Carbohydrate-binding module variants

Abstract

Carbohydrate-binding module variants and glycoside hydrolase variants comprising the carbohydrate-binding module variants are disclosed. The variants with glycoside hydrolase activity comprising the carbohydrate-binding module variants have improved stability in the presence of proteases and are useful in detergent applications such as laundry or dishwashing.

Description

[Technical Field]

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

[0002] The present invention relates to carbohydrate binding module variants, more preferably family 1 carbohydrate binding module variants. [Background technology]

[0003] Glycoside hydrolases, such as cellulases, generally contain a catalytic domain and one or more carbohydrate-binding modules (CBMs) connected by a linker region.

[0004] Cellulases have been used in detergents for many years for their observed benefits in the laundry process, such as color clarification, anti-redeposition, anti-pilling / pilling removal, and improved whiteness, and are characterized by their ability to cleave 1,4-beta-glycosidic bonds in cellulose molecules to smaller molecules.

[0005] In some applications, multiple cellulase enzyme compositions are used, which include two or more cellulolytic enzymes selected from endoglucanases, cellobiohydrolases, and beta-glucosidases, while other applications use enzyme compositions that primarily include one or more endoglucanases.

[0006] WO 1996 / 029397 discloses family 45 endoglucanases for detergent applications. Most commercially available detergent compositions contain proteases, which improve the removal of many common stains. However, proteases also degrade other proteins available in aqueous reaction mixtures containing other enzymes, such as cellulases and other glycoside hydrolases. Therefore, it would be desirable to provide glycoside hydrolases, such as cellulases and their variants, that have increased stability in the presence of proteases. Summary of the Invention

[0007] The present invention relates to carbohydrate binding module variants, more preferably carbohydrate binding module variants of Family 1. The present invention also relates to modified glycoside hydrolase enzymes comprising carbohydrate binding module variants.

[0008] The present invention further relates to polynucleotides and expression constructs comprising the polynucleotides; host cells comprising the polynucleotides or expression constructs and the use of such host cells to produce carbohydrate binding module variants and glycoside hydrolase enzymes comprising the carbohydrate binding module variants of the invention.

[0009] Compositions, particularly detergent compositions such as liquid detergent compositions, comprising carbohydrate-binding module variants and glycoside hydrolase enzymes comprising carbohydrate-binding module variants, and the use of such compositions for laundering fabrics are also disclosed.

[0010] definition As used in the context of describing the present invention, the terms "a," "an," "the," and similar referents are to be construed to encompass both the singular and the plural unless otherwise indicated or clearly contradicted by context.

[0011] Allelic variant: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally through mutation and can result in polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence. An allelic variant of a polypeptide is one encoded by an allelic variant of a gene.

[0012] Anti-Pilling: The term "anti-pilling" refers to the removal of pills from and / or the prevention of pill formation on a textile surface.

[0013] Carbohydrate-binding module: The term "carbohydrate-binding module" refers to a region in a carbohydrate-active enzyme that confers carbohydrate-binding affinity (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.

[0014] Exemplary CBM families useful in accordance with the present invention are CBM families 1, 4, 17, 28, 30, 44, 72, and 79. See also cazy.org / Carbohydrate-Binding-Modules. CBM family 1 contains a module of approximately 40 residues found almost exclusively in fungi. Cellulose-binding function has been demonstrated in many cases and appears to be mediated by three aromatic residues separated by approximately 10.4 angstroms and forming a flat surface. CBM family 4 contains a module of approximately 150 residues found in bacterial enzymes. Binding of these modules to xylan, beta-1,3-glucan, beta-1,3-1,4-glucan, beta-1,6-glucan, and amorphous cellulose has been demonstrated, but binding to crystalline cellulose has not. CBM family 17 contains a module of approximately 200 residues. Binding to amorphous cellulose, cellooligosaccharides, and derivatized cellulose has been demonstrated. For CBM family 28, a module derived from the endo-1,4-glucanase of Bacillus sp. 1139 binds to amorphous cellulose, cellooligosaccharides, and β-(1,3)(1,4)-glucan. For CBM family 30, binding to cellulose has been demonstrated for the N-terminal module of Fibrobacter succinogenes CelF. The C-terminal CBM44 module of the Clostridium thermocellum enzyme has been demonstrated to bind equally well to cellulose and xyloglucan. CBM family 72 contains modules of 130–180 residues found in C-terminal glycoside hydrolases from various families, sometimes as tandem repeats. CBM72, found on endoglucanases from uncultivated microorganisms, was found to bind a wide range of polysaccharides, including soluble and insoluble cellulose, beta-1,3 / 1,4-mixed-linkage glucans, xylans, and beta-mannans.CBM family 79 contains a module of approximately 130 residues that has so far only been found in Ruminococcus proteins. Binding to a variety of beta-glucans has been demonstrated for the R. flavefaciens GH9 enzyme.

[0015] CBM family 1, also referred to as "CBM1", is most preferred.

[0016] Catalytic domain: The term "catalytic domain" refers to the region of an enzyme that contains the enzyme's catalytic machinery.

[0017] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as the mature, spliced ​​mRNA.

[0018] Cellulolytic enzymes or cellulases: The terms "cellulolytic enzymes" or "cellulases" refer to one or more (e.g., several) enzymes that hydrolyze cellulose-derived materials. Such enzymes include endoglucanases (e.g., EC 3.2.1.4), cellobiohydrolases, beta-glucosidases, or combinations thereof. As outlined in Zhang et al., 2006, Biotechnology Advances 24:452-481, basic approaches to measuring cellulolytic enzyme activity include (1) measuring total cellulolytic enzyme activity and (2) measuring individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases). 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).

[0019] Cellulose-derived materials: The term "cellulose-derived materials" refers to any material containing cellulose. The predominant polysaccharide in the primary cell walls of biomass is cellulose, with hemicellulose being the second most abundant and pectin being the third. Secondary cell walls, produced after cells cease growth, also contain polysaccharides and are reinforced by the polymer lignin covalently crosslinked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicellulose contains a variety of compounds, such as xylan, xyloglucan, arabinoxylan, and mannan, in complex branched structures with a range of substituents. Cellulose is generally polymorphic, but in plant tissues it is primarily found as an insoluble crystalline matrix of aligned glucan chains. Hemicellulose typically hydrogen bonds to cellulose and other hemicelluloses, helping to stabilize the cell wall matrix.

[0020] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0021] Color Clarification: During washing and wearing, loose or broken fibers may accumulate on the surface of a fabric. As a result, the color of the fabric may become less bright or intense due to surface soiling. Removal of the loose or broken fibers from the fabric will partially restore the original color and appearance of the fabric. As used herein, the term "color clarification" refers to the partial restoration of the original color of the fabric.

[0022] Detergent Component: The term "detergent component" is defined herein to mean a class of chemicals that can be used in a detergent composition. Examples of detergent components 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, perfumes, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. A detergent component can include one or more of any type of detergent component.

[0023] Control sequence: The term "control sequence" refers to a nucleic acid sequence required for expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., derived from the same gene) or foreign (i.e., derived from different genes) to the polynucleotide encoding the variant, or may be 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 variant.

[0024] Detergent composition: The term "detergent composition" refers to a composition that is used to remove undesirable compounds from cleaned items, 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 and / or fabric care. 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, plastic, ceramic, and metal countertops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile and laundry pre-spotters, and dishwashing detergents).In addition to containing the enzymes of the present invention, detergent formulations may also contain one or more additional enzymes (e.g., amylase, 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, xylanase, pectin acetylesterase, polyglycerol, hydroxybenzoate ... lacturonase, rhamnogalacturonase, other endo-beta-mannanases, exo-beta-mannanases (GH5 and / or GH26), licheninanases, phosphodiesterases, pectin methylesterases, cellobiohydrolases, transglutaminases, nucleases, and combinations thereof, or any mixture thereof), and / or components such as surfactants, builders, chelators or chelating agents, bleaching systems or components, polymers, fabric hueing agents, suds boosters, suds suppressors, dyes, fragrances, tan inhibitors, optical brighteners, bactericides, fungicides, soil suspension agents, anticorrosives, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0025] Dishwashing: The term "dishwashing" refers to all forms of washing dishes, for example, by manual dishwashing (HDW) or automatic dishwashing (ADW). 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.

[0026] Dishwashing compositions: The term "dishwashing compositions" refers to all forms of compositions intended for cleaning dishes, tableware, pots, pans, cutlery, and compositions for cleaning hard surface areas of the kitchen. The present invention is not limited to any particular type of dishwashing composition or any particular detergent.

[0027] Endoglucanase: The term "endoglucanase" refers to an enzyme that catalyzes the endohydrolysis of beta-1,4 linkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethylcellulose), lichenin, mixed beta-1,3-beta-1,4 glucans such as cereal beta-D-glucans or xyloglucans, and 1,4-beta-D-glycosidic linkages in other plant materials containing cellulosic components. For purposes of the present invention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257-268. One unit of endoglucanase activity is defined as 1.0 μmole of reducing sugars produced in 1 minute at 50°C and pH 4.8.

[0028] One particularly preferred class of endoglucanases is that of "family GH45," which are classified as glycoside hydrolase family 45 according to the terminology of Henrissat et al., Biochem. J. 280:309-316 (1991) and the Carbohydrate Active enZYmes database available at cazy.org. GH45 enzymes are endoglucanases in EC 3.2.1.4.

[0029] Expression: The term "expression" includes all steps involved in the production of a variant (for example, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0030] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule containing a polynucleotide encoding a variant, operably linked to a control sequence that provides for its expression.

[0031] Fabric Care: The term "fabric care", also referred to as textile care, refers to treatments that preserve or partially or completely restore the properties of textiles, for example, by clarifying the color, preventing pilling or preventing the formation of pills on the textile surface.

[0032] Fragment: The term "fragment" refers to a polypeptide having one or more (e.g., several) amino acids not present at the amino and / or carboxyl terminus of the mature polypeptide; the fragment retains the enzymatic activity of the full-length polypeptide.

[0033] Engineered: The term "engineered" refers to a synthetic construct.

[0034] Glycoside hydrolase: The term "glycoside hydrolase" (GH) refers to an enzyme that catalyzes the hydrolysis of glycosidic bonds between two or more carbohydrates or between a carbohydrate moiety and a non-carbohydrate moiety. For further details, see, e.g., Henrissat B., "A classification of glycosyl hydrolases based on amino-acid sequence similarities," Biochem. J. 280:309-316 (1991), and the Carbohydrate Active enZYmes database available at cazy.org. Some glycoside hydrolases contain one or more catalytic domains and one or more carbohydrate-binding modules (CBMs), which are connected by one or more linker regions connecting these domains.

[0035] Exemplary glycoside hydrolase families with reported cellulase activity useful according to the present disclosure include those of families GH5, GH6, GH7, GH8, GH9, GH12, GH44, GH45, GH48, GH51, GH124, with family GH45 being particularly preferred.

[0036] 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 of plates, cups, glasses, bowls, cutlery such as spoons, knives, forks, serving utensils, ceramics, plastics, metals, china, glass, and acrylic.

[0037] 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 any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0038] Hybrid polypeptide: The term "hybrid polypeptide" refers to a polypeptide comprising domains from two or more polypeptides from different sources, e.g., a binding module from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or C-terminus. Of particular interest herein are polypeptides comprising a binding module from one polypeptide (which may be naturally occurring or further modified), an engineered linker region, such as a proline-rich linker region that is a synthetic construct, and a catalytic domain from another polypeptide (which may be naturally occurring or further modified).

[0039] Hybridization: The term "hybridization" refers to the substantial pairing of complementary strands of nucleic acid 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 in 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.

[0040] Improved property: The term "improved property" refers to a characteristic associated with a variant that is improved compared to a reference / parent enzyme. Some embodiments of the invention relate to variants that have an improvement factor of greater than 1 when the variant is tested for the property of interest in an appropriate assay, where the property of the reference / parent enzyme is assigned a value of 1.

[0041] Improved stability: The term "improved stability" refers to an enzyme that has better stability in the presence of a protease compared to the stability of a reference / parent enzyme, including, for example, proteolytic stability, stability during storage in a detergent, improved stability during formulation of a detergent composition, and stability during washing. Improved stability can be quantified by determining stability according to the assays described herein in Example 2 (Linker Stability Assay in the Presence of Proteases) and / or Example 7 (Linker Stability Assay During Washing with Proteases).

[0042] Improved cleaning performance: The term "improved cleaning performance" is defined herein as an enzyme that exhibits increased cleaning performance in a detergent composition, e.g., increased color clarification and / or anti-pilling effect, when evaluating fresh samples and / or when samples are stored under the same conditions, compared to the cleaning performance of a reference / parent enzyme. The term "improved cleaning performance" includes not only cleaning performance during laundry, but also cleaning performance in hard surface cleaning, e.g., automatic dishwashing (ADW).

[0043] Isolated: The term "isolated" refers to a substance in a form or setting not found in nature. Non-limiting examples of isolated substances include: (1) any non-naturally occurring substance; (2) any substance, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor, from which one or more or all of the naturally occurring components with which it is naturally associated have been at least partially removed; (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., multiple copies of the gene encoding the substance, use of a stronger promoter than that naturally associated with the gene encoding the substance). An isolated substance may also be present in a fermentation broth sample.

[0044] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its mature form after N-terminal processing (eg, removal of a signal peptide).

[0045] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having cellulase, such as endoglucanase, activity.

[0046] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0047] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that has been isolated from a naturally occurring gene, or that has been modified to contain a segment of nucleic acid in a way not normally found in nature, or that is synthetic and includes one or more regulatory sequences.

[0048] Operably linked: The term "operably linked" refers to a configuration in which a control sequence is positioned in relation to a coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0049] Parent or Parent Cellulase: The term "parent" or "parent cellulase" refers to any polypeptide having glycoside hydrolase activity, in particular cellulolytic or even endoglucanase activity, to which modification is made to generate a glycoside hydrolase variant comprising a carbohydrate-binding module variant of the present invention.

[0050] Proline-rich linker: The term "proline-rich linker" refers to a sequence comprising one or more Pro-Pro, Pro-Xaa (or Xaa-Pro), Xaa-Pro-Xaa, or Xaa-Xaa-Pro (or Pro-Xaa-Xaa) units, where Pro is the three-letter notation for the amino acid proline and Xaa is the three-letter notation for any amino acid. Preferably, the proline-rich linker comprises the above repeating units, such as PP, PPP, PPPP (SEQ ID NO: 27), PX, PXP, PS, PSP, PXPX (SEQ ID NO: 98), XP, XPX, SP, SPS, XPXP (SEQ ID NO: 99), XPXXPX (SEQ ID NO: 100), XXPXXP (SEQ ID NO: 101), in combination and / or consecutively.

[0051] In one aspect, the proline-rich linker comprises one or more of the following optional repeating motifs: [P / S / T / R / K / D / E]P and P[S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E] (SEQ ID NO: 102). In one aspect, the proline-rich linker comprises the following optional repeating motifs: [S / T / R / K / D / E]P[S / T / R / K / D / E / N / Q], [P / S / T / R / K / D / E][P / S / T / R / K / D / E]P, and / or P[P / S / T / R / K / D / E][P / S / T / R / K / D / E]. In one aspect, the proline-rich linker comprises an optionally repeated motif of the same or different amino acids within square brackets as shown: [P / S / T]P and P[S / E]PT (SEQ ID NO: 109).

[0052] In one aspect, the proline-rich linker is (a) (SP)a, a=2 to 10; (b) (PS)a, a=2 to 10; (c) Pb, b=4 to 20, preferably 4 to 15; (d) (PEPT (SEQ ID NO: 125))c, c=2 to 5; (e) (PSPT (SEQ ID NO: 104))d, d=2 to 5; (f) (P[S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E] (SEQ ID NO: 102))e, e=2 to 5; (g) ([S / T / R / K / D / E]P)f, Combinations of each monomer unit are contemplated, including (h) ([S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E])g, g = 2 to 6; (i) ([S / T / R / K / D / E / N / Q][S / T / R / K / D / E / N / Q]P)h, h = 2 to 5; (j) (TP)i, i = 2 to 10; (k) ([S / T / P][S / T / P][S / T / P])j, j = 2 to 11; (l) and / or combinations thereof.

[0053] In one aspect, the proline-rich linker comprises a linker in Table A below, such as PPPPPPP (SEQ ID NO: 31), PPPPPPPG (SEQ ID NO: 30), SPSPSPSPSP (SEQ ID NO: 58) or SPSPSPSPSPG (SEQ ID NO: 25).

[0054] Preferably, the proline-rich linker comprises at least 25% proline, e.g., at least 28% proline, at least 30% proline, at least 35% proline, at least 40% proline, at least 50% proline, such as at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% proline. Preferably, the proline-rich linker comprises at least 4 amino acids, such as 4 to 28 amino acids, and up to 30 amino acids, preferably 4 to 20 amino acids, or 4 to 10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.

[0055] Additionally, the linker region as defined herein may comprise an additional 1-2 amino acid interface at the point of attachment to the catalytic domain and / or carbohydrate binding module.

[0056] Purified: The term "purified" refers to a nucleic acid or polypeptide that is substantially free from 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, in a chromatographic eluate, and / or in a medium subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, and typically at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more pure (e.g., weight percent on a molar basis). In a related sense, a composition is enriched for a molecule if the concentration of the molecule is substantially increased after applying a purification or concentration procedure. The term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other particular material or component that is present in a composition at a higher relative or absolute concentration than in the starting composition.

[0057] Recombinant: The term "recombinant," when used with respect to a cell, nucleic acid, protein, or vector, means altered from the natural state. Thus, for example, a recombinant cell expresses a gene not found in the native form of the cell (non-recombinant), or expresses a native gene at a level or under conditions different from that 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 term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0058] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0059] For purposes of the present invention, sequence homology between two amino acid sequences is preferably determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), 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 opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output 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)

[0060] For 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 in 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 opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. The Needle output 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).

[0061] 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, 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 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.

[0062] Variant: The term "variant" refers to a polypeptide that contains substitutions at one or more (e.g., several) positions and retains the activity of the parent. Substitution refers to the replacement of an amino acid occupying a position with a different amino acid. Carbohydrate binding module variants of the invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the cellulose-binding activity of a parent CBM, such as the polypeptide of SEQ ID NO: 173. Variants with glycoside hydrolase activity, including CBM variants, have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the glycoside hydrolase activity of a parent, such as the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. "Variant," as used herein, can also include hybrid polypeptides.

[0063] Washing liquor: The term "washing liquor" refers to an aqueous solution containing a detergent composition in diluted form, such as, but not limited to, a detergent solution containing a laundry detergent composition in diluted form, such as a wash liquor in a laundry process.

[0064] Whiteness: The term "whiteness" is defined herein as a broad term that has different meanings in different regions and to different consumers. Loss of whiteness can be due, for example, to graying, yellowing, or removal of optical brighteners / hues. Graying and yellowing can be due to soil redeposition, coloring or transfer from body soils, such as iron and copper ions. Whiteness can include one or more issues from the following list: colorant or dye action; incomplete soil removal (e.g., body soils, sebum, etc.); redeposition (graying, yellowing, or other discoloration of objects) (reattachment of removed soil to other parts of the fabric, soiled or unsoiled); chemical changes in the fabric during use; and color clarification or lightening.

[0065] 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).

[0066] Conventional methods for designating variants For the purposes of the present invention, the carbohydrate binding module of SEQ ID NO: 173 is used to determine the corresponding amino acid residue in another carbohydrate binding module. The amino acid sequence of another carbohydrate binding module is aligned with the polypeptide disclosed in SEQ ID NO: 173, and based on this alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 173 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 opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0067] The identification of corresponding amino acid residues in different carbohydrate-binding modules can be determined by alignment of multiple polypeptide sequences using several computer programs with their respective default parameters, including, but not limited to, MUSCLE (multiple sequence comparison with logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA employing ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0068] Other carbohydrate-binding modules may be used in cases where the polypeptide of SEQ ID NO: 173 is divergent, and therefore traditional sequence-based comparisons do not detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615). Higher sensitivity in sequence-based searches may be achieved using search programs that use probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program can generate profiles through an iterative database search process to detect distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity may be achieved when a polypeptide family or superfamily has more than one representative in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potential) as input to a neural network that predicts the structural fold of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models of the polypeptide, which can be assessed for accuracy using a variety of tools developed for this purpose.

[0069] For proteins with known structures, several tools and resources are available for searching and creating structural alignments. For example, proteins of the SCOP superfamily have been structurally aligned, and this alignment can be accessed and downloaded. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747). Implementations of these algorithms can also be used to search structural databases with a structure of interest to find possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0070] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference: Accepted IUPAC single-letter or three-letter amino acid abbreviations are employed.

[0071] 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 a plus sign ("+" or ","), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" or "Gly205Arg,Ser411Phe" or "G205R,S411F" represent substitutions of glycine (G) at position 205 with arginine I and serine (S) at position 411 with phenylalanine (F), respectively.

[0072] Deletions. For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Thus, a deletion of glycine at position 195 is designated as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+" or ","), e.g., "Gly195*+Ser411*" or "Gly195*,Ser411*" or "G195*,S411*".

[0073] 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."

[0074] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. In the above example, the sequence would be:

[0075] [Table 1]

[0076] Multiple modifications. Variants containing multiple modifications are separated by a plus sign "+", for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" or "Arg170Tyr,Gly195Glu" or "R170Y,G195E" represent substitutions of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.

[0077] Different modifications. When different modifications can be introduced at a position, the different modifications are separated by a comma, for example, "Arg170Tyr,Glu" represents a 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".

[0078] nomenclature For purposes of the present invention, square brackets are used to indicate alternative amino acids (using their single-letter codes) at a particular position in a sequence. For example, the nomenclature [S / E] means that the amino acid at this position can be serine (Ser, S) or glutamic acid (Glu, E). Similarly, the nomenclature [P / S / T] means that the amino acid at this position can be proline (Pro, P), serine (Ser, S), or threonine (Thr, T), etc., for other combinations as described herein. Amino acids shown in square brackets using this nomenclature can be separated by a vertical line or, in some cases, without a line; for example, [P / S / T] can also be designated [PST].

[0079] In some cases, a sequence motif includes two or more sets of square brackets, each of which independently represents a position in the sequence. Thus, P[S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E] (SEQ ID NO: 102) means that P, a conservative amino acid, is in the first position; either S, T, R, K, D, E, N, or Q is in the second position; P, a conservative amino acid is in the third position; and either S, T, R, K, D, or E is in the fourth position. The motif represented by this designation can then be any of PSPS (SEQ ID NO: 103), PSPT (SEQ ID NO: 104), PSPR (SEQ ID NO: 105), PSPK (SEQ ID NO: 106), PSPD (SEQ ID NO: 107), PSPE (SEQ ID NO: 108), etc.

[0080] Unless further limited otherwise, the amino acid X (or Xaa) is used herein to represent any of the 20 naturally occurring amino acids. DETAILED DESCRIPTION OF THE INVENTION

[0081] Many proteins are composed of structured domains connected by linkers. For example, cellulases and other glycoside hydrolases (GHs) are often found as modular enzymes with one or more catalytic domains, which may be linked to one or more CBMs via peptides known as linkers, and which may be partially glycosylated. The catalytic domains are involved in the hydrolytic degradation of cellulose, while the CBMs, when present, function by increasing the effective concentration of the enzyme near the substrate surface. In contrast, linkers are generally flexible connectors that provide connectivity between structured domains, although their functional role is largely unknown.

[0082] Cellulases, in particular, are often cleaved (nicked) in exposed regions or partially or completely degraded by proteases in liquid detergents. Most commonly, proteases cleave in the unstructured linker region of cellulases, thereby reducing the ability of cellulases to bind to insoluble cellulose substrates, for example, in detergent applications, thereby reducing their ability to remove fluff and pilling and maintain or restore color to fabrics. Because reduced binding affinity strongly affects cellulase performance, protease-stabilizing molecules are very useful in the laundry / dishwashing liquid detergent field and fabric softeners.

[0083] Similarly, proteolytic cleavage of peptide bonds in the CBM modules can result in a reduction in the ability of cellulases to bind to insoluble cellulose substrates, thereby affecting their performance.

[0084] The present invention relates to a carbohydrate-binding module variant having cellulose-binding activity. The present invention also relates to a glycoside hydrolase enzyme comprising the carbohydrate-binding module variant of the present invention, particularly a glycoside hydrolase variant having a three-domain structure with one or more catalytic domains linked to one or more carbohydrate-binding modules via linkers. Surprisingly, the inventors have found that the carbohydrate-binding module variant can improve the stability of the resulting glycoside hydrolase enzyme comprising the carbohydrate-binding module variant, making the glycoside hydrolase enzyme more stable, i.e., less susceptible to proteolytic cleavage.

[0085] In some embodiments, the glycoside hydrolase variant comprising the carbohydrate-binding module variant also comprises a stretch of peptide that renders the native linker more stable, i.e., less susceptible to proteolytic cleavage. Preferably, the glycoside hydrolase variant comprises, in addition to the carbohydrate-binding module variant, a linker that is an engineered linker region, such as a proline-rich linker as described herein.

[0086] However, prior to this work, it was not known that modifications in the carbohydrate-binding module of a glycoside hydrolase could make the resulting glycoside hydrolase more stable, i.e., less susceptible to proteolytic cleavage.

[0087] Family 1 CBMs contain a module of approximately 40 residues found almost exclusively in fungi. The cellulose-binding function has been demonstrated in many cases and appears to be mediated by three aromatic residues separated by approximately 10.4 angstroms and forming a flat surface. Family 1 CBMs also have a conserved C-terminal SQCL (SEQ ID NO: 201) that is thought to be important for binding affinity. Surprisingly, the inventors found that modifications in the carbohydrate-binding module, including modifications corresponding to Ser and / or Leu in the C-terminal SQCL (SEQ ID NO: 201), resulted in improved stability without sacrificing cellulose-binding activity.

[0088] The inventors have also surprisingly found that modifications in the carbohydrate binding module, including modifications corresponding to Ser and / or Leu of the C-terminal SQCL (SEQ ID NO: 201), result in improved stability to glycoside hydrolase enzymes containing the carbohydrate binding module variants without sacrificing the glycoside hydrolase activity of the polypeptide.

[0089] Carbohydrate-binding module variants The present invention relates to variants of carbohydrate binding modules comprising substitutions at one or more positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, 37 of the polypeptide of SEQ ID NO: 173. Variants of CBMs comprise an amino acid sequence that is about 60%, e.g., 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% sequence identity, but less than 100%, to the polypeptide of SEQ ID NO: 173, and have cellulose binding activity.

[0090] Exemplary starting CBMs useful for modification to result in variants according to the invention are those provided in any of SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, or SEQ ID NO:200. Thus, in one embodiment, variants of the carbohydrate binding module are selected from the group consisting of SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:1 96, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, or SEQ ID NO:200, having 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 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, but less than 100% sequence identity, to the polypeptide of SEQ ID NO:199, SEQ ID NO:200.

[0091] In one aspect, the number of modifications in a variant of the invention is 1 to 20, for example 1 to 10 and 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications.

[0092] In another embodiment, the variant comprises substitutions at one or more positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37 of SEQ ID NO: 173. In another embodiment, the variant comprises substitutions at two positions corresponding to any of positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at three positions corresponding to any of positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at four positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at five positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at six positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at seven positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at eight positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at nine positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at ten positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37. In another embodiment, the variant comprises substitutions at positions corresponding to 14, 18, 21, 22, 25, 27, 28, 29, 34, and 37.

[0093] The present invention also relates to variants of carbohydrate binding modules comprising substitutions at one or more positions, preferably both positions, corresponding to positions 34 and 37 of the polypeptide of SEQ ID NO: 173. Variants of CBMs comprise an amino acid sequence that is about 60%, e.g., 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% sequence identity, but less than 100%, to the polypeptide of SEQ ID NO: 173, and have cellulose binding activity.

[0094] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at position 14 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at position 14 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Trp. In another embodiment, the variant comprises or consists of the substitution Y14W in the polypeptide of SEQ ID NO:173.

[0095] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at position 18 of the polypeptide of SEQ ID NO: 173. The CBM variant comprises an amino acid sequence that is about 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at position 18 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another aspect, the variant comprises or consists of the substitution T18K or T18R in the polypeptide of SEQ ID NO: 173.

[0096] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at position 21 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at position 21 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Glu. In another embodiment, the variant comprises or consists of the substitution V21E of the polypeptide of SEQ ID NO:173.

[0097] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 22 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 22 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Glu, Asn, or Pro. In another embodiment, the variant comprises or consists of the substitution A22E or A22N or A22P in the polypeptide of SEQ ID NO:173.

[0098] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 25 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 25 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Glu. In another embodiment, the variant comprises or consists of the substitution T25E in the polypeptide of SEQ ID NO:173.

[0099] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 27 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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% sequence identity, but less than 100%, to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 27 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Ile. In another embodiment, the variant comprises or consists of the substitution T27I in the polypeptide of SEQ ID NO:173.

[0100] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at position 28 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at position 28 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro. In another embodiment, the variant comprises or consists of the substitution Q28P in the polypeptide of SEQ ID NO:173.

[0101] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 29 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 29 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro. In another embodiment, the variant comprises or consists of the substitution L29P of the polypeptide of SEQ ID NO:173.

[0102] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 34 of the polypeptide of SEQ ID NO: 173. The CBM variant comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 34 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably His, Tyr, Lys, or Arg. In another embodiment, the variant comprises or consists of the substitutions S34H, S34Y, S34K, S34R of the polypeptide of SEQ ID NO:173.

[0103] The present invention also relates to variants of carbohydrate-binding modules comprising a substitution at a position corresponding to position 37 of the polypeptide of SEQ ID NO: 173. The variant CBM comprises an amino acid sequence that is about 60%, e.g., 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%, but less than 100%, identical to the polypeptide of SEQ ID NO: 173, and has cellulose-binding activity. In one embodiment, the amino acid at the position corresponding to position 37 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another embodiment, the variant comprises or consists of the substitution L37R of the polypeptide of SEQ ID NO:173.

[0104] In another aspect, the variant comprises or consists of one or more substitutions selected from the group consisting of Y14W, T18K, T18R, V21E, A22E, A22N, A22P, T25E, T27I, Q28P, L29P, S34H, S34Y, S34K, S34R, and L37R of the polypeptide of SEQ ID NO: 173.

[0105] A preferred CBM variant is the CBM variant of SEQ ID NO: 173 S34H, S34Y, S34K, S34R, Y14W, T18K, L37R, S34Y+L37R, T18K+S34H, S34H+L37R, T18K+S34Y+L37R, T18R, V21E, A22E, A22P, A22N, T25E, T27I, Q28P, L29P, S34H+L37R+Y14W, S34H+L37R+T18K, S34H+L37R+Y14W+T18K, S34H+L37R+T18R, S34H+L37R+V21E, S34H+L37R+A22E, S34H+L37R+A22P, S34H+L37R+A22N, S34H+L37R+Q28P, S34H+L37R+L29P, S34H+L37R+T25E, S34H+L37R+T27I, S34H+L37R+V21E+A22E, S34H+L37R+V21E+A22Nぁ Y14W+T18R+S34H+L37Rぁ Y14W+V21E+S34H+L37Rぁ Y14W+A22E+S34H+L37Rぁ Y14W+A22P+S34H+L37Rぁ Y14W+A22N+S34H+L37Rぁ Y14W+Q18P+S34H+L37Rぁ Y14W+L29P+S34H+L37Rぁ Y14W+T25E+S34H+L37Rぁ Y14W+T27I+S34H+L37Rぁ Y14W+V21E+A22E+S34H+L37Rぁ Y14W+V21E+A22N+S34H+L37Rぁ T18K+V21E+S34H+L37Rぁ T18K+A22E+S34H+L37Rぁ T18K+A22P+S34H+L37Rぁ T18K+A22N+S34H+L37Rぁ T18K+Q28P+S34H+L37Rぁ T18K+L29P+S34H+L37Rぁ T18K+T25E+S34H+L37Rぁ T18K+T27I+S34H+L37Rぁ T18K+V21E+A22E+S34H+L37Rぁ T18K+V21E+A22N+S34H+L37Rぁ Y14W+T18K+V21E+S34H+L37Rぁ Y14W+T18K+A22E+S34H+L37Rぁ Y14W+T18K+A22P+S34H+L37Rぁ Y14W+T18K+A22N+S34H+L37Rぁ Y14W+T18K+Q28P+S34H+L37Rぁ Y14W+T18K+L29P+S34H+L37Rぁ Y14W+T18K+T25E+S34H+L37Rぁ Y14W+T18K+T27I+S34H+L37R, Y14W+T18K+V21E+A22E+S34H+L37R, Y14W+T18K+A21E+A22N+S34H+L37R and has a substitution selected from the group consisting of:

[0106] Polypeptides with glycoside hydrolase activity containing carbohydrate-binding module variants The present invention also relates to variant polypeptides with glycoside hydrolase activity comprising one or more catalytic domains and carbohydrate-binding module variants from the polypeptides with glycoside hydrolase activity as described above, linked by one or more linkers, wherein the variant polypeptides with glycoside hydrolase activity exhibit improved linker stability and / or improved CBM stability compared to the parent polypeptide in aqueous compositions containing proteases.

[0107] As described above, glycoside hydrolases contain one or more catalytic domains and one or more carbohydrate-binding modules (CBMs), which are connected by one or more linker regions connecting these domains.

[0108] Particularly preferred enzymes are those with cellulase, such as endoglucanase, activity, particularly where the relevant catalytic domain is derived from an enzyme in the glycoside hydrolase family 45 (GH45) using the nomenclature of Henrissat et al., as outlined on the CAZY database available at cazy.org.

[0109] The catalytic domain may comprise a wild-type or a variant thereof.

[0110] In one embodiment, the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequence as set forth at positions 1-212 of SEQ ID NO:1.

[0111] In one embodiment, the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequence as set forth at positions 1 to 211 of SEQ ID NO:2.

[0112] In one embodiment, the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequence as set forth at positions 1-210 of SEQ ID NO:3.

[0113] In one embodiment, the catalytic domain comprises an amino acid sequence having at least 70% sequence identity, e.g., at least 75% sequence identity, at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequence as set forth at positions 1 to 210 of SEQ ID NO:4.

[0114] In one aspect, the catalytic domain further comprises several substitutions in the variants of the invention, between 2 and 20, for example between 2 and 10 and between 2 and 5, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0115] In another embodiment, the variant comprises or consists of two substitutions at a position selected from among positions corresponding to 25, 32, 41, 44, 56, 77, 104, 132, 134, 146, 147, 156, 162, 169, 183, 186, 194, or 201 in SEQ ID NO: 1. In another embodiment, the amino acid at this position is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0116] A preferred catalytic domain comprises SEQ ID NO: 5 with the mutations A32S S56A N134D A146D Q147R Q169Y F183V.

[0117] A polypeptide having glycoside hydrolase activity according to the present invention may comprise a naturally occurring linker.

[0118] In some embodiments, a variant polypeptide with glycoside hydrolase activity according to the invention comprises a proline-rich amino acid sequence linking the CBM1 variant and one or more catalytic cores.

[0119] A proline-rich linker as described herein comprises one or more Pro-Pro, Pro-Xaa (or Xaa-Pro), Xaa-Pro-Xaa or Xaa-Xaa-Pro (or Pro-Xaa-Xaa) units, such as PPPP (SEQ ID NO: 27), PXPX (SEQ ID NO: 98), XPXP (SEQ ID NO: 99), XPXXPX (SEQ ID NO: 100), XXPXXP (SEQ ID NO: 101), etc., and optionally further repeats.

[0120] For example, the linker region can contain at least 25% proline, e.g., at least 28% proline, at least 30% proline, at least 40% proline, at least 50% proline, such as at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% proline. In other embodiments, the linker contains at least 50% proline, such as at least 60%, at least 66%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%, and has an overall negative charge. For example, the linker region includes amino acids that are acidic in nature.

[0121] Preferably, the linker region has a length of at least 4 and no more than 30 amino acids, such as 4 to 28 amino acids, preferably 4 to 20 amino acids, or 4 to 10 amino acids, such as 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, or 10 amino acids.

[0122] An exemplary linker region comprises the following optional repeating motif: [P / S / T / R / K / D / E]P and P[S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E] (Sequence number 102) Contains one or more of the following: Other preferred linker regions include the following optionally repeating motif: [S / T / R / K / D / E]P[S / T / R / K / D / E / N / Q] [P / S / T / R / K / D / E][P / S / T / R / K / D / E]P, and / or P[P / S / T / R / K / D / E][P / S / T / R / K / D / E] Includes: Particularly preferred linkers include optionally repeating motifs of the same or different amino acids within square brackets as shown: [P / S / T]P and P[S / E]PT (sequence number 109). Or more specifically, optional repeating motifs represented by [P / S / T]P would include PPPPPP (SEQ ID NO:29), and PPSPTP (SEQ ID NO:110), PPTPTP (SEQ ID NO:111), PPSPSP (SEQ ID NO:112), SPPPTP (SEQ ID NO:113), SPTPPP (SEQ ID NO:114), SPPPPP (SEQ ID NO:115), SPTPTP (SEQ ID NO:116), TPPPSP (SEQ ID NO:117), TPSPPP (SEQ ID NO:118), TPPPPP (SEQ ID NO:119), TPSPSP (SEQ ID NO:120), and optional repeating motifs represented by P[S / E]PT (SEQ ID NO:109) would include PSPTPEPT (SEQ ID NO:121), PSPTPEPTPSPTPEPT (SEQ ID NO:122), PEPTPSPT (SEQ ID NO:123), PEPTPSPTPSPT (SEQ ID NO:124), etc.

[0123] Exemplary linkers further include: (a)(SP) a , a=2~10; (b)(PS) a , a=2~10; (c)P b , b=4 to 20, preferably 4 to 15; (d) (PEPT (SEQ ID NO: 125)) c , c=2~5; (e) (PSPT (SEQ ID NO: 104)) d , d=2~5; (f)(P[S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E](Sequence number 102)) e , e=2~5; (g)([S / T / R / K / D / E]P) f , f=2 to 10, preferably 2 to 5; (h)([S / T / R / K / D / E / N / Q]P[S / T / R / K / D / E]) g , g = 2~6; (i)([S / T / R / K / D / E / N / Q][S / T / R / K / D / E / N / Q]P) h , h=2~5; (j)(TP) i , i=2~10; (k)([S / T / P][S / T / P][S / T / P]) j , j=2~11; (l) and / or combinations thereof, and combinations of each monomer unit are contemplated.

[0124] When a combination of these motifs is included, the minimum repeating unit is a monomer unit. For example, the linker includes SPPEPT (SEQ ID NO: 126), SPPSPT (SEQ ID NO: 127), PSPEPT (SEQ ID NO: 128), and PSPSPT (SEQ ID NO: 129).

[0125] Additional exemplary linkers include SPSP (SEQ ID NO:130), SPSPSP (SEQ ID NO:131), SPSPSPSP (SEQ ID NO:132), SPSPSPSPSP (SEQ ID NO:58), SPSPSPSPSPSP (SEQ ID NO:133), SPSPSPSPSPSPSP (SEQ ID NO:134), SPSPSPSPSPSPSPSP (SEQ ID NO:135), PPPP (SEQ ID NO:27), PPPPP (SEQ ID NO:28), PPPPPP (SEQ ID NO:29), PPPPPPP (SEQ ID NO:31), PPPPPPPP (SEQ ID NO:136), PPPPPPPPP (SEQ ID NO:137), 137), PPPPPPPPPP (SEQ ID NO: 138), PPPPPPPPPPP (SEQ ID NO: 139), PPPPPPPPPPPP (SEQ ID NO: 140), PPPPPPPPPPPPP (SEQ ID NO: 141), PPPPPPPPPPPPPP (SEQ ID NO: 142), PPPPPPPPPPPPPPP (SEQ ID NO: 143), PEPTPEPT (SEQ ID NO: 144), PEPTPEPTPEPT (SEQ ID NO: 145), PEPTPEPTPEPTPEPT (SEQ ID NO: 146), PEPTPEPTPEPTPEPTPEPT (SEQ ID NO: 79), PSPTPSPT (SEQ ID NO: 147), Sequence number 147), PSPTPSPTPSPT (SEQ ID NO: 148), PSPTPSPTPSPTPSPT (SEQ ID NO: 149), PSPTPSPTPSPTPSPTPSPT (SEQ ID NO: 150), SPSSPS (SEQ ID NO: 151), SPSSPSSPS (SEQ ID NO: 152), SPSSPSSPSSPS (SEQ ID NO: 153), SPSSPSSPSSPSSPS (SEQ ID NO: 154), TPTTPT (SEQ ID NO: 155), TPTTPTTPT (SEQ ID NO: 156), TPTTPTTPTTPT (SEQ ID NO: 157), TPTTPTTPTTPTTPT (SEQ ID NO: 158), PEPTPRPTPEPTPRPT (SEQ ID NO: 159), PEPTPKPTPEPTPKPT (SEQ ID NO: 160), PEPTPQPTPEPTPQPT (SEQ ID NO: 161), PRPTPEPTPRPT (SEQ ID NO: 162), PKPTPEPTPKPT (SEQ ID NO: 163), PEPTPQPT (SEQ ID NO: 164), PEPTPQPTPEPT (SEQ ID NO: 165), PEPTPRPTPEPTPRPTG (SEQ ID NO: 85), PEPTPKPTPEPTPKPTG (SEQ ID NO: 87), PEPTPQPTPEPTPQPTG (SEQ ID NO: 88),PRPTPEPTPRPTG (SEQ ID NO: 89), PKPTPEPTPKPTG (SEQ ID NO: 90), PEPTPQPTG (SEQ ID NO: 91), PEPTPQPTPEPTG (SEQ ID NO: 92), PPPGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 82), TTPPTPTPTPTPTP (SEQ ID NO: 166); TTPTPPTPTPTPTPTP (SEQ ID NO: 167), TTPTPTPPTPTPTPTPTP (SEQ ID NO: 168), TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPP (SEQ ID NO: 169).

[0126] Additional exemplary linkers include the above linkers and a C-terminal glycine, such as SPSPG (SEQ ID NO: 24), SPSPSPG, SPSPSPSPG, SPSPSPSPSPG (SEQ ID NO: 25), SPSPSPSPSPSPG, SPSPSPSPSPSPG, SPSPSPSPSPSPG, SPSPSPSPSPSPSPG, PPPPG, PPPPPG, PPPPPPG, PPPPPPPG (SEQ ID NO: 30), PPPPPPPPG (SEQ ID NO: 32), PPPPPPPPPG (SEQ ID NO: 33), PPPPPPPPPPG (SEQ ID NO: 34), Sequence number 34), PPPPPPPPPPPG (SEQ ID NO: 35), PPPPPPPPPPPPG (SEQ ID NO: 170), PPPPPPPPPPPPPG (SEQ ID NO: 36), PPPPPPPPPPPPPPG (SEQ ID NO: 171), PPPPPPPPPPPPPPPG (SEQ ID NO: 172), PEPTPEPTG (SEQ ID NO: 37), PEPTPEPTPEPTG (SEQ ID NO: 38), PEPTPEPTPEPTPEPTG (SEQ ID NO: 39), PEPTPEPTPEPTPEPTPTG (SEQ ID NO: 40), PSPTP SPTG, PSPTPSPTPSPTTG, PSPTPSPTPSPTPSPTTG (SEQ ID NO: 41), PSPTPSPTPSPTPSPTPSPTTG (SEQ ID NO: 42), SPSSPSG (SEQ ID NO: 94), SPSSPSSPSG (SEQ ID NO: 95), SPSSPSSPSSPSSG (SEQ ID NO: 19), SPSSPSSPSSPSSPSG (SEQ ID NO: 20), TPTTPTG (SEQ ID NO: 96), TPTTPTTPTG (SEQ ID NO: 97), TPTTPTTPTTPTTG (SEQ ID NO: 17), TPTTPTTPTTPT TPTG, PEPTPRPTPEPTPRPTG (SEQ ID NO: 85), PEPTPKPTPEPTPKPTG (SEQ ID NO: 87), PEPTPQPTPEPTPQPTG (SEQ ID NO: 88), PRPTPEPTPRPTG (SEQ ID NO: 89), PKPTPEPTPKPTG (SEQ ID NO: 90), PEPTPQPTG (SEQ ID NO: 91), PEPTPQPTPEPTG (SEQ ID NO: 92), PPPGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 82), TTPPTTPTPTPPG (SEQ ID NO: 12);TTPTPPTPTPTPTPG (SEQ ID NO: 13), TTPTPTPPTPTPTPTPTPPG (SEQ ID NO: 14), TTPTPTPTPPTPTPTPTPTPPG (SEQ ID NO: 15), TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPG (SEQ ID NO: 16);

[0127] Particularly preferred linkers are those that contain predominantly or exclusively proline, such as PPPP (SEQ ID NO:27), PPPPP (SEQ ID NO:28), PPPPPP (SEQ ID NO:29), PPPPPPP (SEQ ID NO:31), PPPPPPPP (SEQ ID NO:136), PPPPPPPPP (SEQ ID NO:137), PPPPPPPPPP (SEQ ID NO:138), PPPPPPPPPPP (SEQ ID NO:139), PPPPPPPPPPPP (SEQ ID NO:140), PPPPPPPPPPPPP (SEQ ID NO:141), PPPPPPPPPPPPPP (SEQ ID NO:142). , PPPPPPPPPPPPPPP (SEQ ID NO: 143), PPPPG, PPPPPG, PPPPPPG, PPPPPPPG (SEQ ID NO: 30), PPPPPPPPG (SEQ ID NO: 32), PPPPPPPPPG (SEQ ID NO: 33), PPPPPPPPPPG (SEQ ID NO: 34), PPPPPPPPPPPG (SEQ ID NO: 35), PPPPPPPPPPPPG (SEQ ID NO: 170), PPPPPPPPPPPPPG (SEQ ID NO: 36), PPPPPPPPPPPPPPG (SEQ ID NO: 171), PPPPPPPPPPPPPPPG (SEQ ID NO: 172).

[0128] With respect to the embodiments contemplated above, one of skill in the art will understand that the goal is to replace the parent linker of interest with a proline-rich linker herein that provides additional stability.

[0129] In an alternative embodiment, the linker can be considered as a variant of the linker of the parent molecule with a stabilizing point mutation, including a mutation to proline.

[0130] In particularly preferred embodiments, the linker is selected from any of the following in Table A: Table A. Preferred Linkers TTPPTPTPTPTPG (SEQ ID NO: 12) TTPTPPTPTPTPTPG (SEQ ID NO: 13) TTPTPTPPTPTPTPTPG (SEQ ID NO: 14) TTPTPTPTPPTPTPTPTPG (SEQ ID NO: 15) TPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPTPPG (SEQ ID NO: 16) TPTTPTTPTTPTG (SEQ ID NO: 17) TPTTPTTPTTPTTPTTPTG (SEQ ID NO: 18) SPSSPSSPSSPSG (SEQ ID NO: 19) SPSSPSSPSSPSSPSG (SEQ ID NO: 20) SPPSPPSPPSPPSPPG (SEQ ID NO: 21) SPPSPPSPPSPPSPPSPPSPPSPPSPPSPPSPPG (SEQ ID NO: 22) PPSSPSSPSSPSSPSSPSSPSG (SEQ ID NO: 23) SPSPG (SEQ ID NO: 24) SPSPSPSPSPG (SEQ ID NO: 25) TPTPTPTPTPG (SEQ ID NO: 26) PPPP (SEQ ID NO: 27) PPPPP (SEQ ID NO:28) PPPPPP (SEQ ID NO: 29) PPPPPPPG (SEQ ID NO: 30) PPPPPPP (SEQ ID NO:31) PPPPPPPPG (SEQ ID NO: 32) PPPPPPPPPG (SEQ ID NO: 33) PPPPPPPPPPG (SEQ ID NO: 34) PPPPPPPPPPPG (SEQ ID NO: 35) PPPPPPPPPPPPPG (SEQ ID NO: 36) PEPTPEPTG (SEQ ID NO: 37) PEPTPEPTPEPTG (SEQ ID NO: 38) PEPTPEPTPEPTPEPTG (SEQ ID NO: 39) PEPTPEPTPEPTPEPTPEPTG (SEQ ID NO: 40) PSPTPSPTPSPTPSPTG (SEQ ID NO: 41) PSPTPSPTPSPTPSPTPSPTPSPTG (SEQ ID NO: 42) PQPTPQPTG (SEQ ID NO: 43) PDPTPDPTG (SEQ ID NO: 44) PRPTPEPTG (SEQ ID NO: 45) PQPTPEPTG (SEQ ID NO: 46) PSPNSPNSPNG (SEQ ID NO: 47) PEPTPRPTG (SEQ ID NO: 48) PQPTPEPTPQPTPEPTPQPTPEPTPQPTG (SEQ ID NO: 49) PDPTPDPTPDPTG (SEQ ID NO: 50) PQPTPQPTPQPTPQPTG (SEQ ID NO: 51) PQPTPEPTPQPTPEPTG (SEQ ID NO: 52) SPSPSPSPPPG (SEQ ID NO: 53) SPSPSPSPDPG (SEQ ID NO: 54) SPSPSPSPKPG (SEQ ID NO: 55) SPSPSPSPAPG (SEQ ID NO: 56) SPSPSPSPSPSG (SEQ ID NO: 57) SPSPSPSPSPSP (SEQ ID NO: 58) SPSPSPSPSPS (SEQ ID NO: 59) SPSPSPSPSPP (SEQ ID NO: 60) SPSPSPSPSPE (SEQ ID NO: 61) SPSPSPSPSPN (SEQ ID NO: 62) SPSPSPSPSPGG (SEQ ID NO: 63) SPSPSPSPSPK (SEQ ID NO: 64) PEPTPEPTP (SEQ ID NO: 65) PEPTPEPTR (SEQ ID NO: 66) PEPTPEPTPEPTP (SEQ ID NO: 67) PEPTPEPTPEPTPEPTPSPTG (SEQ ID NO: 68) PEPTPEPTPEPTPEPTPTPTG (SEQ ID NO: 69) PEPTPEPTPEPTPEPTPGPTG (SEQ ID NO: 70) PEPTPEPTPEPTPEPTPDPTG (SEQ ID NO: 71) PEPTPEPTPEPTPEPTPETG (SEQ ID NO: 72) PEPTPEPTPEPTPEPTPEPTD (SEQ ID NO: 73) PEPTPEPTE (SEQ ID NO: 74) PEPTPEPTPEPTPEPTPEP (SEQ ID NO: 75) PEPTPEPTPEPTPEPTPSPT (SEQ ID NO: 76) PEPTPEPTPEPTPEPTPRPTT (SEQ ID NO: 77) PEPTPEPTPEPTPEPTPEPTT (SEQ ID NO: 78) PEPTPEPTPEPTPEPTPEPT (SEQ ID NO: 79) PEPTPEPTPEPTPEPTPEPTS (SEQ ID NO: 80) PEPTPEPTPEPTPEPTPEPTR (SEQ ID NO: 81) PPPGGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 82) PPPGGPGGTGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 83) PPSGGPGGPGTPTSTAPGSGPTSPGGGSG (SEQ ID NO: 84) PEPTPRPTPEPTPRPTG (SEQ ID NO: 85) PKPTPEPTPKPTPEPTG (SEQ ID NO: 86) PEPTPKPTPEPTPKPTG (SEQ ID NO: 87) PEPTPQPTPEPTPQPTG (SEQ ID NO: 88) PRPTPEPTPRPTG (SEQ ID NO: 89) PKPTPEPTPKPTG (SEQ ID NO: 90) PEPTPQPTG (SEQ ID NO: 91) PEPTPQPTPEPTG (SEQ ID NO: 92) TPPTPPG (SEQ ID NO: 93) SPSSPSG (SEQ ID NO: 94) SPSSPSSPSG (SEQ ID NO: 95) TPTTPTG (SEQ ID NO: 96) TPTTPTTPTG (SEQ ID NO: 97)

[0131] In certain embodiments, the linker is PPPPPPP (SEQ ID NO: 31), PPPPPPPG (SEQ ID NO: 30), SPSPSPSPSP (SEQ ID NO: 58), or SPSPSPSPSPG (SEQ ID NO: 25).

[0132] In some embodiments, the variants of the invention have improved properties relative to the reference / parent enzyme.

[0133] In one aspect, the improved property is increased stability, e.g., improved proteolytic stability, improved detergent stability, improved stability during washing, or improved thermal stability. In another aspect, the improved property is increased stability during production of a detergent composition or increased performance after storage in a detergent composition relative to the performance of the parent molecule stored under similar conditions. Some aspects of the invention relate to cellulase variants that have an improvement factor of greater than 1 when the cellulase variants are tested for the property of interest in a suitable assay, where the property of the reference enzyme / parent enzyme is assigned a value of 1. In some aspects, the property is stability, such as improved proteolytic stability. Some aspects of the invention relate to cellulase variants that have an improvement factor of greater than 1 when the cellulase variants are tested for the property of interest in the assay described in Example 2, where the property of the reference enzyme / parent enzyme is assigned a value of 1. In some aspects, the property is stability, such as proteolytic stability.

[0134] In some embodiments, the improved property is increased stability, e.g., improved detergent stability, improved stability during washing, and improved thermal stability. Some embodiments of the invention relate to cellulase variants that have an improvement factor of greater than 1 when the cellulase variant is tested for the property of interest in an appropriate assay, where the property of the reference enzyme / parent enzyme is assigned a value of 1, such as when the cellulase variant is tested for the property of interest in the assay described in Example 7.

[0135] In some embodiments, the improved property is improved thermal stability.

[0136] In some embodiments, the improved property is improved stability in detergents.

[0137] In some embodiments, the improved property is improved proteolytic stability.

[0138] In some embodiments, the improved properties are one or more or all of improved thermal stability, improved detergent stability, and improved proteolytic stability.

[0139] A variant according to the invention is improved under the conditions measured when the residual activity ratio, defined as the residual activity ratio (RAR) = (RA, variant) / (RA, reference), is greater than 1.0 compared to the reference cellulase.

[0140] In certain preferred aspects, variants according to the invention provide improved stability (eg, thermal stability, detergent stability, proteolytic stability, or two or more or all of these) and have an RAR>1.0. In some embodiments, a variant according to the invention has a β-glucanase activity of at least 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.1; 2.2; 2.3; 2.4; 2.5; 2.6; 2.7; 2.8; 2.9; 3.0, 3.1; 3.2; 3.3; 3.4; 3.5, 3.6, 3.7, 3.8, 3.9; 4.0, 4.1; 4.2; 4.3; 4.4; 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1; 5.2; 5.3; 5.4; 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.1, 11.2, 5.6, 5.7, 5.8, 5.9; 3.0, 6.1; 6.2; 6.3; 6.4; 6.5, 6.6, 6.7, 6.8, 6.9; 7.0, 7.1; 7.2; 7.3; 7.4; 7.5, 7.6, 7.7, 7.8, 7.9; 8.0, 8.1; 8.2; 8.3; 8.4; 8.5, 8.6, 8.7, 8.8 , 8.9; 9.0, 9.1; 9.2; 9.3; 9.4; 9.5, 9.6, 9.7, 9.8, 9.9; 10.0, 10.1; 10.2; 10.3; 10.4; 10.5, 10.6, 10.7, 10.8, 10.9; 12, 15, 16, 20, 25 or 30.

[0141] One preferred embodiment relates to cellulase variants with improved stability, wherein the RAR is >1.0 compared to SEQ ID NO: 1. One preferred embodiment relates to cellulase variants with improved stability, wherein the residual activity ratio (RAR) is at least 1.5 compared to SEQ ID NO: 1, as measured as described in Example 2.

[0142] A variant may further include one or more additional modifications at one or more (eg, several) other positions.

[0143] 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.

[0144] 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, in H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0145] 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.

[0146] 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). In the latter technique, single alanine mutations are introduced at every residue in the molecule to identify amino acid residues critical to the molecule's activity, and the resulting mutant molecules are tested for cellulolytic activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of 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 identity of essential amino acids can also be inferred from alignments with related polypeptides.

[0147] Table B provides exemplary preferred glycoside hydrolase variants incorporating CBM variants according to the present invention and is provided in tabular form for ease of comparative reference. As used in tables herein, variants are represented as a whole in N- to C-terminal order, with no additional linkers or further modifications between the sequences specified in each row. Thus, variant 1 represented in the table below can equivalently be represented as the following sequence:

[0148] [ka]

[0149] [Table 2]

[0150] [Table 3]

[0151] [Table 4]

[0152] [Table 5]

[0153] [Table 6]

[0154] Stability in the presence of proteases In some embodiments, variants with glycoside hydrolase activity, including CBM variants, have improved stability in the presence of proteases compared to the parent enzyme. Preferably, the variants have improved stability in the presence of proteases and surfactants, such as detergent compositions, compared to the parent cellulase.

[0155] Stability in the presence of proteases is beneficial for cellulases used in the presence of, for example, proteases, because it extends the time over which the cellulases remain functional, active, and capable of performing their intended function.

[0156] One preferred use of the variants of the present invention is in detergents where proteases are typically included to improve detergent performance. The improved stability of the variants of the present invention means that the variants can exert cellulolytic activity for a longer period during the laundry process compared to the parent cellulase, thereby providing the benefit of improved cleaning performance compared to the parent cellulase.

[0157] With respect to liquid detergent compositions, the variants of the present invention have the further advantage that their improved stability in the presence of proteases means that liquid detergent compositions comprising a protease and further comprising a variant of the present invention will have a longer shelf life compared to the same liquid detergent composition comprising the parent cellulase.

[0158] Stability in the presence of a protease can be determined by incubating a given cellulase under defined conditions in the presence of the protease, measuring the cellulolytic activity after incubation, and comparing it to a sample of cellulase that has not been incubated with the protease.

[0159] Another method for determining stability in the presence of proteases involves preparing two identical test tubes containing a given cellulase to be tested in a defined solution containing the protease, and incubating one test tube at an elevated temperature, e.g., between 30 and 90°C (stressed), while the other tube is incubated at a lower temperature, e.g., between 0 and 5°C (unstressed). The tubes are incubated for a predetermined time, e.g., between 1 and 24 hours, typically 16 hours. After incubation, both samples are analyzed for cellulolytic activity, and the remaining activity is Residual activity (%) = (active, loaded / active, unloaded) * 100 is determined as follows.

[0160] For example, residual activity can be determined in 50% liquid detergent A containing 0.166 v / v-% protease, with samples incubated for 16 h at elevated temperature (loaded) and 5°C (unloaded) before activity is determined. The temperature should be chosen so that the residual activity of the parent molecule is in the range of 10-50%.

[0161] This core stability method is shown in more detail in Example 1.

[0162] The variants of the present invention have higher residual activity than the parent cellulase, in one embodiment, the variants of the present invention have at least 10% higher residual activity compared to the parent cellulase, such as at least 20% higher residual activity compared to the parent, such as at least 30% higher residual activity, for example, at least 40% higher residual activity, for example, at least 50% higher residual activity, such as at least 60% higher residual activity, for example, at least 70% higher residual activity, for example, at least 80% higher residual activity, for example, at least 90% higher residual activity or at least 100% higher residual activity.

[0163] However, in traditional enzyme stability assays used to test thermal stability, activity measurements of loaded and unloaded samples typically focus on measuring changes affecting the catalytic site of the enzyme molecule, for example, by using small synthetic substrates such as 4-methylumbelliferyl-β-cellopentaoside or soluble carboxymethylcellulose (CMC).

[0164] Importantly, however, changes in other properties of the enzyme of interest due to loading (properties that do not directly affect the enzyme's active site but are important for the enzyme's function in the application) are not necessarily detected in these assays. One such example is a glycosyl hydrolase that has a separate catalytic domain and CBM connected by a linker, such as the case of cellulases used to remove fluff and pilling in laundry detergents and fabric care products. If loading only affects the linker and / or CBM portions of the molecule and not the catalytic domain portion, these changes will not be detected by conventional assays such as those described above and / or in Example 1. When using sample substrates such as CMC or 4-methylumbelliferyl-β-cellopentaoside, activity appears to be maintained during loading, but performance is significantly affected because the CBM portion of the enzyme molecule plays a key role in directing the enzyme to the appropriate location on the textile to be treated.

[0165] Alternatively, the importance of the CBM with respect to performance can be tested by comparing the performance of the catalytic domain with that of a catalytic domain with an intact linker and CBM.

[0166] To detect changes in the linker and / or CBM after storage under stressed conditions, special measures must be taken when testing if stress is affecting enzyme performance. This can be done by comparing the performance of the enzyme before and after stressing. Alternatively, it can be tested by ensuring that binding of the enzyme to its native insoluble substrate, such as cotton linters, is included as part of the assay used to test stability and / or first probe for binding of the enzyme to crystalline cellulose or cotton linters, and then measure the activity of the enzymes that have lost their ability to bind cellulose compared to total activity.

[0167] Thus, the stability of the linker and / or CBM is measured by incubating the cellulase in a detergent containing a protease and then determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose binding domain is affected by the protease, the binding affinity of the cellulase to cellulose fibers will be reduced.

[0168] An assay specific for this linker and CBM is illustrated by the conditions described in Example 2.

[0169] The parent polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or C-terminus to a region of another polypeptide.

[0170] The parent may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are 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 joining 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 generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0171] The fusion polypeptide may further comprise a cleavage site between the two polypeptides that is cleaved to release the two polypeptides upon secretion of the fusion protein. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 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.

[0172] The parent may be obtained from any genus of microorganism. For purposes of the present invention, the term "obtained from," when used herein in reference to a given source, means that the parent encoded by the polynucleotide is produced by the source or produced by a strain into which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly. The parent can be a bacterial cellulase. For example, the parent can be a Gram-positive cellulase such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cellulase. The cellulase may be a gram-negative bacterial polypeptide, such as a bacterial polypeptide or a gram-negative bacterial polypeptide, such as a cellulase from Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma.

[0173] In one aspect, the parent is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus The cellulase is derived from Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

[0174] In another embodiment, the parent is a cellulase from Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus.

[0175] In another embodiment, the parent is a cellulase from Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans.

[0176] The parent can be a fungal cellulase, for example, a yeast cellulase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cellulase; or a cellulase from the Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, or the like. Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria ctria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe naporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia,The cellulase may be a filamentous fungal cellulase such as a cellulase from the genus Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

[0177] In another embodiment, the parent is a cellulase from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0178] In another embodiment, the parent is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, or the like. keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia phimeci fimeti), Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosasetosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0179] In another aspect, the parent is a Thielavia terrestris cellulase, such as the cellulase of SEQ ID NO: 1 or a mature polypeptide thereof.

[0180] With respect to the aforementioned species, it will be understood that the present invention encompasses other taxonomic equivalents, e.g., anamorphs, in both perfect and imperfect states and regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0181] Strains of these species are readily publicly available in several culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0182] Parents may also be identified and obtained using the probes described above from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural sources (e.g., soil, compost, water, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Polynucleotides encoding the parents may then be obtained by similarly screening genomic DNA or cDNA libraries of other microorganisms or mixed DNA samples. After detecting the polynucleotides encoding the parents using one or more probes, the polynucleotides can be isolated or cloned using techniques known to those skilled in the art (see, e.g., Sambrook et al., 1989, supra).

[0183] Preparation of variants The present invention also relates to a method for obtaining a variant having glycoside hydrolase activity, comprising: (a) introducing into a parent glycoside hydrolase one or more substitutions in the mature polypeptide of the parent polypeptide at positions corresponding to positions 14, 18, 21, 22, 25, 27, 28, 29, 34 and / or 37 of CBM1 using SEQ ID NO: 173 for alignment; and (b) recovering the variant.

[0184] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0185] Site-directed mutagenesis is a technique in which one or more (eg, several) mutations are introduced at one or more defined sites in a parent coding polynucleotide.

[0186] Site-directed mutagenesis can be achieved in vitro by PCR, involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, involving restriction enzyme cleavage at a site in a plasmid containing a parent encoding polynucleotide, followed by ligation of an oligonucleotide containing the mutation in this polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, and the cohesive ends of the plasmid and insert can be ligated together. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0187] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, e.g., U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16).

[0188] Any site-directed mutagenesis procedure can be used in the present invention. There are many commercially available kits that can be used to prepare variants.

[0189] Synthetic gene construction involves the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, including the multiplexed microchip-based technology described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques in which oligonucleotides are synthesized and assembled on an optically programmable microfluidic chip.

[0190] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods followed by associated screening procedures such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30; 10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46; 145; Ner et al., 1988, DNA 7; 127).

[0191] The mutagenesis / shuffling method can be combined with a high-throughput automated screening method to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and easily sequenced using standard methods in the art. This method allows for the rapid determination of the importance of individual amino acid residues in the polypeptide.

[0192] Semisynthetic gene construction is achieved by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semisynthetic construction is typified by a process that utilizes synthesized polynucleotide fragments combined with PCR technology. Thus, defined regions of a gene may be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, and still other regions may be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.

[0193] Polynucleotides The present invention also relates to polynucleotides encoding the variants of the invention.

[0194] nucleic acid construct The present invention also relates to a nucleic acid construct comprising a polynucleotide encoding a variant of the invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0195] Polynucleotides can be manipulated in a variety of ways to result in expression of variants. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0196] The control sequence may be a promoter (a polynucleotide recognized by a host cell for expression of a polynucleotide). A promoter contains transcriptional control sequences that mediate expression of the variant. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including variant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host.

[0197] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in bacterial host cells include the α-amylase gene (amyQ) of Bacillus amyloliquefaciens, the α-amylase gene (amyL) of Bacillus licheniformis, the penicillinase gene (penP) of Bacillus licheniformis, the maltogenic amylase gene (amyM) of Bacillus stearothermophilus, the levansucrase gene (sacB) of Bacillus subtilis, the xylA and xylB genes of Bacillus subtilis, the xylB gene of Bacillus thuringiensis, and the α-amylase gene (amyL) of Bacillus amyloliquefaciens. These promoters include those derived from the cryIIIA gene of Bacillus thuringiensis (Agaisse and Lereclus, 1994, Molecular Microbiology 13:97-107), the Lac operon of Escherichia coli (E. coli), the trc promoter of E. coli (Egon et al., 1988, Gene 69:301-315), the agarase gene (dagA) of Streptomyces coelicolor, and prokaryotic β-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in "Useful proteins from recombinant bacteria" by Gilbert et al., 1980, Scientific American 242:74-94; and Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0198] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell include those encoding Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease from Fusarium oxysporum (WO 96 / 00787), amyloglucosidase from Fusarium venenatum (WO 00 / 56900), Daria from Fusarium venenatum (WO 00 / 56900), Quinn from Fusarium venenatum (WO 00 / 56900), lipase from Rhizomucor miehei, aspartic proteinase from Rhizomucor miehei, beta-glucosidase from Trichoderma reesei, Trichoderma Cellobiohydrolase I from Trichoderma reesei, Cellobiohydrolase II from Trichoderma reesei, Endoglucanase I from Trichoderma reesei, Endoglucanase II from Trichoderma reesei,promoters obtained from Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, and the NA2-tpi promoter (a modified promoter from the Aspergillus neutral alpha-amylase gene in which the non-translated leader has been replaced by the non-translated leader from the Aspergillus triose phosphate isomerase gene; a non-limiting example is the NA2-tpi promoter, which is a modified promoter from the Aspergillus neutral alpha-amylase gene in which the non-translated leader has been replaced by the non-translated leader from the Aspergillus triose phosphate isomerase gene; a non-limiting example is the NA2-tpi promoter, which is a modified promoter from the Aspergillus neutral alpha-amylase gene in which the non-translated leader has been replaced by the non-translated leader from the Aspergillus triose phosphate isomerase gene; modified promoters derived from the neutral alpha-amylase gene of Aspergillus niger replaced by the untranslated leader derived from the triose phosphate isomerase gene of Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated and hybrid promoters thereof.

[0199] In yeast hosts, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0200] The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the polynucleotide encoding the variant. Any terminator functional in the host cell may be used.

[0201] Preferred terminators for bacterial host cells are obtained from the genes for alkaline protease (aprH) of Bacillus clausii, α-amylase (amyL) of Bacillus licheniformis, and ribosomal RNA (rrnB) of Escherichia coli.

[0202] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0203] Preferred terminators for yeast hosts are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0204] A regulatory sequence can also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0205] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0206] The control sequence may also be a leader, untranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0207] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0208] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0209] The regulatory sequence may also be a polyadenylation sequence operably linked to the 3' end of the variant coding sequence, which, when transcribed, is recognized as a signal by the host cell and adds polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0210] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0211] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0212] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant into the secretory pathway of the cell. The 5' end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence that is naturally linked in translation reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0213] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0214] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0215] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0216] The regulatory sequence may also be a polypeptide coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes a proenzyme). Propolypeptides are generally inactive and can be converted to active polypeptides by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences may be obtained from the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, laccase of Myceliophthora thermophila (WO 95 / 33836), aspartic proteinase of Rhizomucor miehei, and α-factor of Saccharomyces cerevisiae.

[0217] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the variant and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0218] It may also be desirable to add regulatory sequences that control expression of the variant relative to growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein gene, which is amplified by heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked to the regulatory sequence.

[0219] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences may be ligated together to create a recombinant expression vector that may contain one or more convenient restriction sites, allowing for the insertion or substitution of a polynucleotide encoding a variant at such sites. Alternatively, a polynucleotide may be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed within a vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0220] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0221] The vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal element independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids may be used that together contain the total DNA or transposon to be introduced into the genome of the host cell.

[0222] Vectors preferably contain one or more selectable markers which permit easy selection of transformed, transfected, transduced or like cells. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0223] Examples of bacterial selectable markers include the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance, such as ampicillin resistance, chloramphenicol resistance, kanamycin resistance, neomycin resistance, spectinomycin resistance, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and their equivalents. Preferred for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0224] The vector preferably contains elements that allow the vector to integrate into the genome of the host cell or to replicate autonomously within the cell independently of the genome.

[0225] For integration into the host cell genome, the vector may rely on the sequence of a polynucleotide encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides to direct integration into the host cell genome by homologous recombination at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration element should contain a sufficient number of nucleic acids (e.g., 100-10,000 base pairs, 400-10,000 base pairs, and 800-10,000 base pairs) that have high sequence identity to the corresponding target sequence to increase the likelihood of homologous recombination. The integration element may be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integration element may or may not encode a polynucleotide. Alternatively, the vector may integrate into the host cell genome by non-homologous recombination.

[0226] In the case of autonomous replication, the vector may further comprise an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid origin of replication that mediates autonomous replication and functions in cells. The term "origin of replication" or "plasmid origin of replication" refers to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0227] Examples of bacterial origins of replication are those of pBR322, pUC19, pACYC177, and pACYC184 plasmids capable of replicating in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 plasmids capable of replicating in Bacillus.

[0228] Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0229] Examples of replication origins useful in filamentous fungal cells include AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing this gene can be achieved according to the methods disclosed in WO 00 / 24883.

[0230] Two or more copies of the polynucleotide of the present invention can be inserted into a host cell to increase the production of variants. Increasing the copy number of the polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide, and culturing the cells in the presence of an appropriate selection agent allows selection of cells containing an amplified copy of the selectable marker gene, and therefore cells containing additional copies of the polynucleotide.

[0231] The procedures used to ligate the above elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (eg, Sambrook et al., 1989, supra).

[0232] host cell The present invention also relates to recombinant host cells comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that direct the production of the variant of the invention. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector as described above. The term "host cell" includes any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will largely depend on the gene encoding the variant and its source.

[0233] The host cell can be any cell useful in the recombinant production of the variant, for example, a prokaryote or a eukaryote.

[0234] Prokaryotic host cells can be any gram-positive or gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0235] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus spp. ... The cell may be any Bacillus cell, including cells of Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis.

[0236] The bacterial host cell can also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0237] The bacterial host cell can also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0238] DNA can be introduced into Bacillus cells by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli can be by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells can be by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294).Introduction of DNA into Pseudomonas cells can be by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57). DNA can be introduced into Streptococcus cells by natural transformation (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0239] The host cell can also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0240] The host cell may be a fungal cell. As used herein, "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming fungi (as defined by Hawksworth et al., in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0241] The fungal host cell may be a yeast cell. As used herein, "yeast" includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Because the classification of yeasts may change in the future, for purposes of the present invention, yeasts shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0242] Yeast host cells include cells of the genera Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, and the like. The cell may be a cell of Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica.

[0243] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Euglena and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0244] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nelumbo nucifera, and others. The cell may be a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma.

[0245] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girubescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolorThe cell may be a cell of Trichoderma versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0246] Fungal cells can be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transformation of Fusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using the procedures described by Becker and Guarente, in Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0247] Generation method The present invention also relates to a method for producing a variant, the method comprising: (a) culturing a host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

[0248] The host cells are cultured in a nutrient medium suitable for the production of the variant using methods known in the art. For example, the cells can be cultured in shake flask cultures in laboratory or industrial fermentors, or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) carried out in a suitable medium and under conditions that allow for the expression and / or isolation of the variant. The culture is carried out in a suitable nutrient medium containing a carbon source, a nitrogen source, and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., compositions published in catalogs of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0249] Variants can be detected using methods known in the art that are specific for the variants. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the variant.

[0250] The variants may be recovered using methods known in the art, for example, the variants may be recovered from the nutrient medium by conventional procedures including, but not limited to, harvesting, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0251] Variants can be purified by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), solubility differences (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.

[0252] In an alternative embodiment, the variants are not recovered, but rather host cells of the invention that express the variants are used as a source of the variants.

[0253] Detergent Composition In one embodiment, the present invention relates to detergent compositions comprising the enzymes of the present invention in combination with one or more additional cleaning composition components, the selection of which is within the skill of one in the art and includes conventional ingredients, including the exemplary, non-limiting components set forth below.

[0254] The selection of components may include consideration of the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning will occur, and the detergent product formulation for fabric care. The components below are categorized under general headings according to specific functionality, but this should not be construed as limiting, as the components may include additional functionality, as will be understood by those skilled in the art.

[0255] In one embodiment, the present invention relates to a liquid laundry detergent composition comprising an enzyme of the present invention in combination with one or more additional laundry detergent composition components, particularly a protease. In another embodiment, the present invention includes ancillary products used in laundry, such as pre-spotters or stain removal boosters. The present invention also relates to ADW (automatic dishwashing) compositions comprising an enzyme of the present invention in combination with one or more additional ADW composition components. The selection of additional components is within the skill of one in the art and includes conventional ingredients, including the exemplary, non-limiting components listed below.

[0256] In one embodiment of the present invention, a variant having glycoside hydrolase activity, including a CBM1 variant of the present invention, may be added to a detergent composition in an amount corresponding to 0.001 to 200 mg of protein, such as 0.005 to 100 mg of protein per liter of wash liquor, preferably 0.01 to 50 mg of protein, more preferably 0.05 to 20 mg of protein, and even more preferably 0.1 to 10 mg of protein.

[0257] The enzymes of the detergent compositions of the invention may be stabilized using conventional stabilisers, for example polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives, for example aromatic boric acid esters or phenyl boric acid derivatives such as 4-formylphenyl boric acid, and the compositions may be formulated as described, for example, in WO 92 / 19709 and WO 92 / 19708.

[0258] The polypeptides of the present invention may also be incorporated into detergent formulations as disclosed in WO 97 / 07202, which is incorporated herein by reference.

[0259] surfactants The detergent composition may include one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In certain embodiments, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactants are typically present at a level of about 5% to 60% by weight, such as about 5% to about 50%, or about 10% to about 50%, or about 20% to about 50%. The surfactant is selected based on the desired cleaning application and may include any conventional surfactant known in the art.

[0260] When included, detergents will typically contain from about 5% to about 60% by weight, such as from about 5% to about 40%, including from about 10% to about 25%, of one or more anionic surfactants, non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FA), and the like. S), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, α-sulfofatty acid methyl esters (α-SFMe or SES) including methyl ester sulfonates (MES), alkyl- or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, salts (soaps) of sulfosuccinic acid or fatty acids or di- and monoesters of fatty acids, and combinations thereof.

[0261] When included, detergents will typically contain from about 0.1% to about 10% by weight of a cationic surfactant, e.g., from about 0.1% to about 5%, non-limiting examples of cationic surfactants include alkyldimethylethanol quaternary amines (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, quaternary esters, and combinations thereof.

[0262] When included, detergents will typically contain from about 0.2% to about 60% by weight of a nonionic surfactant, for example, from about 1% to about 40%, particularly from about 5% to about 20%, or from about 3% to about 15%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters, such as ethoxylated fatty acid alkyl esters and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates, and the like. N-acyl N-alkyl derivatives of glucosamine (glucosamides, GA or fatty acid glucamides, FAGA), methyl ester ethoxylates (MEE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamides, GA or fatty acid glucamides, FAGA), methyl ester ethoxylates (MEE), and products available under the trade names SPAN and TWEEN, and combinations thereof.

[0263] When included, detergents will typically contain from about 0.1% to about 10% by weight of semi-polar surfactants. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) such as alkyl dimethyl amine oxide, N-(cocoalkyl)-N,N-dimethyl amine oxide, and N-(tallow-alkyl)-N,N-bis(2-hydroxyethyl) amine oxide, and combinations thereof.

[0264] When included, detergents will typically contain from about 0.1% to about 10% by weight of zwitterionic surfactants. Non-limiting examples of zwitterionic surfactants include betaines, such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.

[0265] Solvent system: A solvent system is required for dissolving surfactants and other detergent ingredients. The solvent is usually water, alcohol, polyol, sugar and / or a mixture thereof. Preferred solvents are water, glycerol, sorbitol, propylene glycol (MPG, 1,2-propanediol or 1,3-propanediol), dipropylene glycol (DPG), polyethylene glycol family (PEG 300-600), hexylene glycol, inositol, mannitol, ethanol, isopropanol, n-butoxypropoxypropanol, ethanolamines (monoethanolamine, diethanolamine and triethanolamine), sucrose, dextrose, glucose, ribose, xylose, and related mono- and dipyranosides and mono- and difuranosides.

[0266] The solvent system is typically present at 5-90 wt%, 5-60 wt%, 5-40 wt%, 10-30 wt% overall.

[0267] The water content for a unit dose wrapped in a PVA film typically ranges from 1-15%, 2-12%, 3-10%, 5-10%.

[0268] The polyol content for a unit dose wrapped in a PVA film is typically in the range of 5-50%, 10-40%, or 20-30%.

[0269] In certain embodiments, the surfactant is a non-naturally occurring surfactant.

[0270] Hydrotrope Hydrotropes are compounds that solubilize hydrophobic compounds in aqueous solutions (or substances of opposite polarity in a nonpolar environment). Hydrotropes typically possess both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants). However, the molecular structure of hydrotropes typically does not favor spontaneous self-aggregation; see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not exhibit a critical concentration at which self-aggregation occurs, as is found for surfactants and lipids that form micellar, lamellar, or other distinct mesophases. In fact, many hydrotropes exhibit a sustained aggregation process in which aggregate size increases with increasing concentration. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing polar and nonpolar substances, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries ranging from pharmaceuticals, personal care, and food to technical applications. The use of hydrotropes in detergent compositions allows for, for example, more concentrated formulations of surfactants (as in the process of concentrating liquid detergents by removing water) without inducing undesirable phenomena such as phase separation or high viscosity.

[0271] The detergent may contain 0 to 10% by weight of a hydrotrope, e.g., about 0 to 5% by weight, e.g., about 0.5 to about 5%, or about 3% to 5%. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate, sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0272] Builders and co-builders Detergent compositions may contain about 0-65%, 0-20%, or 0.5-5% detergent builder or co-builder, or a combination thereof. In dishwashing detergents, builder levels are typically 10-65%, particularly 20-40%. The builder and / or co-builder may be a chelating agent, particularly one that forms a water-soluble complex with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples include citrate, sodium carbonate, sodium bicarbonate, and sodium citrate. Examples of phosphonates include 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP, etidronic acid), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), ethylenediaminetetra(methylenephosphonic acid) (EDTMPA), aminotris(methylenephosphonic acid) (ATMP), nitrilotrimethylenephosphonic acid (NTMP), and 2-aminoethylphosphonic acid. (AEPn), dimethylmethylphosphonate (DMPP), tetramethylenediaminetetra(methylenephosphonic acid) (TDTMP), hexamethylenediaminetetra(methylenephosphonic acid) (HDTMP), phosphonobutanetricarboxylic acid (PBTC), N-(phosphonomethyl)iminodiacetic acid (PMIDA), 2-carboxyethylphosphonic acid (CEPA), 2-hydroxyphosphonocarboxylic acid (HPAA), and amino-tris-(methylenephosphonic acid) (AMP). L-glutamic acid N,N-diacetic acid tetrasodium salt (GLDA), methylglycinediacetic acid (MGDA). Non-limiting examples of builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl or alkenyl succinic acids.Additional specific examples include 2,2',2''-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetate (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra(methylenephosphoric acid) diethylenetriaminepentakis(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid N-(2-sulfomethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid Examples of suitable builders and / or co-builders include hydroxypropyltrimethylsilyl-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N,N',N''-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and U.S. Pat. No. 5,977,053.

[0273] In some embodiments, the builder or co-builder is a non-naturally occurring builder or co-builder.

[0274] Bleaching type The detergent may contain 0 to 30% by weight, e.g., about 1% to about 20%, of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleach catalysts, photobleachers, bleach activators, sources of hydrogen peroxide such as sodium percarbonate, sodium perborate and hydrogen peroxide-urea (1:1), preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, diperoxycarboxylic acids, perimidic acids and salts, peroxymonosulfuric acid and salts, e.g., Oxone®, and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may include inorganic salts, including, for example, sodium perborates (usually mono- or tetrahydrate), alkali metal salts such as percarbonates, persulfates, perphosphates, and persilicates, in combination with a peracid-forming bleach activator. The term bleach activator, as used herein, refers to a compound that reacts with hydrogen peroxide to form a peracid via perhydrolysis. The peracid thus formed constitutes activated bleach. Suitable bleach activators to be used herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable examples include tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of bleach activators of interest is disclosed in EP 624154, and among this family, acetyl triethyl citrate (ATC) is particularly preferred. ATC, or the short-chain triglyceride-like triacetin, has the advantage of being environmentally friendly. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in products during storage and are efficient bleach activators.Finally, ATC is multifunctional because the citrate released during the perhydrolysis reaction can function as a builder. Alternatively, the bleaching system can include, for example, an amide, imide, or sulfone type peroxyacid. The bleaching system can also include a peracid such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system can also include a bleaching catalyst. In some embodiments, the bleaching component can be an organic catalyst selected from the group consisting of organic catalysts having the following formula:

[0275] [ka]

[0276] (iii) and mixtures thereof (In the formula, each R 1 are independently a branched alkyl group containing 9 to 24 carbon atoms or a linear alkyl group containing 11 to 24 carbon atoms, and preferably each R 1 are independently a branched alkyl group containing 9 to 18 carbon atoms or a linear alkyl group containing 11 to 18 carbon atoms, and more preferably, each R 1 are independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, dodecyl, tetradecyl, hexadecyl, octadecyl, isononyl, isodecyl, isotridecyl, and isopentadecyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, EP 1 867 708 (vitamin K), and WO 2007 / 087242. Suitable photobleaches may be, for example, sulfonated zinc or aluminum phthalocyanine.

[0277] Preferably, the bleaching component comprises a source of peracid in addition to a bleach catalyst, particularly an organic bleach catalyst, which may be selected from (a) preformed peracid; (b) percarbonate, perborate, or persulfate (a hydrogen peroxide source), preferably in combination with a bleach activator; and (c) perhydrolase enzymes and esters for in situ formation of peracid in the presence of water in textile or hard surface treatment processes.

[0278] In some embodiments, the bleaching system is a non-naturally occurring bleaching system.

[0279] polymer The detergent may contain 0-10% by weight of polymer, e.g., 0.5-5%, 2-5%, 0.5-2%, or 0.2-1%. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as described above, or may provide anti-redeposition, fabric protection, soil release, dye transfer prevention, grease cleaning, and / or defoaming properties. Some polymers may have two or more of the above properties and / or two or more of the following motifs: Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, polyaspartic acid and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethylene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxides and polypropylene oxides (PEO-PPO), and diquaternium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the above polymers are also contemplated.

[0280] In some embodiments, the polymer is a non-naturally occurring polymer.

[0281] Fabric color toning agent The detergent compositions of the present invention may also contain fabric hueing agents, such as dyes or pigments, which, when incorporated into the detergent composition, can be deposited on fabrics when the fabrics come into contact with a wash liquor containing the detergent composition, thereby altering the color of the fabric by absorbing / reflecting visible light. Optical brighteners emit at least some visible light. In contrast, fabric hueing agents absorb at least a portion of the visible light spectrum, thereby altering the color of the surface. Suitable fabric hueing agents include dyes and dye-clay conjugates, and may include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include, for example, small molecule dyes selected from the group consisting of dyes classified in the Color Index (CI) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, as described in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276, and EP 1 876 226 (incorporated herein by reference). The detergent composition preferably contains from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or from about 0.0001 wt% to about 0.04 wt% of a fabric hueing agent. The composition may contain from 0.0001 wt% to about 0.2 wt% of the fabric hueing agent, which may be particularly preferred when the composition is in the form of a unit-dose pouch. Suitable hueing agents are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0282] Additional enzymes The detergent additives and detergent compositions may contain one or more [additional] enzymes such as hydrolases (EC 3.-.-), glycosidases (EC 3.2.-.-), and hydrolases (EC 3.4.-.-) acting on ester bonds, oxidoreductases (EC 1.-.-.-), such as laccases (EC 1.10.-.-) or peroxidases (EC 1.11.-.-), or lyases (EC 4.-.-.-), such as carbon-oxygen lyases (EC 4.2.-.-). In certain embodiments, the detergent compositions may contain one or more [additional] enzymes such as proteases, lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases, e.g., laccases, and / or peroxidases.

[0283] In general, the properties of the selected enzyme should be compatible with the selected detergent (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.), and the enzyme should be present in an effective amount.

[0284] cellulase Suitable cellulases include those of bacterial or fungal origin, including chemically modified or protein engineered variants. Suitable cellulases include cellulases derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and WO 89 / 09259.

[0285] Particularly suitable cellulases are alkaline or neutral cellulases that provide or maintain whiteness and prevent redeposition or provide color protection benefits. Examples of such cellulases are those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0531315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and WO 99 / 001544.

[0286] Other cellulases are endo-β-1,4-glucanase enzymes having a sequence that is at least 97% identical to the amino acid sequence of positions 1 to 773 of SEQ ID NO:2 of WO 2002 / 099091, or family 44 xyloglucanases, where the xyloglucanase enzyme has a sequence that is at least 60% identical to positions 40 to 559 of SEQ ID NO:2 of WO 2001 / 062903.

[0287] Commercially available cellulases include Celluzyme™ and Carezyme™ (Novozymes A / S), Carezyme Premium™ (Novozymes A / S), Celluclean™ (Novozymes A / S), Celluclean Classic™ (Novozymes A / S), Cellusoft™ (Novozymes A / S), Whitezyme™ (Novozymes A / S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0288] Mannanase Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified variants are also included. The mannanase may be an alkaline mannanase of family 5 or 26. It may be a wild-type mannanase derived from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 1999 / 064619. A commercially available mannanase is Mannaway (Novozymes A / S).

[0289] Proteases Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Those of microbial origin are preferred. They include chemically modified variants or protein-engineered variants. They may be alkaline proteases, such as serine proteases or metalloproteases. The serine proteases may be, for example, from the S1 family, such as trypsin, or from the S8 family, such as subtilisin. The metalloprotease proteases may be, for example, thermolysin from the M4 family or other metalloproteases, such as those from the M5, M7, or M8 families.

[0290] 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.

[0291] Examples of subtilases include Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus, and Bacillus gibsonii, which are described in U.S. Pat. No. 7,262,042 and WO 09 / 021867, and subtilisin lentus, subtilisin novo, subtilisin Carlsberg, and Bacillus licheniformis, which are described in WO 89 / 06279. licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168, and protease PD138 described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372, and the chymotrypsin protease from Cellumonas described in WO 05 / 052161 and WO 05 / 052146.

[0292] Further preferred proteases are, for example, the alkaline protease from Bacillus lentus DSM 5483 as described in WO 95 / 23221 and variants thereof as described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148.

[0293] Examples of metalloproteases are neutral metalloproteases such as those from Bacillus amyloliquefaciens and as described in WO 07 / 044993 (Genencor Int.).

[0294] Examples of useful proteases are described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263. and variants described in WO 11 / 036264, in particular variants having substitutions at one or more of the following positions using BPN' numbering: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274. More preferred subtilase variants are those with the mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R These may include S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

[0295] Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Blaze®, Blaze® Evity®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress Excel®, and Progress Uno® (Novozymes A / S), and those sold under the trade names Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect®, These include those sold under the names Prime®, Preferenz™, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco / DuPont), Axapem™ (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of U.S. Pat. No. 5,352,604) and related variants (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0296] Lipase and cutinase: Suitable lipases and cutinases include those of bacterial or fungal origin. These include chemically modified or protein engineered variant enzymes. Examples include those derived from Thermomyces, such as 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 can be used include lipases from Bacillus stearothermophilus (WO 11 / 084417), lipases from Bacillus subtilis (WO 11 / 084599), and lipases from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0297] Other examples are lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0298] Preferred commercially available lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

[0299] Further examples are lipases, sometimes called 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), as well as variants of M. smegmatis perhydrolase, in particular the S54V variant used in the product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0300] amylase: Suitable amylases for use with the variants of the present invention may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin, including chemically modified or protein-engineered variants. Amylases include, for example, alpha-amylases obtained from specialized strains of Bacillus, such as Bacillus licheniformis, which is described in more detail in GB Patent No. 1,296,839.

[0301] Suitable amylases include those having SEQ ID NO: 2 in WO 95 / 10603 or variants thereof having 90% sequence identity with SEQ ID NO: 3. Preferred variants are those set forth in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

[0302] A different suitable amylase includes the amylase having SEQ ID NO: 6 in WO 02 / 010355 or a variant thereof having 90% sequence identity with SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 is one having deletions at positions 181 and 182 and a substitution at position 193.

[0303] Another suitable amylase is a hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the alpha-amylase from B. licheniformis as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or a variant thereof with 90% sequence identity. Preferred variants of this hybrid alpha-amylase have substitutions, deletions or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. The most preferred variant of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 comprises the substitution: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S It has the following characteristics.

[0304] Further suitable amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions at positions R181 and G182, or H183 and G184.

[0305] Additional amylases that may be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those with substitutions, deletions or insertions at one or more of the following positions, using SEQ ID NO: 2 of WO 96 / 023873 for numbering: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those with deletions at two positions selected from 181, 182, 183 and 184, for example, 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 have deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0306] Other amylases that may be used are amylases having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 of WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity with SEQ ID NO: 10 of WO 01 / 66712. Preferred variants of SEQ ID NO: 10 of WO 01 / 66712 are those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

[0307] Further suitable amylases are those having SEQ ID NO: 2 of WO 09 / 061380 or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those with C-terminal truncations and / or substitutions, deletions or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those with substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K, and / or deletions at positions R180 and / or S181 or T182 and / or G183. Most preferred amylase variants of SEQ ID NO: 2 are those with substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, the variant being C-terminally truncated and optionally further comprising a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0308] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 of WO 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having substitutions R118K, N195F, R320K and R458K, as well as variants further having substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, with variants further having substitutions at all of these positions being most preferred.

[0309] Other examples are amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0310] Commercially available amylases include Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, and BAN™ (manufactured by Novozymes A / S), and Rapidase™, Purastar™ / Effectenz™, Powerase™, Preferenz S1000™, Preferenz S110™, and Preferenz S100™ (manufactured by Genencor International Inc. / DuPont).

[0311] Peroxidase / Oxidase: Peroxidase is a peroxidase enzyme included in the enzyme classification EC 1.11.1.7 as described by the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom that exhibits peroxidase activity.

[0312] Suitable peroxidases include those of plant, bacterial, or fungal origin, and include chemically modified or protein-engineered variants. Examples of useful peroxidases include peroxidases from Coprinopsis species, such as C. cinerea (EP 179,486), and variants thereof, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0313] Peroxidases can also include haloperoxidase enzymes, such as chloroperoxidase, bromoperoxidase, and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (EC 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

[0314] In certain embodiments, the haloperoxidase is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase. In preferred methods of the invention, the vanadate-containing haloperoxidase is combined with a source of chloride ions.

[0315] Haloperoxidases have been isolated from a variety of fungi, particularly from the fungal group of dematiaceous hyphomycetes, e.g., Caldariomyces such as C. fumago, Alternaria, Curvularia such as C. verruculosa and C. inaequalis, Drechslera, Ulocladium, and Botrytis.

[0316] Haloperoxidases have also been isolated from bacteria such as Pseudomonas, eg, P. pyrrocinia, and Streptomyces, eg, S. aureofaciens.

[0317] In a preferred embodiment, the haloperoxidase is derived from a Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, for example C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or Drechslera hartlevii as described in WO 01 / 79459. hartlebii, Dendrophiella salina as described in WO 01 / 79458, Phaeotrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium sp. as described in WO 01 / 79460.

[0318] Oxidases include in particular any laccase enzyme included in the enzyme classification EC 1.10.3.2, or any fragment thereof exhibiting laccase activity, or a compound exhibiting similar activity, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0319] Preferred laccase enzymes are those of bacterial origin. The enzyme may be obtained from plants, bacteria or fungi (including filamentous fungi and yeasts).

[0320] Suitable examples of fungal origin include those from the genus Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes versicolor, and the like. es), such as T. villosa and T. versicolor, Rhizoctonia, such as R. solani, Coprinopsis, such as C. cinerea, C. comatus, C. friesii, and C. pl Examples of laccases that can be used include laccases obtained from strains of the genus Psathyrella, such as P. condoleana, Panaeolus, such as P. papilionaceus, Myceliophthora, such as M. thermophila, Schytalidium, such as S. thermophilum, Polyporus, such as P. pinsitus, Phlebia, such as P. radiata (WO 92 / 01046), or Coriolus, such as C. hirsutus (Japanese Patent Publication No. 2238885).

[0321] A suitable example of bacterial origin is laccase obtained from a strain of the genus Bacillus.

[0322] Laccases obtained from the genus Coprinopsis or Myceliophthora are preferred; in particular, laccases obtained from Coprinopsis cinerea, as disclosed in WO 97 / 08325; or laccases obtained from Myceliophthora thermophila, as disclosed in WO 95 / 33836.

[0323] nuclease Suitable nucleases include deoxyribonucleases (DNases) and ribonucleases (RNases), which are enzymes that catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA or RNA backbone, respectively, thereby degrading DNA and RNA. There are two major classifications based on the site of activity: exonucleases digest nucleic acids from the ends; endonucleases act on the middle region of the target molecule. The nuclease is preferably a DNase, which can be obtained from a microorganism, preferably a fungus or a bacterium. In particular, DNases obtainable from Bacillus species are preferred; in particular, DNases obtainable from Bacillus cibi, Bacillus subtilis, or Bacillus licheniformis are preferred. Examples of such DNases are described in WO 2011 / 098579, WO 2014 / 087011, and WO 2017 / 060475. Also particularly preferred are DNases obtainable from Aspergillus species, in particular Aspergillus oryzae, such as the DNase described in WO 2015 / 155350.

[0324] Licheninase Suitable licheninases include enzymes that catalyze the hydrolysis of beta-1,4-glucosidic bonds to yield beta-glucans. Licheninases (or lichenases) (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. Examples of such licheninases are described in patent applications WO 2017 / 097866 and WO 2017 / 129754.

[0325] Detergent enzymes can be included in detergent compositions by adding separate additives containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additives of the present invention, i.e., separate additives or combined additives, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-shattering granules, liquids, especially stabilized liquids, or slurries.

[0326] Non-shattering granules may be prepared, for example, by methods disclosed in U.S. Patents 4,106,991 and 4,661,452, and may optionally be coated by methods known in the art. Examples of wax coating materials include poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and 15 to 80 ethylene oxide units; fatty alcohols, fatty acids, and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed techniques are given in British Patent No. 1,483,591. Liquid enzyme preparations may be stabilized, for example, by adding polyols, such as propylene glycol, sugars or sugar alcohols, lactic acid, or boric acid, according to established methods. The protected enzyme may be prepared according to the method disclosed in EP 238,216.

[0327] Auxiliary materials Any detergent component known in the art for use in laundry detergents can also be utilized.Other optional detergent components include, alone or in combination, anti-corrosion agents, anti-shrinkage agents, anti-resoiling agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegrating agents, dyes, enzyme stabilizers (boric acid, borate salts, CMC, and / or polyols such as propylene glycol), fabric softeners including clay, fillers / processing aids, optical brighteners / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil suspending agents, softeners, foam suppressors, tanning inhibitors, and wicking agents.Any component known in the art for use in laundry detergents can also be utilized.The selection of such components is well within the skill of those skilled in the art.

[0328] Dispersants—The detergent compositions of the present invention can also contain dispersants. In particular, powder detergents may contain dispersants. Suitable water-soluble organic materials include homo- or copolymeric acids or salts thereof, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series Volume 71, Marcel Dekker, Inc.

[0329] Dye Transfer Inhibitors—The detergent compositions of the present invention may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone and polyvinylimidazole, or mixtures thereof. When present in the subject compositions, the dye transfer inhibitors may be present at levels of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0330] Optical Brighteners—The detergent compositions of the present invention also preferably contain an additional component capable of imparting color to the cleaned article, such as an optical brightener or optical brightener. When present, the brightener is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used optical brighteners belong to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and bisphenyl-distyryl derivatives. Examples of diaminostilbene-sulfonic acid derivative-type fluorescent whitening agents include 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydro)- Examples of suitable fluorescent whitening agents include 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and the sodium salt of sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)-disulfonate. Another preferred optical brightener is Parawhite KX, available from Paramount Minerals and Chemicals, Mumbai, India. Other optical brighteners suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins.

[0331] Suitable fluorescent brightener levels include lower levels of about 0.01, 0.05, about 0.1, or about 0.2 wt %, to upper levels of 0.5 or even 0.75 wt %.

[0332] Soil-releasing polymer—The detergent composition of the present invention may also contain one or more soil-releasing polymers to aid in the removal of soil from fabrics such as cotton and polyester-based fabrics, particularly hydrophobic soils from polyester-based fabrics. The soil-releasing polymer may be, for example, a nonionic or anionic terephthalate-based polymer, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, or polyester polyamides, as described, for example, in Chapter 7, Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc. Another type of soil-releasing polymer is an amphiphilic alkoxylated grease-cleaning polymer comprising a core structure and multiple alkoxylate groups attached to the core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure, as detailed in WO 2009 / 087523 (incorporated herein by reference). Additionally, random graft copolymers are suitable soil-releasing polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (incorporated herein by reference). Other soil-releasing polymers are substituted polysaccharide structures, particularly substituted cellulose structures, such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003 / 040279 (both incorporated herein by reference). Suitable cellulose-based polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose-based polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof.Suitable cellulosic polymers include methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxymethyl cellulose, and mixtures thereof.

[0333] Anti-redeposition Agents—The detergent compositions of the present invention may also include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. Under the soil release polymers mentioned above, the cellulosic polymers may also function as anti-redeposition agents.

[0334] Rheology modifiers are structuring or thickening agents that are distinct from viscosity reducers. Rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, and polymeric rheology modifiers that impart shear-thinning properties to the aqueous liquid matrix of the liquid detergent composition. Detergent rheology and viscosity can be modified and controlled by methods known in the art, for example, as shown in EP 2169040.

[0335] Other suitable adjunct materials include, but are not limited to, anti-shrinkage agents, anti-wrinkle agents, biocides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam control agents, hydrotropes, fragrances, pigments, suds control agents, and solvents.

[0336] Protease inhibitors The protease inhibitor can be any compound that stabilizes or inhibits proteases or other enzymes in the laundry soap bar from being degraded. Examples of protease inhibitors include aprotinin, bestatin, calpain inhibitors I and II, chymostatin, leupeptin, pepstatin, phenylmethanesulfonyl fluoride (PMSF), boric acid, borate salts, borax, boronic acid, phenylboronic acid such as 4-formylphenylboronic acid (4-FPBA), peptide aldehydes or their hydrosulfite adducts or hemiacetal adducts, and peptide trifluoromethyl ketones. One or more protease inhibitors can be present, such as 5, 4, 3, 2, or 1 inhibitor, at least one of which is a peptide aldehyde, its hydrosulfite adduct, or hemiacetal adduct.

[0337] Peptide Aldehyde Inhibitors Peptide aldehydes have the formula P-(A) y -L-(B) x -B 0 -H or its hydrosulfite adduct or hemiacetal adduct, iH is hydrogen; ii.B 0 is a single amino acid residue having the L- or D-configuration of the formula -NH-CH(R)-C(=O)-; iii.x is (B) x and B is independently substituted by a B amino acid C-terminal to the B amino acid. 0 is a single amino acid linked to iv. L is absent or L is independently a linker group of formula -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=O)-; vy is (A) y and A is independently a single amino acid residue linked to L via the N-terminus of the A amino acid (provided that if L is absent, then A is absent); vi. P is selected from the group consisting of hydrogen and an N-terminal protecting group (provided that if L is absent, then P is an N-terminal protecting group); vii. R independently represents a C optionally substituted with one or more identical or different substituents R′. 1~6 Alkyl, C 6~10 Aryl or C 7~10 arylalkyl; viii. R' is independently selected from the group consisting of halogen, -OH, -OR", -SH, -SR", -NH2, -NHR", -NR"2, -CO2H, -CONH2, -CONHR", -CONR"2, -NHC(=N)NH2; and ix.R'' is C 1~6 The alkyl group may have a substituent.

[0338] x can be 1, 2 or 3, and therefore B can be 1, 2 or 3 amino acid residues, respectively. 1 , B 2 -B 1 or B 3 -B 2 -B 1 (In the formula, B 3 , B 2 and B 1 each represents one amino acid residue). y can be 0, 1 or 2, so A can be absent or have the formula A 1 or A 2 -A 1 (In the formula, A 2 and A 1 each represent one amino acid residue).

[0339] B 0 may be a single amino acid residue having an L- or D-configuration, linked to H through the C-terminus of that amino acid, where R is C 1~6 Alkyl, C 6~10 Aryl or C 7~10Arylalkyl side chains are, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, or benzyl, and R can be optionally substituted with one or more identical or different substituents R'. Particular examples are the D- or L-forms of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (Ile), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr), and valine (Val). Particular embodiments are 0 is leucine, methionine, phenylalanine, p-tyrosine and valine.

[0340] B 1 B via the C-terminus of the amino acid 0 B is connected to 1 B can be an aliphatic, hydrophobic and / or neutral amino acid. 1 Examples of B are alanine (Ala), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). 1 Particular examples of B are alanine, glycine, isoleucine, leucine and valine. Particular embodiments include B 1 is alanine, glycine or valine.

[0341] If present, B 2 B via the C-terminus of the amino acid 1 B is connected to 2 B can be an aliphatic, hydrophobic, neutral and / or polar amino acid. 2 Examples of B are alanine (Ala), arginine (Arg), capreomycin (Cpd), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). 2Particular examples of B are alanine, glycine, capreomycin, glycine, isoleucine, leucine, phenylalanine and valine. Particular embodiments are 2 is arginine, glycine, leucine, phenylalanine or valine.

[0342] If present, B 3 B via the C-terminus of the amino acid 2 B is connected to 3 B can be a large aliphatic, aromatic, hydrophobic and / or neutral amino acid. 3 Examples of B are isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). 3 Specific examples are leucine, phenylalanine, tyrosine and tryptophan.

[0343] The linker group L can be absent or selected from the group consisting of -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=O)-. Particular embodiments include when L is absent or is a carbonyl group -C(=O)-.

[0344] If present, A is linked to L via the N-terminus of the amino acid 1 A can be an aliphatic, aromatic, hydrophobic, neutral and / or polar amino acid. 1 Examples of A are alanine (Ala), arginine (Arg), capreomycin (Cpd), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp), and valine (Val). 1 Particular examples of B are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan and valine. 2is leucine, phenylalanine, tyrosine or tryptophan.

[0345] If present, A via the N-terminus of the amino acid 1 A connected to 2 The residues may be large aliphatic, aromatic, hydrophobic and / or neutral amino acids. 2 Examples of A are arginine (Arg), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). 2 Particular examples are phenylalanine and tyrosine.

[0346] The N-terminal protecting group P (if present) may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl, (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc) and adamantyloxycarbonyl; p-methoxybenzylcarbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylaminocarbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramidyl (MeOP(OH)(=O)) and benzylphosphoramidyl (PhCHOP(OH)(=O)).

[0347] The general formula for a peptide aldehyde can also be written as follows: PA 2 -A 1 -LB 3 -B 2 B 1 -B 0 -H(in the formula, P, A 2 , A 1 , L, B 3 , B 2 , B 1 and B 0is as defined above).

[0348] In the case of a tripeptide aldehyde having a protecting group (i.e., x=2, L is absent, and A is absent), P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylaminocarbonyl, methylsulfonyl, benzylsulfonyl, and benzylphosphoramidyl. In the case of a tetrapeptide aldehyde having a protecting group (i.e., x=3, L is absent, and A is absent), P is preferably acetyl, methoxycarbonyl, methylsulfonyl, ethylsulfonyl, and methylphosphoramidyl.

[0349] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO 98 / 13460, WO 98 / 13461, WO 98 / 13462, WO 07 / 141736, WO 07 / 145963, WO 09 / 118375, WO 10 / 055052 and WO 11 / 036153.

[0350] More specifically, the peptide aldehyde is Cbz-Arg-Ala-Tyr-H (L-alaninamide, N2-[(phenylmethoxy)carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Ac-Gly-Ala-Tyr-H (L-Alaninamide, N-acetylglycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-) Cbz-Gly-Ala-Tyr-H (L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Cbz-Gly-Ala-Leu-H (L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-3-methylbutyl]-), Cbz-Val-Ala-Leu-H (L-alaninamide, N-[(phenylmethoxy)carbonyl]-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-), Cbz-Gly-Ala-Phe-H (L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-2-phenylethyl]-), Cbz-Gly-Ala-Val-H (L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-2-methylpropyl]-), Cbz-Gly-Gly-Tyr-H (glycinamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Cbz-Gly-Gly-Phe-H (glycinamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[(1S)-1-formyl-2-phenylethyl]-), Cbz-Arg-Val-Tyr-H (L-valinamide, N2-[(phenylmethoxy)carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Cbz-Leu-Val-Tyr-H (L-Valinamide, N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-) Ac-Leu-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-leucylglycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Ac-Phe-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Ac-Tyr-Gly-Ala-Tyr-H (L-alaninamide, N-acetyl-L-tyrosylglycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Ac-Phe-Gly-Ala-Leu-H (L-alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1S)-1-formyl-3-methylbutyl]-), Ac-Phe-Gly-Ala-Phe-H (L-Alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1S)-1-formyl-2-phenylethyl]-) Ac-Phe-Gly-Val-Tyr-H (L-valinamide, N-acetyl-L-phenylalanylglycyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), Ac-Phe-Gly-Ala-Met-H (L-alaninamide, N-acetyl-L-phenylalanylglycyl-N-[(1S)-1-formyl-3-(methylthio)propyl]-), Ac-Trp-Leu-Val-Tyr-H (L-valinamide, N-acetyl-L-tryptophyl-L-leucyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-), MeO-CO-Val-Ala-Leu-H (L-alaninamide, N-(methoxycarbonyl)-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-) MeNHCO-Val-Ala-Leu-H (L-alaninamide, N-(aminomethylcarbonyl)-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-), MeO-CO-Phe-Gly-Ala-Leu-H (L-alaninamide, N-(methoxycarbonyl)-L-phenylalanylglycyl-N-[(1S)-1-formyl-3-methylbutyl]-), MeO-CO-Phe-Gly-Ala-Phe-H (L-alaninamide, N-(methoxycarbonyl)-L-phenylalanylglycyl-N-[(1S)-1-formyl-2-phenylethyl]-), MeSO2-Phe-Gly-Ala-Leu-H (L-alaninamide, N-(methylsulfonyl)-L-phenylalanylglycyl-N-[(1S)-1-formyl-3-methylbutyl]-), MeSO2-Val-Ala-Leu-H (L-alaninamide, N-(methylsulfonyl)-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-), PhCHO-P(OH)(O)-Val-Ala-Leu-H (L-alaninamide, N-[hydroxy(phenylmethoxy)phosphinyl]-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-), EtSO2-Phe-Gly-Ala-Leu-H (L-alaninamide, N-(ethylsulfonyl)-L-phenylalanylglycyl-N-[(1S)-1-formyl-3-methylbutyl]-), PhCH2SO2-Val-Ala-Leu-H (L-alaninamide, N-[(phenylmethyl)sulfonyl]-L-valyl-N-[(1S)-1-formyl-3-methylbutyl]-), PhCHO-P(OH)(O)-Leu-Ala-Leu-H (L-alaninamide, N-[hydroxy(phenylmethoxy)phosphinyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-), PhCHO-P(OH)(O)-Phe-Ala-Leu-H (L-alaninamide, N-[hydroxy(phenylmethoxy)phosphinyl]-L-phenylalanyl-N-[(1S)-1-formyl-3-methylbutyl]-), or It can be MeO-P(OH)(O)-Leu-Gly-Ala-Leu-H; (L-alaninamide, N-(hydroxymethoxyphosphinyl)-L-leucylglycyl-N-[(1S)-1-formyl-3-methylbutyl]-).

[0351] A preferred example is Cbz-Gly-Ala-Tyr-H.

[0352] Further examples of such peptide aldehydes include: α-MAPI (3,5,8,11-tetraazatridecanoic acid, 6-[3-[(aminoiminomethyl)amino]propyl]-12-formyl-9-(1-methylethyl)-4,7,10-trioxo-13-phenyl-2-(phenylmethyl)-, (2S,6S,9S,12S)- L-Valinamide, N2-[[(1-carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-(1-formyl-2-phenylethyl)-,[1(S),2(S)]-; L-Valinamide, N2-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-phenylethyl]-(9CI); SP-chymostatin B), β-MAPI (L-valinamide,N2-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]-L-arginyl-N-[(1R)-1-formyl-2-phenylethyl]-L-valinamide,N2-[[(1-carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-(1-formyl-2-phenylethyl)-,[1(S),2(R)]-), Phe-C(=O)-Arg-Val-Tyr-H (L-valinamide, N2-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-(9CI)), Phe-C(=O)-Gly-Gly-Tyr-H, (3,5,8,11-tetraazatridecanoic acid, 12-formyl-13-(4-hydroxyphenyl)-4,7,10-trioxo-2-(phenylmethyl)-, (2S,12S)-), Phe-C(=O)-Gly-Ala-Phe-H, (3,5,8,11-tetraazatridecanoic acid, 12-formyl-9-methyl-4,7,10-trioxo-13-phenyl-2-(phenylmethyl)-, (2S,9S,12S)-), Phe-C(=O)-Gly-Ala-Tyr-H(3,5,8,11-tetraazatridecanoic acid, 12-formyl-13-(4-hydroxyphenyl)-9-methyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-), Phe-C(=O)-Gly-Ala-Leu-H, (3,5,8,11-tetraazapentadecanoic acid, 12-formyl-9,14-dimethyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-), Phe-C(=O)-Gly-Ala-Nva-H, (3,5,8,11-tetraazapentadecanoic acid, 12-formyl-9-methyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-), Phe-C(=O)-Gly-Ala-Nle-H(3,5,8,11-tetraazahexadecanoic acid, 12-formyl-9-methyl-4,7,10-trioxo-2-(phenylmethyl)-, (2S,9S,12S)-), Tyr-C(=O)-Arg-Val-Tyr-H (L-valinamide, N2-[[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-(4-hydroxyphenyl)ethyl]-(9CI)), Tyr-C(=O)-Gly-Ala-Tyr-H(3,5,8,11-tetraazatridecanoic acid, 12-formyl-13-(4-hydroxyphenyl)-2-[(4-hydroxyphenyl)methyl]-9-methyl-4,7,10-trioxo-,(2S,9S,12S)-), Phe-C(=S)-Arg-Val-Phe-H, (3,5,8,11-tetraazatridecanoic acid, 6-[3-[(aminoiminomethyl)amino]propyl]-12-formyl-9-(1-methylethyl)-7,10-dioxo-13-phenyl-2-(phenylmethyl)-4-thioxo-, (2S,6S,9S,12S)-), Phe-C(=S)-Arg-Val-Tyr-H, (3,5,8,11-tetraazatridecanoic acid, 6-[3-[(aminoiminomethyl)amino]propyl]-12-formyl-13-(4-hydroxyphenyl)-9-(1-methylethyl)-7,10-dioxo-2-(phenylmethyl)-4-thioxo-, (2S,6S,9S,12S)-), Phe-C(=S)-Gly-Ala-Tyr-H, (3,5,8,11-tetraazatridecanoic acid, 12-formyl-13-(4-hydroxyphenyl)-9-methyl-7,10-dioxo-2-(phenylmethyl)-4-thioxo-, (2S,9S,12S)-), Antipain (L-valinamide, N2-[[(1-carboxy-2-phenylethyl)amino]carbonyl]-L-arginyl-N-[4-[(aminoiminomethyl)amino]-1-formylbutyl]-), GE20372A (L-valinamide,N2-[[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1S)-1-formyl-2-phenylethyl]-L-valinamide,N2-[[[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-(1-formyl-2-phenylethyl)-,[1(S),2(S)]-), GE20372B (L-valinamide,N2-[[[(1S)-1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-[(1R)-1-formyl-2-phenylethyl]-L-valinamide,N2-[[[1-carboxy-2-(4-hydroxyphenyl)ethyl]amino]carbonyl]-L-arginyl-N-(1-formyl-2-phenylethyl)-,[1(S),2(R)]-), Chymostatin A (L-leucinamide,(2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-L-leucinamide,(2S)-2-[(4S)-2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-(9CI); L-Leucinamide, L-2-(2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2-phenylethyl)amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-, stereoisomer), Chymostatin B (L-valinamide,(2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-L-valinamide,(2S)-2-[(4S)-2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-(9CI); L-Valinamide, L-2-(2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2-phenylethyl)amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-, stereoisomer), and Chymostatin C (L-isoleucinamide,(2S)-2-[(4S)-2-amino-3,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-L-isoleucinamide,(2S)-2-[(4S)-2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl]-N-[[[(1S)-1-carboxy-2-phenylethyl]amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-(9CI); L-Isoleucinamide, L-2-(2-amino-1,4,5,6-tetrahydro-4-pyrimidinyl)-N-[[(1-carboxy-2-phenylethyl)amino]carbonyl]glycyl-N-(1-formyl-2-phenylethyl)-, stereoisomer Examples include:

[0353] Peptide aldehyde adducts Instead of a peptide aldehyde, the protease inhibitor can be an adduct of the peptide aldehyde. The adduct has the formula P-(A) y -L-(B) x The adduct may be a hydrosulfite adduct having the formula -N(H)-CHR-CH(OH)-SO3M, where P, A, y, L, B, x, and R are as defined above, and M is H or an alkali metal, preferably Na or K. Alternatively, the adduct may be a hydrosulfite adduct having the formula P-(A) y -L-(B) x It may be a hemiacetal having the formula -N(H)-CHR-CH(OH)-OR, where P, A, y, L, B, x, and R are as defined above. A preferred embodiment is a hydrosulfite adduct, where P = Cbz, B 2 =Gly;B 1 =Ala;B 0= Tyr (so R = PhCH2, R' = OH), x = 2, y = 0, L = A = absent and M = Na (Cbz-Gly-Ala-N(H)-CH(CH2-p-CH4OH)-CH(OH)-SO3Na, L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[2-hydroxy-1-[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1:1)).

[0354] The general formula for the hydrosulfite adduct of a peptide aldehyde may also be written as follows: PA 2 -A 1 -LB 3 -B 2 -B 1 -N(H)-CHR-CH(OH)-SO3M (in the formula, P, A 2 , A 1 , L, B 3 , B 2 , B 1 , R and M are as defined above).

[0355] Alternatively, the peptide aldehyde adduct may be Cbz-Gly-Ala-N(H)-CH(CH2-p-CH4OH)-CH(OH)-SONa ((2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1-hydroxy-3-(4-hydroxyphenyl)propane-1-sulfonate) or Cbz-Gly-Ala-N(H)-CH(CH2Ph)-CH(OH)-SONa ((2S)-[(N-{N-[

[0033] The compound may be "MeO-CO_Val-Ala-N(H)-CH(CHCH(CH))-CH(OH)-SONa ((2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1-hydroxy-3-(phenyl)propane-1-sulfonate) or "MeO-CO_Val-Ala-N(H)-CH(CHCH(CH))-CH(OH)-SONa ((2S)-[(N-{N-[(benzyloxy)carbonyl]glycyl}-L-alaninyl)amino]-1-hydroxy-3-(2-propanyl)propane-1-sulfonate).

[0356] Other preferred peptide aldehyde bisulfites are Cbz-Arg-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Ac-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Cbz-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H, where M = Na(L-alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[2-hydroxy-1-[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1:1)), Cbz-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (in the formula, M=Na), Cbz-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), Cbz-Gly-Ala-NHCH(CH2Ph)C(OH)(SO3M)-H (where M=Na), Cbz-Gly-Ala-NHCH(CH(CH3)2)C(OH)(SO3M)-H (where M=Na), Cbz-Gly-Gly-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Cbz-Gly-Gly-NHCH(CH2Ph)C(OH)(SO3M)-H (where M=Na), Cbz-Arg-Val-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Cbz-Leu-Val-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (where M=Na), Ac-Leu-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Ac-Phe-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Ac-Tyr-Gly-Ala-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (in the formula, M=Na), Ac-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H(in the formula, M=Na), Ac-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(SO3M)-H (in the formula, M=Na), Ac-Phe-Gly-Val-NHCH(CH2C6H4OH)C(OH)(SO3M)-H, (in the formula, M=Na), Ac-Phe-Gly-Ala-NHCH(CH2CH2SCH3)(SO3M)-H (where M=Na), Ac-Trp-Leu-Val-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (where M=Na), MeO-CO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), MeNCO-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), MeO-CO-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), MeO-CO-Phe-Gly-Ala-NHCH(CH2Ph)C(OH)(SO3M)-H (where M=Na), MeSO2-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, (in the formula, M=Na), MeSO2-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), PhCH2O(OH)(O)P-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H(in the formula, M=Na), EtSO2-Phe-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H(in the formula, M=Na), PhCH2SO2-Val-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (in the formula, M=Na), PhCH2O(OH)(O)P-Leu-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (in the formula, M=Na), PhCH2O(OH)(O)P-Phe-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (in the formula, M=Na), MeO(OH)(O)P-Leu-Gly-Ala-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H (where M=Na), and Phe-urea-Arg-Val-NHCH(CH2C6H4OH)C(OH)(SO3M)-H (where M=Na).

[0357] salt The salts used in the bars are salts of monovalent cations and organic anions. The monovalent cations are, for example, Na + , K. + or NH4 + The organic anion can be, for example, formate, acetate, citrate, or lactate. Thus, the salt of the monovalent cation and the organic anion can be, for example, sodium formate, potassium formate, ammonium formate, sodium acetate, potassium acetate, ammonium acetate, sodium lactate, potassium lactate, ammonium lactate, monosodium citrate, disodium citrate, trisodium citrate, sodium potassium citrate, potassium citrate, ammonium citrate, etc. A particular embodiment is sodium formate.

[0358] Detergent product formulation The detergent compositions of the present invention may be in any convenient form, such as, for example, a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a shaped or compacted powder, granules, a paste, a gel, or a shaped, compacted or concentrated liquid.

[0359] The pouch can be configured as a single or multiple compartments. It can be of any form, shape, and material suitable for retaining the composition, e.g., preventing release of the composition from the pouch prior to contact with water. The pouch is made from a water-soluble film that encloses the contents. The internal volume can be divided into the pouch compartments. Preferred films are polymeric materials, preferably polymers that can be formed into films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer, e.g., PVA, in the film is at least about 60%. Preferred average molecular weights are typically about 20,000 to about 150,000. The film may also be a hydrolytically degradable, water-soluble polymer blend, such as a blended composition containing polylactide and polyvinyl alcohol (known under the trade name M8630, sold by MonoSol LLC, Indiana, USA) with a plasticizer such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch may contain a solid laundry cleaning composition or portion of a component and / or a liquid cleaning composition or portion of a component separated by a water-soluble film. The compartment for the liquid component may be different in composition from the compartment containing the solid: see U.S. Patent Application Publication No. 2009 / 0011970 A1.

[0360] The detergent components can be physically separated from each other by compartments in a water-soluble pouch or in different layers of a tablet, which can avoid undesirable interactions between the components during storage. Different dissolution profiles in each of the compartments can also cause delayed dissolution of selected components in the wash solution.

[0361] Non-unit-dose liquid or gel detergents may be water-soluble, typically containing at least 20% by weight and up to 95% water, e.g., up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, or up to about 35% water. Other types of liquids, such as, but not limited to, alkanols, amines, diols, ethers, and polyols, may also be included in the water-soluble liquid or gel. The water-soluble liquid or gel detergent may contain 0-30% organic solvents.

[0362] The liquid or gel detergent may be water-insoluble.

[0363] Methods of Producing Compositions The present invention also relates to a method of producing the composition.

[0364] use The present invention also relates to methods for using the compositions.

[0365] Use in detergents. The polypeptides of the present invention may be added and thereby become components of detergent compositions.

[0366] The detergent compositions of the present invention may be formulated, for example, as hand or machine laundry detergent compositions, as laundry additive compositions suitable for pre-treating soiled fabrics or for restoring textiles to like-new condition (e.g., by removing fluff or pilling) and restoring some of the visual and tactile properties of fabrics after extended use to those comparable to those of new textiles, and as rinse-additive fabric softener compositions, or as detergent compositions for use in household hard surface cleaning operations in general, or for hand or mechanical dishwashing operations.

[0367] In certain aspects, the present invention provides a detergent additive comprising a polypeptide of the invention as described herein. [Example]

[0368] Materials and Methods General methods such as PCR, cloning, and ligation of nucleotides are well known to those skilled in the art and can be found, for example, in "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons, (1995); Harwood, CR, and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", DN Glover ed. (1985); "Oligonucleotide Synthesis", MJ Gait ed. (1984); "Nucleic Acid Hybridization", B.D. Hames & S.J. Higgins eds. (1985); "A Practical Guide To Molecular Cloning", B. Perbal, (1984).

[0369] Assay for cellulolytic activity Cellulolytic activity was determined using a cellulase assay kit (CellG5 method) provided by Megazyme (Wicklow, Ireland; Product-code: K-CellG5-4V) according to the manufacturer's instructions. The CellG5 assay reagent for the measurement of endo-cellulase (endo-1,4-β-glucanase) contains two components: The reaction mixture consisted of 1) 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-D-cellopentanoside (BPNPG5) and 2) thermostable β-glucosidase. The ketone blocking group prevented any hydrolysis by β-glucosidase on BPNPG5. Incubation with endo-cellulase produced unblocked chromophoric oligosaccharides that were rapidly hydrolyzed by the auxiliary β-glucosidase. Thus, the rate of 4-nitrophenol formation was directly related to the hydrolysis of BPNPG5 by endo-cellulase.

[0370] Model Detergent A Composition (Liquid) Composition of Detergent A (liquid): Ingredients: 12% LAS, 11% AEO Biosoft N25-7 (NI), 7% AEOS (SLES), 6% MPG (monopropylene glycol), 3% ethanol, 3% TEA, 2.75% cocoa soap, 2.75% soy soap, 2% glycerol, 2% sodium hydroxide, 2% sodium citrate, 1% sodium formate, 0.2% DTMPA and 0.2% PCA, water to 100% (all percentages are w / w).

[0371] Composition of Model Detergent A2 (Liquid) Composition of detergent A2 (liquid): Ingredients: 12% LAS, 12% AEO Biosoft N25-7 (NI), 4% AEOS (SLES), 2% MPG (monopropylene glycol), 3% ethanol, 2% TEA (triethylamine), 3% soap, 0.5% sodium hydroxide, 3.9% sodium citrate, 1.5% DTMPA, Na7 (diethylenetriamine pentakis(methylene)pentakis(phosphonic acid), heptasodium salt), 0.5% phenoxyethanol, water to 100% (all percentages are w / w).

[0372] Proteases The protease used for the examples is that of SEQ ID NO: 10. Other proteases include that of SEQ ID NO: 11 or that of SEQ ID NO: 11 with the mutations S9E+N42R+N74D+V199I+Q200L+Y203W+S253D+N255W+L256E.

[0373] Washing assay Launder-O-Meter (LOM) model cleaning system The Launder-O-Meter (LOM) is a medium-scale model washing system that can be applied to simultaneously test up to 20 different washing conditions. The LOM is essentially a large, temperature-controlled water bath with 20 enclosed metal beakers that rotate inside. Each beaker corresponds to one small washing machine, and during the experiment, each will contain a solution of the specific detergent / enzyme system to be tested along with the soiled and unsoiled fabrics to be tested. Mechanical loading is achieved by rotating the beakers in the water bath and including metal balls in the beakers.

[0374] The LOM model wash system is primarily used for medium-scale testing of detergents and enzymes under European wash conditions. In LOM experiments, factors such as ballast-to-soil ratio and fabric-to-wash liquor ratio can be varied. Thus, LOM provides a bridge between small-scale experiments such as AMSA and miniwash and full-scale experiments in front-loader washing machines, which require longer wash times.

[0375] Mini Launder-O-Meter (MiniLOM) model cleaning system The MiniLOM is a modified Launder-O-Meter (LOM) miniature washing system, a medium-scale model washing system that can be applied to simultaneously test up to 20 different washing conditions. The LOM is essentially a large, temperature-controlled water bath with 20 enclosed metal beakers that rotate inside. Each beaker corresponds to one small washing machine, and during the experiment, each will contain a solution of the specific detergent / enzyme system to be tested along with the soiled and unsoiled fabrics to be tested. Mechanical loading is achieved by rotating the beakers in the water bath and by including metal balls in the beakers.

[0376] The LOM model wash system is primarily used for medium-scale testing of detergents and enzymes under European wash conditions. In LOM experiments, factors such as ballast-to-soil ratio and fabric-to-wash liquor ratio can be varied. Thus, LOM provides a bridge between small-scale experiments such as AMSA and miniwash and full-scale experiments in front-loader washing machines, which require longer wash times.

[0377] In the miniLOM, washes are performed in 50 ml tubes placed in a Stuart rotor.

[0378] Terg-O-Tometer (TOM) washing assay The Terg-O-Meter (TOM) is a medium-scale model washing system that can be adapted to simultaneously test 12 different washing conditions. The TOM is essentially a large, temperature-controlled water bath with up to 12 open metal beakers immersed inside. Each beaker corresponds to a small top-loading washing machine, and during the experiment, each will contain a solution of a specific detergent / enzyme system, as well as soiled and unsoiled fabrics, whose performance is being tested. Mechanical loading is achieved by a rotating stirring arm that agitates the liquid in each beaker. Because the TOM beakers do not have lids, samples can be removed during the TOM experiment, allowing for online analysis of the information during the wash.

[0379] The TOM model wash system is primarily used for mid-scale testing of detergents and enzymes under US or LA / AP wash conditions. In TOM experiments, factors such as ballast-to-soil ratio and fabric-to-wash liquor ratio can be varied. Thus, TOM provides a bridge between small-scale experiments and full-scale experiments in top-loader washing machines, which require more time.

[0380] Example 1: Determining the stability of cellulase variants (core stability method) The stability of cellulase variants is measured in 90% liquid detergent A containing protease. Stability in detergent is assessed by measuring the activity of the variants with the CellG5 kit after incubation in an enzymatic detergent mixture containing protease.

[0381] Temperature / protease load conditions in 90% detergent A: In a 96-well microplate (polystyrene), 20 μL of 1000 ppm purified endo-cellulase diluted in buffer (100 mM HEPES; 0.01% Tween-20; pH 7.5) is mixed with 180 μL of detergent A containing 0.3 mg / mL of active enzyme protease protein.

[0382] 15 μL of the enzyme / detergent mixture was transferred into two new 384-well microplates and sealed. One of the two identical plates was stored at 5°C (baseline), while the other was incubated at elevated temperature (challenge) for 16 or 17 hours. See the results table for the challenge temperatures used. After incubation, 60 μL of assay buffer (100 mM HEPES; 0.01% Tween-20; pH 7.5) was added to the samples in both plates and mixed vigorously for subsequent activity measurements.

[0383] Assay sample for cellulolytic activity (CellG5 kit): Enzyme activity is measured by mixing 20 μL of the diluted enzyme-detergent mixture with 10 μL of assay buffer (100 mM HEPES; 0.01% Tween-20; pH 7.5) and 10 μL of freshly prepared substrate solution in a UV-transparent 384-well microplate. The substrate solution from the CellG5 assay kit is prepared by mixing 10 μL of bottle #2 with 300 μL of bottle #1.

[0384] UV absorbance (405 nm) was measured kinetically (every 2 minutes for 44 minutes) using a microplate reader (Tecan; Infinite, M1000, pro). The portion of the curve showing a constant increase in absorbance was used to calculate the enzyme activity of the sample (mOD / min). The residual activity was then calculated as the enzyme activity of the sample incubated at elevated temperature for 16 or 17 hours relative to the enzyme activity in the corresponding sample stored at 5°C. Residual activity (%) = (activity, sample incubated at high temperature / activity, sample incubated at 5°C) * 100

[0385] Example 2: Linker stability assay principle Linker stability is measured by (A) incubating cellulase in a detergent containing a protease, and then (B) determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose-binding domain is affected by the protease, the binding affinity of the cellulase to cellulose fibers will be reduced.

[0386] Binding is determined by adding a dilution of the incubated cellulase to a suspension of cellulose fibers. After incubation at 5°C, the cellulase bound to cellulose is removed by centrifugation, and the amount of cellulase not bound to cellulose is determined by measuring the activity of the cellulase in the supernatant (C). The activity of the cellulase not bound to cellulose relative to the activity of a parallel sample incubated under similar conditions but in the absence of cellulose is a measure of linker stability.

[0387] Activity is based on detecting the number of reducing ends formed (D) following the hydrolysis of soluble carboxymethylcellulose (CMC), which is a substrate for both intact cellulases and cellulases lacking the cellulose-binding domain.

[0388] A. Incubation in a detergent containing protease chemicals Detergent: Model Detergent A Protease: SEQ ID NO: 10 HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent Dilution buffer: 50mM HEPES, pH8 Protease stock solution, e.g., protease of SEQ ID NO: 10 Detergent with 0.3 mg / mL protease: 300 ppm in model detergent A Model detergent A as above procedure 1) Prepare a detergent with 0.3 mg / mL of protease by adding the protease stock solution to 100 mL of detergent to a final protease concentration of 300 ppm of active protease protein in the detergent and mixing by magnetic stirring at room temperature for 1 hour. 2) Dilute cellulase to 300 ppm in dilution buffer. 3) Pipette 270 μL of detergent with protease from (1) into well positions A1 through D12 in a 96-well polypropylene microplate (Thermo Scientific™ 249944). 4) Add 30 μL of diluted cellulase from (2) to each well (positions A1-D12). Each cellulase is tested in triplicate; positions D4-D6 are used for blanks, with 30 μL of Milli Q water added instead of cellulase. 5) Add a small magnet to each well (positions A1-D12) and seal the plate with a heat seal (Thermo Scientific™ Adhesive PCR Plate Seal AB0558) and then mix by magnetic stirring for 30 minutes. 6) After mixing, the plates are incubated for the time and temperature indicated in the examples.

[0389] B. Binding to cellulose fibers chemicals Cellulose fibers: Avicel®, PH-101, (Sigma 11365) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent: Binding buffer: 50mM HEPES, pH8 Avicel suspension: 1.25g / 100mL Avicel in binding buffer mixed for 1 hour before use procedure: 1) 180 μL of Avicel suspension was added to positions A1 through D12 and 180 μL of binding buffer was added to positions E1 through H12 in a new 96-well microplate (Thermo Scientific™ cat. No. 269620). 2) Next, 20 μL aliquots of sample from each well in the incubated plate of step A were added to wells at positions A1 to D12 and positions E1 to H12, respectively. 3) The plate is shaken at a speed sufficient to keep the cellulose fibers in suspension for 1 hour at 5° C. to allow cellulase to bind to the cellulose. 4) After binding, the plate is centrifuged at 1500 rpm for 10 seconds and the supernatant is diluted 2.5 times in binding buffer (40 μL sample + 60 μL buffer). Both the supernatant from Avicel and the corresponding wells without Avicel are diluted.

[0390] C. CMC activity assay Chemicals: CMC: Sodium carboxymethylcellulose (Sigma C5678) K-Na-tartrate: Merck 8087 β-Glucosidase: Megazyme (Thermotoga maritima; Accession No. Q08638, Megazyme Cat. No. E-BGOS™) diluted to 0.1 mg / mL (specific activity 70 U / mg and activity in the product approximately 460 U / mL -> 6.57 mg / mL) PAHBAH 4-hydroxybenzhydrazide (Sigma H9882) NaOH Sodium Hydroxide (JTBaker 0402.1000) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent: Assay buffer: 50mM HEPES, pH8 CMC substrate: 1.25g CMC / 100mL assay buffer, mixed for 1 hour before use PAHBAH buffer: 50g / L K-Na-tartrate + 20g / L NaOH PAHBAH Reagent: 15 mg / mL PAHBAH in PAHBAH Buffer β-Glucosidase solution: 0.1 mg / mL β-glucosidase in assay buffer procedure: 1) Pipette 160 μL of CMC substrate into a new 96-well microplate (Thermo Scientific™ cat. No. 269620) 2) Add 20 μL of diluted supernatant from step B along with 20 μL of β-glucosidase solution 3) The plate was sealed with a heat seal (Thermo Scientific™ Adhesive PCR Plate Seal AB0558) and incubated at 40°C for 45 minutes. 4) After the reaction, 100 μL of each well was transferred to a ThermoFast 96 non-skirted PCR plate (Thermo Scientific cat. No. AB-0600), and then 75 μL of PAHBAH reagent was transferred. 5) The plate (4) is then sealed with a sealing foil (Greiner bio-one plate sealer, Cat. No. 676001) and incubated at 95°C for 10 minutes, followed by cooling at 10°C for 5 minutes in a BioRad T100™ thermal cycler PCR device. 6) After cooling, 100 μL aliquots were transferred to a new 96-well microplate (Thermo Scientific™ cat. No. 269620) and the absorbance was measured at 405 nm (A 405nm The absorbance is a measure of the activity of cellulase in the supernatant.

[0391] D. Data Processing 1) From the absorbance readings in step C, the average of three blank wells containing no Avicel, A 405nm (blank_ref) was calculated (position H4->H6), the average of the three blanks of the wells containing Avicel, A 405nm (blank_Avicel) is calculated (position D4->D6). 2) The absorbance readings from the cellulase-containing wells are then corrected for their corresponding blanks (i.e., those from (1)). 3) Linker stability: 1-[Act 405nm (+Avicel) / Act405nm (-Avicel)] (In the formula, Act 405nm (+Avicel) and Act 405nm (-Avicel) is calculated as the activity in wells with supernatant from incubation with Avicel and from incubation without Avicel, respectively (ie, absorbance corrected for blank). 4) Linker stabilities reported in the examples are the average of the triplicates analyzed.

[0392] This assay clearly distinguishes between binding in the presence and absence of the core, as further demonstrated by Example 3 below.

[0393] Example 3: Cellulose binding assay - no protease principle Cellulase is bound to cellulose by incubation with Avicel in a dilute detergent solution for 60 minutes at 5°C (A). After incubation, the activity of cellulase not bound to cellulose is determined in the supernatant (B) and compared to a parallel cellulase sample incubated in the absence of cellulase. The temperature during incubation with Avicel is kept low to ensure that the catalytic activity of cellulase during the binding step does not significantly affect the binding assay.

[0394] A. Binding to cellulose chemicals Detergent: Model Detergent A HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 Cellulose fibers: Avicel®, PH-101, (Sigma 11365) reagent Binding buffer: 50mM HEPES, pH8 Avicel suspension: 1.25g / 100mL Avicel in binding buffer mixed for 1 hour before use Model detergent A as above procedure 1) Dilute cellulase to 300 ppm in binding buffer. 2) Pipette 270 μL of detergent into well positions A1 through D12 in a 96-well polypropylene microplate (Thermo Scientific™ 249944). 3) Add 30 μL of diluted cellulase from (1) to each well (positions A1-D12). Each cellulase is tested in triplicate; positions D4-D6 are used for blanks, with 30 μL of Milli Q water added instead of cellulase. 4) Add a small magnet to each well (positions A1-D12) and seal the plate with a heat seal (Thermo Scientific Adhesive PCR Plate Seal AB0558), then mix by magnetic stirring for 30 minutes. 5) Pipette 160 μL of Binding Buffer into a new 96-well microplate (Thermo Scientific™ Nunc™ 96-well Polypropylene DeepWell™ Storage Plate) (positions A1-D12) and pipette 160 μL of Avicel suspension into positions E1-H12. 6) Add 20 μL of Milli Q water to all wells (A1 to H12). 7) Add 20 μL aliquots of the cellulase detergent sample from (4) to wells containing Avicel (A1-D12) and wells not containing Avicel (E1-H12). 8) The plate is then incubated in a cold room at 5°C for 60 minutes to allow cellulase to bind to the cellulose in a Heidolph Titramax 101 shaker. The shaking speed is adjusted to ensure the cellulose remains suspended during the incubation. 9) After binding, the plate is centrifuged at 1500 rpm for 10 seconds and the supernatant is diluted 2.5 times in binding buffer (40 μL sample + 60 μL buffer). Both the supernatant from Avicel and the corresponding wells without Avicel are diluted.

[0395] B. CMC activity assay Chemicals: CMC: Sodium carboxymethylcellulose (Sigma C5678) K-Na-tartrate: Merck 8087 β-Glucosidase: Megazyme (Thermotoga maritima; Accession No. Q08638, Megazyme Cat. No. E-BGOS™) diluted to 0.1 mg / mL (specific activity 70 U / mg and activity in the product approximately 460 U / mL -> 6.57 mg / mL) PAHBAH 4-hydroxybenzhydrazide (Sigma H9882) NaOH Sodium Hydroxide (JTBaker 0402.1000) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent: Assay buffer: 50mM HEPES, pH8 CMC substrate: 1.25g CMC / 100mL assay buffer, mixed for 1 hour before use PAHBAH buffer: 50g / L K-Na-tartrate + 20g / L NaOH PAHBAH Reagent: 15 mg / mL PAHBAH in PAHBAH Buffer β-Glucosidase solution: 0.1 mg / mL β-glucosidase in assay buffer procedure: 1) Pipette 160 μL of CMC substrate into a new 96-well microplate (Thermo Scientific cat. No. 269620). 2) Add 20 μL of diluted supernatant from step A along with 20 μL of β-glucosidase solution 3) The plate was sealed with a heat seal (Thermo Scientific adhesive PCR plate seal AB0558) and incubated at 40°C for 45 minutes. 4) After the reaction, 100 μL of each well was transferred to a ThermoFast 96 non-skirted (registered trademark) PCR plate (Thermo Scientific cat. No. AB-0600), and then 75 μL of PAHBAH reagent was transferred. 5) The plate (4) is then sealed with a sealing foil (Greiner bio-one plate sealer, Cat. No. 676001) and incubated at 95°C for 10 minutes, followed by cooling at 10°C for 5 minutes in a BioRad T100™ thermal cycler PCR device. 6) After cooling, 100 μL aliquots were transferred to a new 96-well microplate (Thermo Scientific cat. No. 269620) and the absorbance was measured at 405 nm (A 405nm The absorbance is a measure of the activity of cellulase in the supernatant.

[0396] C. Data Processing 1) From the absorbance readings in step B, the average of three blank wells containing no Avicel, A 405nm (blank_ref) was calculated (position H4->H6), the average of the three blanks of the wells containing Avicel, A 405nm (blank_Avicel) is calculated (position D4->D6). 2) The absorbance readings from the cellulase-containing wells are then corrected for their corresponding blanks (i.e., those from (1)). 3) The bond is 1-[Act 405nm (+Avicel) / Act 405nm (-Avicel)] (In the formula, Act 405nm (+Avicel) and Act 405nm (-Avicel) is calculated as the activity in wells with supernatant from incubation with Avicel and from incubation without Avicel, respectively (i.e., absorbance corrected for blank). Linker stability reported in the examples is the average of the triplicates analyzed.

[0397] To demonstrate this, samples of cellulase both with and without a CBM were tested for binding to cellulase as described in this example, with the ratio calculated as the binding of the intact cellulase, i.e., the cellulase with the catalytic domain, linker, and CBM, relative to the catalytic domain alone.

[0398] [Table 7]

[0399] The data clearly demonstrate that in the absence of CBM, binding to cellulase is significantly reduced.

[0400] Example 4: Construction of glycoside hydrolase variants Cellulase variants were constructed from Thielavia terrestris cellulase (SEQ ID NO: 1). The variants were generated by traditional cloning of DNA fragments using PCR with appropriately designed oligonucleotides to introduce the desired mutations into the resulting sequence (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). Alternatively, synthetic gene fragments purchased from vendors such as IDT DNA were used to replace the native DNA sequence with the new desired DNA sequence.

[0401] Oligos are designed corresponding to the DNA sequence flanking the desired mutation site or stretch of DNA to be replaced, separated by DNA base pairs that define the insertion / deletion / substitution / synthetic DNA sequence, and are purchased from an oligo vendor such as IDT DNA. To test variants of the invention, the mutated DNA containing the variant of the invention is introduced into competent A. oryzae by homologous recombination, fermented using standard protocols (yeast extract-based medium, 4-5 days, 30°C), and purified as follows.

[0402] The culture broth was filtered through a Nalgene 0.2 μm filtration unit to remove Aspergillus host cells. The pH of the filtrate was adjusted to pH 4.0 with 20% CH3COOH, and the pH-adjusted filtrate was applied to a Capto MMC column (GE Healthcare) equilibrated in 20 mM CH3COOH / NaOH, 1 mM CaCl2, pH 4.0. After extensive washing of the column with equilibration buffer, cellulase was eluted with 50 mM Tris-base, 1 mM CaCl2, unbuffered. Fractions from the column were analyzed for cellulase activity. The cellulase peak was pooled and applied to a Q-Sepharose FF column (GE Healthcare) equilibrated in 50 mM Tris / HCl, pH 9.0. After extensive washing of the column with equilibration buffer, the cellulase is eluted with a linear NaCl gradient over three column volumes between equilibration buffer and 50 mM Tris / HCl, 5 mM CaCl2, 500 mM NaCl, pH 9.0. Fractions from the column are analyzed for cellulase activity, and the cellulase peak is pooled as the purified product. The purified variants are analyzed by SDS-PAGE. Because the cellulase variants are glycosylated, they yield a diffuse band on a Coomassie-stained SDS-PAGE gel. The purified product is used for further characterization.

[0403] Example 5: Linker stability assay during washes with proteases principle Linker stability is measured by (A) incubating cellulase in a detergent wash solution containing a protease, and then (B) determining the ability of the incubated cellulase to bind to cellulose fibers. If the linker or cellulose binding domain is affected by the protease, the binding affinity of the cellulase to cellulose fibers will be reduced.

[0404] Binding is determined by adding a dilution of the incubated cellulase to a suspension of cellulose fibers. After incubation at 5°C, the cellulase bound to cellulose is removed by centrifugation, and the amount of cellulase not bound to cellulose is determined by measuring the activity of the cellulase in the supernatant (C). The activity of the cellulase not bound to cellulose relative to the activity of a parallel sample incubated under similar conditions but in the absence of cellulose is a measure of linker stability.

[0405] Activity is based on detecting the number of reducing ends formed (D) following the hydrolysis of soluble carboxymethylcellulose (CMC), which is a substrate for both intact cellulases and cellulases lacking the cellulose-binding domain.

[0406] E. Incubation in Detergent Containing Proteases chemicals Detergent: Model Detergent A Protease: Protease having SEQ ID NO: 10 HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent Dilution buffer: 50mM HEPES, pH8 Detergent with protease: 0.3 μg / mL of active protease protein in model detergent A Detergent cleaning solution: 3.3 g / L detergent with protease in water with a water hardness of 15° dH procedure 7) Prepare the detergent cleaning solution 8) Dilute cellulase to 300 ppm in dilution buffer. 9) Pipette 270 μL of Detergent Wash Solution from (1) into well positions A1 through D12 in a 96-well polypropylene microplate (Thermo Scientific™ 249944). 10) Add 30 μL of diluted cellulase from (2) to each well (positions A1-D12). Each cellulase is tested in triplicate; positions D4-D6 are used for blanks, with 30 μL of Milli Q water added instead of cellulase. 11) Add a small magnet to each well (positions A1-D12) and seal the plate with a heat seal (Thermo Scientific™ Adhesive PCR Plate Seal AB0558) and then mix by magnetic stirring for 30 minutes. 12) After mixing, the plate is incubated for the time and temperature indicated in the examples, for example, 2 hours at 40°C.

[0407] F. Binding to cellulose fibers chemicals Cellulose fibers: Avicel®, PH-101, (Sigma 11365) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent: Binding buffer: 50mM HEPES, pH8 Avicel suspension: 1.25g / 100mL Avicel in binding buffer mixed for 1 hour before use procedure: 5) 180 μL of Avicel suspension was added to positions A1 through D12 and 180 μL of binding buffer was added to positions E1 through H12 in a new 96-well microplate (Thermo Scientific™ cat. No. 269620). 6) Next, 20 μL aliquots of sample from each well in the incubated plate from step A were added to wells at positions A1 through D12 and positions E1 through H12, respectively. 7) The plate is shaken at a speed sufficient to keep the cellulose fibers in suspension for 1 hour at 5° C. to allow cellulase to bind to the cellulose. 8) After binding, the plate is centrifuged at 1500 rpm for 10 seconds and the supernatant is diluted 2.5 times in binding buffer (40 μL sample + 60 μL buffer). Both the supernatant from Avicel and the corresponding wells without Avicel are diluted.

[0408] G. CMC activity assay Chemicals: CMC: Sodium carboxymethylcellulose (Sigma C5678) K-Na-tartrate: Merck 8087 β-Glucosidase: Megazyme (Thermotoga maritima; Accession No. Q08638, Megazyme Cat. No. E-BGOS™) diluted to 0.1 mg / mL (specific activity 70 U / mg and activity in the product approximately 460 U / mL -> 6.57 mg / mL) PAHBAH 4-hydroxybenzhydrazide (Sigma H9882) NaOH Sodium Hydroxide (JTBaker 0402.1000) HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Sigma H3375 reagent: Assay buffer: 50mM HEPES, pH8 CMC substrate: 1.25g CMC / 100mL assay buffer, mixed for 1 hour before use PAHBAH buffer: 50g / L K-Na-tartrate + 20g / L NaOH PAHBAH Reagent: 15 mg / mL PAHBAH in PAHBAH Buffer β-Glucosidase solution: 0.1 mg / mL β-glucosidase in assay buffer procedure: 7) Pipette 160 μL of CMC substrate into a new 96-well microplate (Thermo Scientific™ cat. No. 269620) 8) Add 20 μL of diluted supernatant from step B along with 20 μL of β-glucosidase solution 9) The plate was sealed with a heat seal (Thermo Scientific™ Adhesive PCR Plate Seal AB0558) and incubated at 40°C for 45 minutes. 10) After the reaction, transfer 100 μL from each well to a ThermoFast 96 non-skirted PCR plate (Thermo Scientific cat. No. AB-0600), and then transfer 75 μL of PAHBAH reagent. 11) Next, the plate (4) is sealed with a sealing foil (Greiner bio-one plate sealer, Cat. No. 676001), incubated at 95°C for 10 minutes, and then cooled at 10°C for 5 minutes in a BioRad T100™ thermal cycler PCR device. 12) After cooling, 100 μL aliquots were transferred to a new 96-well microplate (Thermo Scientific™ cat. No. 269620) and the absorbance was measured at 405 nm (A 405nm The absorbance is a measure of the activity of cellulase in the supernatant.

[0409] H. Data Processing 5) From the absorbance readings in step C, the average of the three blank wells containing no Avicel, A 405nm (blank_ref) was calculated (position H4->H6), the average of the three blanks of the wells containing Avicel, A 405nm (blank_Avicel) is calculated (position D4->D6). 6) The absorbance readings from the cellulase-containing wells are then corrected for their corresponding blanks (i.e., those from (1)). 7) Linker stability: 1-[Act 405nm (+Avicel) / Act 405nm (-Avicel)] (In the formula, Act 405nm (+Avicel) and Act 405nm (-Avicel) is calculated as the activity in wells with supernatant from incubation with Avicel and from incubation without Avicel, respectively (ie, absorbance corrected for blank). 8) Linker stabilities reported in the examples are the average of the triplicates analyzed.

[0410] Example 6: Variant of CBM SEQ ID NO: 173 with P7G linker (SEQ ID NO: 30) The variants were tested as described in Example 2, Linker Stability Assay, with the following modifications: the detergent was model detergent A2, protease SEQ ID NO: 10. The variants were incubated in the detergent with the protease at 45°C for 3 days before being tested for residual activity and ability to bind to cellulose.

[0411] [Table 8]

[0412] [Table 9]

[0413] [Table 10]

[0414] CBM notation: A "+" following a SEQ ID NO: indicates that the specified mutation has been introduced into the referenced sequence. For example, 173+S34H indicates that S at position 34 of SEQ ID NO: 173 has been replaced with H.

[0415] The data clearly demonstrate the positive impact of replacing the CBM having SEQ ID NO:7 with the CBM having SEQ ID NO:173 or a variant of the CBM having SEQ ID NO:173.

[0416] Example 7: Variants of CBM SEQ ID NO: 173 with different linkers Variants of SEQ ID NO: 173 with different linkers were tested as described in Example 2, Linker Stability Assay, with the following modifications: the detergent was model detergent A2, protease SEQ ID NO: 10. The variants were incubated in the detergent with protease at 45° C. for 3 days before being tested for residual activity and ability to bind to cellulose.

[0417] [Table 11]

[0418] [Table 12]

[0419] [Table 13]

[0420] [Table 14]

[0421] [Table 15]

[0422] [Table 16]

[0423] [Table 17]

[0424] [Table 18]

[0425] CBM notation: A "+" following a SEQ ID NO: indicates that the specified mutation has been introduced into the referenced sequence. For example, 173+S34H,L37R indicates that S at position 34 of SEQ ID NO: 173 has been replaced with H and L at position 37 of SEQ ID NO: 173 has been replaced with R.

[0426] The results demonstrate that improved stability can be obtained when different proline-rich linkers are combined with variants of SEQ ID NO: 173.

[0427] Example 8: Variant of CBM SEQ ID NO: 173 with P7G linker (SEQ ID NO: 30) Variants of CBM having SEQ ID NO: 173, containing the mutations S34H, L37R and at least one additional mutation along with a P7G linker (SEQ ID NO: 30), were tested as described in Example 2, Linker Stability Assay, with the following modifications: the detergent was model detergent A2, protease SEQ ID NO: 10. The variants were incubated in the detergent with the protease at 45°C for 3 days before being tested for residual activity and ability to bind to cellulose.

[0428] [Table 19]

[0429] [Table 20]

[0430] [Table 21]

[0431] [Table 22]

[0432] [Table 23]

[0433] CBM notation: A "+" following a SEQ ID NO: indicates that the specified mutations have been introduced into the referenced sequence. For example, 173+A22E,S34H,L37R indicates that A at position 22 of SEQ ID NO: 173 has been substituted with E, S at position 34 of SEQ ID NO: 173 has been substituted with H, and L at position 37 of SEQ ID NO: 173 has been substituted with R.

[0434] The results demonstrate that improved stability can be obtained relative to a CBM having SEQ ID NO:7 when mutations are introduced into SEQ ID NO:173 at positions selected from 14, 18, 21, 22, 25, 27, 28, 34, and 37.

Claims

1. A variant of the carbohydrate binding module, comprising a substitution at position 34 of the polypeptide of SEQ ID NO: 173 with H, Y, K or R; the variant further comprises one or more catalytic domains and one or more linkers from a polypeptide having at least 90% sequence identity but less than 100% sequence identity to the polypeptide of SEQ ID NO: 173 and having glycoside hydrolase activity; and the variant has carbohydrate-binding activity.

2. The variant of claim 1, which is a variant of carbohydrate binding module family 1 (CBM1).

3. 3. The variant of claim 1 or 2, comprising at least one substitution selected from the group consisting of: S34H; S34Y; S34K; and S34R.

4. Substitution of the amino acid residue at position 34 with H, Y, K, or R; or Substitution of the amino acid residue at position 34 with H, Y, K, or R and substitution of the amino acid residue at position 37 with R The variant according to any one of claims 1 to 3, comprising:

5. S34H; S34Y; S34K; S34R; S34Y, L37R; T18K, S34H; S34H, L37R; T18K, S34Y, L37R; S34H, L37R, Y14W; S34H, L37R, T18K; S34H, L37R, T18R; S34H, L37R, V21E; S34H, L37R, A22E; S34H, L37R, A22P; S34H, L37R, A22N; S34H, L37R, Q28P; S34H, L37R, L29P; S34H, L37R, T25E; and S34H, L37R, T27I 5. The variant of any one of claims 1 to 4, comprising a substitution selected from the group consisting of:

6. A cellulase variant comprising the CBM variant according to any one of claims 1 to 4.

7. A variant having glycoside hydrolase activity, comprising: (a) one or more catalytic domains derived from a polypeptide having glycoside hydrolase activity; (b) one or more linkers; and (c) one or more carbohydrate-binding module variants according to any one of claims 1 to 6.

8. 8. The glycoside hydrolase variant of claim 7, which has improved thermostability in the presence of proteases.

9. A detergent composition comprising the glycoside hydrolase variant according to claim 7 or 8.

10. An isolated polynucleotide encoding the glycoside hydrolase variant of claim 7 or 8.

11. A nucleic acid construct or expression vector comprising the polynucleotide of claim 10.

12. A recombinant host cell transformed with the polynucleotide of claim 10.

13. 1. A method for producing a variant of a glycoside hydrolase variant, comprising: a. culturing the recombinant host cell of claim 12 under conditions suitable for expression of the variant; and b. Recovering the variant. A method comprising:

14. 10. Use of a glycoside hydrolase variant according to claim 7 or 8 or a detergent composition according to claim 9 for fabric or textile care.

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