Mannanase variants and polynucleotides encoding same

Abstract

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

Description

[Technical Field]

[0001] Array list reference This application contains a sequence listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to mannanase variants that exhibit mannanase activity, compositions comprising the mannanase variants, polynucleotides encoding the variants, methods of making the variants, and methods of using the variants. [Background technology]

[0003] 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.

[0004] Mannans are a type of hemicellulose that represent up to 25% of the wood's dry weight in softwoods, but are also found in other plant materials, particularly various seeds. Mannans, including guar gum, are used as stabilizers in many food products.

[0005] Therefore, it may be advantageous to use endomannanase in applications where mannans need to be degraded. Examples of where mannanases can be used include detergents for removing stains containing mannans, the production of bioethanol from softwood (Varnai et al., (2011) "Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood", Bioresource Tech., 102(19), pp. 9096-104) and palm kernel press cake (Joergensen 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. 318-32), and animal feed improvement (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 hydrolysis of coffee extract (Nunes et al., (2006), “Characterization of Galactomannan Derivatives in Roasted Coffee Beverages”, J. Agricultural Food Chem., 54(9), pp. 3428-3439).

[0006] In the household care industry, it is known to use mannanases, for example in laundry detergents. WO 1999 / 064619 discloses alkaline mannanases that exhibit mannanase activity even in the alkaline pH range when applied in cleaning compositions.

[0007] According to CAZy (azy.org), endo-1,4-β-mannanases are found in glycoside hydrolyase families 5, 26, and 113. WO 2019 / 068715 and WO 2019 / 068713 disclose mannanases of family GH 26 that exhibit beta-mannanase activity.

[0008] However, alternative mannanases are desirable and industrially applicable, for example, to the detergent manufacturing industry. The present invention is directed to these and other important goals. Summary of the Invention [Means for solving the problem]

[0009] Described herein are variants of a parent mannanase, the variants comprising at least two modifications, wherein (a) the first modification is at any one of positions 168, 7, 13, 15, 20, 26, 36, 46, 64, 69, 70, 82, 101, 103, 109, 120, 141, 155, 162, 163, 166, 167, 168, 172, 176, 178, 182, 190, 197, 214, 215, 219, 239, 248, 253, 258, 271, 276, 280, 283, 286, 299, 366, 378, 385, 408, 410, 413, 473, 48 6, 491, and (b) at least a second of said modifications is at any other position of said variant, wherein the variant has mannanase activity, and the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least or has at least 98% or at least 99% sequence identity but less than 100% sequence identity, or has 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96% sequence identity, or has 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95%, for example, 94 to 95% sequence identity, or has 85% to 94%, e.g. For example, it has 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%,It has 88 to 91%, 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0010] The present invention also relates to compositions comprising the variants as disclosed herein, the use of such compositions in domestic or industrial cleaning processes, isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of making the variants and methods of cleaning using the compositions disclosed herein.

[0011] Array List Overview SEQ ID NO: 1 is the amino acid sequence of a mature GH26 mannanase from a strain of Paenibacillus illinoisensis having a CBM35 domain. SEQ ID NO:2 is the mannanase of SEQ ID NO:1 further comprising a stabilizing mutation. SEQ ID NO:3 is the amino acid sequence of a truncated mature GH26 mannanase from a strain of Paenibacillus illinoisensis lacking the CBM35 domain. SEQ ID NO: 4 is the amino acid sequence of a truncated mature GH26 mannanase from a strain of Paenibacillus illinoisensis lacking the CBM35 domain. SEQ ID NO: 5 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 6 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 7 Mature polypeptide obtained from Cytophaga sp. SEQ ID NO: 8 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 9 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 10 Mature polypeptide obtained from Bacillus subtilis. SEQ ID NO: 11 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 12 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 13 Synthetic construct SEQ ID NO: 14 Mature polypeptide obtained from Bacillus lentus. SEQ ID NO: 15 Mature polypeptide obtained from Bacillus amyloliquefaciens. SEQ ID NO: 16 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 17 Mature polypeptide obtained from Bacillus gibsonii. SEQ ID NO: 18 Mature polypeptide obtained from Bacillus lentus. SEQ ID NO: 19 Mature polypeptide obtained from Bacillus licheniformis. SEQ ID NO: 20 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 21 Mature polypeptide obtained from Humicola insolens. SEQ ID NO: 22 Mature polypeptide obtained from Humicola insolens. SEQ ID NO: 23 Mature polypeptide obtained from Thielavia terrestris. SEQ ID NO: 24 Mature polypeptide obtained from Paenibacillus polymyxa. SEQ ID NO: 25 Mature polypeptide obtained from Melanocarpus albomyces. SEQ ID NO: 26 Mature polypeptide obtained from Thermomyces lanuginosus. SEQ ID NO: 27 Mature polypeptide obtained from Bacillus agaradhaerens. SEQ ID NO: 28 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 29 Mature polypeptide obtained from Bacillus sp. SEQ ID NO: 30 Mature polypeptide obtained from Bacillus akibai. SEQ ID NO: 31 Mature polypeptide obtained from Bacillus mojavensis. SEQ ID NO: 32 Mature polypeptide obtained from a Thermobacillus species. SEQ ID NO: 33 Mature polypeptide obtained from Paenibacillus species. SEQ ID NO: 34 Mature polypeptide obtained from Cohnella species. SEQ ID NO: 35 Mature polypeptide obtained from Paenibacillus elgii. SEQ ID NO: 36 Mature polypeptide obtained from a Bacillus species. SEQ ID NO: 37 Mature polypeptide obtained from a Bacillus species. SEQ ID NO: 38 Mature polypeptide obtained from Bacillus bogoriensis. SEQ ID NO: 39 Mature polypeptide obtained from Paenibacillus species. SEQ ID NO: 40 Mature polypeptide obtained from Bacillus hemicellulosilyticus. SEQ ID NO: 41 Mature polypeptide obtained from Bacillus subtilis. SEQ ID NO: 42 Mature polypeptide obtained from Bacillus cibi. SEQ ID NO: 43 Mature polypeptide obtained from Aspergillus oryzae. SEQ ID NO:44 is the gene encoding the mature GH26 mannanase of SEQ ID NO:1. SEQ ID NO:45 is the gene encoding the mature GH26 mannanase of SEQ ID NO:2.

[0012] definition In accordance with the detailed description, the following abbreviations and definitions apply. It should be noted that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, a reference to "a dosage" includes a reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0013] Allelic variant: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variants arise naturally through mutation and can result in polymorphism within a population. Genetic variations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0014] Amylase: The term "amylase" (EC 3.2.1) refers to an enzyme that catalyzes the hydrolysis of starch, glycogen, and oligosaccharides, maltose, or related polysaccharides with glucose. Amylases are glycoside hydrolases that act on α-1,4-glycosidic bonds. Amylases suitable in the cleaning compositions of the present invention are preferably alpha amylases. Alpha-amylases (EC 3.2.1.1) include 1,4-α-D-glucan glucanohydrolases and glycogenases and are calcium metalloenzymes. By acting at random positions along the starch chain, alpha amylases cleave long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose, and "limit dextrins" from amylopectin. Suitable amylases of the present invention are preferably microbial, e.g., obtained from bacterial or fungal sources. The term "alpha-amylase activity" refers to the activity of alpha 1,4-glucan 4 glucanohydrolases, EC 3.2.1.1, which constitute a group of enzymes that catalyze the hydrolysis of starch and other linear and branched 1,4 alpha-glycosidic oligosaccharides and polysaccharides. Alpha-amylase activity can be determined by Assay II as described in the Examples herein.

[0015] Beta-glucanase: The term "beta-glucanase," as used herein, refers to an endo-acting enzyme that catalyzes the hydrolysis of beta-1,3-, beta-1,6-, and / or beta-1,4-bonds that link two glucosyl residues in a beta-glucan.

[0016] A subgroup of beta-glucanases as defined herein, also known as licheninases (e.g., EC 3.2.1.73), can be used to catalyze the hydrolysis of beta-1,4-glucosidic bonds to obtain beta-glucans. Licheninases (e.g., EC 3.2.1.73) hydrolyze (1,4)-beta-D-glucosidic bonds in beta-D-glucans containing (1,3)- and (1,4)-linkages and can act on lichenin and cereal beta-D-glucans, but cannot act on beta-D-glucans containing only 1,3- or 1,4-linkages. As used herein, the term "beta-glucanase activity" includes licheninase (e.g., EC 3.2.1.73) activity. Licheninase activity can be determined as in Assay VII herein.

[0017] Another further subgroup of beta-glucanases as defined herein, also known as laminarinases (e.g., EC 3.2.1.6, EC 3.2.1.39, and / or EC 3.2.1.58), can also be used to catalyze the hydrolysis of beta-1,3-glucosidic bonds, or beta-1,4-glucosidic bonds when the glucose residue involved in the bond where the reducing group is to be hydrolyzed is replaced at C3 to release glucose or an oligosaccharide. As used herein, the term "beta-glucanase activity" also includes laminarinase (e.g., EC 3.2.1.6, EC 3.2.1.39, and / or EC 3.2.1.58) activity. Laminarinase activity can be determined as in Assay VI herein.

[0018] Biofilm: The term "biofilm" refers to any group of microorganisms in which cells adhere to one another on a surface, such as fabric, dishware, or hard surfaces. These adherent cells are often embedded in a self-produced matrix of extracellular polymeric substances (EPS). Biofilm EPS is generally a polymeric mass composed of extracellular DNA, proteins, and polysaccharides. Biofilms can form on viable or non-viable surfaces. Microbial cells growing within a biofilm are physiologically distinct from planktonic cells of the same organism, which, in contrast, are single cells that can float or swim in a liquid medium.

[0019] Bacteria living within biofilms typically have properties that are significantly different from planktonic bacteria of the same species, as the dense and protected environment of the film allows them to cooperate and interact in diverse ways. One effect of this environment is increased resistance to detergents and antibiotics, as the dense extracellular matrix and outer layer of cells protect the interior of the population.

[0020] On laundry, biofilm-producing bacteria can be found in the following species: Acinetobacter sp., Aeromicrobium sp., Brevundimonas sp., Microbacterium sp., Micrococcus luteus, Pseudomonas sp., Staphylococcus epidermidis, and Stenotrophomonas sp.

[0021] Carbohydrate-binding module: The term "carbohydrate-binding module" refers to a region in a carbohydrate-active enzyme that confers carbohydrate-binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). Most known carbohydrate-binding modules (CBMs) are consecutive amino acid sequences with distinct folds. Carbohydrate-binding modules (CBMs) are usually found at either the N- or C-terminus of the enzyme. Some CBMs are known to have specificity for cellulose. In one embodiment, the CBM is a family 35 CBM (Pfam PF16990), such as that disclosed in Tunnicliffe RB, Bolam DN, Pell G, Gilbert HJ, Williamson MP; J Mol Biol. 2005;347:287-296.

[0022] Catalytic domain: The term "catalytic domain" refers to the region of an enzyme that contains the enzyme's catalytic machinery.

[0023] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before emerging as a spliced ​​mature RNA.

[0024] Cellulolytic enzymes or cellulases: The terms "cellulolytic enzymes" or "cellulases" refer to one or more (e.g., several) enzymes that hydrolyze cellulosic materials. Such enzymes include endoglucanases, cellobiohydrolases, beta-glucosidases, or combinations thereof. Two basic approaches for measuring cellulolytic enzyme activity include (1) measuring overall cellulolytic enzyme activity and (2) measuring individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as outlined in Zhang et al., 2006, Biotechnology Advances 24:452-481. Overall cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman No. 1 filter paper, crystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common overall cellulolytic activity assay is the filter paper assay, which uses Whatman No. 1 filter paper as a substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59:257-68).

[0025] Cellulolytic enzyme activity may be determined by measuring the increase in sugar production / liberation during hydrolysis of cellulosic material with cellulolytic enzymes under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3-7 days at a suitable temperature such as 40-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C, and a suitable pH such as 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0, compared to a control hydrolysis without added cellulolytic enzyme protein. Typical conditions are: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnSO, ​​50°C, 55°C, or 60°C, 72 hours, sugar analysis on an AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and as described in the Examples herein.

[0026] Chimeric polypeptide: The term "chimeric polypeptide" refers to a polypeptide having mannanase activity that is created by replacing the amino acid sequence of one polypeptide having mannanase activity with one derived from the homologous position of one or more other polypeptides having mannanase activity.

[0027] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which generally 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 can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0028] Control sequence: The term "control sequence" refers to a nucleic acid sequence necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence can be native (i.e., from the same gene) or foreign (i.e., from different genes) to the polynucleotide encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter and transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the polypeptide.

[0029] Deep Cleaning: The term "deep cleaning" is defined herein to mean the reduction, destruction, or removal of organic matter, such as polysaccharides, proteins, DNA, dirt, or other components present in the organic matter, e.g., components that may be contained in biofilms. The organic matter may be referred to as poly-organic stains, which contain two or more organic components, such as starch, fat, protein, DNA, and mannan.

[0030] Detergent ingredient: The term "detergent ingredient" is defined herein to mean a class of chemicals that can be used in a detergent composition. Examples of detergent ingredients are surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching systems or components, polymers, fabric hueing agents, fabric softeners, suds boosters, suds suppressors, dispersants, dye transfer inhibitors, optical brighteners, fragrances, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. A detergent ingredient can include one or more of any type of detergent ingredient.

[0031] Detergent composition: The term "detergent composition" refers to a composition that is used to remove undesirable compounds from items to be cleaned, such as textiles, dishware, and hard surfaces. Detergent compositions can be used, for example, to clean textiles, dishware, and hard surfaces for both domestic and industrial cleaning. This term encompasses any material / compound selected for a specific type of desired cleaning composition and product form (e.g., liquid, gel, powder, granule, paste, or spray composition), including, but not limited to, detergent compositions (e.g., liquid and / or solid laundry detergents and fine fabric detergents; cleaning formulations for hard surfaces, such as glass, wood, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, and dishwashing detergents).

[0032] In addition to containing the mannanase variants of the present invention, the detergent composition may also contain one or more additional enzymes (e.g., amylase, protease, protease, peroxidase, cellulase, beta-glucanase, xyloglucanase, hemicellulase, xanthanase, xanthan lyase, lipase, acyltransferase, phospholipase, esterase, laccase, catalase, arylesterase, amylase, alpha-amylase, glucoamylase, cutinase, pectinase, pectate lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, carrageenase, pullulanase, tannase, arabinosidase, hyaluronidase, chondroitinase, xyloglucanase, xylase, hydroxybenzoate ... beta-mannanase, pectin acetylesterase, polygalacturonase, rhamnogalacturonase, other endo-beta-mannanases, exo-beta-mannanases, pectin methylesterase, cellobiohydrolase, transglutaminase, licheninanase, laminarinase, DNase, and combinations thereof, or any mixture thereof), and / or surfactants, builders, chelators or chelating agents, bleaching systems or ingredients, polymers, fabric hueing agents, suds boosters, suds suppressors, dyes, fragrances, tan inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anticorrosives, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing and fluorescent dyes, antioxidants, and solubilizers.

[0033] Dishwashing: The term "dishwashing" refers to all forms of washing dishes, for example, by manual or automatic dishwashing. Washing dishware includes, but is not limited to, cleaning all forms of ceramics, such as plates, cups, glasses, bowls, etc., all forms of cutlery, such as spoons, knives, forks, and serving utensils, as well as ceramics, plastics, metals, china, glass, and acrylic.

[0034] Dishwashing Composition: The term "dishwashing composition" or "dishwashing composition" refers to all forms of compositions intended for cleaning dishes, tableware, pots, pans, cutlery, and compositions for cleaning hard surfaces in the kitchen. The present invention is not limited to any particular type of dishwashing composition or any particular detergent.

[0035] DNase: A DNase is a polypeptide with DNase (deoxyribonuclease) activity that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. Exodeoxyribonucleases cleave or cleave residues at the ends of the DNA backbone, while endodeoxyribonucleases cleave or cleave within the DNA backbone. DNases may cleave only double-stranded DNA, or may cleave double- and single-stranded DNA. The terms "DNase" and "polypeptide with DNase activity" may be used interchangeably throughout this application. For purposes of the present invention, DNase activity can be determined according to the procedures described in Assay IV or Assay V of the Examples herein. Preferably, the DNase is selected from the enzyme class EC3.1, preferably EC3.1.21. Preferably, the polypeptide with DNase activity is obtained from a microorganism, and the DNase is a microbial enzyme. Preferably, the DNase is of fungal or bacterial origin.

[0036] Expression: The term "expression" includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0037] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to a control sequence that provides for its expression.

[0038] Fragment: The term "fragment" refers to a polypeptide or catalytic or carbohydrate-binding module lacking one or more (e.g., several) amino acids at the amino and / or carboxy terminus of a mature polypeptide or domain; the fragment retains mannanase activity. In one embodiment, the fragment comprises at least 90% of the length of the mature polypeptide, e.g., at least 441 amino acids of SEQ ID NO: 1, at least 441 amino acids of SEQ ID NO: 2, at least 280 amino acids of SEQ ID NO: 3, or at least 307 amino acids of SEQ ID NO: 4. In one embodiment, the fragment comprises at least 92% of the length of the mature polypeptide, e.g., at least 451 amino acids of SEQ ID NO: 1, at least 451 amino acids of SEQ ID NO: 2, at least 287 amino acids of SEQ ID NO: 3, or at least 314 amino acids of SEQ ID NO: 4. In one embodiment, the fragment comprises at least 94% of the length of the mature polypeptide, e.g., at least 461 amino acids of SEQ ID NO: 1, at least 461 amino acids of SEQ ID NO: 2, at least 293 amino acids of SEQ ID NO: 3, or at least 321 amino acids of SEQ ID NO: 3. In one embodiment, a fragment comprises at least 96% of the length of the mature polypeptide, e.g., at least 471 amino acids of SEQ ID NO: 1, at least 471 amino acids of SEQ ID NO: 2, at least 299 amino acids of SEQ ID NO: 3, or at least 28 amino acids of SEQ ID NO: 4. In one embodiment, a fragment comprises at least 98% of the length of the mature polypeptide, e.g., at least 481 amino acids of SEQ ID NO: 1, at least 481 amino acids of SEQ ID NO: 2, at least 305 amino acids of SEQ ID NO: 3, or at least 335 amino acids of SEQ ID NO: 4. In one embodiment, a fragment comprises at least 99% of the length of the mature polypeptide, e.g., at least 486 amino acids of SEQ ID NO: 1, at least 486 amino acids of SEQ ID NO: 2, at least 308 amino acids of SEQ ID NO: 3, or at least 338 amino acids of SEQ ID NO: 4.

[0039] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which a polypeptide is fused at the N-terminus or C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and involve linking coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is formed post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). A fusion polypeptide can further include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved to release the two polypeptides. Examples of cleavage sites include Martin et al.,2003,J.Ind.Microbiol.Biotechnol.3:568-576;Svetina et al.,2000,J.Biotechnol.76:245-251;Rasmussen-Wilson et al. al.,1997,Appl.Environ.Microbiol.63:3488-3493;Ward et al.,1995,Biotechnology 13:498-503;and Contreras et al.,1991,Biotechnology 9:378-381;Eaton et al.,1986,Biochemistry 25:505-512;Collins-Racie et al.,1995,Biotechnology 13:982-987;Carter et al. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0040] Hard Surface Cleaning: The term "hard surface cleaning" is defined herein as cleaning of hard surfaces, where hard surfaces may include floors, desks, walls, roofs, etc., as well as the surfaces of hard objects such as automobiles (car washing) and dishes (dishwashing). Dishwashing includes, but is not limited to, cleaning plates, cups, glasses, bowls, and cutlery such as spoons, knives, and forks, serving utensils, ceramic, plastic, metal, china, glass, and acrylic.

[0041] Hemicellulase or Hemicellulase: The term "hemicellulase" or "hemicellulase" refers to one or more (e.g., several) enzymes that hydrolyze cellulosic materials. See, for example, Shallom and Shoham, Current Opinion in Microbiology, 2003, 6(3):219-228. Hemicellulases are important components in the decomposition of plant biomass. Examples of hemicellulases include, but are not limited to, acetylmannan esterase, acetylxylan esterase, arabinanase, arabinofuranosidase, coumaric acid esterase, feruloyl esterase, galactosidase, glucuronidase, glucuronoyl esterase, mannanase, mannosidase, xylanase, and xylosidase. The substrates for these enzymes, hemicellulases, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to cellulose microfibrils in plant cell walls, cross-linking them into a robust network. Hemicellulose is also covalently linked to lignin, forming highly complex structures with cellulose. The variable structure and organization of hemicellulose requires the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs), which hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester bonds in acetate or ferulic acid side chains. Based on primary sequence homology, these catalytic modules can be assigned to GH and CE families. Some families with an overall similar fold can be further divided into alphabetically labeled clans (e.g., GH-A). The most informative and up-to-date classification of these and other carbohydrate-related enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activity can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature between 40°C and 80°C, for example, 50°C, 55°C, 60°C, 65°C, or 70°C, and at a suitable pH between 4 and 9, for example, 5.0, 5.5, 6.0, 6.5, or 7.0.

[0042] Host cell: The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term "host cell" encompasses progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication, as well as recombinant host cells, isolated host cells (e.g., isolated recombinant host cells), and heterologous host cells (e.g., a host cell that is not a Myrothecium roridum host cell).

[0043] Hybrid polypeptide: The term "hybrid polypeptide" refers to a polypeptide that contains domains from two or more polypeptides, for example, a binding domain from one polypeptide and a catalytic domain from another polypeptide. The domains can be fused at the N-terminus or C-terminus.

[0044] Hybridization: The term "hybridization" refers to the pairing of substantially complementary strands of nucleic acids using standard Southern blotting procedures. Hybridization can be performed under moderate, moderate-high, high, or very high stringency conditions. Moderate stringency conditions refer to prehybridization and hybridization in 5x SSPE, 0.3% SDS, 200 micrograms / ml fragmented, denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours, followed by three washes using 0.2x SSC, 0.2% SDS at 55°C for 15 minutes each. Medium-high stringency conditions refer to prehybridization and hybridization in 5x SSPE, 0.3% SDS, 200 micrograms / ml fragmented denatured salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours, followed by three washes in 0.2x SSC, 0.2% SDS for 15 minutes each at 60°C. High stringency conditions refer to prehybridization and hybridization in 5x SSPE, 0.3% SDS, 200 micrograms / ml fragmented denatured salmon sperm DNA, and 50% formamide at 42°C for 12-24 hours, followed by three washes in 0.2x SSC, 0.2% SDS for 15 minutes each at 65°C. High stringency conditions meant prehybridization and hybridization in 5× SSPE, 0.3% SDS, 200 micrograms / ml fragmented denatured salmon sperm DNA, and 50% formamide at 42°C for 12 to 24 hours, followed by three washes in 0.2× SSC, 0.2% SDS at 70°C for 15 minutes each.

[0045] Improved properties: The term "improved properties" refers to characteristics associated with a variant that are improved compared to the parent. Such improved properties include, but are not limited to, detergent stability, thermostability, protease stability, surfactant stability, and pH stability.

[0046] Detergent Stability: The term "detergent stability" or "detergent stability" refers to the stability of a mannanase enzyme, either wild-type, parent, or variant, incubated in detergent. For purposes of the present invention, detergent stability can be determined as shown in the Examples, particularly Example 6.

[0047] Isolated: The term "isolated" refers to a substance in a form that does not occur in nature or in an environment that does not naturally occur. Examples of isolated substances include: (1) any substance that does not occur in nature; (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature, including, but not limited to, any enzymes, variants, nucleic acids, proteins, peptides, or cofactors; (3) any substance that has been modified by the hand of man relative to the substance as found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., by recombinant production in a host cell; multiple copies of the gene encoding the substance; and use of a stronger promoter than that naturally associated with the gene encoding the substance).

[0048] Laundry: The term "laundry" refers to the process of treating textiles with a solution containing the cleaning or detergent composition of the present invention, both in relation to domestic and industrial laundering. For example, the laundering process may be carried out using, for example, a domestic or industrial washing machine, or may be carried out by hand.

[0049] Lipase: The term lipase includes enzymes that catalyze the hydrolysis of fats (lipids). Lipases are a subclass of esterases. Lipases suitable for the present invention include phospholipases, acyltransferases, or perhydrolases, such as acyltransferases with homology to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782), perhydrolases from the CE 7 family (WO 09 / 67279), and variants of M. smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028). Suitable lipases and cutinases include those of bacterial or fungal origin. These include chemically modified or protein engineered mutant enzymes, such as those from the genus Thermomyces, e.g., T. lanuginosus (formerly Humicola lanuginosa), as described in EP 258068 and EP 305216. lanuginosa), for example cutinases from Humicola species such as H. insolens (WO 96 / 13580), for example P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (P.Lipases from strains of Pseudomonas (some of which have now been renamed Burkholderia) such as Pseudomonas wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), cutinases from Magnaporthe grisea (WO 10 / 107560), cutinases from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipases from Thermobifida fusca (WO 11 / 084412), Geobacillus stearothermophilus (Geobacillus stearothermophilus), and the like. Examples of lipases that may be used include lipases from Bacillus stearothermophilus (WO 11 / 084417), Bacillus subtilis (WO 11 / 084599), and Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147). Lipase activity may be determined as described in Assay VIII in the Examples herein.

[0050] Mannanase: The term "mannanase" refers to a polypeptide having mannan endo-1,4-beta-mannosidase activity (EC 3.2.1.78), which catalyzes the hydrolysis of 1,4-β-D-mannose linkages in mannans, galactomannans, and glucomannans. Other names for mannan endo-1,4-beta-mannosidase include 1,4-β-D-mannan mannohydrolase; endo-1,4-β-mannanase; endo-β-1,4-mannase; β-mannanase B; β-1,4-mannan 4-mannanohydrolase; endo-β-mannanase; and β-D-mannanase. For purposes of the present invention, mannanase activity can be determined using a reducing end assay as described in Example 1 herein. In one embodiment, a polypeptide of the invention has at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mannanase activity of the polypeptide of SEQ ID NO: 1. In one embodiment, a polypeptide of the invention has at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mannanase activity of the polypeptide of SEQ ID NO: 2.

[0051] Mature Polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one embodiment, a mature polypeptide is amino acids 1 to 491 of SEQ ID NO: 1. In one embodiment, a mature polypeptide is amino acids 1 to 491 of SEQ ID NO: 2. In one embodiment, a mature polypeptide is amino acids 1 to 312 of SEQ ID NO: 3. In one embodiment, a mature polypeptide is amino acids 1 to 342 of SEQ ID NO: 4.

[0052] It is known in the art that host cells may produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide. It is also known in the art that different host cells may process polypeptides differently, such that one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having different C-terminal and / or N-terminal amino acids) compared to another host cell expressing the same polynucleotide.

[0053] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having mannanase activity.

[0054] Malodor: The term "malodor" refers to an undesirable odor on a clean item. A cleaned item should smell fresh and clean, free of malodors adhering to the item. One example of a malodor is an unpleasant-smelling compound that can be produced by microorganisms. Another example is sweat or body odor that has adhered to an item that has been in contact with a human or animal. Another example of a malodor could be the odor from spices, e.g., curry or other exotic spices, that have adhered to an item, such as a piece of fabric. One way to measure an item's ability to adsorb malodors is through the use of a malodor assay.

[0055] Modified: The term "modified" in the context of a polypeptide of the invention means that one or more amino acids in a reference amino acid sequence (i.e., SEQ ID NO: 1 or 2) are changed by insertion or deletion of an amino acid, preferably by substitution with a different amino acid followed by at least one deletion. The terms "modified," "altered," and "mutated" may be used interchangeably and have the same meaning and purpose.

[0056] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0057] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that contains one or more regulatory sequences, that is isolated from a naturally occurring gene, or that is modified or synthetic so as to contain segments of nucleic acid in a manner that would not otherwise occur in nature.

[0058] Operably linked: The term "operably linked" refers to a configuration in which control sequences are appropriately positioned relative to a polypeptide coding sequence so as to direct the expression of the coding sequence.

[0059] Parent or Parent Mannanase: As used herein, the terms "parent" or "parent mannanase" or "parent polypeptide" refer to any polypeptide having mannanase activity that has been modified to generate an enzyme variant of the present invention. In the present invention, a parent polypeptide is understood to refer to a naturally occurring polypeptide that does not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, the term "parent" with respect to a polynucleotide refers to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. However, a polynucleotide encoding a parent polypeptide is not limited to naturally occurring polynucleotides, but includes any polynucleotide encoding a parent polypeptide. A parent mannanase can be any mannanase that has at least 60%, for example, at least 62%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:2.

[0060] Pectinase: The term "pectinase" refers to a pectinase enzyme as defined in the art and includes enzymes that cleave the polysaccharide and / or oligosaccharide chains in pectin substances, such as poly(1,4-alpha-D-galacturonide) and its derivatives (see reference Sakai et al., Pectin, pectinase and protopectinase: production, properties and applications, pp. 213-294: Advances in Applied Microbiology, vol. 39, 1993). Non-limiting examples of pectinases include hydrolase-type pectinases (e.g., rhamnogalacturonan hydrolase) and lyase-type pectinases (e.g., pectate lyase). Preferably, the pectinase of the present invention is a pectinase enzyme that catalyzes the random cleavage of alpha-1,4-glycosidic bonds in pectic acid, also called polygalacturonic acid, by trans-elimination, such as polygalacturonate lyases (EC 4.2.2.2) (PGL), also known as poly(1,4-alpha-D-galacturonides), also known as pectate lyases, of the enzyme class. Pectinase activity can be determined as described in Assay IX in the Examples herein.

[0061] Protease: The term "protease" includes enzymes that hydrolyze peptide bonds, including peptidases and proteinases. Serine proteases (or serine endopeptidases), EC 3.4.21, are enzymes that cleave peptide bonds in proteins, in which serine serves as the nucleophilic amino acid at the active site. Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, e.g., plant or microbial origin. Microbial origin is preferred. Chemically modified variants or engineered protein variants are included. The most suitable protease for laundry may be an alkaline protease, such as a serine protease. The serine protease may be, for example, an S1 family protease, such as trypsin, or an S8 family protease, such as subtilisin. The metalloprotease protease may be, for example, thermolysin from the M4 family or other metalloproteases, such as those from the M5, M7, or M8 families. The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein 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 classified into six subdivisions: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family. Protease activity can be determined as described in Assay X in the Examples herein.

[0062] pH Stability: The term "pH stability" refers to the stability of a mannanase enzyme, whether wild-type, parent, or variant, incubated at a particular pH, e.g., high pH (above pH 7), such as pH 8-8.5, pH 9-9.5, pH 10-10.5, etc., as in detergent compositions. For purposes of the present invention, pH stability may be determined as shown in the Examples.

[0063] Polypeptide or Enzyme: The terms "polypeptide" and "enzyme" may be used interchangeably and refer to a polymer of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein. A polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified, naturally or interveningly; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the scope of this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art.

[0064] Protease stability: The term "protease stability" refers to the stability of a mannanase enzyme, whether wild-type, parent, or variant, incubated in the presence of a protease, e.g., in the presence of a protease in a detergent. For purposes of the present invention, protease stability can be determined as shown in the Examples. Exemplary proteases are those described in detail below.

[0065] Purified: The term "purified" refers to a nucleic acid or polypeptide that is substantially free of other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, a chromatographic eluate, and / or the medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or molar concentration). In a related sense, a composition is enriched with respect to a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or concentration technique. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0066] Recombinant: The term "recombinant" when used with respect to a cell, nucleic acid, protein, or vector means that it has been altered from its natural 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 conditions that are different from those found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides and / or is operably linked to a heterologous sequence, e.g., a heterologous promoter in an expression vector. A recombinant protein may differ from the native sequence by one or more amino acids and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding a polypeptide is a recombinant vector. The terms "recombinant" are synonymous with "genetically modified" and "transgenic."

[0067] Sequence identity: The relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity." For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 6.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical residues × 100) / (length of alignment−total number of gaps in the alignment)

[0068] For the purposes of the present invention, sequence identity between two deoxyribonucleotide acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment−total number of gaps in the alignment).

[0069] Subsequence: The term "subsequence" refers to a polynucleotide having one or more (e.g., several) nucleotides missing from the 5' and / or 3' end of the mature polypeptide coding sequence, wherein the subsequence encodes a fragment that has lysozyme activity.

[0070] Detergent stability: The term "detergent stability" refers to the stability of a mannanase enzyme, whether wild-type, parent, or variant, incubated in the presence of a surfactant, e.g., in a detergent. Exemplary surfactants are described in detail below and in specific embodiments, and detergent stability refers to stability in the presence of an anionic surfactant, such as LAS. For purposes of the present invention, detergent stability may be determined as shown in the Examples.

[0071] Textile: The term "textile" refers to any textile material, including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made from these materials, and products made from fabrics (e.g., garments and other articles). Textiles or fabrics can be in the form of knits, woven fabrics, denim, nonwoven fabrics, felts, yarns, and towels. Textiles can be cellulosic, such as natural cellulose, including cotton, flax / linen, jute, ramie, sisal, or coir, or man-made cellulose (e.g., derived from wood pulp), including viscose / rayon, ramie fibers, tricell, lyocell, or blends thereof. Textiles or fabrics can also be non-cellulosic, such as natural polyamides, such as wool, camel, cashmere, mohair, rabbit, and silk, or synthetic polymers, such as nylon, aramid, polyester, acrylic, polypropylene, and spandex / elastane, or blends thereof, as well as blends of cellulosic and non-cellulosic fibers. Examples of blends are blends of cotton and / or rayon / viscose with one or more companion materials such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and / or cellulose-containing fibers (e.g., rayon / viscose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell). The fabric may be conventional washable laundry, for example, soiled household laundry. When the terms fabric or garment are used, it is intended to include the broader term textiles.

[0072] Thermostability: The term "thermostable" refers to the stability of mannanase enzymes, whether wild-type, parental, or variant, incubated at elevated temperatures. For purposes of the present invention, thermostability can be determined using nDSF (Prometheus, Nanotemper) by measuring the melting temperature (Tm) of the variant in the presence of a detergent (e.g., Model A). Samples are prepared to reach a 10% Model A concentration and a protein concentration of 200 ppm. After brief mixing, the sample is loaded into a capillary and placed in the nDSF sample tray. Samples are analyzed in Tm estimation mode with a temperature range of 25-85°C with a 1°C / min ramp. After analysis is complete, the Tm is estimated in the software using the signal at 350 nm.

[0073] Variant: The term "variant" refers to a polypeptide having mannanase activity that contains a modification, i.e., a substitution, insertion, and / or deletion of one or more (several) amino acid residues at one or more (multiple) positions. A substitution refers to the replacement of an amino acid at a position with a different amino acid; a deletion refers to the removal of an amino acid at a position; and an insertion refers to the addition of 1 to 3 amino acids adjacent to the amino acid at a position. A variant of the present invention has at least 50%, e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the mannanase activity of the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2.

[0074] Wild-type: The term "wild-type" in reference to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modifications of a wild-type sequence).

[0075] Variant Naming Conventions For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid position in another mannanase. The amino acid sequence of the other mannanase is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0076] In describing the variants of the present invention, the following nomenclature is adapted for ease of reference: Recognized IUPAC single-letter or three-letter amino acid abbreviations are utilized.

[0077] Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine with alanine at position 226 is designated "Thr226Ala" or "T226A." Multiple mutations are separated by an additional mark ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent substitutions of arginine (R) with glycine (G) and phenylalanine (F) with serine (S) at positions 205 and 411, respectively.

[0078] Deletions. For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 is designated "Gly195*" or "G195*." Multiple deletions are separated by additional markers ("+"), e.g., "Gly195*+Ser411*" or "G195*+S411*."

[0079] Insertions. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of lysine after glycine at position 195 would be designated "Gly195GlyLys" or "G195GK." Multiple amino acid insertions are designated [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, an insertion of lysine and alanine after glycine at position 195 would be designated "Gly195GlyLysAla" or "G195GKA."

[0080] In such cases, the inserted amino acid residue is numbered by adding a lowercase letter to the position number of the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence would become:

[0081] [Table 1]

[0082] Multiple modifications Variants containing multiple alterations are separated by additional marks ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+Gly195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0083] Different modifications Where different modifications can be introduced at a position, the different changes are separated by commas, for example, "Arg170Tyr,Glu" denotes the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" designates the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention relates to variants of a parent mannanase, said variants comprising at least two substitutions, wherein (a) the first of said substitutions is at a position selected from: 168, 7, 13, 15, 20, 26, 36, 46, 64, 69, 70, 82, 101, 103, 109, 120, 141, 155, 162, 163, 166, 167, 168, 172, 176, 178, 182, 190, 197, 214, 215, 219, 239, 248, 253, 258, 271, 276, 280, 283, 286, 299, 366, 378, 385, 408, 410, 413, 473, 486, 491 (b) at least a second of said substitutions is at any other position of said variant, wherein the variant has mannanase activity, and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to the polypeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or a fragment thereof having a mannanase activity.

[0085] variant The inventors of the present invention have found that modifications at one of the above-listed positions result in mannanase variants with improved stability compared to the parent mannanase, i.e., a mannanase that does not contain a modification at any one of the listed positions. In particular, stability can be observed as stability in detergent compositions, thermal stability, protease stability, detergent stability, and / or pH stability.

[0086] Stability in a detergent composition may also be referred to herein as "stability in detergent" and is subject to definitions set forth elsewhere herein. These terms may be used interchangeably and may have the same meaning and purpose with respect to the present invention. Stability has been determined as described in the Examples.

[0087] Therefore, an improvement in a mannanase variant is observed when the variant has at least one of the positions listed in (a). It has been observed that stability is maintained when the positions listed in (a) are combined with any other modifications. Therefore, these positions are believed to be important for the stability of the mannanase. In particular, stability can be observed as stability in a detergent composition. Such detergent stability can be determined as described in Example 3, i.e., by incubating the variant in a detergent and then measuring the residual activity. The residual activity is related to / compared to the residual activity of the parent mannanase incubated at 4°C for the same time. The improvement in a mannanase variant is given as a half-life improvement factor (HIF).

[0088] Thus, the present invention provides variants of the parent mannanase, wherein the variants are selected from the group consisting of: 168, 7, 13, 15, 20, 26, 36, 46, 64, 69, 70, 82, 101, 103, 109, 120, 141, 155, 162, 163, 166, 167, 168, 172, 176, 178, 182, 190, 197, 214, 215, 219, 23 9, 248, 253, 258, 271, 276, 280, 283, 286, 299, 366, 378, 385, 408, 410, 413, 473, 486, 491 (numbering according to SEQ ID NO: 1), and at least one second (or further) modification at any other position in the amino acid sequence. The term "second modification" or "further modification" as used herein is to be understood as a substitution or deletion in addition to, and not at, any of the following positions: 168, 7, 13, 15, 20, 26, 36, 46, 64, 69, 70, 82, 101, 103, 109, 120, 141, 155, 162, 163, 166, 167, 168, 172, 176, 178, 182, 190, 197, 214, 215, 219, 239, 248, 253, 258, 271, 276, 280, 283, 286, 299, 366, 378, 385, 408, 410, 413, 473, 486, 491. However, variants according to the invention may contain two or more substitutions at any one of the following positions: 168, 7, 13, 15, 20, 26, 36, 46, 64, 69, 70, 82, 101, 103, 109, 120, 141, 155, 162, 163, 166, 167, 168, 172, 176, 178, 182, 190, 197, 214, 215, 219, 239, 248, 253, 258, 271, 276, 280, 283, 286, 299, 366, 378, 385, 408, 410, 413, 473, 486, 491. Furthermore, the term "secondary substitution" is to be understood as "at least one secondary substitution", ie the secondary substitution may be at one or more positions.

[0089] As can be seen from the examples, all disclosed variants have improved stability, especially when tested under stress, which according to the present invention can be increased by changing (i.e. increasing pH), prolonged / extended incubation, temperature change (i.e. increasing) and / or the presence of proteases.

[0090] It should be understood that a variant of the invention contains at least two alterations, such as substitutions, meaning that the variant may contain a substitution at one of the positions listed in (a) and then a second / further substitution at any other position disclosed herein. However, it should also be understood that a variant of the invention can be a variant that contains substitutions at more than one of the positions listed in (a), i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all 13 of these positions.

[0091] The present invention also provides a mannanase having multiple modifications, e.g., at least 19 mutations, such as at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 mutations compared to a parent mannanase. and wherein the resulting variant has at least 85% but less than 96%, e.g., 86-96%, 87-96%, 88-96%, 89-96%, 90-96%, 91-96%, 92-96%, 93-96%, 94-96%, e.g., 95-96%, sequence identity with the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, or a fragment thereof comprising a mannanase; or at least 85% but less than 95%, e.g., 86-95%, 87-95%, 88-95%, 89-95%, 90-95%, 91-95%, 92-95%, 93-95%, 94-95%, e.g., 95-95%, or at least 85% but less than 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity; or at least 85% but less than 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity. or at least 85% but less than 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, 90 to 92%, for example, 91 to 92% sequence identity; or at least 85% but less than 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, 89 to 91%, for example, 90 to 91% sequence identity; or at least 85% but less than 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0092] In another embodiment, the variant comprises or consists of a deletion or substitution at a position corresponding to position 7 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 7*, 7A, 7D, 7G, 7I, or 7P. In certain embodiments, the deletion or substitution is F7*, F7A, F7D, F7G, F7I, or F7P, preferably F7G.

[0093] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 13 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 13I, 13M, 13N, 13W, 13Y, or 13E. In certain embodiments, the substitution is T13I, T13M, T13N, T13W, T13Y, or T13E, preferably T13I.

[0094] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 15 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 15K, 15C, 15D, 15M, 15Q, or 15T. In certain embodiments, the substitution is S15K, S15C, S15D, S15M, S15Q, or S15T, preferably S15Q.

[0095] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 20 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 20S, 20P, 20T, 20G, or 20V. In certain embodiments, the substitution is A20S, A20P, A20T, A20G, or A20V, preferably A20P.

[0096] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 26 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 26D, 26E, or 26H. In certain embodiments, the substitution is K26D, K26E, or K26H, preferably K26E.

[0097] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 36 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 36A, 36C, 36D, 36E, 36M, or 36T. In certain embodiments, the substitution is K36A, K36C, K36D, K36E, K36M, or K36T, preferably K36E.

[0098] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 46 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 46I, 46F, 46M, or 46Y. In certain embodiments, the substitution is E46I, E46F, E46M, or E46Y, preferably E46M or E46Y.

[0099] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 64 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further wherein the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 64P. In some embodiments, the substitution is E64P.

[0100] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 69 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 69P, 69R, 69S, or 69T. In certain embodiments, the substitution is Q69P, Q69R, Q69S, or Q69T, preferably Q69P.

[0101] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 70 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 70A, 70E, 70I, 70M, 70P, or 70T. In certain embodiments, the substitution is K70A, K70E, K70I, K70M, K70P, or K70T, preferably K70P.

[0102] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 82 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 82T, 82A. In some embodiments, the substitution is N82T, N82A.

[0103] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 101 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 101R, 101C, 101E, 101I, 101L, 101N, 101Q, or 101V. In one embodiment the substitution is A101R, A101C, A101E, A101I, A101L, A101N, A101Q, A101V, preferably A101R or A101L.

[0104] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 103 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 103R, 103A, 103C, 103D, 103E, 103I, 103L, 103N, 103Q, 103V, or 103Y. In one embodiment, the substitution is S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103N, S103Q, S103V, S103Y, preferably S103V.

[0105] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 109 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 109E, 109D. In some embodiments, the substitution is S109E, S109D.

[0106] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 120 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 120S, 120A, 120G, 120I, or 120P. In certain embodiments, the substitution is K120S, K120A, K120G, K120I, or K120P, preferably K120A or K120P.

[0107] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 141 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 141R, 141I, 141Q, or 141A. In certain embodiments, the substitution is S141R, S141I, S141Q, or S141A, preferably 141R.

[0108] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 155 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 155H. In some embodiments, the substitution is Y155H.

[0109] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 162 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 162D, 162G, or 162Q. In certain embodiments, the substitution is E162D, E162G, or E162Q, preferably E162D.

[0110] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 163 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 163*, 163A, 163E, 163T, or 163N. In certain embodiments, the substitution is P163*, P163T, P163A, P163E, or P163N, preferably P163A.

[0111] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 166 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 166E, 166G, 166I, 166L, 166N, 166P, 166Q, 166R, 166V, or 166W. In certain embodiments, the substitution is T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R, T166V, T166W, preferably T166P.

[0112] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 167 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 167P, 167A, 167R, or 167K. In certain embodiments, the substitution is T167P, T167A, T167R, or T167K, preferably T167P.

[0113] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 168 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 168D, 168F, 168M, or 168P. In certain embodiments, the substitution is Q168D, Q168F, Q168M, or Q168P, preferably 168D.

[0114] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 172 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 172I. In certain embodiments, the substitution is M172I.

[0115] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 176 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 176N. In some embodiments, the substitution is S176N.

[0116] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 178 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 178R, 178E, 178H, 178P, or 178T. In certain embodiments, the substitution is S178R, S178E, S178H, S178P, or S178T, preferably S178P.

[0117] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 182 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 182R, 182E. In some embodiments, the substitution is P182R, P182E.

[0118] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 190 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further wherein the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 190Y. In some embodiments, the substitution is F190Y.

[0119] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 197 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 197A, 197D, 197F, 197M, 197N, or 197Y. In certain embodiments, the substitution is K197A, K197D, K197F, K197M, K197N, or K197Y, preferably K197D.

[0120] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 214 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 214R or 214W. In certain embodiments, the substitution is Q214R or Q214W, preferably Q214R.

[0121] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 215 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 215E. In some embodiments, the substitution is Q215E.

[0122] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 219 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further wherein the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 219W. In some embodiments, the substitution is Y219W.

[0123] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 239 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 239A. In some embodiments, the substitution is S239A.

[0124] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 248 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 248I. In certain embodiments, the substitution is M248I.

[0125] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 253 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 253A or 253W. In certain embodiments, the substitution is R253A or R253W, preferably R253A.

[0126] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 258 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 258G. In some embodiments, the substitution is S258G.

[0127] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 271 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 271G or 271Y. In certain embodiments, the substitution is S271G or S271Y, preferably S271G.

[0128] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 276 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 276C, 276N, or 276Y. In certain embodiments, the substitution is H276C, H276N, or H276Y, preferably H276C.

[0129] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 280 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 280L or 280K. In certain embodiments, the substitution is R280L or R280K, preferably R280K.

[0130] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 283 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 283M or 283Y. In some embodiments, the substitution is N283M or N283Y.

[0131] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 286 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further wherein the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 285C. In some embodiments, the substitution is F286C.

[0132] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 299 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 299K, 299D, or 299W. In certain embodiments, the substitution is E299K, E299D, or E299W, preferably E299W.

[0133] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 366 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 366N, 366H, or 366I. In certain embodiments, the substitution is G366N, G366H, or G366I, preferably G366N.

[0134] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 378 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 378D, 378H, 378Q, or 378R. In certain embodiments, the substitution is K378D, K378H, K378Q, or K378R, preferably K378Q.

[0135] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 385 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 385A, 385F, 385H, 385P, or 385S. In certain embodiments, the substitution is D385A, D385F, D385H, D385P, or D385S, preferably D385H.

[0136] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 408 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 408A, 408C, 408D, 408E, 408Q, 408S, 408T, or 408V. In certain embodiments, the substitution is K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K408V, preferably K408M.

[0137] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 410 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 410L or 410M. In certain embodiments, the substitution is D410L or D410M, preferably D410M.

[0138] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 413 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 413Q, 413A, 413T, or 413V. In certain embodiments, the substitution is L413Q, L413A, L413T, or L413V, preferably L413T.

[0139] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 473 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further, the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 473Y. In some embodiments, the substitution is N473Y.

[0140] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 486 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 486E, 486K, or 486V. In certain embodiments, the substitution is D486E, D486K, or D486V, preferably D486E.

[0141] In another embodiment, the variant comprises or consists of a substitution at a position corresponding to position 491 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 491E, 491P, or 491R. In certain embodiments, the substitution is K491E, K491P, or K491R, preferably K491R.

[0142] In one embodiment, the modification is a substitution.

[0143] In one embodiment, the modification is a deletion.

[0144] In one embodiment, the modification is a substitution, wherein said substitution of a naturally occurring amino acid residue at one position with a different amino acid residue produces a mannanase variant having an improvement factor of >1.0 with respect to the stability measure.

[0145] When the improvement factor (IF) is greater than 1.0, it indicates that the tested variant has an improved property, such as improved stability, compared to a reference enzyme, e.g., a parent or wild-type mannanase.

[0146] In one embodiment, the improved stability is stability in detergent or thermal stability.

[0147] Unless specifically disclosed herein, numbering of amino acid residues or positions is done according to SEQ ID NO:1.

[0148] In some embodiments, the variant has at least 85%, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence of the parent mannanase.

[0149] In another embodiment, the variant has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or the polypeptide of SEQ ID NO: 2.

[0150] In another embodiment, the variant has at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4, i.e., a truncated mannanase lacking a CBM.

[0151] As mentioned above, stability, such as stability in detergent or thermal stability, may be determined as described in the Examples. In particular, stability may be determined at a pH of 8 to 8.5.

[0152] Thus, in one embodiment, the variants of the present disclosure have improved stability when measured at pH 8 to 8.5.

[0153] Stability may be measured at pH 8-8.5, but may also be measured at either alkaline or acidic pH. The pH at which stability is measured may vary depending on the detergent composition in which the variant (or enzyme) needs to be stable. Thus, the present invention also encompasses stability at other pHs, such as pH 10-11, e.g., pH 10.5. Thus, in one embodiment, the variants of the present disclosure have improved stability when measured at pH 10-11.

[0154] In one embodiment, the improved stability of the variants of the invention is at least 1.0 on the stability scale at pH 8-8.5 and / or pH 10-11.

[0155] In one embodiment, the improved stability of the variants of the invention is measured at either pH 8-8.5 or 10-11, or improved stability is seen at both pHs, i.e., pH 8 and pH 10.8. Thus, in one embodiment, the variants have improved stability compared to the parent mannanase when stability is measured at pH 8-8.5 and / or pH 10-11.

[0156] In one embodiment the variant has an improvement factor of at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 2.0, at least 2.5, at least 3.0 on the stability scale at pH 8 and / or pH 10.5.

[0157] Amino acid changes can be minor, being conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions such as polyhistidine tracts, antigenic epitopes, or binding domains that facilitate purification by altering net charge or another function.

[0158] Examples of conservative substitutions are included in the following groups: 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 the specific activity are known in the art and are described, for example, by H. Neurath and R.L. 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.

[0159] Alternatively, the amino acid changes may be of such a nature that the physicochemical properties of the polypeptide are altered, for example, the amino acid changes may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0160] Essential amino acids in a polypeptide can be identified using art-known procedures such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). The latter technique involves introducing a single alanine mutation at every residue in the molecule, and the resulting mutant molecules are tested for mannanase activity to identify amino acid residues critical to the molecule's activity. See Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0161] In one embodiment, the variant comprises a second modification at one or more of the following positions compared to the parent mannanase: 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 14, 17, 18, 19, 21, 22, 23, 25, 30, 35, 37, 38, 42, 45, 48, 49, 53, 57, 61, 63, 65, 68, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 72, 74, 76, 78, 83, 86, 89, 94, 96, 97, 100, 105, 110, 111, 112, 115, 117, 118, 121, 122, 123, 126, 129, 130, 134, 137, 139, 142, 143, 148, 160, 161, 162, 164, 165, 169, 170, 171, 174, 179, 181, 183, 184, 185, 188, 189, 192, 193, 195, 198, 210, 213, 217, 218, 228, 235, 242, 249, 252, 254, 259, 260, 267, 270, 272, 296, 301, 304, 312, 315, 321, 322, 323, 324, 342, 344, 347, 351, 352, 353 4, 355, 358, 363, 368, 377, 379, 380, 391, 398, 403, 414, 417, 419, 422, 423, 437, 438, 440, 442, 443, 452, 454, 462, 463, 466, 467, 470, 472, 477, 479, 480, 482, 483, 484, 487, 488, 489, 490.

[0162] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 1 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and further wherein the variant has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 1*, 1S, 1T, 1G, 1D, 1I, 1V, or 1Y. In some embodiments, the deletion or substitution is A1*, A1S, A1T, A1V, A1G, A1D, A1I, or A1Y.

[0163] In another embodiment, the second modification is a deletion at a position corresponding to position 2 of the polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 2*. In some embodiments, the deletion or substitution is 12*.

[0164] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 3 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 3*, 3A, 3E, or 3G. In some embodiments, the deletion or substitution is T3*, T3A, T3E, or T3G.

[0165] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 4 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 4*, 4D, 4N, 4G, 4M, or 4Y. In some embodiments, the deletion or substitution is V4*, V4D, V4N, V4G, V4M, or V4Y.

[0166] In another embodiment, the second modification is a deletion at a position corresponding to position 5 of the polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion is 5*. In some embodiments, the deletion is P5*.

[0167] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 6 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 6*, 6P. In some embodiments, the deletion or substitution is G6*, G6P.

[0168] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 8 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 8*, 8A, 8Q, 8F, 8D, or 8W. In some embodiments, the deletion or substitution is V8*, V8A, V8Q, V8F, V8D, or V8W.

[0169] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 9 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 9*, 9A, 9D, 9G, 9M, or 9P. In some embodiments, the deletion or substitution is V9*, V9D, V9A, V9G, V9M, or V9P.

[0170] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 10 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 10**, 10Y, or 10G. In certain embodiments, the deletion or substitution is E10*, E10Y, or E10G.

[0171] In another embodiment, the second modification is a substitution at a position corresponding to position 11 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 11A, 11L, 11E, 11I, or 11N. In some embodiments, the substitution is P11A, P11L, P11E, P11I, or P11N.

[0172] In another embodiment, the second modification is a substitution at a position corresponding to position 12 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 12A, 12D, 12E, 12G, 12L, 12P, 12Q, 12R, or 12V. In certain embodiments, the substitution is H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R, or H12V.

[0173] In another embodiment, the second modification is a substitution at a position corresponding to position 14 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 14Q, 14R, 14E, 14H, 14N, or 14V. In certain embodiments, the substitution is S14Q, S14R, S14E, S14H, S14N, or S14V.

[0174] In another embodiment, the second modification is a substitution at position 17 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 17K, 17A. In some embodiments, the substitution is Q17K, Q17A.

[0175] In another embodiment, the second modification is a substitution at position 18 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 18S. In some embodiments, the substitution is N18S.

[0176] In another embodiment, the second modification is a substitution at a position corresponding to position 19 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 19N. In some embodiments, the substitution is Q19N.

[0177] In another embodiment, the second modification is a substitution at a position corresponding to position 21 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 21L. In some embodiments, the substitution is I21L.

[0178] In another embodiment, the second modification is a substitution at a position corresponding to position 22 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 22V, 22Q, or 22T. In certain embodiments, the substitution is I22V, I22Q, or I22T.

[0179] In another embodiment, the second modification is a substitution at a position corresponding to position 23 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 23I, 23T, 23V, 23M, or 23S. In some embodiments, the substitution is A23I, A23T, A23V, A23M, or A23S.

[0180] In another embodiment, the second modification is a substitution at position 25 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 25W, 25Y. In some embodiments, the substitution is F25W, F25Y.

[0181] In another embodiment, the second modification is a substitution at a position corresponding to position 30 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 30L. In some embodiments, the substitution is I30L.

[0182] In another embodiment, the second modification is a deletion at a position corresponding to position 35 of the polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion is 35*. In some embodiments, the deletion is I35*.

[0183] In another embodiment, the second modification is a substitution at a position corresponding to position 37 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 37V. In some embodiments, the substitution is K37V.

[0184] In another embodiment, the second modification is a substitution at a position corresponding to position 38 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 38C, 38F, 38L, 38M, or 38T. In certain embodiments, the substitution is R38C, R38F, R38L, R38M, or R38T.

[0185] In another embodiment, the second modification is a substitution at a position corresponding to position 42 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 42E. In some embodiments, the substitution is T42E.

[0186] In another embodiment, the second modification is a substitution at a position corresponding to position 45 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 45M, 45L, or 45R. In some embodiments, the substitution is A45L, A45M, or A45R.

[0187] In another embodiment, the second modification is a substitution at a position corresponding to position 48 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 48A, 48P, or 48S. In some embodiments, the substitution is D48A, D48P, or D48S.

[0188] In another embodiment, the second modification is a substitution at a position corresponding to position 49 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 49M. In some embodiments, the substitution is L49M.

[0189] In another embodiment, the second modification is a substitution at a position corresponding to position 53 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 53C, 53E, 53H, 53K, 53P, or 53Q. In certain embodiments, the substitution is T53C, T53E, T53H, T53K, T53P, or T53Q.

[0190] In another embodiment, the second modification is a substitution at a position corresponding to position 57 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 57P, 57R. In some embodiments, the substitution is S57P, S57R.

[0191] In another embodiment, the second modification is a substitution at a position corresponding to position 61 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 61D. In some embodiments, the substitution is E61D.

[0192] In another embodiment, the second modification is a substitution at a position corresponding to position 63 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 63C. In some embodiments, the substitution is D63C.

[0193] In another embodiment, the second modification is a substitution at a position corresponding to position 65 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 65C, 65F, 65H, 65N, 65Q, 65S, or 65T. In some embodiments, the substitution is K65C, K65F, K65H, K65N, K65Q, K65S, or K65T.

[0194] In another embodiment, the second modification is a substitution at a position corresponding to position 68 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 68M, 68T. In some embodiments, the substitution is V68M, V68T.

[0195] In another embodiment, the second modification is a substitution at a position corresponding to position 69 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 69P, 69R, 69S, or 69T. In certain embodiments, the substitution is Q69P, Q69R, Q69S, or Q69T.

[0196] In another embodiment, the second modification is a substitution at a position corresponding to position 72 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 72F. In some embodiments, the substitution is S72F.

[0197] In another embodiment, the second modification is a substitution at a position corresponding to position 74 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 74A, 74Q, or 74E. In certain embodiments, the substitution is T74A, T74Q, or T74E.

[0198] In another embodiment, the second modification is a substitution at a position corresponding to position 76 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 76T or 76E. In some embodiments, the substitution is Q76T or Q76E.

[0199] In another embodiment, the second modification is a substitution at a position corresponding to position 83 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 83E or 83S. In some embodiments, the substitution is G83E or G83S.

[0200] In another embodiment, the second modification is a substitution at a position corresponding to position 86 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 86D. In some embodiments, the substitution is E86D.

[0201] In another embodiment, the second modification is a substitution at a position corresponding to position 89 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 89F. In some embodiments, the substitution is L89F.

[0202] In another embodiment, the second modification is a substitution at a position corresponding to position 94 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 94M. In some embodiments, the substitution is P94M.

[0203] In another embodiment, the second modification is a substitution at a position corresponding to position 96 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 97E, 97D, 96E, 96D, or 96R. In some embodiments, the substitution is G96E, G96D, or G96R.

[0204] In another embodiment, the second modification is a substitution at a position corresponding to position 97 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 97Y. In certain embodiments, the substitution is N97E, N97D, or N97Y.

[0205] In another embodiment, the second modification is a substitution at a position corresponding to position 100 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 100R. In some embodiments, the substitution is K100R.

[0206] In another embodiment, the second modification is a substitution at a position corresponding to position 105 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 105V. In some embodiments, the substitution is S105V.

[0207] In another embodiment, the second modification is a substitution at a position corresponding to position 110 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 110R, 110D, 110P, 110C, or 110S. In some embodiments, the substitution is G110R, G110D, G110P, G110C, or G110S.

[0208] In another embodiment, the second modification is a substitution at a position corresponding to position 111 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 111L. In some embodiments, the substitution is A111L.

[0209] In another embodiment, the second modification is a substitution at a position corresponding to position 112 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 112V. In some embodiments, the substitution is G112V.

[0210] In another embodiment, the second modification is a substitution at a position corresponding to position 115 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 115W, 115Y, 115V, 115I, or 115F. In certain embodiments, the substitution is T115W, T115Y, T115V, T115I, or T115F.

[0211] In another embodiment, the second modification is a substitution at a position corresponding to position 117 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 117P. In some embodiments, the substitution is S117P.

[0212] In another embodiment, the second modification is a substitution at a position corresponding to position 118 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 118V. In some embodiments, the substitution is A118V.

[0213] In another embodiment, the second modification is a substitution at a position corresponding to position 121 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 121A. In some embodiments, the substitution is P121A.

[0214] In another embodiment, the second modification is a substitution at a position corresponding to position 122 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 122P. In some embodiments, the substitution is G122P.

[0215] In another embodiment, the second modification is a substitution at a position corresponding to position 123 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 123R. In some embodiments, the substitution is T123R.

[0216] In another embodiment, the second modification is a substitution at a position corresponding to position 126 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 126D, 126S, or 126T. In some embodiments, the substitution is A126D, A126S, or A126T.

[0217] In another embodiment, the second modification is a substitution at a position corresponding to position 129 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 129R, 129L, or 129M. In certain embodiments, the substitution is K129R, K129L, or K129M.

[0218] In another embodiment, the second modification is a substitution at a position corresponding to position 130 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 130I, 130V. In some embodiments, the substitution is M130I, M130V.

[0219] In another embodiment, the second modification is a substitution at a position corresponding to position 134 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 134I, 134V, 134P, or 134T. In some embodiments, the substitution is L134I, L134V, L134P, or L134T.

[0220] In another embodiment, the second modification is a substitution at a position corresponding to position 137 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 137A, 137D, or 137W. In some embodiments, the substitution is S137A, S137D, or S137W.

[0221] In another embodiment, the second modification is a substitution at a position corresponding to position 139 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 139I, 139K, or 139A. In some embodiments, the substitution is N139I, N139K, or N139A.

[0222] In another embodiment, the second modification is a substitution at a position corresponding to position 143 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 143T, 143S, or 143R. In certain embodiments, the substitution is Q143T, Q143S, or Q143R.

[0223] In another embodiment, the second modification is a substitution at a position corresponding to position 155 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 155H. In some embodiments, the substitution is Y155H.

[0224] In another embodiment, the second modification is a substitution at a position corresponding to position 160 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 160I, 160F, 160K, 160L, 160N, or 160V. In certain embodiments, the substitution is R160I, R160F, R160K, R160L, R160N, or R160V.

[0225] In another embodiment, the second modification is a substitution at a position corresponding to position 161 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 161S, 161T. In some embodiments, the substitution is V161S, V161T.

[0226] In another embodiment, the second modification is a deletion or substitution at a position corresponding to position 164 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the deletion or substitution is 164**, R164Q. In some embodiments, the deletion is R164**, R164Q.

[0227] In another embodiment, the second modification is a substitution at a position corresponding to position 165 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 165E, 165D, 165T, or 165L. In certain embodiments, the substitution is I165E, I165D, I165T, or I165L.

[0228] In another embodiment, the second modification is a substitution at a position corresponding to position 169 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 169P, 169Q, 169T, or 169V. In certain embodiments, the substitution is K169P, K169Q, K169T, or K169V.

[0229] In another embodiment, the second modification is a substitution at a position corresponding to position 170 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 170E, 170H. In some embodiments, the substitution is T170E, T170H.

[0230] In another embodiment, the second modification is a substitution at a position corresponding to position 171 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 171E, 171N, 171I, 171K, or 171Q. In certain embodiments, the substitution is M171E, M171I, M171K, M171N, or M171Q.

[0231] In another embodiment, the second modification is a substitution at a position corresponding to position 174 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 174K. In some embodiments, the substitution is P174K.

[0232] In another embodiment, the second modification is a substitution at a position corresponding to position 179 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 179S, 179H, 179Q, 179N, or 179R. In certain embodiments, the substitution is K179S, K179H, K179Q, K179N, or K179R.

[0233] In another embodiment, the second modification is a substitution at a position corresponding to position 181 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 181T. In some embodiments, the substitution is S181T.

[0234] In another embodiment, the second modification is a substitution at a position corresponding to position 183 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 183E. In some embodiments, the substitution is Q183E.

[0235] In another embodiment, the second modification is a substitution at a position corresponding to position 184 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 184T. In some embodiments, the substitution is A184T.

[0236] In another embodiment, the second modification is a substitution at a position corresponding to position 185 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 184N, 185Q. In some embodiments, the substitution is K185N, K185Q.

[0237] In another embodiment, the second modification is a substitution at a position corresponding to position 188 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 188F, 188I, or 188Y. In certain embodiments, the substitution is M188F, M188I, or M188Y.

[0238] In another embodiment, the second modification is a substitution at a position corresponding to position 189 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 189S. In some embodiments, the substitution is K189S.

[0239] In another embodiment, the second modification is a substitution at a position corresponding to position 192 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 192A, 192V. In some embodiments, the substitution is T192A, T192V.

[0240] In another embodiment, the second modification is a substitution at a position corresponding to position 193 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 193W, 193S. In some embodiments, the substitution is N193W, N193S.

[0241] In another embodiment, the second modification is a substitution at a position corresponding to position 195 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 195H. In some embodiments, the substitution is S195H.

[0242] In another embodiment, the second modification is a substitution at a position corresponding to position 197 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 197A, 197D, 197F, 197M, 197N, or 197Y. In certain embodiments, the substitution is K197A, K197D, K197F, K197M, K197N, or K197Y.

[0243] In another embodiment, the second modification is a substitution at a position corresponding to position 210 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 210E, 210F, 210T, 210Q, or 210S. In some embodiments, the substitution is A210E, A210Q, A210F, A210T, or A210S.

[0244] In another embodiment, the second modification is a substitution at a position corresponding to position 213 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 213K, 213Q, or 213R. In certain embodiments, the substitution is Y213K, Y213Q, or Y213R.

[0245] In another embodiment, the second modification is a substitution at a position corresponding to position 217 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 217Q. In some embodiments, the substitution is G217Q.

[0246] In another embodiment, the second modification is a substitution at a position corresponding to position 218 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 218R. In some embodiments, the substitution is K218R.

[0247] In another embodiment, the second modification is a substitution at a position corresponding to position 228 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 228I. In some embodiments, the substitution is M228I.

[0248] In another embodiment, the second modification is a substitution at a position corresponding to position 235 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 235L. In some embodiments, the substitution is V235L.

[0249] In another embodiment, the second modification is a substitution at a position corresponding to position 242 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 242T. In some embodiments, the substitution is N242T.

[0250] In another embodiment, the second modification is a substitution at a position corresponding to position 249 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 249H, 249L, or 249Q. In certain embodiments, the substitution is E249H, E249L, or E249Q.

[0251] In another embodiment, the second modification is a substitution at a position corresponding to position 252 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 252G or 252E. In some embodiments, the substitution is K252G or K252E.

[0252] In another embodiment, the second modification is a substitution at a position corresponding to position 254 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 254S. In some embodiments, the substitution is G254S.

[0253] In another embodiment, the second modification is a substitution at a position corresponding to position 259 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 259V. In some embodiments, the substitution is L259V.

[0254] In another embodiment, the second modification is a substitution at a position corresponding to position 260 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 260C. In some embodiments, the substitution is S260C.

[0255] In another embodiment, the second modification is a substitution at a position corresponding to position 267 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 267L. In some embodiments, the substitution is K267L.

[0256] In another embodiment, the second modification is a substitution at a position corresponding to position 270 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 270C. In some embodiments, the substitution is G270C.

[0257] In another embodiment, the second modification is a substitution at a position corresponding to position 272 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 272D. In some embodiments, the substitution is N272D.

[0258] In another embodiment, the second modification is a substitution at a position corresponding to position 285 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 285D. In some embodiments, the substitution is E285D.

[0259] In another embodiment, the second modification is a substitution at a position corresponding to position 296 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 296A. In some embodiments, the substitution is N296A.

[0260] In another embodiment, the second modification is a substitution at a position corresponding to position 301 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 301K. In some embodiments, the substitution is E301K.

[0261] In another embodiment, the second modification is a substitution at a position corresponding to position 304 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 304A, 304F, 304Q, 304R, or 304S. In some embodiments, the substitution is K304A, K304F, K304Q, K304R, or K304S.

[0262] In another embodiment, the second modification is a substitution at a position corresponding to position 312 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 312V. In some embodiments, the substitution is A312V.

[0263] In another embodiment, the second modification is a substitution at a position corresponding to position 315 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 315A, 315V. In some embodiments, the substitution is T315A, T315V.

[0264] In another embodiment, the second modification is a substitution at a position corresponding to position 321 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 321K, 321W. In some embodiments, the substitution is Q321K, Q321W.

[0265] In another embodiment, the second modification is a substitution at a position corresponding to position 322 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 322R, 322A, or 322D. In some embodiments, the substitution is E322R, E322A, or E322D.

[0266] In another embodiment, the second modification is a substitution at a position corresponding to position 323 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 323A. In some embodiments, the substitution is H323A.

[0267] In another embodiment, the second modification is a substitution at a position corresponding to position 324 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 324K or 324N. In some embodiments, the substitution is H324N.

[0268] In another embodiment, the second modification is a substitution at a position corresponding to position 342 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 342G. In some embodiments, the substitution is W342G.

[0269] In another embodiment, the second modification is a substitution at a position corresponding to position 344 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 344C. In some embodiments, the substitution is A344C.

[0270] In another embodiment, the second modification is a substitution at a position corresponding to position 347 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 347D. In some embodiments, the substitution is P347D.

[0271] In another embodiment, the second modification is a substitution at a position corresponding to position 351 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 351V. In some embodiments, the substitution is K351V.

[0272] In another embodiment, the second modification is a substitution at a position corresponding to position 352 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 352Q. In some embodiments, the substitution is K352Q.

[0273] In another embodiment, the second modification is a substitution at a position corresponding to position 354 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 354W. In some embodiments, the substitution is Y354W.

[0274] In another embodiment, the second modification is a substitution at a position corresponding to position 355 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 355Q. In some embodiments, the substitution is R355Q.

[0275] In another embodiment, the second modification is a substitution at a position corresponding to position 358 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 358W. In some embodiments, the substitution is Y358W.

[0276] In another embodiment, the second modification is a substitution at a position corresponding to position 363 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 363H. In some embodiments, the substitution is E363H.

[0277] In another embodiment, the second modification is a substitution at a position corresponding to position 364 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 364D, 364R, or 364Y. In some embodiments, the substitution is K364D, K364R, or K364Y.

[0278] In another embodiment, the second modification is a substitution at a position corresponding to position 368 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 368D. In some embodiments, the substitution is N368D.

[0279] In another embodiment, the second modification is a substitution at a position corresponding to position 377 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 377E. In some embodiments, the substitution is V377E.

[0280] In another embodiment, the second modification is a substitution at a position corresponding to position 379 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 379K or 379P. In some embodiments, the substitution is E379K or E379P.

[0281] In another embodiment, the second modification is a substitution at a position corresponding to position 380 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 380T or 380D. In some embodiments, the substitution is E380T or E380D.

[0282] In another embodiment, the second modification is a substitution at a position corresponding to position 391 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 391L. In some embodiments, the substitution is V391L.

[0283] In another embodiment, the second modification is a substitution at position 398 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 398F or 398E. In some embodiments, the substitution is P398F or P398E.

[0284] In another embodiment, the second modification is a substitution at a position corresponding to position 403 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 403Q, 403R. In some embodiments, the substitution is S403Q, S403R.

[0285] In another embodiment, the second modification is a substitution at a position corresponding to position 414 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 414T, 414S, or 414P. In some embodiments, the substitution is F414T, F414S, or F414P.

[0286] In another embodiment, the second modification is a substitution at a position corresponding to position 417 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 417G, 417E, or 417Q. In certain embodiments, the substitution is K417G, K417E, or K417Q.

[0287] In another embodiment, the second modification is a substitution at a position corresponding to position 419 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 419V. In some embodiments, the substitution is K419V.

[0288] In another embodiment, the second modification is a substitution at a position corresponding to position 422 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 422R, 422I. In some embodiments, the substitution is V422R, V422I.

[0289] In another embodiment, the second modification is a substitution at a position corresponding to position 423 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 423G. In some embodiments, the substitution is A423G.

[0290] In another embodiment, the second modification is a substitution at a position corresponding to position 437 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 437I, 437A. In some embodiments, the substitution is Q437I, Q437A.

[0291] In another embodiment, the second modification is a substitution at a position corresponding to position 438 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 438A. In some embodiments, the substitution is T438A.

[0292] In another embodiment, the second modification is a substitution at a position corresponding to position 440 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 440Q. In some embodiments, the substitution is G440Q.

[0293] In another embodiment, the second modification is a substitution at a position corresponding to position 442 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 442N. In some embodiments, the substitution is H442N.

[0294] In another embodiment, the second modification is a substitution at a position corresponding to position 443 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 443G. In some embodiments, the substitution is W443G.

[0295] In another embodiment, the second modification is a substitution at a position corresponding to position 452 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 452N. In some embodiments, the substitution is D452N.

[0296] In another embodiment, the second modification is a substitution at a position corresponding to position 454 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 454I. In some embodiments, the substitution is L454I.

[0297] In another embodiment, the second modification is a substitution at a position corresponding to position 462 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 462A, 462R, 462L, or 462P. In certain embodiments, the substitution is K462A, K462R, K462L, or K462P.

[0298] In another embodiment, the second modification is a substitution at a position corresponding to position 463 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 463Q. In some embodiments, the substitution is E463Q.

[0299] In another embodiment, the second modification is a substitution at a position corresponding to position 466 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 466F, 466H, 466N, or 466Q. In certain embodiments, the substitution is K466F, K466H, K466N, or K466Q.

[0300] In another embodiment, the second modification is a substitution at a position corresponding to position 467 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 467H or 467Y. In some embodiments, the substitution is K467H or K467Y.

[0301] In another embodiment, the second modification is a substitution at a position corresponding to position 470 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 470Y. In some embodiments, the substitution is N470Y.

[0302] In another embodiment, the second modification is a substitution at a position corresponding to position 472 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 472G, 472Y. In some embodiments, the substitution is D472G, D472Y.

[0303] In another embodiment, the second modification is a substitution at a position corresponding to position 477 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 477F. In some embodiments, the substitution is L477F.

[0304] In another embodiment, the second modification is a substitution at a position corresponding to position 479 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 479R. In some embodiments, the substitution is E479R.

[0305] In another embodiment, the second modification is a substitution at a position corresponding to position 480 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 480I. In some embodiments, the substitution is L480I.

[0306] In another embodiment, the second modification is a substitution at a position corresponding to position 482 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 482A, 482P, 482D, 482G, 482Q, or 482R. In certain embodiments, the substitution is K482A, K482P, K482D, K482G, K482Q, or K482R.

[0307] In another embodiment, the second modification is a substitution at a position corresponding to position 483 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, and the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 483A or 483D. In some embodiments, the substitution is G483A or G483D.

[0308] In another embodiment, the second modification is a substitution at a position corresponding to position 484 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 484Q. In some embodiments, the substitution is L484Q.

[0309] In another embodiment, the second modification is a substitution at a position corresponding to position 487 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 487R, 487G. In some embodiments, the substitution is S487R, S487G.

[0310] In another embodiment, the second modification is a substitution at a position corresponding to position 488 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 488I, 488L. In some embodiments, the substitution is P488I, P488L.

[0311] In another embodiment, the second modification is a substitution at a position corresponding to position 489 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 489F, 489K, 489N, or 489P. In certain embodiments, the substitution is R489K, R489F, R489N, or R489P.

[0312] In another embodiment, the second modification is a substitution at a position corresponding to position 490 of a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a polypeptide of SEQ ID NO: 1 having mannanase activity, wherein the variant further has improved stability compared to the mannanase of SEQ ID NO: 1. In one embodiment, the substitution is 490A. In some embodiments, the substitution is W490A.

[0313] In another aspect, the variants are at the positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E, K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S10 3I, S103L, S103N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P163A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R, T166V, T166 W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S178H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K 197M, K197N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S271G, S271Y, H276C, H276N, H276Y, R280L , R280K, N283M, N283Y, F286C, E299K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K4 08A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K408V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K,and a second modification is a substitution or deletion, and is selected from the group consisting of: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9 A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H1 2R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0314] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0315] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0316] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0317] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0318] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0319] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0320] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, and is at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V 9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R , H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0321] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0322] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0323] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0324] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0325] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0326] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0327] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0328] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0329] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0330] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0331] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12 R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R , D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65 F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E8 6D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T1 15Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K12 9M, M130I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143 T, Q143S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E,I165D, I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K 179R, S181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S , T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A21 0S, Y213K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E 249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D , E285D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T31 5A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A 344C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P3 98E, S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452 N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A.

[0332] In another aspect, the variants are at positions: Q168D, Q168F, Q168M, Q168P, F7*, F7A, F7D, F7G, F7I, F7P, T13I, T13M, T13N, T13W, T13Y, T13E, S15K, S15C, S15D, S15M, S15Q, S15T, A20S, A20P, A20T, A20G, A20V, K26D, K26E, K26H, K36A, K36C, K36D, K36E, K36M, K36T, E46I, E46F, E46M, E46Y, E64P, Q69P, Q69R, Q69S, Q69T, K70A, K70E , K70I, K70M, K70P, K70T, N82T, N82A, A101R, A101C, A101E, A101I, A101L, A 101N, A101Q, A101V, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103 N, S103Q, S103V, S103Y, S109E, S109D, K120S, K120A, K120G, K120I, K120P, S141R, S141I, S141Q, S141A, Y155H, E162D, E162G, E162Q, P163*, P163T, P16 3A, P163E, P163N, T166E, T166G, T166I, T166L, T166N, T166P, T166Q, T166R , T166V, T166W, T167P, T167A, T167R, T167K, M172I, S176N, S178R, S178E, S1 78H, S178P, S178T, P182R, P182E, F190Y, K197A, K197D, K197F, K197M, K197 N, K197Y, Q214R, Q214W, Q215E, Y219W, S239A, M248I, R253A, R253W, S258G, S 271G, S271Y, H276C, H276N, H276Y, R280L, R280K, N283M, N283Y, F286C, E29 9K, E299D, E299W, G366N, K378D, K378H, K378Q, K378R, D385A, D385F, D385H, D385P, D385S, K408A, K408C, K408D, K408E, K408M, K408Q, K408S, K408T, K40 8V, D410L, D410M, L413Q, L413A, L413T, L413V, N473Y, D486E, D486K, K491E,and the second modification is a substitution or deletion, at positions: A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G. , V9M, V9P, E10*, E10Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R, H12V, S14Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25Y, I30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, K65C, K65F, K65H, K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, A101R, A101C, A101E, A101I, A101L, A101 N, A101Q, A101V, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y, T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M1 30I, M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143 S, Q143R, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D,I165T, I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M171K, M171Q, P174K, K179S, K179H, K179Q, K179N, K179R, S 181T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y2 13K, Y213Q, Y213R, G217Q, K218R, M228I, V235L, N242T, E249H, E249L, E249Q, K252G, K252E, G254S, L259V, S260C, K267L, G270C, N272D, E28 5D, N296A, K304A, E301K, K304F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E322D, H323A, H324N, W342G, A344 C, P347D, K351V, K352Q, Y354W, R355Q, Y358W, E363H, K364D, K364R, K364Y, N368D, V377E, E379K, E379P, E380T, E380D, V391L, P398F, P398E , S403Q, S403R, F414T, F414S, F414P, K417G, K417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442N, W443G, D452N, At least one or more positions corresponding to L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, L477F, E479R, L480I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, and W490A.

[0333] In one embodiment the variant comprises substitutions at positions corresponding to positions: L413T, K491R, K482D, R489N, and the variant is at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least and the like have 98% or at least 99% sequence identity but less than 100% sequence identity, or have 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96% sequence identity, or have 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity; or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity. or has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or has 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, 89 to 91%, or for example, 90 to 91% sequence identity, or has 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0334] In one aspect, the variants are at positions: Q168D+ L454I, Q168D+ L413A, K26E+ Q168D, E64P+ Q168D, K26H+ Q168D, Q17A+ Q168D, A23M+ Q168D, S103V+ Q168D, S103E+ Q168D, Q69P+ Q168D, H12A+ Q168D, K65S+ Q168D, N97Y+ Q168D, Q168D+ K304F, Q168D+ K179V, M130V+ Q168D, D410M+ and comprising a substitution at the position corresponding to L413T, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0335] In one aspect, the variants are at positions: K26E+ Q168D+ N296A, Q168D+ D385P+ L413A, Q168D+ Q183E+ L413A, R38Q+ Q168D+ D385F, K26E+ Q168D+ N283Y, R38Q+ Q168D+ L413T, R38Q+ Q168D+ T192V, R38Q+ Q168D+ S239A, K26E+ Q168D+ K408T, K26E+ Q168D+ K197F, K26E+ Q168D+ L413T, K26E+ Q168D+ D385A, K26E+ Q168D+ R355Q, K26E+ Q168D+ P347D, K26E+ Q168D+ G483D, K26E+ Q168D+ T192V, K26E+ Q168D+ A210E, K26E+ Q168D+ N193S, K26E+ Q168D+ S239A, K26E+ Q69P+ Q168D, K26E+ E61D+ Q168D, K26E+ K36E+ Q168D, K26E+ R38M+ Q168D, K26E+ K36T+ Q168D, K26E+ E64P+ Q168D, H12D+ K26E+ Q168D, K26E+ K70E+ Q168D, K26E+ T42E+ Q168D, K26E+ T53C+ Q168D, K26E+ T53P+ Q168D, K26E+ K70M+ Q168D, K26E+ K70P+ Q168D, K26E+ S72F+ Q168D, K26E+ A101I+ Q168D, K26E+ A101L+ Q168D, K26E+ S103E+ Q168D, K26E+ S103I+ Q168D, K26E+ Q168D+ Y354W, K26E+ Q168D+ L413V, K26E+ Q168D+ E249L, K26E+ R38L+ Q168D, K26E+ Q168D+ R489P, K26E+ Q168D+ L477F, K26E+ Q168D+ K491P, K26E+ Q168D+ N283M, K26E+ Q168D+ Y195H, D410M+ L413T+ K466F, K408M+ D410M+ L413T, D410M+ L413T+and comprising a substitution at the position corresponding to N473Y, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0336] In one aspect, the variants are at positions: Q168D+ E249Q+ N283Y+ L413A, K26E+ Q168D+ N283Y+ N296A, K26E+ Q168D+ Q183E+ L413A, K26E+ Q168D+ S271Y+ L454I, K26E+ Q168D+ E249L+ P347D, K26E+ Q168D+ L259F+ S271G, K26E+ Q168D+ L259F+ N283Y, K26E+ T53E+ E61D+ Q168D, K26E+ T53E+ E64P+ Q168D, K26E+ T53E+ K65N+ Q168D, K26E+ R38T+ Q168D+ D385A, K26E+ S103V+ Q168D+ D385A, K26E+ K36T+ Q168D+ D385A, K26E+ R38M+ Q168D+ D385A, K26E+ K36A+ Q168D+ D385A, K26E+ R38L+ Q168D+ D385A, K26E+ S103C+ Q168D+ D385A,K26E+ K70M+ Q168D+ D385A, K26E+ S103I+ Q168D+ D385A, K26E+ K70I+ Q168D+ D385A, K26E+ R38F+ Q168D+ D385A, K26E+ S103Q+ Q168D+ D385A, K26E+ A101C+ Q168D+ D385A, K26E+ K70E+ Q168D+ D385A, K26E+ A101L+ Q168D+ D385A, K26E+ K36C+ Q168D+ D385A, K26E+ S103D+ Q168D+ D385A, K26E+ K70T+ Q168D+ D385A, K26E+ A101Q+ Q168D+ D385A, K26E+ K65N+ Q168D+ D385A, K26E+ K100R+ Q168D+ D385A, K26E+ S103L+ Q168D+ D385A, K26E+ K36M+ Q168D+ D385A, K26E+ T53P+ Q168D+ D385A, K26E+ A101N+ Q168D+ D385A, K26E+ K36D+ Q168D+ D385A, K26E+ T53C+ Q168D+ D385A, K26E+ K65T+ Q168D+ D385A, K26E+ R38L+ K70P+Q168D、K26E+ T42E+ Q69P+ Q168D、K26E+ Q168D+ N283Y+ K408S、K26E+ K70P+ Q168D+ D385A、K26E+ K70P+ Q168D+ E249L、K26E+ K70P+ Q168D+ Y354W、K26E+ K70P+ Q168D+ L413V、K26E+ S103E+ Q168D+ D385A、K26E+ S103E+ Q168D+ L413V、K26E+ Q168D+ N283Y+ D385A、K26E+ K36E+ Q168D+ D385A、D410M+ L413T+ D472G+ W490A、D385A+ K408M+ D410M+ L413T、D410M+ L413T+ K417Q+and comprising a substitution at the position corresponding to K462P, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0337] In one aspect, the variants are at positions: Q168D+ A210E+ N283Y+ L413A+ R489K, K26E+ Q168D+ S181T+ K408C+ K482Q, K26E+ R164A+ Q168D+ S181T+ N296A, K26E+ Q168D+ N283Y+ N296A+ S403Q, K169P+ Q183E+ S260C+ N283Y+ L413A, K26E+ Q168D+ N296A+ D385F+ K482Q, K26E+ Q168D+ A210Q+ S271Y+ V377E, K26E+ Q168D+ A210Q+ S271Y+ L454I, K26E+ Q168D+ F190Y+ N242T+ T315A, K26E+ Q168D+ Y213K+ S260C+ G366H, K26E+ Q168D+ K252E+ R253W+ K378H, K26E+ Q168D+ A210E+ S271G+ N283Y, K26E+ Q168D+ A210E+ L259F+ S271G, K26E+ R38L+ K70P+ A101L+ Q168D, K26E+ R38C+ A126S+ Q168D+ D385A, K26E+ A101V+ S103Y+ Q168D+ D385A, V4D+ K26E+ K65T+ K120P+ Q168D, H12D+ Q17A+ K26E+ E64P+ Q168D, K26E+ A101E+ Q168D+ K197A+ K408S, K26E+ T42E+ Q69P+ Q168D+ D385A, K26E+ Q168D+ N283Y+ D385A+ K408S, K26E+ Q168D+ K197A+ N283Y+ K408S, K26E+ K36E+ E64P+ Q168D+ D385A, K26E+ K36E+ S103V+ Q168D+ D385A, K26E+ K36E+ Q168D+ D385A+ K417Q, K26E+ K70A+ S103V+ Q168D+ D385A, K26E+ K70M+ S103V+ Q168D+ D385A, K26E+ K70I+ S103V+ Q168D+ D385A, K26E+ S103V+ K120P+ Q168D+ D385A, K26E+ K36E+ T53P+ Q168D+ D385A, K26E+ K36T+S103V+ Q168D+ D385A、K26E+ R38M+ S103V+ Q168D+ D385A、K26E+ T42E+ S103V+ Q168D+ D385A、K26E+ T53P+ S103V+ Q168D+ D385A、K26E+ K36E+ K70A+ Q168D+ D385A、K26E+ K36E+ K70P+ Q168D+ D385A、K26E+ K36E+ S72F+ Q168D+ D385A、K26E+ K36E+ A101L+ Q168D+ D385A、K26E+ K70P+ Q168D+ K197F+ N283Y、K26E+ K70P+ S103V+ Q168D+ D385A、N283Y+ D385A+ K408M+ D410M+ L413T、T315V+ D385A+ K408M+ D410M+ L413T、D385A+ K408M+ D410M+ L413T+ P488I、D385A+ K408M+ D410M+ L413T+ P488L、K378Q+ D385A+ K408M+ D410M+and comprising a substitution at the position corresponding to L413T, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0338] In one aspect, the variants are at positions: K26E+ Q168D+ A210E+ S271G+ N283Y+ N296A, K26E+ Q168D+ A210Q+ E249Q+ S271Y+ N296A, K26E+ Q168D+ K252E+ N296A+ A344C+ D385P, K26E+ R164H+ Q168D+ F190Y+ S271G+ N296A, K26E+ Q168D+ F190Y+ S239A+ S271G+ N296A, K26E+ Q168D+ N283Y+ N296A+ S403Q+ L413T, K26E+ K36N+ Q168D+ K198N+ L413A+ N473F, Q168D+ Q183E+ K252E+ R253W+ K408T+ L413A, K26E+ Q168D+ A210Q+ S239A+ E249Q+ K364Y, K26E+ Q168D+ A184T+ F190Y+ N242T+ T315A, K26E+ Q168D+ K197N+ K252E+ R253W+ K408T, K26E+ Q168D+ K197N+ K252E+ R253W+ K378H, V4D+ K26E+ T53P+ K120P+ Q168D+ D385A, K26E+ K36E+ S72F+ Q168D+ D385A+ W443G, K26E+ K36E+ K70P+ K120P+ Q168D+ D385A, K26E+ T42E+ Q69P+ S103V+ Q168D+ D385A, K26E+ K36E+ S103V+ Q168D+ F190Y+ D385A, K26E+ K36E+ S103V+ Q168D+ S271G+ D385A, K26E+ K36E+ S103V+ Q168D+ D385A+ L413V, K26E+ K36E+ S103V+ Q168D+ D385A+ K417Q, K26E+ K36E+ S103V+ Q168D+ D385A+ K462L, K26E+ K36E+ S103V+ Q168D+ D385A+ K408T, K26E+ K36E+ S103V+ K120P+ Q168D+ D385A, K26E+ R38L+ K70P+ A101L+ Q168D+ N283Y, K26E+ R38L+ K70P+ A101L+ Q168D+K408D、K26E+ R38L+ K70P+ A101L+ Q168D+ L413A、K26E+ R38L+ K70P+ A101L+ Q168D+ L413T、K26E+ R38L+ K70P+ A101L+ S109D+ Q168D、K26E+ R38L+ K70P+ A101L+ Q168D+ D385A、K26E+ K70P+ Q168D+ N283Y+ K408S+ L413T、K26E+ K70P+ Q168D+ N283Y+ D385A+ S403Q、K26E+ K70A+ S103V+ Q168D+ K197A+ D385A、K26E+ K70A+ S103V+ Q168D+ D385A+ K462P、K26E+ E64P+ K70A+ S103V+ Q168D+ D385A、K26E+ K36T+ K70A+ S103V+ Q168D+ D385A、K26E+ K70A+ S103V+ Q168D+ K304R+ D385A、N283Y+ D385A+ K408M+ D410M+ L413T+ R489N、N283Y+ D385A+ K408M+ D410M+ L413T+ K482D、N283Y+ D385A+ K408M+ D410M+ L413T+ S487G、N283Y+ K378D+ D385A+ K408M+ D410M+ L413T、N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T、N283Y+ D385A+ K408M+ D410M+ L413T+ N473Y、S271G+ N283Y+ D385A+ K408M+ D410M+ L413T、K378Q+ D385A+ K408M+ D410M+ L413T+ N473Y、K378Q+ D385A+ K408M+ D410M+ L413T+and comprising a substitution at the position corresponding to R489N, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0339] In one aspect, the variants are at positions: K26E+ Q168D+ A210E+ S260C+ N283Y+ N296A+ K482Q, K26E+ Q168D+ A210E+ S260C+ S271G+ N283Y+ N296A, K26E+ Q168D+ A210Q+ S239A+ E249Q+ S271Y+ N296A, K26E+ Q168D+ A184T+ N242T+ N296A+ E380D+ T438A, K26E+ R164A+ Q168D+ S181T+ N296A+ K408S+ L413V, K26E+ R164A+ Q168D+ S181T+ N296A+ K408S+ L413V, K26E+ R164A+ Q168D+ S181T+ N296A+ Q321W+ K408S, K26E+ Q168D+ Y213K+ K252E+ E285D+ N296A+ D385P, K26E+ Q168D+ T192A+ Y213K+ N296A+ A344C+ D385P, K26E+ Q168D+ S271G+ N296A+ Y354W+ V377E+ K408A, K26E+ Q168D+ N283Y+ N296A+ D385S+ S403Q+ K408C, K26E+ Q168D+ N283Y+ N296A+ S403Q+ K408C+ L413T, K26E+ Q168D+ S181T+ E249Q+ N283Y+ D385A+ L413A, K26E+ Q168D+ A184T+ F190Y+ N242T+ E322D+ H323A, K26E+ Q168D+ A184T+ F190Y+ N242T+ T315A+ E380D, K26E+ Q168D+ F190Y+ N242T+ N283Y+ E322D+ H323A, K26E+ Q168D+ A210E+ N283Y+ D385A+ L413A+ R489K, K26E+ Q168D+ S181T+ A210E+ N283Y+ L413A+ R489K, K26E+ Q168D+ A210E+ L259F+ S271G+ N283Y+ R489K, K26E+ Q168D+ N283Y+ D385A+ K408S+ D452N+ K466Q, V4D+ K26E+ T53P+ K65Q+ K120P+ Q168D+ D385A, H12D+ Q17A+ K26E+ E64P+ A111L+ Q168D+D385A、H12D+ Q17A+ K26E+ E64P+ V68T+ Q168D+ D385A、K26E+ K36E+ K70P+ S103V+ K120P+ Q168D+ D385A、F25Y+ K36E+ T53P+ S103V+ Q168D+ T192V+ D385A、K26E+ R38L+ K70P+ A101L+ A126T+ Q168D+ E379P、K26E+ K70P+ Q168D+ K197A+ K408S+ K462L+ K466Q、K26E+ K70P+ Q168D+ K197F+ N283Y+ K408S+ L413T、H12D+ K26E+ R38L+ T42E+ K70P+ A101L+ Q168D、K26E+ K36E+ S103V+ Q168D+ D385A+ L413V+ K462L、K26E+ K36E+ S103V+ Q168D+ K197D+ D385A+ K462L、N283Y+ K378D+ D385A+ K408M+ D410M+ L413T+ K482D、S258G+ S271G+ N283Y+ D385A+ K408M+ D410M+ L413T、N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T+ P488L、S271G+ N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T、K378Q+ D385A+ K408M+ D410M+ L413T+ H442N+ N473Y、E299W+ K378Q+ D385A+ K408M+ D410M+ L413T+ N473Y、N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T+ N473Y、N283Y+ E380D+ D385A+ K408M+ D410M+ L413T+ G440Q、N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T+ H442N、G366H+ K378Q+ D385A+ K408M+ D410M+ L413T+and comprising a substitution at the position corresponding to N473Y, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0340] In one aspect, the variants are at positions: K26E+ Q168D+ A184T+ N296A+ E322D+ H323A+ E380D+ T438A, K26E+ R164A+ Q168D+ S181T+ N296A+ Q321W+ K408S+ L413V, K26E+ Q168D+ K252E+ E285D+ N296A+ A344C+ G366H+ D385P, K26E+ Q168D+ N283Y+ N296A+ D385S+ S403Q+ K408C+ L413T, K26E+ Q168D+ S181T+ K197F+ V235L+ E249Q+ N283Y+ L413A H12D+ Q17A+ K26E+ E64P+ V68T+ A111L+ Q168D+ D385A, H12D+ K26E+ T42E+ E64P+ K70P+ S103V+ Q168D+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ Q168D+ N283Y+ D385A, K26E+ K36E+ T53P+ K65Q+ Q168D+ F190Y+ K197A+ D385A, N283Y+ K378D+ D385A+ S403Q+ K408M+ D410M+ L413T+ K482D, K378Q+ D385A+ K408M+ D410M+ L413T+ N473Y+ K482R+ K491E, S258G+ S271G+ N283Y+ K378Q+ D385A+ K408M+ D410M+ L413A, S271G+ N283Y+ K378Q+ D385A+ K408M+ D410M+ L413T+ H442N, S258G+ S271G+ N283Y+ K378Q+ D385A+ K408M+ D410M+and comprising a substitution at the position corresponding to L413T, the variant has at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the polypeptide of SEQ ID NO: 1. However, the sequence identity is less than 100%, or the sequence identity is 85% to 96%, for example, 86 to 96%, 87 to 96%, 88 to 96%, 89 to 96%, 90 to 96%, 91 to 96%, 92 to 96%, 93 to 96%, 94 to 96%, for example, 95 to 96%, or the sequence identity is 85% to 95%, for example, 86 to 95%, 87 to 95%, 88 to 95%, 89 to 95%, 90 to 95%, 91 to 95%, 92 to 95%, 93 to 95% , for example, 94 to 95% sequence identity, or 85% to 94%, for example, 86 to 94%, 87 to 94%, 88 to 94%, 89 to 94%, 90 to 94%, 91 to 94%, 92 to 94%, for example, 93 to 94% sequence identity, or 85% to 93%, for example, 86 to 93%, 87 to 93%, 88 to 93%, 89 to 93%, 90 to 93%, 91 to 93%, for example, 92 to 93% sequence identity, or It has 85% to 92%, for example, 86 to 92%, 87 to 92%, 88 to 92%, 89 to 92%, or 90 to 92%, for example, 91 to 92% sequence identity, or 85% to 91%, for example, 86 to 91%, 87 to 91%, 88 to 91%, or 89 to 91%, for example, 90 to 91% sequence identity, or 85% to 90%, for example, 86 to 90%, 87 to 90%, 88 to 90%, for example, 89 to 90% sequence identity.

[0341] In one aspect, the variants are at positions: K26E+ Q168D+ A184T+ N242T+ N296A+ E322D+ H323A+ E380D+ T438A, H12D+ K26E+ K36E+ T42E+ E64P+ K70P+ A101L+ Q168D+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ Q168D+ T192V+ N283Y+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ E162D+ Q168D+ N283Y+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ S109D+ Q168D+ N283Y+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ Q168D+ K197Y+ N283Y+ D385A, K26E+ K36E+ E64P+ K70P+ S103V+ Q168D+ N283Y+ D385A+ L477F, K26E+ K36E+ E64P+ K70P+ S103V+ Q168D+ N283Y+ D385A+ L413T, F25Y+ K26E+ K36E+ T53P+ S103V+ Q168D+ T192V+ D385A+ K462L, K26E+ K36E+ E64P+ S103V+ K120P+ Q168D+ N283Y+ D385A+ K462L, K26E+ K36E+ E64P+ K70P+ S103V+ K169T+ N283Y+ D385A+ L477F, K26E+ K36E+ E64P+ Q69P+ S103V+ Q168D+ N283Y+ D385A+ L477F, S271G+ N283Y+ E299W+ K378Q+ D385A+ K408M+ D410M+ L413T+ N473Y, S271G+...

Claims

Claim 1: A variant of a parent mannanase having SEQ ID NO: 1, wherein the variant comprises at least two modifications: (a) the first said modification is a substitution at position 168; (b) at least a second of said modifications is selected from the group consisting of A1*, A1S, A1T, A1V, A1G, A1D, A1I, A1Y, I2*, T3*, T3A, T3E, T3G, V4*, V4N, V4D, V4G, V4M, V4Y, P5*, G6*, G6P, V8*, V8A, V8Q, V8F, V8D, V8W, V9*, V9D, V9A, V9G, V9M, V9P, E10*, E10 Y, E10G, P11A, P11L, P11E, P11I, P11N, H12A, H12D, H12E, H12G, H12L, H12P, H12Q, H12R, H12V, S14 Q, S14R, S14E, S14H, S14N, S14V, Q17K, Q17A, N18S, Q19N, I21L, I22V, I22Q, I22T, A23I, A23T, A23V A23M, A23S, F25W, F25YI30L, I35*, K37V, R38C, R38F, R38L, R38M, R38T, T42E, A45L, A45M, A45R, D48A, D48P, D48S, L49M, T53C, T53E, T53H, T53K, T53P, T53Q, S57P, S57R, E61D, D63C, E64P, K65C, K65F, K65H , K65N, K65Q, K65S, K65T, V68M, V68T, S72F, T74A, T74Q, T74E, Q76T, Q76E, G83E, G83S, E86D, L89F, P94 M, G96E, G96D, G96R, N97E, N97D, N97Y, K100R, S103R, S103A, S103C, S103D, S103E, S103I, S103L, S103N , S103Q, S103V, S103Y, Q105V, G110R, G110D, G110P, G110C, G110S, A111L, G112E, G112V, T115W, T115Y , T115V, T115I, S117P, A118V, P121A, G122P, T123R, A126D, A126S, A126T, K129R, K129L, K129M, M130I , M130V, L134I, L134V, L134P, L134T, S137A, S137D, S137W, N139I, N139K, N139A, Q143T, Q143S, Q143R , Y155H, R160I, R160F, R160K, R160L, R160N, R160V, V161S, V161T, R164*, R164Q, I165E, I165D, I165T,I165L, K169P, K169Q, K169T, K169V, T170E, T170H, M171E, M171N, M171I, M1 71K, M171Q, M172I, P174K, S176N, K179S, K179H, K179Q, K179N, K179R, S181 T, Q183E, A184T, K185N, K185Q, M188F, M188I, M188Y, K189S, F190Y, T192A, T192V, N193W, N193S, Y195H, A210E, A210Q, A210F, A210T, A210S, Y213K, Y21 3Q, Y213R, Q214R, Q214W, Q215E, G217Q, K218R, Y219W, M228I, V235L, S239A , N242T, M248I, E249H, E249L, E249Q, K252G, K252E, R253A, R253W, G254S, L2 59V, S260C, K267L, G270C, N272D, E285D, F286C, N296A, K304A, E301K, K304 F, K304Q, K304R, K304S, A312V, T315A, T315V, Q321K, Q321W, E322R, E322A, E 322D, H323A, H324N, W342G, A344C, P347D, K351V, K352Q, Y354W, R355Q, Y35 8W, E363H, K364D, K364R, K364Y, G366N, N368D, V377E, E379K, E379P, E380T , E380D, V391L, P398F, P398E, S403Q, S403R, F414T, F414S, F414P, K417G, K 417E, K417Q, K419V, V422R, V422I, A423G, Q437I, Q437A, T438A, G440Q, H442 N, W443G, D452N, L454I, K462A, K462R, K462L, K462P, E463Q, K466F, K466H, K466N, K466Q, K467H, K467Y, N470Y, D472G, D472Y, N473Y, L477F, E479R, L48 0I, K482A, K482P, K482D, K482G, K482Q, K482R, G483A, G483D, L484Q, S487R, P488I, P488L, R489F, R489K, R489N, R489P, W490A, K491E, K491P, and K491R,and combinations thereof, the variant has mannanase activity, the variant has an improved half-life improvement factor, detergent stability or thermal stability compared to the parent mannanase; A variant, wherein the variant has at least 90% sequence identity with SEQ ID NO: 1 or SEQ ID NO:

2.

2. 2. The variant of claim 1, wherein the first modification comprises a substitution selected from the group consisting of Q168D, Q168F, Q168M, and Q168P.

3. 3. The variant of claim 1 or 2, wherein the variant has an improvement factor (IF) of >1.0 compared to the parent in terms of a stability measure, wherein the IF is at least 1.4 compared to the parent.

4. 4. The variant of any one of claims 1 to 3, wherein the variant has an improvement factor (IF) of >1.0 on a measure of stability at pH 8 to 8.5, wherein the IF is at least 1.4 compared to the parent.

5. An isolated polynucleotide encoding the variant of any one of claims 1 to 4.

6. A nucleic acid construct or expression vector comprising the polynucleotide of claim 5.

7. A recombinant host cell transformed with the polynucleotide of claim 5.

8. A method for producing a mannanase variant, comprising: culturing a recombinant host cell described in claim 7 under conditions suitable for expression of the variant; and recovering the variant.

9. A detergent composition comprising a variant according to any one of claims 1 to 4 and at least one detergent component.

10. Use of a detergent composition according to any of the variants of claims 1 to 4 or claim 9 for washing, washing, cleaning and / or deep cleaning of textiles and / or hard surfaces.

Images