μm or less than 40 μm.

[0211] The salt may be added from a salt solution (wherein the salt is completely dissolved), or from a salt suspension (wherein the fine particles are less than 50 μm, such as less than 10 μm or less than 5 μm).

[0212] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, particularly having a solubility of at least 0.1 g in 100 g water at 20°C, preferably at least 0.5 g / 100 g water, such as at least 1 g / 100 g water, such as at least 5 g / 100 g water.

[0213] The salt can be an inorganic salt, for example a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or a salt of a simple organic acid (less than 10 carbon atoms, for example 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions or metal ions of the first transition series, for example sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chlorine, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0214] The salt in the coating may have a constant humidity of 60% or more, in particular 70% or more, 80% or more or 85% or more at 20°C, or it may be another hydrate form (e.g., anhydrous) of such a salt. The salt coating may be as described in WO00 / 01793 or WO 2006 / 034710.

[0215] A specific example of a suitable salt is NaCl (CH 20 ℃=76%)、Na2CO3(CH 20 ℃=92%)、NaNO3(CH 20℃ =73%), Na2HPO4(CH 20℃ =95%), Na3PO4(CH 25℃ =92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93,0%)、NH4H2PO4(CH 20℃ =93.1%), (NH4)2SO4(CH 20℃ =81.1%), KCl(CH 20℃ =85%), K2HPO4(CH 20℃ =92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ =93.5%), Na2SO4(CH 20℃ =93%), K2SO4(CH 20℃ =98%), KHSO4(CH 20℃ =86%), MgSO4(CH 20℃ =90%), ZnSO4(CH 20℃ =90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0216] The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate having one or more bound waters of crystallization, as described, for example, in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0217] Preferably, the salt is used as a salt solution, for example using a fluidized bed.

[0218] The coating material can be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1483591.

[0219] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxy-propylcellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with various coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0220] The core can be prepared by granulating a blend of ingredients, for example by a process comprising granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction, drum granulation and / or high shear granulation.

[0221] Methods for preparing the cores can be found in Handbook of Powder Technology; CE Capes, Particle size enlargement; Vol. 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0222] (a) Spray-dried products, in which a liquid enzyme-containing solution is atomized in a spray-drying tower to form small droplets which dry as they descend along the drying tower to form a particulate material containing the enzyme. Very small particles can be produced in this way (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0223] (b) layered products, wherein the enzyme is coated in layers around preformed inert core particles, wherein the enzyme solution is typically atomized in a fluidized bed apparatus, in which the preformed core particles are fluidized and the enzyme solution is attached to the core particles and dried until the dry enzyme layer is left on the surface of the core particles. If a useful core particle with a desired size can be found, particles with a desired size can be obtained in this way. This type of product is described in, for example, WO 97 / 23606.

[0224] (c) Absorbed core particles, wherein instead of the variant being coated as a layer around the core, the enzyme is absorbed on and / or in the surface of the core. Such a method is described in WO 97 / 39116.

[0225] (d) Extruded or pelletized products, in which the variant-containing paste is pressed into pellets or extruded under pressure through small openings and cut into particles, and the pellets are subsequently dried. Such particles are usually of considerable size, since the material through which the extrusion openings are opened (usually a flat plate with drilled holes) limits the permissible pressure drop across the extrusion openings. Furthermore, when small openings are used, very high extrusion pressures increase the heat generation in the enzyme paste, which is detrimental to the enzymes (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0226] (e) Spray granulated products, in which a powder containing the variant is suspended in molten wax and the suspension is sprayed (e.g. by a rotating disk atomizer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The product obtained is one in which the variant is uniformly distributed throughout the inert material rather than concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

[0227] (f) mixer granulation product, wherein the variant liquid is added to the dry powder composition of the conventional granulation component.Liquid and powder are mixed in a suitable ratio, and because the moisture of the liquid is absorbed by the dry powder, the components of the dry powder begin to adhere and agglomerate, and the particles will accumulate to form particles comprising enzymes. Such methods are described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In the particular aspects of this process, various high shear mixers can be used as granulators. The particles composed of variants, fillers and adhesives etc. are mixed with cellulose fibers to strengthen particles, thereby producing so-called T-particles. Strengthened particles are more solid, and release less enzyme dust.

[0228] (g) Particle size reduction, wherein the core is produced by milling or crushing larger particles, pellets, platelets, briquettes, etc. containing the enzyme. The desired core particle fraction is obtained by sieving the milled or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0229] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending microparticles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. The particles hit by the sprayed droplets are wetted and become sticky. The sticky particles collide with other particles and attach to them to form granules.

[0230] (i) The cores may be subjected to drying, for example in a fluid bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by the skilled person. Drying is preferably carried out at a product temperature of from 25°C to 90°C. For some enzymes, it is important that the cores comprising the variant contain a small amount of water before coating with salt. If a water-sensitive enzyme is coated with salt before the excess water is removed, the excess water will be trapped in the core and may have a negative impact on the activity of the enzyme. After drying, the cores preferably contain 0.1% w / w-10% w / w of water.

[0231] Non-dusting granules may be produced, for example, as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.

[0232] The granules may further comprise one or more additional enzymes. Each enzyme will then be present in more granules, ensuring a more even distribution of the enzymes and also reducing the physical separation of different enzymes due to different particle sizes. A method for producing multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0233] Another example of formulating enzymes by using co-granules is disclosed in WO 2013 / 188331.

[0234] The present invention also relates to a protected enzyme prepared according to the method disclosed in EP 238216.

[0235] In an embodiment, the granule further comprises one or more additional enzymes selected from the group consisting of amylase, protease, peroxidase, cellulase, beta-glucanase, xyloglucanase, hemicellulase, xanthan endoglucanase, xanthan lyase, lipase, acyltransferase, phospholipase, esterase, laccase, catalase, arylesterase, amylase, alpha-amylase, glucoamylase, cutinase, pectinase, pectin lyase, keratinase, Reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenase, pullulanase, tannase, arabinosidase, hyaluronidase, chondroitinase, xylanase, pectin acetylesterase, polygalacturonidase, rhamnogalacturonidase, other endo-β-mannanase, exo-β-mannanase, pectin methylesterase, cellobiohydrolase, transglutaminase, lichenase, laminarinase and DNase or any combination thereof.

[0236] Liquid preparations

[0237] The present invention also relates to a liquid composition comprising a variant of the present invention. The composition may comprise an enzyme stabilizer (examples of the enzyme stabilizer include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic boric acid esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid).

[0238] In certain embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates and silicates, as well as talcum, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In certain embodiments, compositions contain from about 5% to about 90% of such materials.

[0239] In one aspect, the liquid formulation comprises 20%-80% w / w of a polyol. In one embodiment, the liquid formulation comprises 0.001%-2% w / w of a preservative.

[0240] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0241] (A) 0.001%-25% w / w of a variant of the invention;

[0242] (B) 20% to 80% w / w of a polyol;

[0243] (C) optionally 0.001%-2% w / w preservative; and

[0244] (D) Water.

[0245] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0246] (A) 0.001%-25% w / w of a variant of the invention;

[0247] (B) 0.001%-2% w / w preservative;

[0248] (C) optionally 20% to 80% w / w of a polyol; and

[0249] (D) Water.

[0250] In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0251] In another embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% polyol, such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0252] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w preservative, 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0253] In another embodiment, the liquid formulation further comprises one or more additional enzymes selected from the group consisting of amylase, alpha-amylase, beta-amylase, beta-glucanase, protease, oxidoreductase, peroxidase, cellulase, beta-glucanase, xyloglucanase, hemicellulase, xanthan endoglucanase, xanthan lyase, lipase, acyltransferase, phospholipase, esterase, laccase, catalase, arylesterase, amylase, alpha-amylase, glucoamylase, cutinase, , pectinases, pectin lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenase, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xylanases, pectin acetylesterases, polygalacturonidases, rhamnogalacturonidases, other endo-β-mannanases, exo-β-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, lichenases, laminarinases and DNases, or any combination thereof.

[0254] Other enzymes

[0255] In one embodiment, the mannanase variants of the invention are combined with one or more enzymes, such as at least two enzymes, more preferably at least three, four or five enzymes. Preferably, these enzymes have different substrate specificities, such as proteolytic activity, amylolytic activity, lipolytic activity, hemicellulolytic activity, mannanase activity or pectinase activity.

[0256] The detergent additive together with the detergent composition may comprise one or more enzymes, such as proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, additional mannanases, arabinanases, galactanases, xylanases, oxidases (e.g., laccases) and / or peroxidases, lichenases, laminarinases, DNases.

[0257] Generally, the properties of the enzyme(s) selected should be compatible with the selected detergent (ie, pH optimum, compatibility with other enzymes and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in an effective amount.

[0258] Cellulase:

[0259] Suitable cellulases include those of animal, plant or microbial origin. Particularly suitable cellulases include those of bacterial or fungal origin. Chemically modified variants or protein engineered variants are included. Suitable cellulases include cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, such as those disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89 / 09259 produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum.

[0260] Especially suitable cellulases are alkaline or neutral cellulases with color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are those cellulase variants as described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307 and WO 1999 / 001544.

[0261] Commercially available cellulases include Celluzyme TM , and Carezyme TM (Novozymes A / S), Clazinase TM , and Puradax HA TM (Genencor International Inc.), and KAC-500(B) TM (Kao Corporation).

[0262] Protease:

[0263] Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, such as microbial or plant origin. Preferably microbial origin. Chemically modified variants or protein engineered variants are included. It can be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease can be, for example, a S1 family (such as trypsin) or a S8 family (such as subtilisin). Metalloproteases can be, for example, thermolysin or other metalloproteases from, for example, the M4 family, such as those from the M5, M7 or M8 families.

[0264] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Engng. [Protein Engineering] 4 (1991) 719-737 and Siezen et al., Protein Science [Protein Science] 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 subclasses, namely, the subtilisin family, the thermophilic protease family, the proteinase K family, the lanthionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0265] Examples of subtilases are those derived from the genus Bacillus, such as Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US7262042 and WO09 / 021867; and subtilisin lentus protease, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89 / 06279 and protease PD138 described in (WO93 / 18140). Other useful proteases may be those described in WO92 / 175177, WO01 / 016285, WO02 / 026024 and WO02 / 016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease (described in WO89 / 06270, WO94 / 25583 and WO05 / 040372), and chymotrypsin from Cellumonas (described in WO05 / 052161 and WO05 / 052146).

[0266] Further preferred proteases are the alkaline proteases from Bacillus lentus DSM 5483 (as described, for example, in WO 95 / 23221) and variants thereof (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0267] Examples of metalloproteases are neutral metalloproteases as described in WO 07 / 044993 (Genencor International), for example those derived from Bacillus amyloliquefaciens.

[0268] Examples of useful proteases are variants described in WO92 / 19729, WO96 / 034946, WO98 / 20115, WO98 / 20116, WO99 / 011768, WO01 / 44452, WO03 / 006602, WO04 / 03186, WO04 / 041979, WO07 / 006305, WO11 / 036263, WO11 / 036264, especially those having substitutions at one or more of the following positions: Variants: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, numbered using BPN'. More preferred protease variants may comprise the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (use BPN' for numbering).

[0269] Suitable commercially available proteases include those sold under the following trade names: Duralase TM 、Durazym TM , Ultra, Ultra, Ultra, Ultra, and (Novozymes), those sold under the following trade names: Purafect Prefer TM 、Purafect Purafect Purafect EffectTM , and (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in FIG. 29 of US5352604) and its variants (Henkel AG), and KAP (alkaliphilic Bacillus subtilisin) from Kao Corporation.

[0270] Lipase:

[0271] Suitable lipases include those of animal, plant or microbial origin. Particularly suitable lipases include those of bacterial or fungal origin. Chemically modified variants or protein engineered variants are included. Examples of useful lipases include lipases from the genus Humicola (synonymous with Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216, or from Humicola insolens as described in WO 96 / 13580; Pseudomonas lipases, e.g., from Pseudomonas alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (P. stutzeri) (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95 / 06720 and WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus lipase, for example from Bacillus subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta [Biochemistry and Biophysics Acta], 1131:253-360), Bacillus stearothermophilus (JP 64 / 744992) or Bacillus pumilus (WO 91 / 16422).

[0272] Other examples are lipase variants, such as those described in WO 92 / 05249, WO 94 / 01541, EP 407225, EP 260105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079 and WO 97 / 07202.

[0273] Preferred commercially available lipases include Lipolase TM , Lipolase Ultra TM and Lipex TM (Novozymes).

[0274] Amylase:

[0275] Suitable amylases that can be used with the mannanase of the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified variants or protein engineered variants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, such as a particular strain of Bacillus licheniformis (described in more detail in GB 1,296,839). Suitable amylases include amylases having SEQ ID NO:3 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO:3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and in SEQ ID NO: 4 of WO 99 / 019467, such as variants having substitutions in 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. Different suitable amylases include an amylase having SEQ ID NO: 6 in WO 02 / 010355, or a variant thereof having 90% sequence identity thereto. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193. Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens as shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of a Bacillus licheniformis alpha-amylase as shown 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 derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 is a variant having the following substitutions:

[0276] M197T;

[0277] H156Y+A181T+N190F+A209V+Q264S; or

[0278] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S。

[0279] Another suitable amylase is an amylase having SEQ ID NO:6 in WO 99 / 019467 or a variant thereof having 90% sequence identity with 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, 1206, E212, E216, and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184. Another amylase that can be used is one having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 of WO 96 / 023873, or a variant thereof having 90% sequence identity with 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. More preferred variants are those having deletions at positions 181 and 182 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those having deletions in positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476. Other amylases that can be used are those having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or a variant thereof having 90% sequence identity with SEQ ID NO: 2 of WO 08 / 153815, or a variant thereof having 90% sequence identity with SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264. Another suitable amylase is an amylase having SEQ ID NO: 2 of WO 09 / 061380, or a variant thereof having 90% sequence identity with SEQ ID NO: 2.Preferred variants of SEQ ID NO:2 are those with 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 are those with 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 those with deletions in positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those with the following substitutions:.

[0280] N128C+K178L+T182G+Y305R+G475K;

[0281] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0282] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0283] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C-terminally truncated and optionally further comprise a substitution at position 243 and / or a deletion at position 180 and / or position 181. Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, deletion or insertion at one or more of the following positions of SEQ ID NO: 12 in WO01 / 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 with a deletion of D183 and G184 and with substitutions R118K, N195F, R320K and R458K, and variants with substitutions in addition at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, most preferably variants with substitutions in addition at all of these positions. Other examples are amylase variants such as those described in WO2011 / 098531, WO2013 / 001078 and WO2013 / 001087. Commercially available amylases are Duramyl TM Termamyl TM 、Fungamyl TM 、Stainzyme TM 、Stainzyme Plus TM 、Natalase TM , Liquozyme X and BAN TM (from Novozymes), and Rapidase TM 、Purastar TM / Effect TM , Powerase and Preferenz S100 (from Genencor International / DuPont).

[0284] Peroxidase / Oxidase:

[0285] Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified variants or protein engineered variants are included. Examples of useful peroxidases include peroxidases from Coprinus, such as C. cinereus, and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602 and WO 98 / 15257.

[0286] Commercially available peroxidases include Guardzyme TM (Novozymes).

[0287] Pectinase

[0288] Pectin lyases (pectinases) can be classified according to their preferred substrates (highly methyl-esterified pectin or lowly methyl-esterified pectin and polygalacturonic acid (pectic acid)) and their reaction mechanism (β-elimination or hydrolysis). Pectinases can be primarily endo-acting, cleaving the polymer at random sites within the chain to produce a mixture of oligomers; or they can be exo-acting, attacking from one end of the polymer and producing monomers or dimers. The enzyme classification provided by Enzyme Nomenclature (1992) includes several pectinase activities that act on the smooth zone of pectin, such as pectin lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), exopolygalacturonase (EC 3.2.1.67), exopolygalacturonate lyase (EC 4.2.2.9) and exopoly-α-galacturonidase (EC 3.2.1.82).

[0289] Pectin lyases have been cloned from different bacterial genera such as Erwinia, Pseudomonas, Klebsiella and Xanthomonas. Pectin lyases have also been described from Bacillus subtilis (Nasser et al. (1993) FEBS 335:319-326) and Bacillus species YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem. [Bioscience Biotechnology and Biochemistry] 58:947-949). Pectin lyases are generally characterized by an alkaline optimum pH and an absolute requirement for divalent cations, Ca. 2+ The most stimulating.

[0290] Variants of the pectin lyase from Bacillus subtilis are disclosed in, inter alia, patent applications WO 2002 / 092741, WO 2003 / 095638 and WO 2018 / 007435.

[0291] Xanthan Endoglucanase

[0292] Xanthan gum endoglucanase is an endoglucanase exhibiting endo-β-1,4-glucanase activity, which can catalyze the hydrolysis of 1,4-connected β-D-glucose polymeric backbones of xanthan gum together with a suitable xanthan lyase. Such endoglucanase is disclosed in WO 2018 / 037062. Xanthan gum endoglucanase activity can be determined using a colorimetric assay developed by Lever (1972), Anal. Biochem. [Analytical Biochemistry] 47: 273-279, 1972.

[0293] An example of a xanthan endoglucanase is the polypeptide having SEQ ID NO:31.

[0294] Xanthan Gum Lyase

[0295] Xanthan lyases are enzymes that cleave the β-D-mannosyl-β-D-1,4-glucuronyl bonds of xanthan gum and have been described in the literature. Xanthan lyases are known in the art, for example, two xanthan lyases have been isolated from Paenibacillus alginolyticus XL-1 (e.g., Ruijssenaars et al. (1999) 'A pyruvated mannose-specific xanthan lyase involved in xanthandegradation by Paenibacillus alginolyticus XL-1', Appl. Environ. Microbiol. 65(6):2446-2452, and Ruijssenaars et al. (2000), 'A novel gene encoding xanthan lyase of Paenibacillus alginolyticus strain XL-1 [A novel gene encoding xanthan lyase from Vibrio alginolyticus Paenibacillus strain XL-1]', Appl. Environ. Microbiol. [Application and Environmental Microbiology] 66(9): 3945-3950).

[0296] Xanthan lyase is classified according to enzyme nomenclature as EC 4.2.2.12. This enzyme belongs to the family of lyases, particularly those carbon-oxygen lyases acting on polysaccharides.

[0297] WO 2017 / 046260 discloses polypeptides having xanthan gum degrading activity.

[0298] An example of a xanthan lyase is the polypeptide having SEQ ID NO:30.

[0299] Xyloglucanase

[0300] Xyloglucanase can catalyze xyloglucan to be dissolved into xyloglucan oligosaccharides.Some xyloglucanases only show xyloglucanase activity, while other xyloglucanases show both xyloglucanase activity and cellulase activity.Xyloglucanase can be classified as EC 3.2.1.4 or EC.3.2.1.151.Enzymes with xyloglucanase activity are for example described in Vincken et al. (1997) Carbohydrate Research [Carbohydrate Research] 298 (4): 299-310, wherein characterize three different endoglucanases EndoI, EndoV and EndoVI from Trichoderma viride (similar to T. reesei). EndoI, EndoV and EndoVI belong to glycosyl hydrolase families 5, 7 and 12, respectively, see Henrissat, B. (1991) Biochem. J. [Journal of Biological Chemistry] 280: 309-316 and Henrissat, B. and Bairoch, A. (1993) Biochem. J. [Journal of Biological Chemistry] 293: 781-788. WO 94 / 14953 discloses a Family 12 xyloglucanase (EG II) cloned from the fungus Aspergillus aculeatus. WO 99 / 02663 discloses a Family 12 and a Family 5 xyloglucanase cloned from Bacillus licheniformis and Bacillus agaradhaerens, respectively. WO 01 / 062903 discloses a Family 44 xyloglucanase.

[0301] WO 2009 / 147210 discloses xyloglucanase variants. WO 99 / 02663 and WO 01 / 062903 indicate that xyloglucanases can be used in detergents.

[0302] Lichenase / β-Glucanase:

[0303] Suitable lichenases (lichenases) include those of bacterial or fungal origin. They may be chemically modified or protein engineered. Examples of useful beta-glucanases include those described in WO 2015 / 144824 (Novozymes) and WO 99 / 06516 (Henkel KGAA, Germany).

[0304] Nuclease:

[0305] Suitable nucleases include deoxyribonucleases (DNA enzymes) and ribonucleases (RNA enzymes), which are any enzymes that catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA or RNA backbone to degrade DNA and RNA, respectively. There are two main classifications according to the active site. Exonucleases digest nucleic acids from the ends. Endonucleases act on the region in the middle of the target molecule. Nucleases are preferably DNA enzymes, which are preferably available from microorganisms, preferably fungi or bacteria. In particular, DNA enzymes available from species of Bacillus are preferred; in particular, DNA enzymes available from food Bacillus (Bacillus cibi), Bacillus subtilis or Bacillus licheniformis are preferred. Examples of such DNA enzymes are described in WO 2011 / 098579, WO2014 / 087011 and WO2017 / 060475. Also particularly preferred is a DNase obtainable from Aspergillus species; in particular a DNase obtainable from Aspergillus oryzae, such as the DNase described in WO 2015 / 155350.

[0306] One or more detergent enzymes may be included in the detergent composition by adding a single additive containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additive of the present invention, i.e., the single additive or the combined additive, may be formulated, for example, as a granule, a liquid, a slurry, etc. Preferred detergent additive formulations are granules, especially non-dusting granules as described above; liquids, especially stabilized liquids; or slurries.

[0307] Mannanase

[0308] Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. Mannanases may be alkaline mannanases of family 5 or 26. It may be a wild type from Bacillus or Humicola, particularly from Bacillus viscidus, Bacillus licheniformis, Bacillus halodurans, Bacillus clausii, or Humicola insolens. Suitable mannanases are described in WO 1999 / 064619. Commercially available mannanases are Mannaway (Novozymes). Other commercially available mannanases are disclosed in SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

[0309] Xanthanase

[0310] Complete enzymatic degradation of xanthan gum requires enzyme activity, which includes xanthan lyase activity and xanthan endoglucanase activity as described above. Xanthan lyases and endoglucanases for degrading xanthan gum and the use of such enzymes for cleaning purposes (e.g., removal of stains containing xanthan gum) and in the drilling and oil industries are known in the art, for example, from WO 2013 / 167581 A1.

[0311] In the context of the present invention, the term xanthanase or xanthanase means a combination of enzyme activities including xanthan lyase activity and xanthan endoglucanase activity.

[0312] A commercially available xanthanase is the product Caledonia 100L from Novozymes.

[0313] In preferred aspects of the invention, the mannanase variants of the invention can be combined with at least one additional enzyme (e.g., at least two, three, four or five enzymes), which are described in detail in the "Other enzymes" section. Preferably, these enzymes have different substrate specificities, such as carbohydrate decomposition activity, proteolytic activity, amylolytic activity, lipolytic activity, hemicellulose decomposition activity, pectin decomposition activity, or mannanases with different substrate specificities compared to the mannanase variants of the invention, such as GH5 mannanases. Enzyme combinations can be, for example, mannanases of the invention with another detergent enzyme, such as mannanases of the invention with proteases (e.g., serine proteases), mannanases of the invention with amylases, mannanases of the invention with cellulases, mannanases of the invention with lipases, mannanases of the invention with cutinases, mannanases of the invention with pectinases. More particularly preferred are mannanases of the invention with another enzyme having carbohydrate decomposition activity, such as cellulases, such as endoglucanases and beta-glucanases.

[0314] More preferably, the mannanase of the present invention is combined with at least two other detersive enzymes, such as a mannanase, a lipase and an amylase of the present invention; or a mannanase, a protease and an amylase of the present invention; or a mannanase, a protease and a lipase of the present invention; or a mannanase, a protease and a pectinase of the present invention; or a mannanase, a protease and a cellulase such as an endoglucanase or a β-glucanase of the present invention; or a mannanase, a protease and a hemicellulase of the present invention; or a mannanase, a protease and a cutinase of the present invention; or or mannanase, amylase and hemicellulase of the present invention; or mannanase, lipase and pectinase of the present invention; or mannanase, lipase and cutinase of the present invention; or mannanase, lipase and cellulase such as endoglucanase or β-glucanase of the present invention; or mannanase, lipase and hemicellulase of the present invention; or mannanase, lipase and pectinase of the present invention; or mannanase, lipase and cutinase of the present invention; or mannanase, lipase and cellulase such as endoglucanase or β-glucanase of the present invention; or mannanase, lipase and hemicellulase of the present invention. Even more preferably, the mannanase of the present invention can be combined with at least three other detersive enzymes, such as mannanase, protease, lipase and amylase of the present invention; or mannanase, protease, amylase and pectinase of the present invention; or mannanase, protease, amylase and cutinase of the present invention; or mannanase, protease, amylase and cellulase of the present invention; or mannanase, protease, amylase and hemicellulase of the present invention; or mannanase, amylase, lipase and pectinase of the present invention; or mannanase, amylase, lipase and cutinase of the present invention; or mannanase, amylase, lipase and cellulase of the present invention; or mannanase, amylase, lipase and hemicellulase of the present invention; or mannanase, amylase, lipase and pectinase of the present invention; or mannanase, protease, lipase and cutinase of the present invention; or mannanase, protease, lipase and cellulase of the present invention; or mannanase, protease, lipase and hemicellulase of the present invention. The mannanase according to the invention may be combined with any enzyme selected from the non-exhaustive list comprising: a carbohydrase, such as an amylase, a hemicellulase, a pectinase, a cellulase, a xanthan lyase, a xanthan endoglucanase or a pullulanase, a peptidase, a protease or a lipase.

[0315] In a preferred embodiment, the mannanase of the invention is combined with a serine protease such as an S8 family protease (e.g. )combination.

[0316] In another embodiment of the present invention, the mannanase of the present invention can be combined with one or more metalloproteases (such as M4 metalloproteases, including Such a combination may further comprise a combination of other detergent enzymes as outlined above.

[0317] In a particularly preferred embodiment, the mannanase of the invention is combined with a GH5 mannanase, in particular a GH5 mannanase selected from the group consisting of a mannanase having at least 60%, such as at least 70% or at least 80% identity to the mannanase of any one of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, such as a mannanase having at least 85%, at least 90%, at least 95% or even 100% identity to the mannanase of any one of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

[0318] In a more particular embodiment, the mannanase variant is combined with a GH5 mannanase, which has at least 80% identity to the mannanase of SEQ ID NO:3, such as at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identity to the mannanase of SEQ ID NO:3, and the GH5 mannanase is selected from the group consisting of a mannanase having at least 80% identity to the mannanase of any one of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, such as a mannanase having at least 85%, 90%, 95% or even 100% identity to the mannanase of any one of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

[0319] In another embodiment, the mannanase variant of the invention is combined with a xanthan lyase or a xanthan endoglucanase, or both. A preferred xanthan lyase has at least 80% identity to SEQ ID NO: 30, such as at least 85%, 90%, 95% or even 100% identity to the xanthan lyase of SEQ ID NO: 30. A preferred xanthan endoglucanase has at least 80% identity to SEQ ID NO: 31, such as at least 85%, at least 90%, at least 95% or even 100% identity to the xanthan endoglucanase of SEQ ID NO: 31.

[0320] In more particular embodiments, a mannanase variant having at least 80% identity to the mannanase of SEQ ID NO:3, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the mannanase of SEQ ID NO:3, is combined with a xanthan lyase having at least 80% identity to SEQ ID NO:30, such as at least 85%, 90%, 95% or even 100% identity to the xanthan lyase of SEQ ID NO:30.

[0321] In another specific embodiment, a mannanase variant having at least 80% identity to the mannanase of SEQ ID NO:3, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the mannanase of SEQ ID NO:3, is combined with a xanthan endoglucanase having at least 80% identity to SEQ ID NO:31, such as at least 85%, 90%, 95% or even 100% identity to the xanthan lyase of SEQ ID NO:31.

[0322] In more particular embodiments, a mannanase variant is combined with a xanthan lyase and a xanthan endoglucanase, wherein the mannanase variant has at least 80% identity to the mannanase of SEQ ID NO:3, such as at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the mannanase of SEQ ID NO:3, the xanthan lyase has at least 80% identity to SEQ ID NO:30, such as at least 85%, 90%, 95% or even 100% identity to the xanthan lyase of SEQ ID NO:30, and the xanthan endoglucanase has at least 80% identity to SEQ ID NO:31, such as at least 85%, 90%, 95% or even 100% identity to the xanthan endoglucanase of SEQ ID NO:31.

[0323] Detergent ingredients

[0324] Surfactants

[0325] Typically, detergent compositions contain (by weight of the composition) one or more surfactants in the following range: 0% to 50%, preferably 2% to 40%, more preferably 5% to 35%, more preferably 7% to 30%, most preferably 10% to 25%, even most preferably 15% to 20%. In a preferred embodiment, the detergent is a liquid or powder detergent containing less than 40%, preferably less than 30%, more preferably less than 25%, even more preferably less than 20% surfactant by weight. The composition may contain 1% to 15%, preferably 2% to 12%, 3% to 10%, most preferably 4% to 8%, even most preferably 4% to 6% of one or more surfactants. Preferred surfactants are anionic surfactants, nonionic surfactants, cationic surfactants, zwitterionic surfactants, amphoteric surfactants and mixtures thereof. Preferably, the major part of the surfactant is anionic. Suitable anionic surfactants are well known in the art and can include fatty acid carboxylates (soaps), side chain, straight chain and random chain alkyl sulfates or fatty alcohol sulfates or primary alcohol sulfates or alkylbenzene sulfonates (such as LAS and LAB) or phenylalkanesulfonates (phenylalknesulfonate) or alkenylsulfonates or alkenylbenzenesulfonates or alkylethoxysulfates or fatty alcohol ether sulfates or alpha-olefin sulfonates or dodecenyl / tetradecnyl succinic acid. Anionic surfactants can be alkoxylated. Detergent compositions can also include 1wt% to 10wt% of nonionic surfactants, preferably 2wt% to 8wt%, more preferably 3wt% to 7wt%, even more preferably less than 5wt% of nonionic surfactants. Suitable nonionic surfactants are well known in the art and may include alcohol ethoxylates, and / or alkyl ethoxylates, and / or alkylphenol ethoxylates, and / or glucamides (such as fatty acid N-glucosyl N-methylamides), and / or alkyl polyglucosides and / or mono- or diethanolamides or fatty acid amides. The detergent composition may also include 0 wt% to 10 wt% of a cationic surfactant, preferably 0.1 wt% to 8 wt%, more preferably 0.5 wt% to 7 wt%, even more preferably less than 5 wt% of a cationic surfactant. Suitable cationic surfactants are well known in the art and may include alkyl quaternary ammonium compounds, and / or alkyl pyridine compounds and / or alkyl quaternary phosphonium compounds and / or alkyl ternary sulfide compounds. The composition preferably includes a surfactant in an amount of 100 ppm to 5,000 ppm of surfactant in the wash liquor during the laundry washing process. The composition typically forms a wash liquor after contact with water, the wash liquor containing 0.5 g / l to 10 g / l of the detergent composition.Many suitable surface-active compounds are available and are well described in the literature, for example by Schwartz, Perry and Berch in "Surface-Active Agents and Detergents", Volumes 1 and 11. Also preferred are bio-based surfactants, which may be entirely bio-based (bio-based carbon in the total carbon>95% according to European Standard EN 17035). As used herein, bio-based surfactants are commercial or industrial products (other than food or feed) that consist entirely or largely of bioproducts or renewable agricultural or forestry materials and / or are established by European Standard EN 16575:2014. In particular, rhamnolipids and sophorolipids can be used as detergent ingredients.

[0326] Detergent

[0327] The main function of the builder is to chelate divalent metal ions (such as calcium and magnesium ions) from the wash solution, which would otherwise interact negatively with the surfactant system. Builders can also effectively remove metal ions and inorganic soils from the fabric surface, thereby improving the removal of particulate and beverage stains. Builders are also a source of alkalinity and buffer the pH of the wash water to a level of 9.5 to 11. The buffering capacity is also called reserve alkalinity and should preferably be greater than 4.

[0328] The detergent composition of the present invention can comprise one or more detergent builders or builder systems. Many suitable builder systems are described in the literature, for example, in Powdered Detergents, Surfactant science series, Volume 71, Marcel Dekker, Inc. By the weight of the subject composition, the builder can comprise 0% to 60%, preferably 5% to 45%, more preferably 10% to 40%, most preferably 15% to 35%, even more preferably 20% to 30% builder. By the weight of the subject composition, the composition can comprise 0% to 15%, preferably 1% to 12%, 2% to 10%, most preferably 3% to 8%, even most preferably 4% to 6% builder.

[0329] Builders include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates (e.g., tripolyphosphate STPP), alkali metal silicates, alkaline earth metal salts and alkali metal carbonates, aluminosilicate builders (e.g., zeolites), as well as polycarboxylic acid compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethoxysuccinic acid, various alkali metal, ammonium and substituted ammonium salts of polyacetic acid (e.g., ethylenediaminetetraacetic acid and nitrilotriacetic acid), together with polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethoxysuccinic acid and soluble salts thereof. Ethanolamines (MEA, DEA and TEA) can also contribute to buffering capacity in liquid detergents.

[0330] Bleach

[0331] The detergent compositions of the present invention may contain one or more bleaching agents. In particular, powdered detergents may contain one or more bleaching agents. Suitable bleaching agents include other photobleaches, preformed peracids, hydrogen peroxide sources, bleach activators, hydrogen peroxide, bleach catalysts, and mixtures thereof. Typically, when a bleaching agent is used, the compositions of the present invention may contain from about 0.1% to about 50%, or even from about 0.1% to about 25%, of the bleaching agent by weight of the subject cleaning composition. Examples of suitable bleaching agents include:

[0332] (1) Other photobleaching agents, such as vitamin K3;

[0333] (2) Preformed peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts (e.g., potassium hydrogen persulfate (Oxone)), and mixtures thereof. Suitable peroxycarboxylic acids include hydrophobic and hydrophilic peracids of the formula R-(C=O)OOM, wherein R is an alkyl group (optionally branched), having from 6 to 14 carbon atoms or from 8 to 12 carbon atoms when the peracid is hydrophobic, and having less than 6 carbon atoms or even less than 4 carbon atoms when the peracid is hydrophilic; and M is a counterion, such as sodium, potassium or hydrogen;

[0334] (3) A source of hydrogen peroxide, such as an inorganic perhydrate salt, including alkali metal salts such as perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, sodium persilicate, and mixtures thereof. In one aspect of the invention, the inorganic perhydrate salt is selected from the group consisting of perborate, sodium percarbonate, and mixtures thereof. When used, the inorganic perhydrate salt is typically present in an amount of 0.05 wt % to 40 wt % or 1 wt % to 30 wt % of the overall composition, and is typically incorporated into such a composition as a crystalline solid that can be coated. Suitable coatings include inorganic salts such as alkali metal silicates, carbonates or borates, or mixtures thereof, or organic materials such as water-soluble or water-dispersible polymers, waxes, oils or fatty soaps. Useful bleaching compositions are described in U.S. Pat. Nos. 5,576,282 and 6,306,812;

[0335] (4) Bleach activators having R-(C=O)-L, wherein R is an alkyl group (optionally branched), having from 6 to 14 carbon atoms or from 8 to 12 carbon atoms when the bleach activator is hydrophobic, and having less than 6 carbon atoms or even less than 4 carbon atoms when the bleach activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives - especially benzene sulfonate. Suitable bleach activators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulfonate, decanoyl oxybenzoic acid or its salts, 3,5,5-trimethylhexanoyl oxybenzene sulfonate, tetraacetylethylenediamine (TAED) and nonanoyl oxybenzene sulfonate (NOBS). Suitable bleach activators are also disclosed in WO 98 / 17767. While any suitable bleach activator may be employed, in one aspect of the present invention, the subject cleaning compositions may comprise NOBS, TAED, or mixtures thereof; and

[0336] (5) Bleach catalysts capable of accepting an oxygen atom from a peroxyacid and transferring the oxygen atom to an oxidizable substrate are described in WO 2008 / 007319. Suitable bleach catalysts include, but are not limited to, iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonyl imides; N-phosphoryl imides; N-acyl imides; thiadiazole dioxides; perfluoro imides; cyclic sugar ketones and mixtures thereof. Bleach catalysts will typically be included in the detergent composition at a level of 0.0005% to 0.2%, 0.001% to 0.1%, or even 0.005% to 0.05% by weight.

[0337] When present, the peracid and / or bleach activator is typically present in the composition in an amount of from about 0.1 wt % to about 60 wt %, from about 0.5 wt % to about 40 wt %, or even from about 0.6 wt % to about 10 wt %, based on the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof.

[0338] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0339] Supplementary Materials

[0340] Dispersants - The detergent compositions of the present invention may also contain dispersants. In particular, powdered detergents may contain dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0341] Dye transfer inhibitors - The detergent 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, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles or mixtures thereof. When present in the subject compositions, the dye transfer inhibiting agents can be present at a level of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0342] Fluorescent whitening agents - The detergent compositions of the present invention will also preferably contain additional components which may impart color to the article being cleaned, such as fluorescent whitening agents or optical brighteners. Any fluorescent whitening agent suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used fluorescent whitening agents are those belonging to the following classes: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.

[0343] Preferred fluorescent whitening agents 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 disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulfonate.

[0344] A further preferred optical brightener is commercially available as Parawhite KX supplied by Paramount Minerals and Chemicals, Mumbai, India.

[0345] Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-aminoalkylcoumarins.

[0346] Suitable fluorescent brightener levels include lower levels of about 0.01 wt%, 0.05 wt%, about 0.1 wt% or even about 0.2 wt% to higher levels of 0.5 wt% or even 0.75 wt%.

[0347] Fabric hueing agents - The detergent compositions of the present invention may also include fabric hueing agents, e.g., dyes or pigments that, when formulated in a detergent composition, can be deposited on a fabric when it is contacted with a wash liquor comprising the detergent composition, thereby changing the color of the fabric by visible light absorption. Fluorescent brighteners emit at least some visible light. Conversely, when fabric hueing agents absorb at least a portion of the visible spectrum, they change the color of a surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may also 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 the following dyes classified into the Colour Index (CI) classification: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet and basic red, or mixtures thereof, such as described in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276 and EP 1 876 226. The detergent composition preferably comprises from about 0.00003wt% to about 0.2wt%, from about 0.00008wt% to about 0.05wt%, or even from about 0.0001wt% to about 0.04wt% of fabric hueing agent. The composition may comprise from 0.0001wt% to 0.2wt% of fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit dose bag.

[0348] Soil release polymers - The detergent compositions of the present invention may also include one or more soil release polymers that help remove soil from fabrics, such as cotton and polyester-based fabrics, particularly remove hydrophobic soils from polyester-based fabrics. Soil release polymers may be, for example, polymers based on nonionic or anionic terephthalates, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Powdered Detergents, Surfactant science series, Vol. 71, Chapter 7, Marcel Dekker, Inc. Another type of soil release polymer is an amphiphilic alkoxylated grease cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may include a polyalkyl imine structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523. In addition, any graft copolymer is a suitable soil release polymer. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314. Other soil release polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, such as those described in EP 1 867 808 or WO2003 / 040279. Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0349] Anti-redeposition agents - The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described above under soil release polymers may also function as anti-redeposition agents.

[0350] Other suitable auxiliary materials include, but are not limited to, shrink-proofing agents, wrinkle-proofing agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, foam suppressants, solvents, structurants for liquid detergents and / or structural elastic agents.

[0351] In one aspect, the detergent is a compressed fluid laundry detergent composition comprising: a) at least about 10%, preferably 20% to 80%, by weight of the composition, of a surfactant selected from anionic surfactants, nonionic surfactants, soaps, and mixtures thereof; b) from about 1% to about 30%, preferably 5% to 30%, by weight of the composition, of water; c) from about 1% to about 15%, preferably 3% to 10%, by weight of the composition, of a non-amino functional solvent; and d) from about 5% to about 20%, by weight of the composition, of a performance additive selected from chelating agents, soil release polymers, enzymes, and mixtures thereof; wherein the compressed fluid laundry detergent composition comprises at least one of:

[0352] (i) the surfactant has a weight ratio of anionic surfactant to nonionic surfactant of from about 1.5:1 to about 5:1, the surfactant comprising from about 15% to about 40% anionic surfactant by weight of the composition and comprising from about 5% to about 40% soap by weight of the composition; (ii) comprising from about 0.1% to about 10% foam booster by weight of the composition selected from foam boosting polymers, cationic surfactants, zwitterionic surfactants, amine oxide surfactants, amphoteric surfactants, and mixtures thereof; and (ii) both (i) and (ii). All ingredients are described in WO 2007 / 130562. Additional polymers useful in detergent formulations are described in WO 2007 / 149806.

[0353] In another aspect, the detergent is a compressed granular (powdered) detergent comprising: a) at least about 10%, preferably 15% to 60%, by weight of the composition, of a surfactant selected from anionic surfactants, nonionic surfactants, soaps and mixtures thereof; b) about 10% to 80%, preferably 20% to 60%, by weight of the composition, of a builder, wherein the builder may be a mixture of builders selected from: i) phosphate builders, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% of the total builder being phosphate builders; ii) zeolite builders, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% of the total builder being phosphate builders; builder; iii) citrate, preferably 0 to 5% of the total builder is citrate builder; iv) polycarboxylate, preferably 0 to 5% of the total builder is polycarboxylate builder; v) carbonate, preferably 0 to 30% of the total builder is carbonate builder and vi) sodium silicate, preferably 0 to 20% of the total builder is sodium silicate builder; c) about 0% to 25% filler, such as sulfate, by weight of the composition, preferably 1% to 15%, more preferably 2% to 10%, more preferably 3% to 5% filler by weight of the composition; and d) about 0.1% to 20% enzymes, by weight of the composition, preferably 1% to 15%, more preferably 2% to 10% enzymes by weight of the composition.

[0354] Dirt and stains that are important to detergent formulators consist of many different substances, and a range of different enzymes with different substrate specificities have been developed for use in detergents involving both laundry and hard surface cleaning (such as dishwashing). These enzymes are believed to provide enzymatic washing benefits because they specifically improve stain removal in the same process to which they are applied, compared to a cleaning process without enzymes. Detergent enzymes known in the art include the following enzymes, such as carbohydrases, amylases, proteases, lipases, cellulases, hemicellulases, xylanases, cutinases, and pectinases.

[0355] The cleaning process or textile care process can be, for example, a laundry process, a dishwashing process or a hard surface such as bathroom tiles, floors, desktops, drains, sinks and wash basins. The laundry process can be, for example, a household laundry process, but it can also be an industrial laundry process. In addition, the present invention relates to a method for washing fabrics and / or clothing, wherein the method comprises treating fabrics with a washing solution containing a detergent composition and at least one mannanase of the present invention. For example, a cleaning process or a textile care process can be carried out in a machine washing process or in a manual washing process. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0356] The fabrics and / or garments subjected to the washing, cleaning or textile care process of the present invention may be conventional washable garments, such as household garments. Preferably, the major portion of the garments are garments and fabrics, including knitwear, woven fabrics, denim, non-woven fabrics, felt, yarn, and terry cloth. These fabrics may be cellulose-based, such as natural cellulose, including cotton, flax, linen, jute, ramie, sisal or coconut shell fibers; or artificial cellulose (e.g., derived from wood pulp), including viscose / rayon, ramie, cellulose acetate fibers (tricell), lyocell fibers or blends thereof. These fabrics may also be non-cellulose-based, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair and silk; or synthetic polymers, such as nylon, aromatic polyamides, polyesters, acrylates, polypropylene and spandex / elastic fibers; or blends thereof together with blends of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and / or rayon / viscose fibers with one or more accompanying materials such as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aromatic polyamide fibers) and cellulose-containing fibers (e.g. rayon / viscose fibers, ramie, flax, linen, jute, cellulose acetate fibers, lyocell fibers).

[0357] In recent years, there has been an increasing interest in replacing components in detergents, which stems from replacing petrochemicals with renewable biocomponents such as enzymes and polypeptides without compromising washing performance. When the components of detergent compositions are changed, new enzyme activities or new enzymes with alternative and / or improved properties compared to commonly used detergent enzymes (such as proteases, lipases and amylases) are required to achieve similar or improved washing performance when compared to traditional detergent compositions.

[0358] Typical detergent compositions include various components in addition to enzymes, which have different roles, some components like surfactants reduce the surface tension of the detergent, which allows the stains being cleaned to be lifted and dispersed and subsequently washed out, other components like bleaching systems generally remove color by oxidation and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Still other components like builders and chelating agents soften the wash water, for example by removing metal ions from the liquid.

[0359] In a particular embodiment, the present invention relates to the use of a composition comprising a mannanase of the present invention in laundry washing or dishwashing, wherein the composition further comprises at least one or more of the following: a surfactant, a builder, a chelating agent or a chelating agent, a bleaching system or a bleaching component.

[0360] In a preferred embodiment of the present invention, the amount of surfactant, builder, chelant or chelating agent, bleaching system and / or bleaching component is reduced compared to the amount of surfactant, builder, chelant or chelating agent, bleaching system and / or bleaching component used without the addition of the mannanase of the present invention. Preferably, at least one component as a surfactant, builder, chelant or chelating agent, bleaching system and / or bleaching component is present in an amount that is 1% less, such as 2% less, such as 3% less, such as 4% less, such as 5% less, such as 6% less, such as 7% less, such as 8% less, such as 9% less, such as 10% less, such as 15% less, such as 20% less, such as 25% less, such as 30% less, such as 35% less, such as 40% less, such as 45% less, such as 50% less than the amount of the component in the system without the addition of the mannanase of the present invention (such as the conventional amount of such a component). In one aspect, the mannanase of the invention is used in a detergent composition, wherein the composition is free of at least one component which is a surfactant, a builder, a chelant or chelating agent, a bleaching system or bleaching component and / or a polymer.

[0361] Also preferred are bio-based surfactants, which may be entirely bio-based (>95% bio-based carbon in total carbon according to European standard EN 17035). As used herein, a bio-based surfactant is a commercial or industrial product (other than food or feed) that consists entirely or largely of bio-products or renewable agricultural or forestry materials and / or is established by European standard EN 16575:2014.

[0362] Additional bio-based surfactants may be used, for example where the surfactant is a sugar-based nonionic surfactant, which may be hexyl-β-D-maltopyranoside, thiomaltopyranoside or cyclic maltopyranoside, such as described in EP 2516606 B1. Other bio-based surfactants may include rhamnolipids and sophorolipids.

[0363] When included in a detergent, the amount of bio-based surfactant is 1% - 25% (and / or below the levels of surfactant specified above).

[0364] In an embodiment, a detergent composition is provided comprising a mannanase variant as described herein and an additional mannanase, and wherein the additional mannanase is a GH5 mannanase, e.g., a mannanase having at least 50%, e.g., 60%, 70%, 80%, 90%, 95% or even 100% identity to any one of SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID: 20 or SEQ ID NO: 21. Preferably, the ratio between the mannanase variant of the invention and the GH5 mannanase is in the range of 1:3 to 3:1.

[0365] use

[0366] Mannanase variants of the present invention can be used for applications where it is desired to degrade mannans. Examples where mannanase can be used are in the production of bioethanol from cork and palm kernel filter cakes, for improving animal feed and in the hydrolysis of coffee. In addition, guar gum is used in many foods and is used in the oil and gas industry, so mannanase of the present invention can be used in detergents to remove mannan-containing stains, for hydraulic fracturing to produce underground fracturing extending from a borehole into a stratum, thereby increasing the rate of fluids that can be produced by the stratum or for cleaning borehole filter cakes. Therefore mannans can be used in the fracturing of underground strata caused by a borehole, or mannans can be used as a component in a borehole filter cake.

[0367] In one aspect, the mannanase variant of the first or second aspect, the detergent composition of the third or fourth aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect can be used to degrade mannans, such as linear mannans, galactomannans, glucomannans, and galactoglucomannans. In one aspect, the mannanase variant of the first or second aspect, the detergent composition of the third or fourth aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect can be used to degrade mannans, such as linear mannans, galactomannans, glucomannans, and galactoglucomannans.

[0368] In one aspect, the mannanase variant of the first or second aspect, the detergent composition of the third or fourth aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect can be used to control the viscosity of the drilling fluid. In one aspect, the mannanase variant of the first or second aspect, the detergent composition of the third or fourth aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect can be used in the fracturing of underground formations caused by a wellbore.

[0369] The mannanase variants of the present invention can be used to prevent, reduce or remove the malodor of articles. Therefore, in one embodiment, the mannanase variants of the first or second aspects, the detergent composition of the third or fourth aspects, the granules of the fifth or sixth aspects or the liquid formulations of the seventh or eighth aspects can be used to prevent, reduce or remove the malodor of articles.

[0370] Washing method

[0371] The detergent composition of the present invention is ideally suited for use in laundry applications. Accordingly, the present invention includes a method for washing fabrics. The method includes the step of contacting a fabric to be washed with a clean laundry solution comprising a detergent composition according to the present invention. The fabric may include any fabric that can be washed under normal consumer use conditions. The solution preferably has a pH of about 5.5 to about 8. The composition may be used in a solution at a concentration of about 100 ppm, preferably 500 ppm to about 15,000 ppm. The range of water temperature is typically about 5°C to about 90°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The ratio of water to fabric is typically about 1:1 to about 30:1.

[0372] In particular embodiments, the washing method is carried out at a pH of about 5.0 to about 11.5, or in alternative embodiments, even at a pH of about 6 to about 10.5, such as about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, preferably about 5.5 to about 9, and more preferably about 6 to about 8.

[0373] In a particular embodiment, the washing method is carried out at a hardness of about 0°dH to about 30°dH, such as about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21°dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, and about 30°dH. Under typical European wash conditions, the hardness is about 15° dH, under typical US wash conditions, about 6° dH, and under typical Asian wash conditions, about 3° dH.

[0374] The present invention relates to methods for cleaning fabrics, dishes or hard surfaces using a detergent composition comprising a mannanase variant of the present invention.

[0375] Preferred embodiments relate to a method of cleaning comprising the steps of contacting the object with a cleaning composition comprising a mannanase variant of the invention under conditions suitable for cleaning the object. In preferred embodiments, the cleaning composition is a detergent composition and the process is a laundry or dishwashing process.

[0376] Still another embodiment relates to a method for removing stains from a fabric, comprising contacting the fabric with a composition comprising a mannanase variant of the present invention under conditions suitable for cleaning the object.

[0377] Low temperature use

[0378] One embodiment of the invention relates to a method of performing laundry, dishwashing or industrial cleaning, the method comprising contacting a surface to be cleaned with a mannanase variant of the invention, and wherein the laundry, dishwashing, industrial or institutional cleaning is performed at a temperature of about 40° C. or less. One embodiment of the invention relates to the use of a mannanase in a laundry, dishwashing or cleaning process, wherein the temperature in the laundry, dishwashing, industrial cleaning is about 40° C. or less.

[0379] In another embodiment the invention relates to the use of a mannanase according to the invention in a protein removal process, wherein the temperature in the protein removal process is about 40°C or less.

[0380] In each of the methods and uses identified above, the washing temperature is about 40°C or lower, for example about 39°C or lower, for example about 38°C or lower, for example about 37°C or lower, for example about 36°C or lower, for example about 35°C or lower, for example about 34°C or lower, for example about 33°C or lower, for example about 32°C or lower, for example about 31°C or lower, for example about 30°C or lower, for example about 29°C or lower, for example about 28°C or lower, for example about 27°C or lower, for example about 26°C or lower, for example about 25°C or lower, for example about 24°C or lower, for example about 23°C or lower, for example about 22°C or lower. ℃ or lower, for example, about 21 ℃ or lower, for example, about 20 ℃ or lower, for example, about 19 ℃ or lower, for example, about 18 ℃ or lower, for example, about 17 ℃ or lower, for example, about 16 ℃ or lower, for example, about 15 ℃ or lower, for example, about 14 ℃ or lower, for example, about 13 ℃ or lower, for example, about 12 ℃ or lower, for example, about 11 ℃ or lower, for example, about 10 ℃ or lower, for example, about 9 ℃ or lower, for example, about 8 ℃ or lower, for example, about 7 ℃ or lower, for example, about 6 ℃ or lower, for example, about 5 ℃ or lower, for example, about 4 ℃ or lower, for example, about 3 ℃ or lower, for example, about 2 ℃ or lower, for example, about 1 ℃ or lower.

[0381] In another preferred embodiment, the wash temperature is in the range of about 5° C.-40° C., such as about 5° C.-30° C., about 5° C.-20° C., about 5° C.-10° C., about 10° C.-40° C., about 10° C.-30° C., about 10° C.-20° C., about 15° C.-40° C., about 15° C.-30° C., about 15° C.-20° C., about 20° C.-40° C., about 20° C.-30° C., about 25° C.-40° C., about 25° C.-30° C., or about 30° C.-40° C. In a particularly preferred embodiment, the wash temperature is about 20° C., about 30° C., or about 40° C.

[0382] Use of the mannanase of the present invention in preventing, reducing or removing biofilm

[0383] Biofilm can develop on textiles when there are microorganisms on articles and when they stick together on articles. Some microorganisms tend to adhere to the surface of articles (such as textiles). Some microorganisms adhere to such surfaces and form biofilm on the surface. Biofilm can be sticky and adhered microorganisms and / or biofilm can be difficult to remove. In addition, due to the sticky nature of biofilm, biofilm adheres to dirt. Commercial laundry detergent compositions available on the market do not remove such adhered microorganisms or biofilm.

[0384] Use of the mannanase of the present invention in food processing and animal feed

[0385] Several anti-nutritional factors may limit the use of specific plant materials in the preparation of animal feed and human food. For example, plant materials containing oligomannans (such as mannans, galactomannans, glucomannans and galactoglucomannans) reduce the digestibility and absorption of nutritional compounds (such as minerals, vitamins, sugars and fats) by animals. In particular, these negative effects are due to the high viscosity of mannan-containing polymers and the ability of mannan-containing polymers to absorb nutritional compounds. These effects can be reduced by using enzymes that degrade mannan-containing polymers, i.e., endo-β-mannanases (such as mannanases described herein), which allow a higher proportion of mannan-containing polymers containing cheap plant materials to be included in the feed, thereby reducing feed costs. In addition, through the activity of the mannanase of the present invention, the mannan-containing polymer is broken down into simpler sugars, which can be more easily assimilated to provide additional energy. Accordingly, the present invention further relates to the use of the mannanase of the present invention in the processing and / or manufacture of food or animal feed.

[0386] Accordingly, the present invention relates to an animal feed composition and / or an animal feed additive composition and / or a pet food comprising the mannanase variant of the present invention.

[0387] The present invention further relates to a method for preparing such an animal feed composition, and / or an animal feed additive composition and / or a pet food, which method comprises mixing a mannanase variant of the present invention with one or more animal feed ingredients and / or animal feed additive ingredients and / or pet food ingredients.

[0388] Furthermore, the present invention also relates to the use of the mannanase variant of the present invention in the preparation of an animal feed composition and / or an animal feed additive composition and / or a pet food.

[0389] Use of the mannanase of the present invention in fermented beverages

[0390] In one aspect, the present invention relates to a method for preparing a fermented beverage such as beer or wine, the method comprising mixing the mannanase variant of the first aspect or the second aspect, the granules of the fifth aspect or the sixth aspect or the liquid formulation of the seventh aspect or the eighth aspect with malt and / or an adjuvant.

[0391] Another aspect relates to a method of providing a fermented beverage, the method comprising the step of contacting mash and / or wort with the mannanase variant of the first or second aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect.

[0392] In the context of the present invention, the term "fermented beverage" is intended to include any beverage, such as wine or beer, produced by a process involving a fermentation process, such as microbial, bacterial and / or yeast fermentation.

[0393] In one aspect of the present invention, the fermented beverage is beer. The term "beer" is meant to include any fermented wort produced by fermenting / brewing starch-containing plant material. Typically, beer is produced from malt or adjuvants or any combination of malt and adjuvants as starch-containing plant material. As used herein, the term "malt" is understood to mean any germinated cereal grains, such as germinated barley or wheat.

[0394] As used herein, the term "adjuvant" refers to any starch and / or sugar-containing plant material other than malt (such as barley or wheat malt). As examples of adjuvants, materials that can be used as starch sources, such as common corn grits, refined corn grits, brewery milling yeast, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, cassava, tapioca and syrups (such as corn syrup, cane syrup, invert syrup, barley and / or wheat syrup) and the like can be mentioned.

[0395] As used herein, the term "mash" refers to an aqueous slurry of any starch and / or sugar-containing plant material such as grist (e.g., including crushed barley malt, crushed barley) and / or other adjuvants, or a combination thereof, which is subsequently mixed with water to separate into wort and spent grains.

[0396] As used herein, the term "wort" refers to the unfermented liquid run-off after extraction of grist during mashing.

[0397] Use of the mannanase of the present invention for treating coffee extract

[0398] The mannanase variants of the present invention can also be used to hydrolyze galactomannans present in liquid coffee extracts. In certain preferred embodiments, the mannanase variants of the present invention are used to inhibit gel formation during freeze drying of liquid coffee extracts. The reduction in extract viscosity reduces energy consumption during drying. In certain other preferred embodiments, in order to reduce enzyme consumption and avoid contamination of coffee extracts, the mannanase variants of the present invention are used in immobilized form. Such use is further disclosed in EP 676 145.

[0399] Typically, the coffee extract is incubated in the presence of the isolated mannanase variant of the invention, or a fragment or variant thereof, under conditions suitable for hydrolyzing galactomannans present in the liquid coffee extract.

[0400] Thus, in one embodiment, the present invention relates to a method for producing a coffee extract, the method comprising the following steps:

[0401] (a) providing roasted and ground coffee beans;

[0402] (b) adding water and the polypeptide of the first aspect or the second aspect, the particles of the fifth aspect or the sixth aspect, or the liquid formulation of the seventh aspect or the eighth aspect to the coffee beans;

[0403] (c) incubating to prepare an aqueous coffee extract; and

[0404] (d) separating the coffee extract from the extracted coffee beans.

[0405] Use of the mannanase of the present invention in baked foods

[0406] In another aspect, the present invention relates to a method for preparing a baked product, the method comprising adding the mannanase variant of the first aspect or the second aspect, the granules of the fifth aspect or the sixth aspect, or the liquid formulation of the seventh aspect or the eighth aspect to dough, and then baking the dough.

[0407] Examples of baked products are well known to those skilled in the art and include bread, rolls, puff pastry, sweet yeast dough, buns, cakes, crackers, biscuits, rolls, waffles, wafers, tortillas, breakfast cereals, extruded products, and the like.

[0408] Mannanase variants of the present invention can be added to dough as part of a bread improver composition. Bread improvers are compositions containing multiple ingredients that improve the quality of dough properties and baked products (e.g., bread and cakes). Bread improvers are usually added to industrial baking processes due to their beneficial effects (e.g., dough stability and bread texture and volume). Bread improvers usually contain fats and oils together with additives (like emulsifiers, enzymes, antioxidants, oxidants, stabilizers, and reductants). In addition to mannanase variants of the present invention, other enzymes may also be present in bread improvers, including amylases, hemicellulases, amylolytic complexes, lipases, proteases, xylanases, pectinases, pullulanases, non-starch polysaccharide degrading enzymes, and oxidoreductases (like glucose oxidase, lipoxygenase, or ascorbic acid oxidase).

[0409] In one aspect, the mannanase variants of the invention can be added to the dough as part of a bread improver composition which also comprises a source of glucomannan and / or galactomannan, such as konjac gum, guar gum, locust bean gum (Ceratonia siliqua), copra meal, ivory nut mannan (Phyteleohas macrocarpa), seaweed mannan extract, coconut meal, and cell walls of brewer's yeast (which can be dried or used in the form of brewer's yeast extract).

[0410] A further aspect of the invention relates to the use of the mannanase variants of the invention in dough for improving dough tolerance, flexibility and stickiness. Preferably, the dough to which the mannanase variants of the invention can be added is not a pure wheat flour dough, but contains bran or oat, rice, millet, corn or pulse flour in addition to or instead of pure wheat flour.

[0411] Yet a further aspect of the invention relates to the use of any of the mannanase variants of the invention in dough to improve the crumb structure and delay staling of the final baked product, such as bread.

[0412] Use of the mannanase of the present invention in dairy foods

[0413] In one aspect of the invention, the mannanase variants of the invention can be added to milk or any other dairy product to which glucomannan and / or galactomannan are also added. Typical sources of glucomannan and / or galactomannan are listed above in terms of baking and include guar gum or konjac gum. When found in the large intestine or colon at favorable population densities, the combination of the mannanase variants of the invention and glucomannan and / or galactomannan releases mannanase hydrolysates (mannooligosaccharides) that act as soluble prebiotics by promoting the selective growth and proliferation of probiotics (particularly bifidobacteria and lactobacilli) that are typically associated with good health.

[0414] In one aspect, the present invention relates to a method for preparing milk or a dairy product, the method comprising adding to the milk or dairy product (a) glucomannan, galactomannan and / or galactoglucomannan and (b) the mannanase variant of the first or second aspect, the granules of the fifth or sixth aspect, or the liquid formulation of the seventh or eighth aspect.

[0415] In one aspect of the invention, the mannanase variants of the invention are used in combination with any glucomannan or galactomannan before or after addition to a dairy-based food to produce a dairy-based food containing prebiotic mannan hydrolysate. In another aspect of the invention, the manno-oligosaccharide-containing dairy product produced in this way is capable of increasing the population of beneficial human intestinal flora, and in yet another aspect of the invention, a dairy-based food may comprise the mannanase variants of the invention and any source of glucomannan and / or galactomannan and / or galactoglucomannan, and the dosage is sufficient for inoculation with at least one bacterial strain known to be beneficial in the human large intestine (such as bifidobacteria or lactobacilli). Preferably, the dairy-based food is a yogurt or a milk drink.

[0416] Use of the mannanase of the present invention for pulp bleaching

[0417] The mannanase variants of the present invention can further be used for enzyme-assisted bleaching of pulp (such as chemical pulp, semi-chemical pulp, kraft pulp, mechanical pulp or pulp prepared by the sulfite process). Therefore, the present invention relates to a method for bleaching pulp, which method comprises incubating the pulp with the polypeptide of the first aspect or the second aspect, the detergent composition of the third aspect or the fourth aspect, the granules of the fifth aspect or the sixth aspect or the liquid formulation of the seventh aspect or the eighth aspect.

[0418] In some embodiments, the pulp is a chlorine-free pulp bleached with oxygen, ozone, peroxide or peroxyacid. In some embodiments, the mannanase variants of the present invention are used for enzyme-assisted bleaching of pulp exhibiting low lignin content, which pulp is produced by a modified pulping process or a continuous pulping process. In some other embodiments, the mannanase variants of the present invention are used alone or preferably in combination with xylanase and / or endoglucanase and / or α-galactosidase and / or cellobiohydrolase.

[0419] Example

[0420] The present invention is further defined by the following numbered paragraphs:

[0421] 1) A variant of the polypeptide of SEQ ID NO: 2, wherein the amino acids at positions 490 and 491 are deleted, wherein the variant has at least 60% sequence identity with the polypeptide of SEQ ID NO: 3, and wherein the variant has mannanase activity.

[0422] 2) A variant as described in Example 1, wherein the variant comprises an extension of one or more amino acids at the N-terminus and / or C-terminus, or a truncation of one or more amino acids at the N-terminus and / or C-terminus, with the proviso that the insertion at the C-terminus is not *490W and *491R.

[0423] 3) A variant as described in Example 1 or 2, which comprises a deletion of at least 11 amino acids from the N-terminus, such as amino acids 1 to 12, such as amino acids 1 to 13, such as amino acids 1 to 14, or such as amino acids 1 to 15 of SEQ ID NO: 2.

[0424] 4) A variant as described in any of Examples 1 to 3, which comprises the following deletion: A1*+I2*+G3*+V4*+P5*+G6*+G7*+V8*+A9*+E10*+P11*+H12*+T13*+S14*+Q15*.

[0425] 5) The variant of any one of embodiments 1 to 4, further comprising the substitution D16A.

[0426] 6) A variant as described in any one of Examples 1 to 5, further comprising one or more modifications selected from the group consisting of: I30L, D48P, Y155H, T167P, Q215E, H276C, R280K, F286C, G366N and D486E.

[0427] 7) A variant as described in Example 6, which further comprises one or more modifications selected from the group consisting of: G20P, A101L, A111P, A118E, S137A, Q143R, R160L, V161A, E162D, R164S, R164P, R164A, R164K, I165G, I165A, I165P, I165T, I165E, M171I, N176S, P182R, Q183E, V244A, H324K and D385H.

[0428] 8) A variant as described in any one of embodiments 1 to 7, wherein the variant is a polypeptide of SEQ ID NO: 3, a polypeptide of SEQ ID NO: 4, a polypeptide of SEQ ID NO: 5, a polypeptide of SEQ ID NO: 6, or a polypeptide of SEQ ID NO: 7, a polypeptide of SEQ ID NO: 22, a polypeptide of SEQ ID NO: 23, a polypeptide of SEQ ID NO: 24, a polypeptide of SEQ ID NO: 25, a polypeptide of SEQ ID NO: 26, a polypeptide of SEQ ID NO: 27, a polypeptide of SEQ ID NO: 28, or a polypeptide of SEQ ID NO: 29. 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 99.9% or even 100% sequence identity to the polypeptide of NO:29, wherein the variant has mannanase activity.

[0429] 9) An enzyme composition comprising the variant as described in any one of claims 1 to 8 and GH5 mannanase, wherein the enzyme composition is a granulated product or a liquid product, wherein the ratio between the GH5 mannanase and the variant as described in any one of embodiments 1 to 9 is a ratio of 1:3 to 3:1 based on the weight of enzyme protein.

[0430] 10) A detergent composition comprising the variant of any one of embodiments 1 to 8 and at least one detergent adjunct ingredient.

[0431] 11) The detergent composition of embodiment 10, further comprising the enzyme composition of embodiment 9.

[0432] 12) A detergent composition as described in embodiment 11, wherein the GH5 mannanase has at least 60%, 70% or 80% identity with the mannanase of any one of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21, for example, it has at least 85%, 90%, 95% or even 100% identity with the mannanase of any one of SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21.

[0433] 13) The detergent composition of any one of embodiments 10 to 12, further comprising a xanthan lyase having at least 80% identity to SEQ ID NO:30 and a xanthan endoglucanase having at least 80% identity to SEQ ID NO:31.

[0434] 14) Use of the variant of any one of embodiments 1 to 8, the enzyme composition of embodiment 9 or the detergent composition of embodiments 10 to 12 in a cleaning process, such as laundry washing or hard surface cleaning, such as dishwashing.

[0435] 15) A method of cleaning an item, the method comprising exposing the item to a wash liquor comprising a variant as described in any one of embodiments 1 to 8, an enzyme composition as described in embodiment 9, or a detergent composition as described in embodiment 10 or 12, for example, wherein the item is a textile or a hard surface.

[0436] 16) A method for washing textiles, the method comprising:

[0437] a) exposing the textile to a wash liquor comprising the variant of any one of embodiments 1 to 8, the enzyme composition of embodiment 9, or the detergent composition of embodiment 10 or 12;

[0438] b) completing at least one wash cycle; and optionally

[0439] c) rinsing the textile article.

[0440] 17) A detergent composition, use or method as described in any of embodiments 10 to 14, wherein the detergent composition comprises one or more enzymes selected from the group consisting of amylase, protease, peroxidase, cellulase, β-glucanase, xyloglucanase, hemicellulase, xanthanase, xanthan lyase, lipase, acyltransferase, phospholipase, esterase, laccase, catalase, arylesterase, amylase, α-amylase, glucoamylase, cutinase, pectinase, pectinase, Glutamic acid lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, carrageenase, pullulanases, tannases, arabinosidases, hyaluronidases, chondroitinases, xylanases, pectin acetylesterases, polygalacturonidases, rhamnogalacturonidases, other endo-β-mannanases, exo-β-mannanases, pectin methylesterases, cellobiohydrolases, transglutaminases, lichenases, laminarinases and DNases, or any combination thereof.

[0441] 18) A polynucleotide, a nucleic acid construct or an expression vector, or a recombinant host cell, wherein the polynucleotide encodes a variant as described in any one of Examples 1 to 8, the nucleic acid construct or the expression vector comprises the polynucleotide, and the recombinant host cell is transformed with the polynucleotide.

[0442] 19) A method for producing a variant as described in any one of embodiments 1 to 8, the method comprising:

[0443] a) culturing the recombinant host cell as described in Example 17 under conditions suitable for expressing the variant; and

[0444] b) recovering the variant.

[0445] Examples

[0446] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention.

[0447] strains

[0448] DNA encoding the GH26 mannanase gene was isolated from Paenibacillus iliniflora (isolated from a soil sample collected in Virginia, USA in 2014) and Paenibacillus species (isolated from a sand sample collected in the United States in 1991) and sequenced as described in WO 2019 / 068715.

[0449] Material

[0450] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0451] Standard detergent system

[0452] Table 1A: Standard Detergent 1 Composition (Liquid)

[0453]

[0454]

[0455] Table 1B: Standard Detergent 2 Composition (Liquid)

[0456]

[0457] Small fabric swatch

[0458] The swatches included a combination of food stains and technical stains.

[0459] Material source DN-33 Fenugreek Seeds CFT CS-43 Guar Gum CFT CS-73 Locust Bean Gum CFT

[0460] The above commercial test materials are available from Center for Testmaterials BV, Stoomloggerweg 11, 3133 KT, Vlaardingen, The Netherlands.

[0461] Washing performance

[0462] Terg-O-tometer (TOM) washing test

[0463] The terg-o-tometer is the industry standard. 1L of wash solution was incubated in a water bath temperature controlled environment. The solution was mixed for 5 min and then 1L was added to each beaker. The temperature in the beaker was measured to be 20.0°C. The washed and rinsed swatches were dried in a drying oven overnight and measured as shown in Table 2 below.

[0464] Table 2: Conditions for the Terg-O-tometer washing test

[0465]

[0466] Washing performance is expressed as Δ reflectance value (ΔRem). After washing and rinsing, the small piece of cloth sample is spread and flattened and allowed to air dry overnight at room temperature. The light reflection evaluation of the small piece of cloth sample is carried out using a Macbeth Color Eye7000 reflection spectrophotometer with a large aperture. Measure under the condition of no UV in the incident light, and extract the reflectivity at 460nm. Measure the dry small piece of cloth sample with ColorEye 2. Small aperture measurement is carried out by 3 layers (3 pieces in the small piece of cloth sample of the same type from the same beaker), and 2 measurements are carried out on each small piece of cloth sample on the front side marked with beaker number and small piece of cloth sample number. The reflectance value of a single small piece of cloth sample is calculated by subtracting the reflectance value of the small piece of cloth sample of the control from the reflectance value of the washed small piece of cloth sample. The enzyme effect for each stain is calculated by obtaining the measured value from the small piece of cloth sample washed with enzyme and subtracting the measured value from the small piece of cloth sample not washed with enzyme. The total enzyme performance is calculated as the mean value of a single ΔRem.

[0467] Example 1: Reducing End Assay for Determining Mannanase Activity

[0468] To estimate the mannose yield after substrate hydrolysis, the reducing end assay developed by Lever (1972), Anal. Biochem. [Analytical biochemistry] 47: 273-279 was used. The assay is based on 4-hydroxybenzoic acid hydrazide, which reacts with the reducing end of the sugar under alkaline conditions. The product is a strong yellow anion that absorbs at 410 nm.

[0469] method

[0470] 4-Hydroxyphenylhydrazide (PAHBAH) (Sigma, H9882) was diluted to a concentration of 15 mg / ml in PAHBAH buffer. PAHBAH buffer contains: 50 g / L K-Na-tartrate (Merck, 1.08087) and 20 g / L sodium hydroxide (Sigma, S8045). This PAHBAH mixture was made before use.

[0471] Mix 70 μl of PAHBAH mixture and MiliQ water in a 96-well PCR plate (Thermo Scientific). Add samples from the hydrolysis experiment. Samples and MiliQ always reach a total volume of 150 μl, but the dilution of the samples is different. Seal the plate with a sticky PCR sealing foil (Thermo Scientific). In a PTC-200 thermal cycler (MJ Research), the plate is incubated at 95 ° C for 10 min, cooled and maintained at 10 ° C for 1 min. 100 μl samples are transferred to a flat-bottomed 96-well microtiter plate (NuncTM), and on a SpectraMax 190 absorbance microplate reader (Molecular Devices), color development is measured at 405 nm. The results are compared with mannose standards, which have been subjected to the same treatment and dilution of the samples compared thereto.

[0472] Example 2: Generation of variants by site-directed mutagenesis

[0473] Site-directed variants of SEQ ID NO: 2 were generated by adding one or more mutations, expression regulatory elements, and Bacillus genome homology regions to the PCR product simultaneously for site-directed integration by SOE (splicing by overlap extension). Bacillus subtilis was transformed with the PCR product; the correct gene sequence was confirmed by NGS.

[0474] Example 3: Determination of in-detergent stability

[0475] The variants generated as described above were tested for in-detergent stability under relevant conditions.In-detergent stability was determined by incubating the variants in a detergent containing 490 ppm protease (SEQ ID NO: 17) and subsequently calculating the half-life of the variants.

[0476] The following procedure was applied (conditions are shown in Table 3):

[0477] 1) Dissolve the mannanase variant in 0.01% Triton x-100 buffer (Triton X-100, Sigma Aldrich product number: 93426, CAS number 9036-19-5)

[0478] 2) Add the mannanase variant to a standard detergent containing protease to obtain the desired final mannanase concentration and stir for 30 minutes

[0479] 3) Incubate the enzyme / detergent solution at the desired temperature

[0480] 4) Samples of the enzyme / detergent solution were analyzed for residual mannanase activity after 0, 24, 72 and 144 hours and the half-life was calculated.

[0481] Residual mannanase activity was measured by using Mannazyme Tablets (a substrate for endo-1,4-β-mannanase) from Megazyme. The substrate for the Mannanase Detection Tablets was dissolved in 100 mM MOPS (3-(N-morpholino) propanesulfonic acid, CAS No.: 1132-61-2) and incubated with the enzyme / detergent solution for 60 minutes at room temperature with shaking at 800 rpm. The sedimentation was spun at 2000 rpm for 2 minutes to allow the insoluble substrate to settle, and the mannanase activity was measured by reading the optical density of the supernatant at 590 nm.

[0482] Calculate the half-life (T 1 / 2 ), which is the time at which 50% of the mannanase activity is retained.

[0483] The half-lives of SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 were obtained by the manner disclosed above and the results are provided in Table 3. It is clear that the mannanase variants having SEQ ID NO:3 and SEQ ID NO:4 have significantly improved in-detergent stability compared to the mannanase variant having SEQ ID NO:2.

[0484] Table 3: Half-life of variants of the invention

[0485]

[0486] Example 4: Residual Activity

[0487] 600 ppm of protease (SEQ ID NO: 17) and mannanase were added to Standard Detergent 1 and Standard Detergent 2. The samples for stability testing were stored at 37°C for 4 weeks with the lid closed, while the reference samples were stored at -18°C for 4 weeks. The residual activity was calculated as:

[0488] Activity (incubation sample) / Activity (reference sample) * 100%

[0489] For activity measurement, the principle of the reducing end assay described in Example 1 was applied with the following particularities: 5 g of detergent sample was dissolved in 400 mL of buffer (0.1 M NaH2PO4, 2H2O and 0.05% (w / v) 20, pH 7.3) and further diluted in 0.05 M HEPES buffer (pH 8.0) to the relevant concentration range.

[0490] The residual activity was measured and the results are listed in Table 4.

[0491] Table 4: Residual activity after storage

[0492]

[0493] It is clear from the data in Table 4 that the storage stability of detergents with the mannanase variants of SEQ ID NOs: 3 to 7 is greatly improved compared to the storage stability of the detergent with the mannanase known from the prior art (SEQ ID NO: 2).

[0494] Example 5: Residual Wash Performance (RWP) of Mannanase Variants Using Terg-O-tometer

[0495] The RWP of the variants generated as described above was tested under relevant conditions. RWP was determined by incubating the variants in a detergent containing 490 ppm protease (SEQ ID NO: 17) and then assessing the RWP using the TOM wash test method as described above. RWP was calculated as

[0496] Washing performance (incubation sample) / washing performance (reference sample) * 100%

[0497] The reference samples were prepared in the same way as the incubation samples but stored at -18°C for 4 weeks. The RWP reflects the average of the individual ΔREM values ​​of the three types of swatches.

[0498] Table 5: RWP after storage

[0499]

[0500]

[0501] It is clear from the data in Table 5 that the residual wash performance after storage of detergents having the mannanase variants of SEQ ID NOs: 3 to 7 is greatly improved compared to the mannanase known in the prior art (SEQ ID NO: 2).

[0502] Example 6: Half-life improvement factors of variants of SEQ ID NO: 3

[0503] Stability testing was performed by incubating the variants in detergent for different time periods and temperatures and comparing the activity to a control having SEQ ID NO: 3 incubated at 4°C for the same time period.

[0504] For the variants in Table 6, stress conditions included incubation of the variants in Standard Detergent 1 and protease having SEQ ID NO: 32 (1471 μM) at 42°C for 24 hours.

[0505] The residual activity was measured using the mannanase assay using an insoluble azo-carob-galactomannan substrate from Magzyme. The substrate was incubated with the enzyme at 30°C for 30 min, accompanied by shaking at 800 rpm. Thereafter, the reaction mixture was kept static for 10 min to allow the insoluble substrate to settle. The enzyme activity was measured by reading the optical density of the supernatant at 590 nm. The residual activity (RA) was calculated by taking the ratio of the stress response to the stress-free response, and the residual activity was then used to calculate the half-life. The half-life was calculated according to the following equation, where RA = residual activity, t = incubation time in hours, and the half-life was defined in hours:

[0506]

[0507] The half-life improvement factor (HIF) was calculated by taking the ratio of the half-life of the sample variant to the mannanase having SEQ ID NO: 3 also grown in duplicate microtiter plates.

[0508] Table 6: Half-life improvement factors of variants of SEQ ID NO: 3

[0509]

[0510]

[0511]

[0512] Examples 3 and 4 show that variants with SEQ ID NO: 3 are improved compared to the mannanase known in the prior art (SEQ ID NO: 2). From the data in Table 6 it is clear that the stability of the variants of SEQ ID NO: 3 in the presence of proteases is further improved.

[0513] Example 7: Synergistic effect of mannanase and other enzymes in stain removal

[0514] In a wash test assay, the synergistic effect of mannanase with 4 different laundry enzymes on 9 different stains was tested.

[0515] Material

[0516] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

[0517] Standard detergent system

[0518] Standard detergent 1 as disclosed in Table 1A was used.

[0519] Enzymes

[0520] Table 7: Enzymes tested

[0521]

[0522] *The above commercial enzymes are from Novozymes

[0523] 100L contains xanthan lyase and xanthan endoglucanase having SEQ ID NO: 31 and SEQ ID NO: 32, respectively.

[0524] Stains

[0525] Stains include a combination of food stains and technical stains.

[0526] Table 8: Types of stains

[0527]

[0528] CFT: Center for Testing Materials BV, Stoomloggerweg 11, 3133 KT, Vlaardingen, The Netherlands.

[0529] Equest: Warwick Equest, UK

[0530] Pre-washed white ballast

[0531] A ballast consisting of 50%:50% (polyester:cotton) fabric was pre-washed with amylase and cellulase to reduce / remove starch, carboxymethyl cellulose (CMC) and other textile additives. 3 kg of the textile ballast was washed in water with a hardness of 15° dH [Ca 2+ Mg 2+ :CO3 2- The mixture was washed three times in water [ratio, 4:1:7.5] and 78.6 g of W-ECE-2 detergent (wfk Testgewebe GmbH, Germany) containing the following enzymes for each of the three pre-wash steps.

[0532] Table 9: Pre-wash steps

[0533]

[0534] 3 kg of textiles were pre-washed with 13-15 liters of water using a standard wash program in a Miele Softtronic W1935 WTL machine at 40° C. After the third pre-wash step, rinsing was performed in deionized water and the textile fabrics were then line dried and cut into 5 cm x 5 cm pieces.

[0535] Washing performance

[0536] Under the above conditions, the synergy test of enzyme was tested in the Terg-O-tometer (TOM) washing test to determine the washing performance (stain removal efficacy). General washing conditions are given in Table 2, and enzymes are added to the specified dosage (Table 7) for enzyme washing. Two stains of each type of stain (Table 8) are included in each beaker together with the pre-washed ballast, so the total weight is about 30g / beaker. At room temperature, the washed and rinsed stains are dried overnight in a drying oven, and stain removal is evaluated using a Datacolor 800V spectrophotometer (Datacolor, Lawrenceville, New Jersey, the United States). The light reflection of the stain is evaluated under CIE standard illuminant D65 and CIE 1964 10 degree standard observer. Measured without UV in the incident light, and the reflectance value of each stain at 460nm using enzyme and not using enzyme washing is recorded. Wash performance is expressed as the average reflectance at 460 nm (REM) of two stains per stain type.

[0537] result

[0538] This example shows that the total stain removal efficacy (Table 10, "Total of all 9 stains") is higher when the GH26 mannanase is used only with the xanthanase (Table 10, "M / X") compared to the controls in the combination ("B", "M" and "X"). An increase in the total stain removal efficacy is also observed if the GH26 mannanase is used in a multi-enzyme combination with an amylase / protease (Table 10, "A / P"), an amylase / protease / lipase (Table 10, "A / P / L") and an amylase / protease / lipase / xanthanase (Table 10, "A / P / L / X") when compared to their respective controls. The observed synergistic effect of the GH26 mannanase and laundry enzymes together in stain removal depends on the stain type and, therefore, on the soil substrate. For example, no synergy of GH26 mannanase together with the tested enzymes was detected on "Grass / Mud", "Cooked Tallow" and "Cooked Butter", while it was apparent that certain stains were more effective in demonstrating synergy of GH26 mannanase together with specific enzymes in stain removal.

[0539] Table 10: Reflectance values ​​at 460 nm (stain removal efficacy)

[0540]

[0541]

[0542] Values ​​represent the average of two stains of each type.

[0543] *The sum is the sum of the average reflectance values ​​for each stain type

[0544] B: Black - Detergent Only

[0545] A:Amylase 100L)M: Mannanase SEQ ID NO: 3

[0546] P:Protease (Liquananse 3.5L)L: Lipase (Lipex 200L)X: Xanthanase ( 100L)

[0547] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to serve as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications of the invention, in addition to those shown and described herein, become apparent to those skilled in the art due to the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure including definitions shall prevail.

Claims

1. A variant of the polypeptide of SEQ ID NO: 2, wherein the amino acids at positions 490 and 491 are deleted, wherein the variant has at least 60% sequence identity with the polypeptide of SEQ ID NO: 3, and wherein the variant has mannanase activity.

2. The variant of claim 1, wherein the variant comprises an extension of one or more amino acids at the N-terminus and / or C-terminus, or a truncation of one or more amino acids at the N-terminus and / or C-terminus, with the proviso that the insertion at the C-terminus is not *490W and *491R.

3. The variant of claim 1 or 2, comprising a deletion of at least 11 amino acids from the N-terminus, such as amino acids 1 to 12, such as amino acids 1 to 13, such as amino acids 1 to 14 or such as amino acids 1 to 15 of SEQ ID NO:

2.

4. The variant of any one of claims 1 to 3, further comprising one or more modifications selected from the group consisting of I30L, D48P, Y155H, T167P, Q215E, H276C, R280K, F286C, G366N and D486E.

5. The variant of claim 4, further comprising one or more modifications selected from the group consisting of G20P, A101L, A111P, A118E, S137A, Q143R, R160L, E162D, M171I, N176S, P182R, Q183E, V244A, H324K and D385H.

6. The variant of any one of claims 1 to 5, wherein the variant is a polypeptide of SEQ ID NO: 3, a polypeptide of SEQ ID NO: 4, a polypeptide of SEQ ID NO: 5, a polypeptide of SEQ ID NO: 6, a polypeptide of SEQ ID NO: 7, a polypeptide of SEQ ID NO: 22, a polypeptide of SEQ ID NO: 23, a polypeptide of SEQ ID NO: 24, a polypeptide of SEQ ID NO: 25, a polypeptide of SEQ ID NO: 26, a polypeptide of SEQ ID NO: 27, a polypeptide of SEQ ID NO: 28, or a polypeptide of SEQ ID NO:

3. 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 99.9% or even 100% sequence identity to the polypeptide of NO:29, wherein the variant has mannanase activity.

7. An enzyme composition comprising any variant according to any one of claims 1 to 6 and a GH5 mannanase.

8. A detergent composition comprising a variant as claimed in any one of claims 1 to 6 and at least one detergent adjunct ingredient.

9. The detergent composition of claim 8, further comprising a GH5 mannanase.

10. The detergent composition of claim 9, wherein the ratio between the GH5 mannanase and the variant of any one of claims 1 to 6 is a ratio of 1:3 to 3:1 based on the weight of enzyme protein.

11. The detergent composition of any one of claims 8 to 10, further comprising a xanthan lyase and a xanthan endoglucanase, wherein the xanthan lyase has at least 80% identity with SEQ ID NO: 30 and the xanthan endoglucanase has at least 80% identity with SEQ ID NO:

31.

12. Use of a variant as claimed in any one of claims 1 to 6 or a detergent composition as claimed in claims 8 to 11 in a cleaning process, such as laundry or hard surface cleaning, such as dishwashing.

13. A method of cleaning an item, the method comprising exposing the item to a wash liquor comprising a variant as claimed in any one of claims 1 to 6 or a detergent composition as claimed in claims 8 to 11, for example wherein the item is a textile or a hard surface.

14. A polynucleotide, a nucleic acid construct or an expression vector, or a recombinant host cell, wherein the polynucleotide encodes the variant as described in any one of claims 1 to 6, the nucleic acid construct or the expression vector comprises the polynucleotide, and the recombinant host cell is transformed with the polynucleotide.

15. A method for producing a variant as claimed in any one of claims 1 to 6, the method comprising: a) culturing the recombinant host cell according to claim 14 under conditions suitable for expressing the variant; as well as b) recovering the variant.

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