Mannanase variants and methods of use

Abstract

Disclosed herein are one or more mannase variants, polynucleotides encoding mannase, compositions containing mannase, and methods of use thereof, including one or more mannase variants having improved stability as compared to one or more reference mannase. Compositions containing mannase are suitable for use as detergents and for cleaning fabrics and hard surfaces, as well as in a variety of other industrial applications.

Description

[0001] Disclosed herein are one or more mannanase variants, polynucleotides encoding mannanases, compositions containing mannanases, and methods of use thereof, including one or more mannanase variants having improved stability compared to one or more reference mannanases. Compositions containing mannanases are suitable for use as detergents and for cleaning fabrics and hard surfaces, as well as in various other industrial applications.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 374,387, filed on September 2, 2022, which is incorporated by reference in its entirety.

[0004] Reference to a sequence listing submitted electronically

[0005] The contents of the sequence listing submitted electronically with the application in an XML file (file name: NB41943PCT_SequenceListing; size: 8,148 bytes; created on: August 22, 2023) constitute a part of this application and are hereby incorporated by reference in their entirety. Background Art

[0006] Mannanases (including endo-β-mannanases) have been applied to detergent cleaning compositions for removing colloidal stains by hydrolyzing mannans. A variety of mannans are found in nature, such as linear mannans, glucomannans, galactomannans, and glucogalactomannans. Each such mannan consists of a polysaccharide containing a β-1,4-linked backbone of mannose residues, which can be substituted by up to 33% of glucose residues (Yeoman et al., Adv Appl Microbiol [Advances in Applied Microbiology], 70:1, 2010, Elsevier Press (Elsevier)). In galactomannans or glucogalactomannans, the galactose residues are linked to the mannan backbone with α-1,6-links (Moreira and Filho, Appl Microbiol Biotechnol [Applied Microbiology and Biotechnology], 79:165, 2008). Therefore, hydrolysis of mannan into its component sugars requires endo-1,4-β-mannanases that hydrolyze the backbone bonds to produce short-chain manno-oligosaccharides, which are further degraded into monosaccharides by 1,4-β-mannosidases.

[0007] Although mannanases, such as, for example, endo-β-mannanases, are known in the art of industrial enzymes, there is still a need for improved mannanase variants. Summary of the invention

[0008] The variants, compositions and methods disclosed herein relate to recombinant mannanases or recombinant polypeptides produced by conventional molecular biology techniques (see, e.g., Sambrook et al., Molecular Cloning: Cold Spring Harbor Laboratory Press).

[0009] In one embodiment, a mannanase variant is provided, wherein the mannanase variant comprises one or more amino acid substitutions at one or more positions selected from 32, 72, 161 and 172, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0010] In one embodiment, the mannanase variant is a variant comprising amino acid substitutions selected from the group consisting of 19D, 32Y, 34D, 72V, 93Q, 131S, 136P, 139R, 161G, 172F, 225N / Q, 259D, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0011] In one embodiment, the mannanase variant is a variant comprising two or more amino acid substitutions selected from the group consisting of 19D, 32Y, 72V, 93Q, 131S, 136P, 139R, 161G, 168T, 172F, 225N / Q, 259D / E, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0012] In one embodiment, the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of 19D-276W, 32Y-259D, 93Q-276W, 131S-276W, 136P-276W, 139R-276W, 161G-276W, 225N / Q-276W, 259D / E-276W and 261D / E-276W.

[0013] In one embodiment, the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of 19D-131S-276W, 32Y-261D-276W, 32Y-259D-276W, 32Y-172F-259D, 168T-259D-276W, 259D-261E-276W and 259Q-261E-276W.

[0014] In one embodiment, the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of Y061W-G259D-R261E-F276W, Y061W-T062E-G259D-R261E-F276W, Y061W-V228T-G259D-R261E-F276W, V228T-G259D-R261E-F276W, F032Y-G259D-R261E-F276W, F032Y-Y061W-Y167F-P168S-G259D-R261E-F276W, F032Y-Y061W-G259D-R261E- F276W, F032Y-Y061W-T062E-G259D-R261E-F276W, F032Y-T062E-R261D- F276W, F032Y-T062E-G259D-F276W, T062E-G259D-R261E-F276W, V059S-Q 060L-G259D-R261E-F276W, V059S-Q060L-Y061W-G259D-R261E-F276W, V 059S-Q060L-T062E-G259D-R261E-F276W, T062E-V228T-G259D-R261E-F2 76W, F032Y-T062E-G259D-R261E-F276W, F032Y-T062E-G259D-R261E-F2 76W-T284E, E019D-A068S-T131S-F276W, F032Y-T062E-G259D-F276W-T28 4E, F032Y-T062E-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-G25 9D-F276W-T284E, F032Y-T062E-A068S-G259D-F276W-T284E, F032Y-Y061 W-T062E-G259D-F276W-T284E, F032Y-V059S-Q060L-T062E-G259D-F276 W-T284E, N010T-F032Y-T062E-G259D-F276W-T284E, F032Y-T062E-Y167F -P168S-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-V228T-G259D -F276W-T284E, F032Y-T062E-I072V-Y167F-P168S-G259D-F276W-T284E,F032Y-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-V228T-G259D-F276W-T284E、F032Y-V059S、Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-G259D-F276W-T284E、F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E and N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E, F032Y-Y061W-T062E-G259D-R261E-F276W-T284E, F032Y-T062E-A068S-G259D-R261E-F276W-T284 E. F032Y-T062E-T131S-G259D-R261E-F276W-T284E, F032Y-T062E-N150D -G259D-R261E-F276W-T284E, F032Y-T062E-P168S-G259D-R261E-F276W- T284E, F032Y-T062E-L235K-G259D-R261E-F276W-T284E, N010T-F032Y-Y 061W-T062E-P168A-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T06 2E-Y167F-P168S-V228T-G259E-F276W-T284E, F032Y-Y061W-T062E-T131 S-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T062E -A136P-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W- T062E-K093Q-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y 061W-T062E-Y167F-P168S-V228T-L235K-G259D-F276W-T284E, N010T-F0 32Y-Y061W-T062E-D139R-Y167F-P168S-V228T-G259D-F276W-T284E, N01 0T-F032Y-Y061W-T062E-N150D-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-Y167F-P168A-V228T-G259D-F276W-T284E,N010T-F032Y-Y061W-T062E-T131S-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-P168S-V228T-G259D-F276W-T284E, N010T-F03 2Y-Y061W-T062E-A068S-Y167F-P168A-G259D-F276W-T284E, and N010T-F032Y-Y061W-T062E-A068S-Y167F-P168A-G259E-F276W-T284E, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0015] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:1.

[0016] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1.

[0017] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant has improved stability when compared to a parent mannanase or a reference mannanase.

[0018] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant has equal or improved cleaning performance in a detergent when compared to a parent mannanase or a reference mannanase.

[0019] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the mannanase variant has mannanase activity.

[0020] Also provided are polynucleotide compositions comprising a nucleic acid sequence encoding one or more mannanase variants described herein, wherein the polynucleotides are optionally isolated.

[0021] Further provided are enzyme compositions comprising one or more mannanase variants described herein. In one aspect, the enzyme composition is an enzyme granule. In another aspect, the enzyme composition further comprises one or more other enzymes selected from the group consisting of acyltransferases, amylases, α-amylases, β-amylases, α-galactosidases, arabinanases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, hyaluronic acid enzymes, keratinases, laccases, lactases, ligninases, lipases, lipolytic enzymes, lipoxygenases, lysozymes, mannanases, metalloproteinases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, subtilisins, tannases, transglutaminases, xylan acetylesterases, xylanases, and xylosidases; and combinations thereof.

[0022] Further provided are cleaning compositions comprising one or more mannanase variants described herein.In one aspect, the cleaning composition is a detergent composition selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, a medical device cleaning detergent, and a hard surface cleaning detergent.

[0023] The present disclosure also provides a cleaning method. In one embodiment, the method is a cleaning method, which includes contacting a surface or article in need of cleaning with an effective amount of a mannanase variant described herein or an enzyme composition described herein; and optionally further comprising the following steps: rinsing the surface or article after contacting the surface or article with the variant or enzyme composition. In one aspect, the article is a dishware or a fabric.

[0024] In some embodiments, the mannanase variants have cleaning activity in detergent compositions. In some embodiments, the mannanase variants have mannanase activity in the presence of a protease. In some embodiments, the mannanase variants retain at least 50% of the mannanase activity in the presence of a protease. In some embodiments, the mannanase variants are capable of hydrolyzing a substrate selected from the group consisting of guar gum, locust bean gum, and combinations thereof. In some embodiments, the mannanase variants do not further comprise a carbohydrate binding module.

[0025] In some embodiments, the method is a cleaning method, which comprises contacting a surface or item in need of cleaning with an effective amount of a mannanase described herein or an enzyme composition comprising a mannanase variant described herein; and optionally further comprising the step of rinsing the surface or item after contacting the surface or item with the variant or enzyme composition.

[0026] Still further embodiments relate to methods of cleaning comprising contacting a surface or item comprising a soil or stain containing mannan with (i) a mannanase variant or recombinant polypeptide or (ii) a cleaning composition described herein, wherein the mannan contained in the soil or stain is hydrolyzed.

[0027] Some embodiments further relate to nucleic acids or isolated nucleic acids encoding mannanase variants or recombinant polypeptides described herein. Additional embodiments relate to expression vectors comprising nucleic acids or isolated nucleic acids described herein operably linked to regulatory sequences. Even additional embodiments relate to host cells comprising expression vectors as described herein, or nucleic acids encoding mannanase variants or recombinant polypeptides described herein. In some embodiments, the host cell is a bacterial cell or a fungal cell. Still additional embodiments relate to methods for producing mannanase variants described herein, comprising: stably transforming host cells with expression vectors comprising polynucleotides encoding mannanase variants; culturing the transformed host cells under suitable conditions to produce mannanase variants; and recovering mannanase variants. DETAILED DESCRIPTION

[0028] In one embodiment, the disclosure provides one or more mannanase variants comprising one or more amino acid substitutions as described in more detail below. In some embodiments, when compared to a reference mannanase having an amino acid sequence of SEQ ID NO: 1 or 2, the variants provided herein exhibit one or more improved properties, such as improved stability. The mannanase variants provided herein can be used to prepare cleaning compositions (e.g., automatic dishwashing compositions). In addition, the mannanase variants provided herein can also be used in cleaning methods (e.g., dishwashing methods) using such variants or compositions comprising such mannanase variants.

[0029] Unless otherwise indicated herein, one or more of the mannanase variants described herein can be made and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, the recombinant DNA field, and industrial enzyme use and development.

[0030] In one embodiment, one or more of the mannanase variants described herein have glycosyl hydrolase activity and / or are stable in the presence of a protease. These features of the mannanases described herein make them suitable for use in a variety of cleaning and other industrial applications, for example, in which the enzymes can hydrolyze mannans in the presence of surfactants, proteases, and / or other components found in detergent compositions.

[0031] The following terms are defined for clarity. Undefined terms and abbreviations should conform to their conventional meanings used in the art. For example, technical and scientific terms undefined herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs (see, for example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology [microbiology and molecular biology dictionary], 2nd edition, John Wiley and Sons, New York, 1994; and Hale and Marham, The Harper Collins Dictionary of Biology [Harper Collins biological dictionary], Harper Perennial Press (Harper Perennial), New York, 1991). Unless the context clearly indicates otherwise, as used herein, the singular terms "a / an" and "the / the" include plural indicators. Unless otherwise indicated, nucleic acid sequences are written from left to right in a 5' to 3' direction; and amino acid sequences are written from left to right in an amino to carboxyl direction. Every numerical range used herein will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0032] As used herein in conjunction with a numerical value, the term "about" refers to a range of + / -0.5 of the numerical value, unless the term is otherwise specifically defined in the context. For example, the phrase "a pH value of about 6" refers to a pH value of 5.5 to 6.5, unless the pH value is otherwise specifically defined.

[0033] The nomenclature of the amino acid substitution of one or more mannanase variants described herein uses one or more of the following: position; position: one or more amino acid substitutions; or one or more starting amino acids: position: one or more amino acid substitutions. Reference to "position" (e.g., 5, 8, 17, 22, etc.) covers any starting amino acid that may be present at such a position, and any substitution that may be present at such a position. Reference to "position: one or more amino acid substitutions" (e.g., 1S / T / G, 3G, 17T, etc.) covers any starting amino acid that may be present at such a position and one or more amino acids that may replace such a starting amino acid. Reference to position can list several forms, for example, position 003 can also be referred to as position 03 or 3. Reference to starting or substituted amino acids can be further represented as several starting or substituted amino acids separated by a foreslash (" / "). For example, D275S / K means that position 275 is substituted with serine (S) or lysine (K), and P / S197K means that the starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K). References to X as an amino acid at a position refer to any amino acid at the listed position.

[0034] The positions of the amino acid residues in a given amino acid sequence are numbered by corresponding to the amino acid sequence of SEQ ID NO:1. That is, the amino acid sequence of SEQ ID NO:1 is used as a reference sequence for numbering the positions of the amino acid residues. For example, the amino acid sequence of one or more mannanase variants described herein is compared with the amino acid sequence of SEQ ID NO:1 using an alignment algorithm as described herein, and each amino acid residue in a given amino acid sequence aligned (preferably, optimally aligned) with the amino acid residues in SEQ ID NO:1 is conveniently numbered by reference to the numerical position of the corresponding amino acid residue. When compared to a query sequence (sometimes also referred to as a "reference sequence"), one or more positions where insertion or deletion occurs in the subject sequence will be identified, for example, as a sequence alignment algorithm as described herein.

[0035] The term "mannan endo-1,4-β-mannosidase," "endo-1,4-β-mannanase," "endo-β-1,4-mannanase," "β-mannanase B," "β-1,4-mannan 4-mannosylhydrolase," "endo-β-mannanase," "β-D-mannanase," "1,4-β-D-mannan mannohydrolase," or "endo-β-mannanase" (EC 3.2.1.78) refers to an enzyme that is capable of randomly hydrolyzing 1,4-β-D-mannosidic bonds in mannans, galactomannans, and glucomannans. Endo-1,4-β-mannanase is a member of several families of glycosyl hydrolases, including GH26 and GH5. In particular, endo-β-mannanases constitute a group of polysaccharidases that degrade mannans and represent enzymes that are capable of cleaving polysaccharide chains containing mannose units (i.e., capable of cleaving glycosidic bonds in mannans, glucomannans, galactomannans, and galactoglucomannans). The "endo-β-mannanases" described herein may have additional enzymatic activities (e.g., endo-1,4-β-glucanase, 1,4-β-mannosidase, and cellodextrinase activity).

[0036] The term "mannanase", "mannosidic enzyme", "mannolytic enzyme", "mannanase enzyme", "mannanase polypeptide", or "mannanase protein" refers to an enzyme, polypeptide, or protein that can degrade mannan. The mannanase can, for example, be an endo-β-mannanase, an exo-β-mannanase, or a glycosyl hydrolase. As used herein, mannanase activity can be determined according to any procedure known in the art (see, e.g., Lever, Anal. Biochem, 47:273, 1972; Eriksson and Winell, Acta Chem. Scand., (1968), 22:1924; US 6,602,842; and WO 95 / 35362A1).

[0037] As used herein, "mannan" is a polysaccharide having a backbone consisting of β-1,4-linked mannose; "glucomannan" is a polysaccharide having a backbone of more or less regularly alternating β-1,4-linked mannose and glucose; "galactomannans" and "galactoglucomannans" are mannans and glucomannans with α-1,6-linked galactose side branches. These compounds may be acetylated. The degradation of galactomannans and galactoglucomannans is facilitated by total or partial removal of the galactose side branches. In addition, the degradation of acetylated mannans, glucomannans, galactomannans and galactoglucomannans is facilitated by total or partial deacetylation. The acetyl groups may be removed by alkali or by mannan acetylesterase. The oligomers released from mannanase or by a combination of mannanase and α-galactosidase and / or mannan acetylesterase can be further degraded by β-mannosidase and / or β-glucosidase to release free maltose.

[0038] The terms "protease" and "proteinase" refer to enzymes with the ability to decompose proteins and peptides. Proteases have the ability to "proteolyze" by hydrolyzing the peptide bonds that connect amino acids together in the peptide or polypeptide chain that forms a protein. This activity of proteases as protein digestive enzymes is referred to as "proteolytic activity". There are many well-known procedures for measuring proteolytic activity. For example, proteolytic activity can be determined by comparative assays that analyze the ability of each protease to hydrolyze a suitable substrate. Exemplary substrates that can be used to analyze proteases or proteolytic activity include, but are not limited to, dimethyl casein (Sigma C-9801), bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625) and Keratin Azure (Sigma-Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, for example, WO 99 / 34011 and US 6,376,450).

[0039] The term "modification" refers to any change or alteration in an amino acid sequence, including substitution of an amino acid at an identified position of a target amino acid sequence with an amino acid different from the starting amino acid, deletion of an amino acid at an identified position of a target amino acid sequence, insertion of an amino acid at an identified position of a target amino acid sequence, replacement of amino acid side chains in a target amino acid sequence, and / or chemical modification of a target amino acid sequence.

[0040] The term "catalytic activity" or "activity" quantitatively describes the conversion of a given substrate under defined reaction conditions. The term "residual activity" is defined as the ratio of the catalytic activity of an enzyme under a certain set of conditions to the catalytic activity under a different set of conditions. The term "specific activity" quantitatively describes the catalytic activity per unit amount of enzyme under defined reaction conditions.

[0041] The term "pH stability" describes the ability of a protein to withstand limited exposure to pH values ​​that depart significantly from the pH at which its stability is optimal (e.g., more than one pH unit above or below the optimal pH) without losing its activity if its activity is measurable.

[0042] The term "detergent stability" refers to the stability of a particular detergent composition ingredient (eg, a hydrolase) in a detergent composition mixture.

[0043] The term "perhydrolase" refers to an enzyme capable of catalyzing a reaction resulting in the formation of peracids useful in cleaning, bleaching, and disinfecting applications.

[0044] As used in the phrases "aqueous composition" and "aqueous environment", the term "aqueous" refers to a composition composed of at least 50% water. An aqueous composition may contain at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98% or 99% water.

[0045] The term "surfactant" refers to any compound generally recognized in the art as having surface active qualities. Surfactants generally include anionic, cationic, nonionic, and zwitterionic compounds, which are further described herein.

[0046] The term "surface properties" is used to refer to electrostatic charge, and properties such as hydrophobicity and hydrophilicity exhibited by the protein surface.

[0047] The term "chelator stability" refers to the retention of a specified amount of enzyme activity by a mannanase of the present disclosure over a given period of time under conditions prevailing during a mannosidation, hydrolysis, cleaning or other process disclosed herein, such as when exposed to or in contact with a chelator. In some embodiments, after contact with a chelator for a given period of time (e.g., at least about 10 minutes, about 20 minutes, about 40 minutes, about 60 minutes, about 100 minutes, etc.), the mannanase variant retains at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98% or about 99% of the mannanase activity.

[0048] The terms "thermal stability" and "thermostable" mean that the mannanase variant retains a specified amount of enzymatic activity under the conditions prevailing during a mannosidation, hydrolysis, cleaning or other process, e.g., when exposed to elevated temperatures, after exposure to elevated temperatures for a given period of time. In some embodiments, the mannanase retains at least about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%, about 98% or about 99% of the mannanase activity after exposure to an elevated temperature (e.g., at least about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C) for a given period of time (e.g., at least about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 120 minutes, 180 minutes, 240 minutes, 300 minutes, etc.).

[0049] The term "cleaning activity" refers to the cleaning performance achieved by mannanase variants under conditions prevailing during mannosides disclosed herein, hydrolysis, cleaning or other processes. In certain embodiments, cleaning performance is determined by applying various cleaning assays for enzyme-sensitive stains caused by food products, household agents or personal care products. As determined by various chromatography, spectrophotometry or other quantitative methods after the stain is subjected to standard washing conditions, some of these stains include, for example, ice cream, ketchup, barbecue sauce, mayonnaise, soup, chocolate milk, chocolate pudding, frozen desserts, shampoo, lotion, sunscreen products, toothpaste, locust bean gum or guar gum. Exemplary assays include but are not limited to those described in WO99 / 34011, US 6,605,458 and US 6,566,114, and those methods described in the examples.

[0050] The terms "cleaned surface" and "cleaned textile" refer to a surface or textile having a stain removal percentage of at least 10%, preferably at least 15%, 20%, 25%, 30%, 35% or 40% of the soiled surface or textile, respectively.

[0051] When used in conjunction with a mannanase variant, the term "effective amount" refers to the amount of the mannanase variant required to achieve the desired level of enzyme activity in a particular cleaning composition. Such an effective amount is readily determined by one of ordinary skill in the art and is based on many factors, such as the specific mannanase variant used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, stick, powder, solid, liquid, tablet, gel, paste, foam, sheet or unit dose) composition is desired, etc.

[0052] When used in conjunction with a cleaning composition, the term "adjunct ingredient" means any liquid, solid or gaseous material selected for the particular type of cleaning composition and product form desired (e.g., liquid, granular, powder, bar, paste, spray, tablet, gel, unit dose, sheet or foam composition), which is also preferably compatible with the mannanase variant used in the composition. In some embodiments, the granular composition is in a "compacted" form, while in other embodiments, the liquid composition is in a "concentrated" form.

[0053] The terms "cleaning composition" and "cleaning formulation" refer to a mixture of chemical ingredients that can be used to remove unwanted compounds (e.g., soil or stains) from items or surfaces to be cleaned (e.g., such as fabrics, dishes, contact lenses, solid surfaces, hair, skin, and teeth). The composition or formulation can be in the form of a liquid, gel, granules, powder, stick, paste, spray, tablet, gel, unit dose, wafer or foam, depending on the surface or item to be cleaned and the desired form of the composition or formulation.

[0054] The terms "detergent composition" and "detergent formulation" refer to a mixture of chemical and / or biological ingredients intended for use in a washing medium (the washing medium is used to clean contaminated objects). Detergent compositions / preparations typically include at least one surfactant, and may optionally include hydrolases, oxidoreductases, builders, bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, shielding agents, enzyme activators, antioxidants, solubilizers, and one or more microorganisms (microorganism or microbe) or microbial extracts. Microorganisms can be used as the only biologically active ingredient, but they can also be used in combination with one or more enzymes described herein. For example, a bacillus strain with a deposit accession number of PTA-7543 can be used to reduce malodor, as described in WO 2012 / 112718. Other purposes may include in situ production of desired biological compounds, or inoculating / colonizing one or more microorganisms in a field to competitively prevent other undesirable microorganisms from colonizing in the same field (competitive exclusion).

[0055] The term "dishwashing composition" refers to all forms of compositions for cleaning dishware and cutlery, including, for example, granular, unit dose and liquid forms. In some embodiments, the dishwashing composition is an "automatic dishwashing" composition that can be used in an automatic dishwashing machine. The term "dishware" refers to tableware (e.g., plates, cups, glasses, bowls and containers) and cutlery (e.g., utensils (utensil), including, but not limited to, spoons, knives and forks) of any material (including, but not limited to, ceramic, plastic, metal, porcelain, glass and acrylic resin).

[0056] The term "bleaching" refers to treating a material (e.g., fabric, clothing, pulp, etc.) or a surface for a sufficient period of time and under appropriate pH and temperature conditions to achieve whitening (i.e., whitening) and / or cleaning of the material. Examples of chemicals suitable for bleaching include, but are not limited to, ClO2, H2O2, peracids, and NO2.

[0057] The term "wash performance" of a mannanase variant refers to the contribution of the variant to washing providing additional cleaning performance to a detergent composition. Wash performance is compared under relevant wash conditions. The term "relevant wash conditions" as used herein indicates conditions that are actually used for household use in the dishwashing or laundry detergent market segment, particularly wash temperature, time, wash mechanics, soap water concentration, detergent type, and water hardness.

[0058] As used herein, the term "disinfecting" refers to removing contaminants from a surface, as well as inhibiting or killing microorganisms on the surface of an item.

[0059] The "dense" form of the cleaning composition herein is best reflected by density, and in terms of the composition, by the amount of inorganic filler salt. Inorganic filler salts are conventional ingredients of detergent compositions in powder form. In conventional detergent compositions, filler salts are present in a base amount, typically from about 17% to about 35% by weight of the total composition. In contrast, in intensive compositions, filler salts are present in an amount not exceeding about 15% of the total composition. In some embodiments, filler salts are present in an amount not exceeding about 10% or more preferably about 5% by weight of the composition. In some embodiments, the inorganic filler salts are selected from alkali salts and alkaline earth metal salts of sulfates and chlorides. In some embodiments, a preferred filler salt is sodium sulfate.

[0060] The term "fabric" refers to materials such as woven, knitted, and nonwoven materials, as well as staple fibers and filaments that can be converted into, for example, yarns and woven, knitted, and nonwoven fabrics. The term encompasses materials made from natural as well as synthetic (e.g., manufactured) fibers.

[0061] When a nucleic acid or polynucleotide is at least partially or completely separated from other components (including but not limited to, for example, other proteins, nucleic acids and cells), the nucleic acid or polynucleotide is "isolated". Similarly, when a polypeptide, protein or peptide is at least partially or completely separated from other components (including but not limited to, for example, other proteins, nucleic acids and cells), the polypeptide, protein or peptide is "isolated". In terms of moles, the isolated species is more abundant than other species in the composition. For example, the isolated species can contain at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (in terms of moles) of all macromolecular species present. Preferably, the target species is purified to basic homogeneity (i.e., the contaminant species cannot be detected in the composition by conventional detection methods). Many techniques well known in the art such as agarose or polyacrylamide gel electrophoresis of nucleic acid or protein samples can be used, respectively, followed by visualization after staining to determine purity and homogeneity. If desired, the material can be purified using high resolution techniques such as high performance liquid chromatography (HPLC) or similar methods.

[0062] The term "purified" as applied to nucleic acids or polypeptides generally refers to a nucleic acid or polypeptide that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms discrete bands in an electrophoretic gel, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). For example, a nucleic acid or polypeptide that produces essentially one band in an electrophoretic gel is "purified." A purified nucleic acid or polypeptide is at least about 50% pure, 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., percentages by weight on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, polypeptide, cell, nucleic acid, amino acid or other specific material or component in a composition at a relative or absolute concentration greater than that in the starting composition.

[0063] As used herein, "polypeptide" refers to a molecule comprising a plurality of amino acids linked by peptide bonds. The terms "polypeptide," "peptide," and "protein" are used interchangeably. Proteins may optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, and sulfonated) to increase functionality. When such amino acid sequences exhibit activity, they may be referred to as "enzymes." Amino acid sequences are represented using the standard amino-to-carboxyl orientation (i.e., N→C) using conventional single-letter or three-letter codes for amino acid residues.

[0064] The term "polynucleotide" encompasses DNA, RNA, heteroduplexes, and synthetic molecules that can encode polypeptides. Nucleic acids can be single-stranded or double-stranded and can have chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the compositions and methods of the present invention encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5' to 3' orientation.

[0065] As used herein, the terms "wild-type" and "native" refer to a polypeptide or polynucleotide as found in nature.

[0066] With respect to polypeptides, the terms "wild-type" and "parent" refer to naturally occurring polypeptides that do not include artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild-type" and "parent" refer to naturally occurring polynucleotides that do not include artificial substitutions, insertions or deletions at one or more nucleosides. However, it is noted that polynucleotides encoding wild-type or parent polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type or parent polypeptides.

[0067] With respect to polypeptides, the term "reference" refers to naturally occurring polypeptides that do not comprise artificial substitutions, insertions or deletions at one or more amino acid positions, and polypeptides that comprise one or more artificial substitutions, insertions or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the term "reference" refers to naturally occurring polynucleotides that do not comprise artificial substitutions, insertions or deletions at one or more nucleosides, and polynucleotides that comprise one or more artificial substitutions, insertions or deletions at one or more nucleosides. However, it is noted that polynucleotides encoding wild-type or parent polypeptides are not limited to naturally occurring polynucleotides, and encompass any polynucleotides encoding wild-type or parent polypeptides.

[0068] Single letter code "Z" identifies the insertion or deletion in the parent or reference amino acid sequence. For insertion relative to the parent or reference sequence, the single letter code "Z" is located on the left side of the position number, and further includes a number (e.g., .01) to indicate the order of insertion before each amino acid is inserted therein. For example, the insertion of an amino acid glutamine (Q) at position 298 will be depicted as "Z298.01Q"; the insertion of an amino acid X (wherein X can be any amino acid) at position 298 will be depicted as "Z298.01X"; and the insertion of three amino acids (alanine (A), serine (S) and tyrosine (Y)) between positions 87 and 88 will be depicted as "Z87.01A / Z87.02S / Z87.03Y". For deletion, the single letter code "Z" is located on the right side of the position number. For example, the deletion of alanine (A) from position 100 will be depicted as A100Z. Combinations of some of the above insertions and deletions would be depicted as: “G87S / Z87.01A / Z87.02S / Z87.03Y / A100Z”.

[0069] The term "mannanase variant" refers to a polypeptide derived from a reference polypeptide by replacing, adding or lacking one or more amino acids, typically by recombinant DNA technology. Mannanase variants can differ from a reference polypeptide by a small number of amino acid residues and can be defined by the level of primary amino acid sequence homology / identity with the reference polypeptide over the length of the catalytic domain. For example, a mannanase variant has at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with a reference polypeptide. Reference polypeptides include, but are not limited to, naturally occurring mannanases and recombinant mannanases, such as, but not limited to, mannanases within the GH5-8 subfamily of mannanases (endo-1,4β-mannosidase, EC 3.2.1.78) (Aspeborg et al. (2012), "Evolution, substrate specificity and subfamily classification of glycosyl hydrolase family 5 (GH5)", BMC Evolutionary Biology, 12: 186). Exemplary reference GH5-8 bacterial mannanases include, for example, NDL-clade mannanases, such as, for example, PspMan4 (SEQ ID NO: 1), PspMan138 (SEQ ID NO: 2), and PspMan9 (SEQ ID NO: 3); and other mannanases, such as, for example, SEQ ID NOs: 4-6.

[0070] One or more amino acid substitutions of the mannanase variants described herein may be included in any known mannanase, including but not limited to the mannanase of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.

[0071] The term "variant polynucleotide" refers to a polynucleotide encoding a mannanase variant, having a specified degree of homology / identity with a parent polynucleotide, or hybridizing to a parent polynucleotide or its complementary sequence under stringent conditions. For example, a variant polynucleotide has at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity with a parent polynucleotide.

[0072] Sequence identity can be determined from protein sequence alignments using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters. (See, e.g., Altschul et al.

[1990] J. Mol. Biol. 215:403-410; Henikoff et al.

[1989] Proc. Natl. Acad. Sci. USA 89:10915; Karin et al.

[1993] Proc. Natl. Acad. Sci. USA 90:5873; and Higgins et al.

[1988] Gene 73:237-244). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI). Databases can also be searched using FASTA (Pearson et al.

[1988] Proc. Natl. Acad. Sci. USA 85:2444-2448). One indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ in conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, e.g., where the two peptides differ only in conservative substitutions. Another useful algorithm for alignment and comparison of multiple protein sequences is the MUSCLE program (Robert C. Edgar. MUSCLE: multiple sequence alignment with high accuracy and high throughput [MUSCLE: multiple sequence alignment with high accuracy and high throughput] Nucl. Acids Res. [Nucleic Acids Research] (2004) 32(5):1792-1797) available from Geneious software (Biomatters Ltd.).

[0073] The term "derived from" encompasses the terms "originated from," "obtained from," "obtainable from," "isolated from," and "produced from," and generally indicates that one specified material finds its origin in, or has characteristics that can be described with reference to, another specified material.

[0074] The term "hybridization" refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing, as known in the art.

[0075] The term "hybridization conditions" refers to the conditions under which a hybridization reaction is performed. These conditions are typically classified according to the "stringency" of the conditions under which hybridization is measured. Stringency can be based on, for example, the melting temperature (T) of the nucleic acid binding complex or probe. m ). For example, "maximum stringency" typically occurs at about T m -5℃(lower than the probe's T m 5℃); "high stringency" occurs below T m About 5°C-10°C; "moderate stringency" occurs below the probe's T m About 10℃-20℃; and "low stringency" occurs below T m About 20°C-25°C. Alternatively, or in addition, hybridization conditions can be based on the salt or ionic strength conditions of hybridization, and / or one or more stringent washes, for example: 6XSSC = very low stringency; 3X SSC = low to medium stringency; 1X SSC = medium stringency; and 0.5XSSC = high stringency. Functionally, maximum stringency conditions can be used to identify nucleic acid sequences that have strict identity or near strict identity with the hybridization probe; while high stringency conditions are used to identify nucleic acid sequences that have about 80% or more sequence identity with the probe. For applications requiring high selectivity, it is typically desirable to use relatively stringent conditions to form hybridization (e.g., using relatively low salt and / or high temperature conditions).

[0076] The terms "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides mean that the polynucleotides or polypeptides comprise a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a parent or reference sequence, or that includes amino acid substitutions, insertions, deletions or modifications that are intended only to circumvent this specification but do not add functionality.

[0077] The term "expression vector" refers to a DNA construct comprising a DNA sequence encoding a specific polypeptide, and is operably linked to a suitable control sequence that can realize expression of the polypeptide in a suitable host. Such control sequences include promoters that affect transcription, optional operator sequences that control such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences that control transcription and translation termination. The vector can be a plasmid, a phage particle, or simply a potential genome insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or can be integrated into the genome itself in some cases.

[0078] The term "recombinant" refers to genetic material (i.e., nucleic acids, polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating a coding sequence to produce an altered polypeptide, fusing a coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at reduced or elevated levels, conditionally or constitutively expressing a gene in a manner that differs from its native expression profile, etc. Generally, recombinant nucleic acids, polypeptides, and cells based thereon have been artificially manipulated such that they are not identical to the related nucleic acids, polypeptides, and cells found in nature.

[0079] The term "signal sequence" refers to an amino acid sequence that is bound to the N-terminal portion of a polypeptide and promotes secretion of the mature form of the protein from the cell. The mature form of the extracellular protein lacks the signal sequence which is cleaved off during the secretion process.

[0080] The term "selectable marker" or "selectable marker" refers to a gene that can be expressed in a host cell that allows for easy selection of those hosts containing the introduced nucleic acid or vector. Examples of selectable markers include, but are not limited to, antimicrobial substances (e.g., hygromycin, bleomycin, or chloramphenicol) and / or genes that confer a metabolic advantage (e.g., a nutritional advantage) on the host cell. The term "selectable gene product" refers to a gene encoding an enzymatic activity that confers antibiotic or drug resistance on cells expressing the selectable marker.

[0081] As used herein, the term "regulatory element" refers to a genetic element that controls some aspect of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked coding region. Additional regulatory elements include splicing signals, polyadenylation signals, and termination signals.

[0082] The term "host cell" generally refers to a prokaryotic or eukaryotic host transformed or transfected with a vector constructed using recombinant DNA techniques known in the art. The transformed host cell is capable of replicating the vector encoding the protein variant or expressing the desired protein variant. In the case of a vector encoding a pre-form or pro-form of a protein variant, when expressed, these variants are typically secreted from the host cell into the host cell culture medium.

[0083] In the context of inserting a nucleic acid sequence into a cell, the term "introduction" means transformation, transduction or transfection. Transformation means include protoplast transformation, calcium chloride precipitation, electroporation, naked DNA, etc., as known in the art. (See Chang and Cohen

[1979] Mol. Gen. Genet. [Molecular and General Genetics] 168: 111-115; Smith et al.

[1986] Appl. Env. Microbiol. [Applied and Environmental Microbiology] 51: 634; and Ferrari et al. in Harwood, Bacillus [Bacillus], Plenum Publishing Corporation, pp. 57-72, 1989, a review article).

[0084] Any headings used herein are provided for convenience and should not be construed as limiting. The description included under one heading may apply to the entire specification.

[0085] Variants, compositions and methods disclosed herein relate to recombinant mannanases comprising one or more modifications, wherein such variants are generated by conventional molecular biology techniques (see, e.g., Sambrook et al., Molecular Cloning: Cold Spring Harbor Laboratory Press). In one embodiment, the variant mannanase comprises one or more modifications selected from at least one substitution, at least one deletion and at least one insertion. In some embodiments, the modifications comprise a combination of mutations, such as, for example, at least one substitution and at least one deletion, at least one deletion and at least one insertion, at least one insertion and at least one substitution, or a combination of at least one substitution, at least one deletion, and at least one insertion.

[0086] In one embodiment, a mannanase variant is provided, wherein the mannanase variant comprises one or more amino acid substitutions at one or more positions selected from 32, 72, 161 and 172, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0087] In one embodiment, the mannanase variant is a mannanase variant comprising amino acid substitutions selected from the group consisting of 19D, 32Y, 34D, 72V, 93Q, 131S, 136P, 139R, 161G, 172F, 225N / Q, 259D, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0088] In one embodiment, the mannanase variant is a mannanase variant comprising two or more amino acid substitutions selected from the group consisting of: 19D, 32Y, 72V, 93Q, 131S, 136P, 139R, 161G, 168T, 172F, 225N / Q, 259D / E, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0089] In one embodiment, the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of 19D-276W, 32Y-259D, 93Q-276W, 131S-276W, 136P-276W, 139R-276W, 161G-276W, 225N / Q-276W, 259D / E-276W and 261D / E-276W.

[0090] In one embodiment, the mannanase variant is a variant comprising an amino acid substitution selected from the group consisting of 19D-131S-276W, 32Y-261D-276W, 32Y-259D-276W, 32Y-172F-259D, 168T-259D-276W, 259D-261E-276W and 259Q-261E-276W.

[0091] In one embodiment, the mannanase variant is a mannanase variant comprising an amino acid substitution selected from the group consisting of Y061W-G259D-R261E-F276W, Y061W-T062E-G259D-R261E-F276W, Y061W-V228T-G259D-R261E-F276W, V228T-G259D-R261E-F276W, F032Y-G259D-R261E-F276W, F032Y-Y061W-Y167F-P168S-G259D-R261E-F276W. 1E-F276W, F032Y-Y061W-T062E-G259D-R261E-F276W, F032Y-T062E-R26 1D-F276W, F032Y-T062E-G259D-F276W, T062E-G259D-R261E-F276W, V059 S-Q060L-G259D-R261E-F276W, V059S-Q060L-Y061W-G259D-R261E-F276W , V059S-Q060L-T062E-G259D-R261E-F276W, T062E-V228T-G259D-R261E- F276W, F032Y-T062E-G259D-R261E-F276W, F032Y-T062E-G259D-R261E- F276W-T284E, E019D-A068S-T131S-F276W, F032Y-T062E-G259D-F276W-T 284E, F032Y-T062E-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-G2 59D-F276W-T284E, F032Y-T062E-A068S-G259D-F276W-T284E, F032Y-Y06 1W-T062E-G259D-F276W-T284E, F032Y-V059S-Q060L-T062E-G259D-F27 6W-T284E, N010T-F032Y-T062E-G259D-F276W-T284E, F032Y-T062E-Y167 F-P168S-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-V228T-G259D -F276W-T284E, F032Y-T062E-I072V-Y167F-P168S-G259D-F276W-T284E,F032Y-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-V228T-G259D-F276W-T284E、F032Y-V059S、Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-G259D-F276W-T284E、F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E and N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E, F032Y-Y061W-T062E-G259D-R261E-F276W-T284E, F032Y-T062E-A068S-G259D-R261E-F276W-T284 E. F032Y-T062E-T131S-G259D-R261E-F276W-T284E, F032Y-T062E-N150D -G259D-R261E-F276W-T284E, F032Y-T062E-P168S-G259D-R261E-F276W- T284E, F032Y-T062E-L235K-G259D-R261E-F276W-T284E, N010T-F032Y-Y 061W-T062E-P168A-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T06 2E-Y167F-P168S-V228T-G259E-F276W-T284E, F032Y-Y061W-T062E-T131 S-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T062E -A136P-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W- T062E-K093Q-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y 061W-T062E-Y167F-P168S-V228T-L235K-G259D-F276W-T284E, N010T-F0 32Y-Y061W-T062E-D139R-Y167F-P168S-V228T-G259D-F276W-T284E, N01 0T-F032Y-Y061W-T062E-N150D-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-Y167F-P168A-V228T-G259D-F276W-T284E,N010T-F032Y-Y061W-T062E-T131S-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-P168S-V228T-G259D-F276W-T284E, N010T-F03 2Y-Y061W-T062E-A068S-Y167F-P168A-G259D-F276W-T284E, and N010T-F032Y-Y061W-T062E-A068S-Y167F-P168A-G259E-F276W-T284E, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the mannanase variant is a mannanase variant as described herein, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 1. ,

[0092] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1.

[0093] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant has improved stability when compared to a parent mannanase or a reference mannanase.

[0094] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the variant has equal or improved cleaning performance in a detergent when compared to a parent mannanase or a reference mannanase.

[0095] In one embodiment, the mannanase variant is a mannanase variant described herein, wherein the mannanase variant has mannanase activity.

[0096] Another embodiment relates to a mannanase variant comprising an amino acid sequence having at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1-6.

[0097] In one embodiment, the reference polypeptide is selected from SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, one or more of the mannanase variants described herein have at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0098] In some embodiments, the mannanase variants or recombinant polypeptides described herein are separated. In other embodiments, the mannanase variants described herein are endo-β-mannanases. In further embodiments, the mannanase variants described herein have mannanase activity. In still other embodiments, the mannanase variants described herein have mannanase activity in the presence of a surfactant. In some embodiments, the mannanase activity is the activity to mannan gum, locust bean gum galactomannan, and / or konjac glucomannan. In further embodiments, the mannanase variants described herein have cleaning activity in detergent compositions. Still other embodiments relate to mannanase variants or recombinant polypeptides having mannanase activity in the presence of a protease. Further embodiments relate to mannanase variants or recombinant polypeptides that hydrolyze a substrate selected from the group consisting of guar gum, locust bean gum, and combinations thereof. In some embodiments, the mannanase variants or recombinant polypeptides described herein do not comprise a carbohydrate binding module.

[0099] In some embodiments, the mannanase variants have enzymatic activity under a wide range of pH conditions. In certain embodiments, the mannanase variants have enzymatic activity at a pH from about 4.0 to about 11.0. In other embodiments, the mannanase variants or recombinant polypeptides have at least 50%, 60%, 70%, 80%, 90%, 95% or 100% mannanase activity at a pH from about 4.0 to about 11.0, about 4.5 to about 9.0, about 5.5 to about 8.5, or about 6.0 to about 7.5.

[0100] In still further embodiments, the mannanase variant or recombinant polypeptide has mannanase activity in a temperature range from about 20° C. to about 90° C., about 30° C. to about 80° C., about 20° C. to about 50° C., or about 30° C. to about 66° C. In certain embodiments, the mannanase variant or recombinant polypeptide has at least 50%, 60%, 70%, 80%, 90%, 95%, or 100% mannanase activity at a temperature ranging from about 20° C. to about 90° C., about 30° C. to about 80° C., about 20° C. to about 50° C., or about 30° C. to about 66° C.

[0101] Still further embodiments relate to mannanase variants or recombinant polypeptides described herein, wherein the variant or recombinant polypeptide retains at least 70% of its maximum mannanase activity at a pH range of 4.5-9.0, 5.5-8.5, or 6.0-7.5. Some embodiments relate to mannanase variants or recombinant polypeptides described herein, wherein the variant retains at least 70% of its maximum mannanase activity at a pH greater than 3.0, 3.5, 4.0, or 4.5, or at a pH less than 9.0, 9.5, or 10.0.

[0102] In some embodiments, one or more mannanase variants described herein have one or more improved properties compared to a reference polypeptide, wherein the improved properties are selected from improved stability in the presence of a protease, improved stability in a detergent or buffer, improved cleaning performance, and improved aged cleaning performance. Aged cleaning performance refers to the difference in stain removal measured for samples where an aged test sample (wherein the enzyme was pre-incubated in a detergent at an elevated temperature (such as 37° C.) for an extended period of time (such as 3-4 weeks) was compared to a 'fresh' stain cleaning of the same enzyme (without pre-incubation). Thus, an enzyme with improved aged cleaning performance exhibits a smaller difference between an aged sample and a freshly prepared sample when compared to the same evaluation performed with a reference / parent enzyme.

[0103] In another embodiment, one or more of the mannanase variants described herein have one or more improved properties compared to a reference polypeptide, wherein the improved property is selected from improved stability in the presence of a protease, improved stability in a detergent or buffer, improved cleaning performance, wherein the reference polypeptide is selected from SEQ ID NO: 1 or 2.

[0104] In some embodiments, the mannanase variants or recombinant polypeptides are substantially identical to SEQ ID NO: 1 or 2, which means that they may contain amino acid substitutions, insertions or deletions that do not significantly affect the structure, function or expression of the variant or polypeptide. Such mannanase variants or recombinant polypeptides include those designed only to circumvent this specification.

[0105] In some embodiments, the mannanase variant has 1,4-β-D-mannoside hydrolase activity, which includes mannanase, endo-1,4-β-D-mannanase, exo-1,4-β-D-mannanase galactomannanase, and / or glucomannanase activity. 1,4-β-D-mannoside hydrolase activity can be determined and measured using the assays described herein or by other assays known in the art. In some embodiments, the polypeptides of the present invention are active in the presence of a detergent composition.

[0106] In some embodiments, the mannanase variants described herein are produced as N- and / or C-terminal fusion proteins, for example to aid in extraction, detection and / or purification and / or to increase the functional properties of the variant or recombinant polypeptide or its active fragment. Examples of fusion protein partners include, but are not limited to, glutathione-S-transferase (GST), 6XHis, GAL4 (DNA binding and / or transcriptional activation domain), FLAG, MYC, BCE103 (WO 2010 / 044786), or other tags well known to those skilled in the art. In some embodiments, a proteolytic cleavage site is provided between the fusion protein partner and the target protein sequence to allow removal of the fusion protein sequence. Preferably, the fusion protein does not hinder the activity of the mannanase variant or recombinant polypeptide described herein.

[0107] In some embodiments, the mannanase variants or recombinant polypeptides described herein are fused to functional domains including a leader peptide, a propeptide, one or more binding domains (modules) and / or a catalytic domain. Suitable binding domains include, but are not limited to, carbohydrate binding modules (CBMs) with various specificities that provide increased affinity for carbohydrate components present during use of the mannanase variants or recombinant polypeptides described herein. As described herein, the CBM and catalytic domain of the polypeptides of the present invention are operably linked.

[0108] CBM is defined as a continuous amino acid sequence within a carbohydrate-active enzyme that is in an independent fold with carbohydrate binding activity. Some exceptions are the rare cases of CBMs in cellulosome scaffolds and independently putative CBMs in proteins. The need for CBMs to exist as modules within larger enzymes distinguishes such carbohydrate-binding proteins from other non-catalytic sugar-binding proteins (such as lectins and sugar transporters). Based on several modules that initially found to bind cellulose, CBMs were previously classified as cellulose-binding domains (CBDs) (Tomme et al., Eur J Biochem [European Journal of Biochemistry], 170: 575-581, 1988; and Gilkes et al., J Biol Chem [Journal of Biological Chemistry], 263: 10401-10407, 1988). However, it is constantly discovered that additional modules in carbohydrate-active enzymes bind carbohydrates other than cellulose, but otherwise meet the CBM criteria, so it is necessary to use more inclusive terms to reclassify these polypeptides. The previous classification of cellulose-binding domains was based on amino acid similarity. The grouping of CBDs is referred to as "type" and numbered with Roman numerals (e.g., Type I or Type II CBD). In order to be consistent with the classification of glycoside hydrolases, these groups are now referred to as families and numbered with Arabic numerals. Families 1 to 13 are the same as Types I to XIII (Tomme et al., Enzymatic Degradation of Insoluble Polysaccharides [enzymatic degradation of insoluble polysaccharides] (Saddler, JN & Penner, M. ed.), Cellulose-binding domains: classification and properties [cellulose binding domain: classification and properties], pp. 142-163, American Chemical Society [American Chemical Society], Washington, 1995). Detailed comments on CBM structure and binding mode can be found in Boraston et al., Biochem J [Journal of Biological Chemistry], 382: 769-81, 2004. It is expected that the family classification of CBMs will help identify CBMs, predict binding specificity, help identify functional residues, reveal evolutionary relationships, and may predict polypeptide folding. Because the folding of proteins is more conserved than their sequence, some CBM families can be divided into superfamilies or clans. The current CBM families are 1-63. CBDs are found at the N- and C-termini of proteins or are internal.Enzyme hybrids are known in the art (see, e.g., WO 90 / 00609 and WO 95 / 16782) and can be prepared by transforming a DNA construct comprising at least a fragment of DNA encoding a cellulose binding domain, linked with or without a linker to a DNA sequence encoding a mannanase variant described herein, into a host cell and culturing the host cell to express the fusion gene.

[0109] The enzyme hybrid can be described by the following formula:

[0110] CBM-MR-X or X-MR-CBM, wherein CBM is the N-terminal or C-terminal region of an amino acid sequence corresponding to at least a carbohydrate binding module; MR is a middle region (linker) and can be a bond, or a short linker from about 2 to about 100 carbon atoms, from about 2 to about 40 carbon atoms, from about 2 to about 100 amino acids, or from about 2 to about 40 amino acids; and X is the N-terminal or C-terminal region of a mannanase variant having mannanase catalytic activity as described herein. In addition, the mannanase may contain more than one CBM or other modules / domains of non-glycolytic functions. The terms "module" and "domain" are used interchangeably in the present disclosure.

[0111] Other non-limiting examples of catalytic domains include: cellulases; hemicellulases, such as xylanases; exo-mannanases; glucanases; arabinanases; galactosidases; pectinases; and / or other activities, such as proteases, lipases, acid phosphatases, and / or other or functional fragments thereof. The fusion protein is optionally linked to the mannanase variants described herein via a linker sequence that simply links the mannanase variant and the fusion domain without significantly affecting the properties of either component, or the linker optionally has functional importance for the intended application.

[0112] In some embodiments, the enzyme is a mannanase variant as provided herein in combination with one or more additional enzymes selected from the group consisting of: acyltransferase, amylase, α-amylase, β-amylase, α-galactosidase, arabinanase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenanase, catalase, cellulase, chondroitinase, cutinase, dispersin, DNA enzyme (also known as nuclease or dispersin), endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase. 、Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, PETase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthan lyase, xanthanase, xylan acetylesterase, xylanase, xylosidase, and combinations or mixtures thereof.

[0113] In some embodiments, the mannanase variants described herein are fused to a signal peptide for directing extracellular secretion of the variant or polypeptide. For example, in certain embodiments, the signal peptide is a natural signal peptide of the mannanase variants described herein. In other embodiments, the signal peptide is a non-natural signal peptide, such as Bacillus subtilis (B. subtilis) AprE signal peptide.

[0114] In some embodiments, polypeptides of the invention are expressed in a heterologous organism, ie, an organism other than Paenibacillus spp. Exemplary heterologous organisms are Gram-positive (+) bacteria, such as Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, Geobacillus (formerly Bacillus) stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megaterium, B. thuringiensis, S. lividans, or S. murinus; Gram-negative (-) bacteria, such as E. coli; yeast, such as Saccharomyces species. spp.) or Schizosaccharomyces spp., such as S. cerevisiae; and filamentous fungi, such as Aspergillus spp., such as A. oryzae or A. niger, and T. reesei. Methods for transforming nucleic acids into these organisms are well known in the art. Suitable procedures for transforming Aspergillus host cells are described in EP 238023.

[0115] In certain embodiments, the mannanase variants described herein are expressed as secreted polypeptides in a heterologous organism, in which case the compositions and methods encompass methods for expressing the variants as secreted polypeptides in the heterologous organism.

[0116] Additional embodiments relate to methods of producing the mannanase variants described herein, comprising: stably transforming a host cell with an expression vector comprising a polynucleotide encoding the mannanase variant; culturing the transformed host cell under suitable conditions to produce the mannanase variant; and recovering the mannanase variant.

[0117] Yet another embodiment relates to a polynucleotide encoding a mannanase variant described herein. In one aspect, the polynucleotide is contained in an expression vector contained in a heterologous organism (such as those identified herein). The polynucleotide can be operably linked to a regulatory element (e.g., a promoter, terminator, enhancer, etc.) to assist in expressing the encoded variant or recombinant polypeptide described herein.

[0118] Some embodiments relate to polynucleotides encoding mannanase variants having at least 59%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequences of SEQ ID NOs: 1-6. In some embodiments, the polynucleotides are codon optimized for expression in different hosts, mutated to introduce cloning sites, or otherwise altered to add functionality.

[0119] In some embodiments, the polynucleotide encoding the mannanase described herein is fused downstream of the coding sequence of a signal peptide that directs extracellular secretion of the variant. The expression vector can be provided in a heterologous host cell suitable for expressing the variant described herein, or for propagating the expression vector before introducing the expression vector into a suitable host cell.

[0120] DNA encoding the mannanase variants described herein can be chemically synthesized from the disclosed sequences or obtained directly from host cells with the gene (e.g., by cDNA library screening or PCR amplification). In some embodiments, the polynucleotide is contained in an expression cassette and / or cloned into a suitable expression vector by standard molecular cloning techniques. Such expression cassettes or vectors contain sequences that assist in the initiation and termination of transcription (e.g., promoters and terminators), and typically contain selectable markers.

[0121] The expression cassette or vector is introduced into a suitable expression host cell, which then expresses the corresponding mannanase variant described herein. Particularly suitable expression hosts are bacterial expression host genera, including Escherichia (e.g., E. coli), Pseudomonas (e.g., P. fluorescens or P. stutzerei), Proteus (e.g., P. mirabilis), Ralstonia (e.g., R. eutropha), Streptomyces, Staphylococcus (e.g., S. carnosus), Lactococcus (e.g., Lactococcus lactis), or Bacillus (e.g., B. subtilis, B. megaterium, B. licheniformis, etc.). Yeast expression hosts are also particularly suitable, such as Saccharomyces cerevisiae, S. pombe, Yarrowia lipolytica, H. polymorpha, Kluyveromyces lactis, or P. pastoris. Particularly suitable are fungal expression hosts, such as C. lucknowense, Aspergillus (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, etc.), or Trichoderma reesei. Also suitable are mammalian expression hosts, such as mouse (e.g., NSO), Chinese hamster ovary (CHO), or baby hamster kidney (BHK) cell lines. Other eukaryotic hosts such as insect cells or viral expression systems (e.g., bacteriophages such as M13, T7 phage, or lambda, or viruses such as baculovirus) are also suitable for producing the mannanase variants described herein.

[0122] Promoters and / or signal sequences associated with secreted proteins in a particular host of interest are candidates for heterologous production and secretion of mannanases in that host or in other hosts. For example, in filamentous fungal systems, promoters of genes driving cellobiohydrolase I (cbhl), glucoamylase A (glaA), TAKA-amylase (amyA), xylanase (exlA), gpd-promoters (cbhl, cbhlll), promoters driving endoglucanase genes EGI-EGV, Cel61B, Cel74A, egl1-egl5, gpd promoter, Pgkl, pkil, EF-1α, tefl, cDNAl and hexl are particularly suitable and can be derived from many different organisms (e.g., Aspergillus niger, Trichoderma reesei, Aspergillus oryzae, A. awamori, and Aspergillus nidulans). In some embodiments, the polynucleotide is recombined with a polynucleotide encoding a suitable homologous or heterologous signal sequence, which causes the mannanase variants described herein to be secreted into the extracellular (or periplasmic) space, thereby allowing direct detection of the enzymatic activity in the cell supernatant (or periplasmic space or lysate). Particularly suitable signal sequences for Escherichia coli, other Gram-negative bacteria, and other organisms known in the art include those that drive the expression of HlyA, DsbA, Pbp, PhoA, PelB, OmpA, OmpT, or M13 phage Gill genes. Particularly suitable signal sequences for Bacillus subtilis, Gram-positive organisms, and other organisms known in the art further include those that drive the expression of AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for Saccharomyces cerevisiae or other yeasts, include killer toxins, Bar1, Suc2, mating factor α, Inu1A, or Ggplp signal sequences. The signal sequence can be cleaved by many signal peptidases, thereby removing it from the remainder of the expressed protein. In some embodiments, the remaining polypeptide is expressed alone or as a fusion with other peptides, tags or proteins (e.g., 6XHis, HA or FLAG tags) located at the N- or C-terminus. Suitable fusions include tags, peptides or proteins (e.g., BCE103, 6XHis, HA, chitin binding protein, thioredoxin or FLAG tags) that promote affinity purification or detection and those that promote expression, secretion or processing of the target mannanase. Suitable processing sites include enterokinase, STE13, Kex2 or other protease cleavage sites for in vivo or in vitro cleavage.

[0123] The mannanase variants described herein can be introduced into expression host cells by a number of transformation methods, including, but not limited to, electroporation, lipid-assisted transformation or transfection ("lipofection"), chemical-mediated transfection (e.g., CaCl and / or CaP), lithium acetate-mediated transformation (e.g., of host cell protoplasts), biolistic "gene gun" transformation, PEG-mediated transformation (e.g., of host cell protoplasts), protoplast fusion (e.g., using bacterial or eukaryotic protoplasts), liposome-mediated transformation, Agrobacterium tumefaciens, adenovirus or other viral or phage transformation or transduction.

[0124] Alternatively, the mannanase variants described herein can be expressed intracellularly. Optionally, after the intracellular expression of the enzyme variant or the secretion into the periplasmic space using a signal sequence (such as those described above), the polypeptide can be released into the supernatant using a permeabilization or lysis step. The destruction of the membrane barrier is affected by the use of mechanical means such as ultrasound, pressure treatment (French press), cavitation, or by the use of membrane digestive enzymes (such as lysozyme) or enzyme mixtures. As another alternative, the polynucleotides encoding the mannanase variants described herein can be expressed by using a suitable cell-free expression system. In a cell-free system, the target polynucleotide is typically transcribed with the help of a promoter, but the connection to form a circular expression vector is optional. In other embodiments, RNA is generated through exogenous addition or without the need for transcription and translated in a cell-free system.

[0125] Another embodiment relates to cleaning compositions comprising mannanase variants, and methods for using such compositions in cleaning applications. Cleaning applications include, but are not limited to, clothing or textile cleaning, clothing or textile softening, dishwashing (manual and automatic), stain pretreatment, and the like. Specific applications are those in which mannans (e.g., locust bean gum, guar gum, etc.) are components of the soil or stain to be removed.

[0126] The cleaning compositions typically include an effective amount of a mannanase variant described herein, such as at least 0.0001 weight percent, from about 0.0001 weight percent to about 1 weight percent, from about 0.001 weight percent to about 0.5 weight percent, from about 0.01 weight percent to about 0.1 weight percent, or even from about 0.1 weight percent to about 1 weight percent, or more. The effective amount of the mannanase variant in the cleaning composition results in the mannanase variant having sufficient enzymatic activity to hydrolyze a mannan-containing substrate (e.g., locust bean gum, guar gum, or a combination thereof).

[0127] Some embodiments relate to cleaning compositions in a form selected from the group consisting of powders, liquids, granules, sticks, solids, semisolids, gels, pastes, emulsions, tablets, capsules, unit doses, flakes, and foams. In some embodiments, the cleaning composition is a detergent composition. In other embodiments, the cleaning composition or detergent composition is selected from laundry detergents, fabric softening detergents, dishwashing detergents, medical device cleaning detergents, and hard surface cleaning detergents.

[0128] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a detergent composition selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, a medical device cleaning detergent, and a hard surface cleaning detergent.

[0129] In some embodiments, the present invention relates to detergent compositions comprising at least two proteases in combination with one or more additional cleaning composition components, such as but not limited to the liquid laundry compositions described in WO 2022106404.

[0130] In some embodiments, one or more mannanase variants described herein may be part of or added to a liquid laundry detergent composition, such as, but not limited to, liquid laundry compositions described in US11046919B2, WO 2021 / 223552, WO 2022 / 167251, WO 2022 / 074037, WO 2021 / 123184, WO 2021 / 037895, WO 2022 / 10372, WO 2020 / 264077, WO 2022 / 106404 and / or WO2017 / 54983; intensive liquid laundry compositions (US10683474B2); water-soluble unit dose preparations comprising fatty alkyl ester alkoxylate nonionic surfactants and alkoxylated alcohol nonionic surfactants (US20220162523A1); liquid laundry detergent compositions comprising improved alkylbenzene sulfonate surfactants (WO 2021 / 108307); liquid laundry detergent compositions comprising benzyl benzoate (WO 2020 / 223959); and / or detergent compositions containing branched surfactants (WO 2021 / 247801), water-soluble unit dose preparations comprising amphiphilic graft polymers and polyester terephthalate (WO 2019 / 032257).

[0131] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition containing an alkyl ether carboxylic acid, a betaine, an anionic surfactant, a nonionic surfactant to provide softening benefits (WO 2013 / 087286).

[0132] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition containing a sulfite radical scavenger, a protease stabilizer / inhibitor, or a combination thereof (WO2022 / 157311).

[0133] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition as described in: US20210317387A1, WO 2021 / 219296, WO 2021 / 127662, WO 2021 / 041685, US11208619, US20220186144.

[0134] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition comprising a dispersin variant, such as, but not limited to, a liquid laundry detergent composition described in US20210317387A1.

[0135] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition that is a high alkaline textile cleaner, such as, but not limited to, the liquid laundry detergent composition described in WO2021 / 219296.

[0136] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition that is a low-density unit dose detergent encapsulated with a perfume, such as, but not limited to, the detergent composition described in WO 2021 / 127662.

[0137] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a liquid laundry detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent composition described in WO 2021 / 041685.

[0138] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a detergent composition containing polyethylene glycol and an organic acid, such as, but not limited to, the detergent composition described in WO 2021 / 041685.

[0139] In some embodiments, the cleaning compositions comprising one or more mannanase variants described herein are detergent compositions that are active on protein stains, such as, but not limited to, the detergent compositions described in US Pat. No. 1,120,8619.

[0140] In some embodiments, the cleaning composition comprising one or more mannanase variants described herein is a detergent composition containing a soil release polymer, such as, but not limited to, the detergent composition described in US 20220186144.

[0141] Unless otherwise noted, all components or composition levels provided herein are given with reference to the activity level of the component or composition, and do not include impurities that may be present in commercially available sources, such as residual solvents or by-products. Enzyme component weight is based on total active protein. Unless otherwise indicated, all percentages and ratios are calculated by weight. Unless otherwise indicated, all percentages and ratios are calculated based on total composition. In exemplary detergent compositions, enzyme levels are represented by pure enzymes by weight of total composition, and unless otherwise noted, detergent ingredients are represented by weight of total composition.

[0142] In some embodiments, the cleaning compositions described herein further comprise a surfactant. In some embodiments, the surfactant is selected from nonionic surfactants, amphoteric surfactants, semi-polar surfactants, anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations and mixtures thereof. In yet another embodiment, the surfactant is selected from anionic surfactants, cationic surfactants, zwitterionic surfactants, and combinations thereof. In some embodiments, the cleaning compositions described herein comprise surfactants from about 0.1% to about 60%, from about 1% to about 50%, or from about 5% to about 40% by weight of the composition. Exemplary surfactants include, but are not limited to, sodium dodecylbenzene sulfonate, C12-14 alcohol polyoxyethylene ether (pareth) -7, C12-15 alcohol polyoxyethylene ether -7, C12-15 alcohol polyoxyethylene ether sodium sulfate, C14-15 alcohol polyoxyethylene ether -4, laureth sulfate (e.g., Steol CS-370), sodium hydrogenated coconut oil, C 12Ethoxylates (Alfonic 1012--6, Hetoxol LA7, Hetoxol LA4), sodium alkylbenzene sulfonate (e.g., Nacconol 90G), and combinations and mixtures thereof. Anionic surfactants include, but are not limited to, linear alkylbenzene sulfonate (LAS), α-olefin sulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxy sulfate (AEOS or AES), secondary alkane sulfonate (SAS), α-sulfo fatty acid methyl ester, alkyl- or alkenyl succinic acid, or soap. Nonionic surfactants include, but are not limited to, alcohol ethoxylates (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylates, alkyl polyglycosides, alkyl dimethyl amine oxides, ethoxylated fatty acid monoethanolamides, fatty acid monoethanolamides or polyhydroxy alkyl fatty acid amides (e.g., as described in WO 92 / 06154), polyoxyethylene esters of fatty acids, polyoxyethylene sorbitan esters (e.g., TWEEN), polyoxyethylene alcohols, polyoxyethylene isoalcohols, polyoxyethylene ethers (e.g., TRITON and BRIJ), polyoxyethylene esters, polyoxyethylene-p-tert-octylphenol or octylphenyl-ethylene oxide condensates (e.g., NONIDE TP40), condensates of ethylene oxide with fatty alcohols (e.g., LUBROL), polyoxyethylene nonylphenols, polyalkylene glycols (SYNPERONIC F108), sugar-based surfactants (e.g., glucopyranosides, thioglucopyranosides), and combinations and mixtures thereof.

[0143] In additional embodiments, the detergent compositions disclosed herein further comprise a surfactant mixture including, but not limited to, 5%-15% anionic surfactants, <5% nonionic surfactants, cationic surfactants, phosphonates, soaps, enzymes, perfumes, butylphenyl methylpropionate, geraniol, zeolites, polycarboxylates, hexyl cinnamaldehyde, limonene, cationic surfactants, citronellol, and benzisothiazolinone.

[0144] The cleaning compositions described herein may additionally comprise one or more detergent builders or builder systems, complexing agents, polymers, bleaching systems, stabilizers, foam builders, foam inhibitors, corrosion inhibitors, soil suspension agents, anti-soil redeposition agents, dyes, bactericides, hydrogen promoters, tarnish inhibitors, fluorescent brighteners, fabric conditioners and perfumes. The cleaning compositions described herein may also comprise additional enzymes selected from the group consisting of proteases, amylases, cellulases, lipases, pectin degrading enzymes, xyloglucanases, or additional carboxylic acid ester hydrolases.

[0145] In some embodiments, the cleaning compositions described herein further comprise from about 1%, from about 3% to about 60%, or even from about 5% to about 40% of a builder, by weight of the cleaning composition. Builders may include, but are not limited to, alkali metal, ammonium and alkanolammonium salts of polyphosphates; alkali metal silicates, alkaline earth metal and alkali metal carbonates; aluminosilicates; polycarboxylic acid compounds; ether hydroxy polycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxybenzene-2,4,6-trisulfonic acid, and carboxymethyloxysuccinic acid; various alkali metal, ammonium and substituted ammonium salts of polyacetic acid, such as ethylenediaminetetraacetic acid and nitrilotriacetic acid; together with polycarboxylates, such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid and soluble salts thereof.

[0146] In some embodiments, builders form water-soluble hardness ion complexes (e.g., chelating builders), such as citrates and polyphosphates (e.g., sodium tripolyphosphate and sodium tripolyphosphate hexahydrate, potassium tripolyphosphate, and mixed sodium tripolyphosphate and potassium tripolyphosphate, etc.). Any suitable builder can be used in the compositions described herein, including those known in the art (see, e.g., EP 2100949).

[0147] As indicated herein, in some embodiments, the cleaning compositions described herein further comprise adjunct ingredients including, but not limited to, surfactants, builders, bleaching agents, bleach activators, bleach catalysts, other enzymes, enzyme stabilization systems, chelating agents, fluorescent brighteners, soil release polymers, dye transfer agents, dye transfer inhibitors, catalytic materials, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removers, structural elastic agents, dispersants, foam inhibitors, dyes, perfumes, colorants, filler salts, hydrotropes, photoactivators, fluorescent agents, fabric conditioners, hydrolyzable surfactants, solvents, preservatives, antioxidants, anti-shrinkage agents, anti-wrinkle agents, bactericides, fungicides, color embellishments, silver care agents, anti-tarnish and / or anti-corrosion agents, alkalinity sources, solubilizers, carriers, processing aids, pigments, and pH control agents (see, e.g., U.S. Patents 4,633,669). 6,610,642; 6,605,458; 5,705,464; 5,710,115; 5,698,504; 5,695,679; 5,686,014; and 5,646,101). In some embodiments, one or more adjunct ingredients are incorporated, for example, to assist or enhance cleaning performance (for treating substrates to be cleaned), or to modify the aesthetics of the cleaning composition (as with fragrances, colorants, dyes, etc.). Any such adjunct ingredients are in addition to the mannanase variants described herein. The precise nature of these additional components and their incorporation levels will depend on the physical form of the composition and the nature of the cleaning operation in which the composition will be used.

[0148] In embodiments where one or more adjunct ingredients are incompatible with the mannanase variant, suitable methods can be employed to keep the cleaning adjunct ingredients and the mannanase separated (i.e., out of contact with each other) until the combination of the two components is appropriate. Such separation methods include any suitable methods known in the art (e.g., encapsulation, tableting, physical separation, etc.). The specific selection of suitable adjunct ingredients is readily made by considering the surface, article, or fabric to be cleaned, and the desired composition form for the cleaning conditions during use (e.g., throughout detergent washing use).

[0149] The cleaning compositions described herein are advantageously used, for example, in laundry applications, hard surface cleaning, dishwashing applications, and cosmetic applications. In addition, the polypeptides of the present invention can be used in granular compositions and liquid compositions.

[0150] Mannanase variants described herein can also be used in cleaning additive products. In some embodiments, additives are encapsulated in dosage forms suitable for addition to the cleaning process. In some embodiments, additives are encapsulated in dosage forms for addition to the cleaning process, in which a peroxide source is used and an increased bleaching effect is desired. The present disclosure can use any suitable single unit dosage form, including but not limited to pills, tablets, capsules, or other single unit dosage forms (such as pre-measured powders or liquids). In some embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include but are not limited to various salts of sulfates, carbonates, and silicates, as well as talcum, clay, etc. Suitable fillers or carrier materials for liquid compositions include but are not limited to water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include but are not limited to methanol, ethanol, propanol, and isopropanol. In some embodiments, the composition comprises from about 5% to about 90% of such materials. Acidic fillers can be used to reduce pH. Alternatively, in some embodiments, the cleaning additive includes one or more auxiliary ingredients.

[0151] In one embodiment, the cleaning composition or cleaning additive contains an effective amount of a mannanase variant described herein, optionally in combination with other mannanases and / or additional enzymes. In certain embodiments, the additional enzymes include, but are not limited to, at least one enzyme selected from the group consisting of acyltransferases, amylases, α-amylases, β-amylases, α-galactosidases, arabinanases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, DNAse (also known as nuclease or dispersin), endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase 、 Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, PETase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthan lyase, xanthanase, xylan acetylesterase, xylanase, xylosidase, and combinations or mixtures thereof. In further embodiments, the cleaning compositions or cleaning additives described herein further comprise a protease and / or an amylase.

[0152] The cleaning compositions herein are typically formulated so that during use in aqueous cleaning operations, the wash water has a pH of from about 3.0 to about 11. Liquid product formulations are typically formulated to have a neat pH of from about 5.0 to about 9.0. Granular laundry products are typically formulated to have a pH of from about 8.0 to about 11.0. Techniques for controlling pH at recommended use levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0153] Suitable low pH cleaning compositions typically have a net pH of from about 3.0 to about 5.0 or even from about 3.5 to about 4.5. Low pH cleaning compositions typically do not contain surfactants that hydrolyze in such pH environments. Such surfactants include sodium alkyl sulfate surfactants, which contain at least one ethylene oxide moiety or even from about 1 to about 16 moles of ethylene oxide. Such cleaning compositions typically contain a sufficient amount of pH adjusters (such as sodium hydroxide, monoethanolamine or hydrochloric acid) to provide such cleaning compositions with a net pH of from about 3.0 to about 5.0. Such compositions typically contain at least one acid-stable enzyme. In some embodiments, the composition is liquid, while in other embodiments, these compositions are solid. The pH of such liquid compositions is typically measured as a net pH. The pH of such solid compositions is measured as a 10% solid solution of the composition, wherein the solvent is distilled water. In these embodiments, unless otherwise stated, all pH measurements are performed at 20°C.

[0154] Suitable high pH cleaning compositions typically have a net pH of from about 9.0 to about 11.0, or even a net pH of from 9.5 to 10.5. Such cleaning compositions typically include a sufficient amount of a pH adjusting agent (such as sodium hydroxide, monoethanolamine or hydrochloric acid) to provide such cleaning compositions with a net pH of from about 9.0 to about 11.0. Such compositions typically include at least one alkali-stable enzyme. In some embodiments, the composition is a liquid, while in other embodiments, these compositions are solid. The pH of such liquid compositions is typically measured with a net pH. The pH of such solid compositions is measured with a 10% solid solution of the composition, wherein the solvent is distilled water. In these embodiments, unless otherwise stated, all pH measurements are performed at 20°C.

[0155] In certain embodiments, the mannanase variant is in the form of encapsulated particles to protect it from the influence of other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the mannanase variant during cleaning. In certain embodiments, encapsulation enhances the performance of the mannanase variant and / or other enzymes. In this regard, the mannanase variant is encapsulated with any suitable encapsulating material known in the art. Typically, the encapsulating material is water-soluble and / or water-dispersible. In certain embodiments, the encapsulating material has a glass transition temperature (Tg) of 0°C or higher. The glass transition temperature is described in more detail in WO 97 / 11151. The encapsulating material is typically selected from carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides and combinations thereof. In some typical embodiments, the encapsulating material is starch (see, for example, EP0922499 and US 4,977,252; 5,354,559; and 5,935,826). In some embodiments, the encapsulating material is microspheres made of plastic (such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof); commercially available microspheres that can be used include, but are not limited to, microspheres made of (Stockviksverken, Sweden), and PM6545, PM6550, PM7220, PM7228, and (PQ Company, Valley Forge, PA).

[0156] The term "granular composition" refers to an agglomeration of discrete solid, macroscopic particles. Powders are a special class of granular materials due to their small particle size, which makes them more cohesive and easier to suspend.

[0157] The concentration of detergent compositions in typical wash solutions throughout the world varies from less than about 800 ppm of detergent composition (a "low detergent concentration geography," e.g., about 667 ppm in Japan), to between about 800 ppm and about 2000 ppm (a "medium detergent concentration geography," e.g., about 975 ppm in the United States and about 1500 ppm in Brazil), to greater than about 2000 ppm (a "high detergent concentration geography," e.g., about 4500 ppm to about 5000 ppm in Europe and about 6000 ppm in high sudsing phosphate builder geography).

[0158] In some embodiments, the detergent compositions described herein can be utilized at temperatures ranging from about 10° C. to about 60° C., or from about 20° C. to about 60° C., or from about 30° C. to about 60° C., from about 40° C. to about 60° C., from about 40° C. to about 55° C., or all ranges within 10° C. to 60° C. In some embodiments, the detergent compositions described herein are used in a “cold water wash” at temperatures ranging from about 10° C. to about 40° C., or from about 20° C. to about 30° C., from about 15° C. to about 25° C., from about 15° C. to about 35° C., or all ranges within 10° C. to 40° C.

[0159] As another example, different geographic locations typically have different water hardnesses. 2+ / Mg 2+ The hardness of water is described by the number of grains. Hardness is the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) in the United States, but the hardness varies. Moderately hard (60-120 ppm) to hard (121-181 ppm) water has a hardness of 60 to 181 ppm (parts per million) of minerals (to convert ppm to grains per U.S. gallon, divide the ppm number by 17.1, which equals grains per gallon).

[0160] Table 1. Water hardness levels

[0161]

[0162]

[0163] Typically, European water hardness is greater than about 10.5 (e.g., about 10.5 to about 20.0) grains / gallon of mixed Ca 2+ / Mg 2+ (For example, about 15 grains / gallon of mixed Ca 2+ / Mg 2+ Typically, the water hardness in North America is greater than the water hardness in Japan, but lower than the water hardness in Europe. For example, the water hardness in North America can be between about 3 and about 10 grains, about 3 to about 8 grains, or about 6 grains. Typically, the water hardness in Japan is lower than the water hardness in North America, typically less than about 4 (e.g., about 3) grains / gallon of mixed Ca 2+ / Mg 2+ .

[0164] In certain embodiments, the mannanase variants described herein are comparable to commercially available mannanases in terms of wash performance. In certain embodiments, the mannanase variants described herein exhibit enhanced wash performance compared to commercially available mannanases. In certain embodiments, the mannanase variants described herein exhibit enhanced cleaning power under various conditions, and / or enhanced chelating agent stability. In addition, the mannanase variants described herein may also be used in detergent-free cleaning compositions alone or in combination with builders and stabilizers.

[0165] In addition to the mannanase variants described herein, any other suitable mannanase can be used in the compositions described herein alone or in combination with the variants described herein. Suitable mannanases include, but are not limited to, mannanases of the GH26 family of glycosyl hydrolases, mannanases of the GH5 family of glycosyl hydrolases, acid mannanases, neutral mannanases, and alkaline mannanases. Examples of alkaline mannanases include those described in the following: US 6,060,299; 6,566,114; and 6,602,842; and WO 9535362, WO 9964573, WO 9964619, and WO2015022428. In addition, suitable mannanases include, but are not limited to, those of animal, plant, fungal, or bacterial origin. Chemically or genetically modified mutants are contemplated in the present disclosure.

[0166] Examples of useful mannanases include Bacillus endo-β-mannanase (such as Bacillus subtilis endo-β-mannanase (see, e.g., U.S. Pat. No. 6,060,299 and WO 9964573), Bacillus sp. I633 endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619), Bacillus sp. AAI12 endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619), Bacillus sp. AA349 endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619), Bacillus agaricus NCIMB 40482 endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619). 9964619), B. halodurans endo-β-mannanase, B. clausii endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619), B. licheniformis endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619A1)), Humicola endo-β-mannanase (such as H. insolens endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619)), and Caldocellulosiruptor endo-β-mannanase (such as Caldocellulosiruptor sp. endo-β-mannanase (see, e.g., U.S. Pat. No. 6,566,114 and WO 9964619A1)). 6,566,114 and WO 9964619)).

[0167] In addition, many identified mannanases (i.e., endo-β-mannanases and exo-β-mannanases) can be used in some embodiments of the present disclosure, including but not limited to A. bisporus mannanase (see, Tang et al.,

[2001] Appl. Environ. Microbiol. [Application and Environmental Microbiology] 67:2298–2303), A. tamarii mannanase (see, Civas et al.,

[2002] Appl. Environ. Microbiol. [Application and Environmental Microbiology] 67:2397–241 ...

[1984] Biochem. J. [Journal of Biological Chemistry] 219: 857–863), A. aculeatus mannanase (see, Christgau et al.,

[1994] Biochem. Mol. Biol. Int. [International Biochemistry and Molecular Biology] 33: 917–925), A. awamori mannanase (see, Setati et al.,

[2001] Protein Express Purif. [Protein Expression and Purification] 21:105–114), A. fumigatus mannanase (see, Puchart et al.,

[2004] Biochimica et biophysica Acta. [Biochemistry and Biophysics] 1674:239–250), A. niger mannanase (see, Ademark et al.,

[1998] J. Biotechnol. [Journal of Biotechnology] 63:199–210), A. oryzae NRRL mannanase (see, Regalado et al.,

[2000] J. Sci. Food Agric. [Journal of Food and Agricultural Sciences] 80:1343–1350), A. sulphureus mannanase (see, Chen et al.,

[2007] J. Biotechnol. [Journal of Biotechnology] 128(3):452–461), A. terrus mannanase (see, Huang et al.,

[2007] Wei Sheng Wu Xue Bao. [Acta Microbiologica Sinica] 47(2):280–284), Paenibacillus and Bacillus species mannanase (see, US 6,376,445.), Bacillus AM001 mannanase (see, Akino et al.,

[1989] Arch. Microbiol. [Archives of Microbiology] 152:10–15), Bacillus brevis mannanase (see, Araujo and Ward,

[1990] J. Appl. Bacteriol.[Journal of Applied Bacteriology] 68:253–261), B. circulans K-1 mannanase (see, Yoshida et al.,

[1998] Biosci. Biotechnol. Biochem. [Bioscience, Biotechnology and Biochemistry] 62(3):514–520), B. polymyxa mannanase (see, Araujo and Ward,

[1990] J. Appl. Bacteriol. [Journal of Applied Bacteriology] 68:253–261), Bacillus sp. JAMB-750 mannanase (see, Hatada et al.,

[2005] Extremophiles. [Extremophiles] 9:497–500), Bacillus sp. M50 mannanase (see, Chen et al.,

[2000] Wei Sheng Wu Xue Bao. [Acta Microbiologica Sinica] 40:62–68), Bacillus sp. N16-5 mannanase (see Yanhe et al.

[2004] Extremophiles [Extremophiles] 8:447–454), B. stearothermophilus mannanase (see Talbot and Sygusch,

[1990] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 56:3505–3510), subtilisin Bacillus subtilis mannanase (see, Mendoza et al.,

[1994] World J. Microbiol. Biotechnol. 10:51–54), Bacillus subtilis B36 mannanase (Li et al.,

[2006] Z. Naturforsch (C) [Natural Research Journal (C Edition)]. 61:840–846), Bacillus subtilis BM9602 mannanase (see, Cui et al.,

[1999] Wei ... Sheng Wu Xue Bao. [Acta Microbiologica Sinica] 39(1):60–63), Bacillus subtilis SA-22 mannanase (see, Sun et al.,

[2003] Sheng Wu Gong Cheng Xue Bao. [Journal of Bioengineering] 19(3):327–330), Bacillus subtilis 168 mannanase (see, Helow and Khattab,

[1996] Acta Microbiol. Immunol. Hung. [Hungarian Journal of Microbiology and Immunology] 43:289–299), Bacteroides ovatus mannanase (see, Gherardini et al.,

[1987] J. Bacteriol.[Journal of Bacteriology] 169:2038–2043), B. ruminicola mannanase (see, Matsushita et al.,

[1991] J. Bacteriol. [Journal of Bacteriology] 173:6919–6926), C. cellulovorans mannanase (see, Sunna et al.,

[2000] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 66:664–670), C. saccharolyticus mannanase (see, Morris et al.,

[2001] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 67:684–697), and C. saccharolyticus mannanase (see, Morris et al.,

[2001] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 66:684–697).

[1995] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 61:2262–2269), C. saccharolyticum mannanase (see, Bicho et al.,

[1991] Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 36:337–343), C. fimi mannanase (see, Stoll et al.,

[1999] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 65(6):2598–2605), Clostridium butyricum (C. butyricum) / C. beijerinckii mannanase (see, Nakajima and Matsuura,

[1997] Biosci. Biotechnol. Biochem. [Biological Science, Biotechnology and Biochemistry] 61: 1739-1742), C. cellulolyticum mannanase (see, Perret et al.,

[2004] Biotechnol. Appl. Biochem. [Biotechnology Application and Biochemistry] 40: 255-259), C. tertium mannanase (see, , Kataoka and Tokiwa,

[1998] J. Appl. Microbiol. [Journal of Applied Microbiology] 84:357–367), C. thermocellum mannanase (see, Halstead et al.,

[1999] Microbiol. [Microbiology] 145:3101–3108), D. thermophilum mannanase (see, Gibbs et al.,

[1999] Curr. Microbiol. [Current Microbiology] 39(6):351–357), Flavobacterium sp.) mannanase (see, Zakaria et al.,

[1998] Biosci. Biotechnol. Biochem. 62:655-660), G. pulmonata mannanase (see, Charrier and Rouland,

[2001] J. Expt. Zool. 290:125-135), L. brevicula mannanase (see, Yamamura et al.,

[1996] Biosci. Biotechnol. Biochem. [Biological Science, Biotechnology and Biochemistry] 60:674-676), edible tomato (L. esculentum) mannanase (see, Filichkin et al.,

[2000] Plant Physiol. [Plant Physiology] 134: 1080–1087), P. curdlanolyticus mannanase (see, Pason and Ratanakhanokchai,

[2006] Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 72: 2483–2490), P. polymyxa mannanase (see, Han et al.,

[2006] Appl. Microbiol. [Applied and Environmental Microbiology] 72: 2483–2490), Biotechnol. 73(3):618–630), P. chrysosporium mannanase (see Wymelenberg et al.

[2005] J. Biotechnol. 118:17–34), Piromyces sp. mannanase (see Fanutti et al.

[1995] J. Biol. Chem. 270(49):29314–29322), P. insulars mannanase (see Yamamura et al.

[1993] Biosci. Biotechnol. Biochem. 7:1316–1319), P. fluorescens subsp. cellulosus (see P. fluorescens subsp. cellulosa) mannanase (see Braithwaite et al.,

[1995] Biochem J. 305:1005–1010), R. marinus mannanase (see Politz et al.,

[2000] Appl. Microbiol. Biotechnol. 53(6):715–721), S.rolfsii) mannanase (see, Sachslehner et al.,

[2000] J. Biotechnol. 80:127–134), S. galbus mannanase (see, Kansoh and Nagieb,

[2004] Anton. van. Leeuwenhoek. 85:103–114), S. lividans mannanase (see, Arcand et al.,

[1993] J. Biochem. 290:857–863), T. Polysaccharolyticum mannanase (see, Cann et al.,

[1999] J. Bacteriol. 181:1643–165 1), T. fusca mannanase (see, Hilge et al.,

[1998] Structure 6:1433–1444), T. maritima mannanase (see, Parker et al.,

[2001] Biotechnol. Bioeng. 75(3):322–333), T. neapolitana mannanase (see, Duffaud et al.,

[1997] Appl. Environ. Microbiol. 63:169–177), T. harzianum strain T4 mannanase (see, Franco et al.,

[2004] Biotechnol. Bioeng. 75(3):322–333), T. neapolitana mannanase (see, Duffaud et al.,

[1997] Appl. Environ. Microbiol. 63:169–177), T. harzianum strain T4 mannanase (see, Franco et al.,

[2004] Biotechnol. Bioeng. 75(3):322–333), T. harzianum strain T4 mannanase (see, Franco et al.,

[2004] Biotechnol. Bioeng. 75(3):322–333), T. harzianum strain T4 mannanase (see, Appl. Biochem. [Applied Biotechnology and Biochemistry] 40:255–259), T. reesei mannanase (see, Stalbrand et al.,

[1993] J. Biotechnol. [Journal of Biotechnology] 29:229–242), and Vibrio sp. mannanase (see, Tamaru et al.,

[1997] J. Ferment. Bioeng. [Journal of Fermentation and Bioengineering] 83:201–205).

[0168] Additional suitable mannanases include commercially available endo-β-mannanases, such as (Chemgen Corporation); and (Novozymes A / S, Denmark); EFFECTENZ TM M 1000, M 100, PURABRITETM and MANNASTAR TM (DuPont); and 160 and 200 (Diversa).

[0169] In other embodiments, the compositions described herein comprise one or more mannanase variants described herein and one or more additional enzymes. The one or more additional enzymes are selected from acyltransferases, α-amylases, β-amylases, α-galactosidases, arabinosidases, arylesterases, β-galactosidases, carrageenase, catalase, cellobiohydrolase, cellulase, chondroitinase, cutinase, endo-β-1,4-glucanase, endo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hyaluronidase, keratinase, laccase, lactase, , lignanases, lipases, lipoxygenases, additional mannanases, metalloproteinases, oxidases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonase, proteases, pullulanases, reductases, rhamnogalacturonase, β-glucanase, tannase, transglutaminase, xylan acetylesterase, xylanase, xyloglucanase, xylosidase, and any combination or mixture thereof. Some embodiments relate to enzyme combinations (i.e., "cocktails") combined with one or more mannanase variants described herein and / or one or more additional mannanases, the enzyme combination comprising conventional enzymes, like amylases, lipases, cutinases, proteases and / or cellulases.

[0170] In some embodiments, the cleaning compositions described herein further comprise a protease. In some embodiments, the composition comprises from about 0.00001% to about 10% protease by weight of the composition. In another embodiment, the cleaning composition comprises from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5% protease by weight of the composition.

[0171] In one embodiment, protease is a serine protease. Suitable proteases include those of animal, plant or microbial origin. In certain embodiments, protease is a microbial protease. In other embodiments, protease is a chemically or genetically modified mutant. In another embodiment, protease is an alkaline microbial protease or a trypsin-like protease. Exemplary alkaline proteases include subtilisin derived from, for example, Bacillus lentus (e.g., subtilisin of Bacillus lentus, subtilisin of Bacillus amyloliquefaciens, subtilisin of Bacillus gibsonii, Carlsberg subtilisin, subtilisin 309, Bacillus species TY-145, subtilisin 147 and subtilisin 168).Exemplary additional proteases include, but are not limited to, WO 92 / 21760, WO 95 / 23221, WO 2008 / 010925, WO 09 / 149200, WO 09 / 149144, WO 09 / 149145, WO10 / 056640, WO 10 / 056653, WO 2010 / 0566356、WO 11 / 072099、WO 2011 / 13022、WO 11 / 140364、WO 12 / 151534、WO 2015 / 038792、WO 2015 / 089447、WO 2015 / 089441、WO 2015 / 143360、WO 2016 / 061438、WO 2016 / 069548、WO 2016 / 069544、WO 2016 / 069557、WO 2016 / 069563、WO 2016 / 069569、WO 2016 / 069552、WO 2016 / 145428、US Publ.No.2008 / 0090747、US 5,801,039, US 5,340,735, US 5,500,364, US 5,855,625, RE 34,606, US 5,955,340, US 5,700,676, US 6,312,936, US 6,482,628, US 8,530,219, US Provisional Application Nos. 62 / 331282, 62 / 332417, 62 / 343618 and 62 / 351649 and PCT Application Nos. PCT / US16 / 32514 and PCT / US2016 / 038245, and WO 1999014341, WO 1999033960, WO 1999014342, WO 1999034003, WO2007044993, WO 2009058303, WO 2009058661, WO 2014071410, WO 2014194032, WO2014194034, WO 2014194054 and WO Metalloproteases as described in 2014 / 194117. Exemplary proteases include, but are not limited to, trypsin (e.g., trypsin of porcine or bovine origin) and the Fusarium protease described in WO89 / 06270. Exemplary commercial proteases include, but are not limited to. MAXACAL TM , MAXAPEM TM , OXP、PURAMAX TM 、EXCELLASE TM 、PREFERENZTM Proteases (e.g. P100, P110, P280), EFFECTENZ TM Proteases (e.g. P1000, P1050, P2000), EXCELLENZ TM Proteases (e.g. P1000), and PURAFAST TM (DuPont); 16L, ULTRA, DURAZYM TM , LIQUANASE PROGRESS and (Novozymes); BLAP TM and BLAP TM Variant (Henkel); LAVERGY TM PRO 104L (BASF), and KAP (B. alkalophilus subtilisin (Kao)).

[0172] In some embodiments, the cleaning compositions described herein further comprise a suitable amylase. In one embodiment, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase by weight of the composition. Any amylase (e.g., alpha amylase and / or beta amylase) suitable for use in alkaline solutions can be used for use in compositions included in such compositions. Exemplary amylases can be chemically or genetically modified mutants. Exemplary amylases include, but are not limited to, those of bacterial or fungal origin, such as, for example, amylases described in GB 1,296,839, WO 9100353, WO 9402597, WO94183314, WO 9510603, WO 9526397, WO 9535382, WO 9605295, WO 9623873, WO 9623874, WO9630481, WO 9710342, WO 9741213, WO 9743424, WO 9813481, WO 9826078, WO 9902702, WO9909183, WO 9919467, WO 9923211, WO 9929876, WO 9942567, WO 9943793, WO 9943794, WO9946399, WO 0029560, WO 0060058, WO 0060059, WO 0060060, WO 0114532, WO 0134784, WO0164852, WO 0166712, WO 0188107, WO 0196537, WO 02092797, WO 0210355, WO 0231124, WO2004055178, WO 2004113551, WO 2005001064, WO 2005003311, WO 2005018336, WO2005019443, WO 2005066338, WO 2006002643, WO 2006012899, WO 2006012902, WO2006031554, WO 2006063594, WO 2006066594, WO 2006066596, WO 2006136161, WO2008000825, WO 2008088493, WO 2008092919, WO 2008101894, WO2008 / 112459, WO2009061380, WO 2009061381, WO 2009100102, WO 2009140504, WO 2009149419, WO2010 / 059413、WO 2010088447、WO 2010091221、WO 2010104675、WO 2010115021、WO10115028、WO2010117511、WO 2011076123、WO 2011076897、WO 2011080352, WO 2011080353, WO2011080354, WO 2011082425, WO 2011082429, WO 2011087836, WO 2011098531, WO2013063460, WO2013184577, WO 2014099523、WO 2014164777 and WO 2015077126. Exemplary commercial amylases include but are not limited to AMPLIFY STAINZYME STAINZYME STAINZYME and BAN TM (Novozymes); EFFECTENZ TM S 1000, POWERASE TM 、PREFERENZ TM S 100, PREFERENZ TM S 110, EXCELLENZ TM S 2000, and P(DuPont).

[0173] In some embodiments, the cleaning compositions described herein further comprise a suitable pectin degrading enzyme. As used herein, "one or more pectin degrading enzymes" encompasses arabinanases (EC 3.2.1.99), galactanases (EC 3.2.1.89), polygalacturonases (EC 3.2.1.15), exo-polygalacturonases (EC 3.2.1.67), exo-poly-α-galacturonosidases (EC 3.2.1.82), pectin lyases (EC 4.2.2.10), pectinesterases (EC 3.1.1.11), pectate lyases (EC 4.2.2.2), exo-polygalacturonate lyases (EC 4.2.2.9), and hemicellulases (e.g., endo-1,3-β-xylosidase (EC 3.2.1.32), xylan-1,4-β-xylosidase (EC 3.2.1.4), and xylan-2,3-β-xylosidase (EC 3.2.1.5). Pectin degrading enzymes are natural mixtures of the enzyme activities mentioned above. Thus, pectinase includes pectin methylesterases that hydrolyze pectin methyl ester bonds, polygalacturonase that cleaves glycosidic bonds between galacturonic acid molecules, and pectin transeliminase or lyase that acts on pectic acid to bring about non-hydrolytic cleavage of α-1,4 glycosidic bonds to form unsaturated derivatives of galacturonic acid.

[0174] Suitable pectin degrading enzymes include those of plant, fungus or microbial origin. In certain embodiments, include chemical or genetically modified mutants. In certain embodiments, pectin degrading enzymes are alkaline pectin degrading enzymes, i.e., enzymes with at least 10%, at least 25% or at least 40% enzymatic activity at a pH from about 7.0 to about 12 of its maximum activity. In certain other embodiments, pectin degrading enzymes are enzymes with at least 7.0 to about 12 of its maximum activity of pH. Alkaline pectin degrading enzymes are produced by alkaliphilic microorganisms, which are, for example, bacteria, fungi and yeast microorganisms, such as bacillus species. In certain embodiments, as described in JP 56131376 and JP 56068393, microorganisms are bacillus firmus, bacillus circulans and bacillus subtilis. Alkaline pectinolytic enzymes may include, but are not limited to, galacturonan-1,4-α-galacturonase (EC 3.2.1.67), poly-galacturonase activity (EC 3.2.1.15), pectinesterase (EC 3.1.1.11), pectate lyase (EC 4.2.2.2) and isozymes thereof. Alkaline pectinolytic enzymes may be produced by Erwinia species. In some embodiments, the alkaline pectinase is produced by E. chrysanthemi, E. carotovora, E. amylovora, E. herbicola, and E. dissolvens as described in JP 59066588, JP 63042988, and World J. Microbiol. Biotechnol. (8, 2, 115-120) 1992. In certain other embodiments, the alkaline pectinase is produced by Bacillus species as disclosed in JP 73006557 and Agr. Biol. Chem. (1972), 36 (2) 285-93. In some embodiments, the cleaning compositions described herein further comprise from about 0.00001% to about 10%, from about 0.0001% to about 10%, from about 0.001% to about 5%, from about 0.001% to about 2%, or from about 0.005% to about 0.5%, by weight of the composition, of a pectin degrading enzyme.

[0175] In some other embodiments, the cleaning compositions described herein further comprise suitable xyloglucanases. Suitable xyloglucanases include, but are not limited to, those of plant, fungal, or bacterial origin. In some embodiments, chemically or genetically modified mutants are included. As used herein, "one or more xyloglucanases" encompass the family of enzymes described by Vincken and Voragen at Wageningen University [Vincken et al. (1994) Plant Physiol. [Plant Physiology], 104, 99-107], and can degrade xyloglucans as described in Hayashi et al. (1989) Annu. Rev. Plant. Physiol. Plant Mol. Biol. [Annual Review of Plant Physiology and Plant Molecular Biology], 40, 139-168. Vincken et al. demonstrated that xyloglucan coatings were removed from the cellulose of the apple cell walls separated by xyloglucanase (endo-IV-glucanase) purified from green mold (Trichoderma viride). This enzyme enhances the enzymatic degradation of the cellulose embedded in the cell wall, and works in synergy with pectinase. Rapidase LIQ+ from DSM contains xyloglucanase activity. In certain embodiments, cleaning compositions as herein described further comprise by the weight of composition from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% xyloglucanase. In certain other embodiments, the xyloglucanase for specific application is an alkaline xyloglucanase, i.e., an enzyme with at least 10%, at least 25% or at least 40% enzymatic activity of its maximum activity at a pH range from 7 to 12. In certain other embodiments, xyloglucanase is an enzyme with maximum activity at a pH from about 7.0 to about 12.

[0176] In some additional embodiments, the detergent compositions described herein further comprise suitable cellulases. In one embodiment, the composition comprises from about 0.00001% to about 10%, 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulases by weight of the composition. Any suitable cellulases can be used in the compositions described herein. Exemplary cellulases can be chemically or genetically modified mutants. Exemplary cellulases include, but are not limited to, those of bacterial or fungal origin, such as those described in WO2005054475, WO 2005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and U.S. Provisional Application No. 62 / 296,678. Exemplary commercial cellulases include, but are not limited to and PREMIUM (Novozymes); REVITALENZ TM 100. REVITALENZ TM 200 / 220, and 2000 (DuPont); and KAC-500 (B) TM (Kao Corporation) In some embodiments, the cellulase is incorporated as a portion or fragment of a mature wild-type or variant cellulase in which a portion of the N-terminus is deleted (see, e.g., US 5,874,276).

[0177] In still other embodiments, the detergent compositions described herein further comprise a suitable lipase. In some embodiments, the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight of the composition. Exemplary lipases can be chemically or genetically modified mutants. Exemplary lipases include, but are not limited to, e.g., those of bacterial or fungal origin, such as, for example, H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T. lanuginosa lipase (see, e.g., WO 2014 / 059360 and WO 2015 / 010009), Rhizomucor miehei lipase (see, e.g., EP 238023), Candida lipases, such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761), Pseudomonas lipases, such as P. alcaligenes and P. pseudoalcaligenes lipases (see, e.g., EP 238023). 218272), P. cepacia lipase (see, e.g., EP 331376), P. stutzeri lipase (see, e.g., GB 1,372,034), P. fluorescens lipase, Bacillus lipase (e.g., B. subtilis lipase (Dartois et al., Biochem. Biophys. Acta [Biochemistry and Biophysics] 1131:253-260 (1993)), B. stearothermophilus lipase (see, e.g., JP 64 / 744992), and B. pumilus lipase (see, e.g., WO 91 / 16422)).Exemplary cloned lipases include, but are not limited to, Penicillium camembertii lipase (see Yamaguchi et al., Gene 103:61-67 (1991)); Geotricum candidum lipase (see Schimada et al., J. Biochem., 106:383-388 (1989)); and various Rhizopus lipases, such as R. delemar lipase (see Hass et al., Gene 109:117-113 (1991)), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)), and R. oryzae lipase. Other lipolytic enzymes (such as cutinases) can also be used in one or more compositions described herein, including but not limited to, for example, cutinases derived from Pseudomonas mendocina (see WO 88 / 09367) and / or Fusarium solani pisi (see WO 90 / 09446). Exemplary commercial lipases include but are not limited to M1 LIPASE. TM 、LUMA FAST TM and LIPOMAX TM (DuPont); and ULTRA (Novozymes); and LIPASE P TM (Amano Pharmaceutical Co. Ltd).

[0178] In certain embodiments, cleaning compositions as herein described further comprise a peroxidase combined with hydrogen peroxide or its source (e.g., percarbonate, perborate or persulfate). In some alternative embodiments, oxidase is used in combination with oxygen. Both types of enzymes are used for "solution bleaching", i.e., preventing textile dyes from transferring to another fabric from a dyed fabric when fabric is washed together in a detergent solution), preferably used together with a synergist (see, e.g., WO 94 / 12621 and WO 95 / 01426). Suitable peroxidase / oxidase includes, but is not limited to, those of plant, bacterial or fungal origin. In certain embodiments, chemical or genetically modified mutants are included. In certain embodiments, cleaning compositions of the present disclosure further comprise peroxidase and / or oxidase by weight of the composition from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, about 0.005% to about 0.5%.

[0179] In some embodiments, the cleaning compositions described herein further comprise additional enzymes, including but not limited to perhydrolases (see, e.g., WO 05 / 056782). Some embodiments relate to mixtures of one or more of the above-mentioned proteases, amylases, lipases, mannanases, and / or cellulases.

[0180] Some embodiments relate to cleaning compositions, such as those described in US 6,605,458. In some embodiments, the cleaning compositions described herein are intensive granular fabric cleaning compositions, while in other embodiments, the compositions are granular fabric cleaning compositions useful in laundry washing of colored fabrics. In further embodiments, the compositions are granular fabric cleaning compositions that provide softening by washing capacity, and in further embodiments, the compositions are heavy-duty liquid (HDL) fabric cleaning compositions. In other embodiments, the cleaning compositions described herein are fabric cleaning compositions, such as those described in US 6,610,642 and 6,376,450. In alternative embodiments, the cleaning compositions described herein are suitable hard surface cleaning compositions. Suitable hard surface cleaning compositions include, for example, US 6,610,642; 6,376,450; and those described in 6,376,450. In yet further embodiments, the cleaning compositions described herein are dishwashing compositions. In some further embodiments, the compositions described herein are oral care compositions, such as, for example, those described in US 6,376,450 and 6,605,458. The formulations and descriptions of compounds and cleaning adjunct materials contained in the aforementioned US 6,376,450, 6,605,458 and 6,610,642 can be used in the polypeptides of the present invention.

[0181] In still further embodiments, the cleaning compositions described herein are fabric softening compositions, such as, for example, those described in GB 400898, GB 514276, EP0011340, EP0026528, EP0242919, EP0299575, EP0313146, and US 5,019,292.

[0182] The cleaning compositions described herein can be formulated into any suitable form and prepared by any process selected by the formulator, non-limiting examples of which are described in the following: US 5,879,584; 5,691,297; 5,574,005; 5,569,645; 5,565,422; 5,516,448; 5,489,392; and 5,486,303. When a low pH cleaning composition is desired, the pH of such a composition is adjusted via the addition of materials such as monoethanolamine or an acidic material such as HCl.

[0183] In certain embodiments, cleaning compositions as described herein are provided in unit dosage form, including tablets, capsules, medicine bags, pouches, thin slices and multi-compartment pouches. In certain embodiments, unit dosage form is designed to provide controlled release of ingredients in multi-compartment pouches (or other unit dosage forms). Suitable unit dosage and controlled release forms are known in the art (see, for example, EP 2100949, EP 2100947, WO 02 / 102955, WO 04 / 111178, WO 2013 / 165725 and US 4,765,916 and 4,972,017). In certain embodiments, unit dosage form is provided by a tablet wrapped with a water-soluble film or a water-soluble pouch.

[0184] In some embodiments, the cleaning compositions described herein further comprise at least one chelating agent. Suitable chelating agents may include, but are not limited to, copper, iron and / or manganese chelating agents, and mixtures thereof. In embodiments using at least one chelating agent, the cleaning compositions of the present disclosure comprise from about 0.1% to about 15% or even from about 3.0% to about 10% of the chelating agent by weight of the cleaning composition.

[0185] In some still further embodiments, the cleaning compositions described herein further comprise at least one deposition aid. Suitable deposition aids include, but are not limited to, polyethylene glycol, polypropylene glycol, polycarboxylates, soil release polymers (such as polyethylene terephthalate), clays such as kaolin, montmorillonite, attapulgite, illite, bentonite, halloysite, and mixtures thereof.

[0186] In some embodiments, the cleaning compositions described herein further comprise at least one anti-redeposition agent. In some embodiments, the anti-redeposition agent is a nonionic surfactant, such as described, for example, in EP 2100949. In some automatic dishwashing embodiments, nonionic surfactants are used as surface modifiers (particularly for flakes) to avoid filming and staining and to improve shine.

[0187] In some embodiments, the cleaning compositions described herein further comprise one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinyl pyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinyl pyrrolidone and N-vinylimidazole, polyvinyl oxazolidones and polyvinylimidazoles, or mixtures thereof. In some embodiments, the cleaning compositions described herein comprise from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% dye transfer inhibitors by weight of the cleaning compositions.

[0188] In some embodiments, the cleaning compositions described herein further comprise one or more silicates. In some such embodiments, sodium silicates (e.g., sodium disilicate, sodium metasilicate, and crystalline phyllosilicates) can be used. In some embodiments, the cleaning compositions described herein comprise from about 1% to about 20% or from about 5% to about 15% silicates by weight of the composition.

[0189] In yet further embodiments, the cleaning compositions described herein further comprise one or more dispersants.Suitable water-soluble organic materials include, but are not limited to, homo- or co-polymeric acids or their salts, wherein the polyacid comprises at least two carboxyl groups separated from each other by not more than two carbon atoms.

[0190] In some further embodiments, the enzyme used in the cleaning composition is stabilized by any suitable technology. In certain embodiments, the enzyme used herein is stabilized by the presence of a water-soluble source of calcium and / or magnesium ions in the finished composition. In certain embodiments, enzyme stabilizers include oligosaccharides, polysaccharides and inorganic divalent metal salts (including alkaline earth metal salts, such as calcium salts). It is envisioned that various technologies for enzyme stabilization will be used in the present disclosure. For example, in certain embodiments, the enzyme used herein is stabilized by the presence of the following in the finished composition: a water-soluble source of zinc (II), calcium (II) and / or magnesium (II) ions, and other metal ions (for example, barium (II), scandium (II), iron (II), manganese (II), aluminum (III), tin (II), cobalt (II), copper (II), nickel (II) and vanadium (IV)). Chlorides and sulfates may also be used in certain embodiments. Examples of suitable oligosaccharides and polysaccharides (for example, dextrins) are known in the art (see, for example, WO 07 / 145964). In some embodiments, reversible protease inhibitors such as boron-containing compounds (eg, borates, 4-formylphenylboronic acid) and / or peptide aldehydes (US Pat. No. 9,181,296 B2) may be used to further improve stability.

[0191] In some embodiments, the cleaning compositions described herein further comprise one or more bleaching agents, bleach activators, and / or bleach catalysts. In some embodiments, the cleaning compositions described herein comprise one or more inorganic and / or organic bleaching compounds. Inorganic bleaching agents may include, but are not limited to, perhydrate salts (e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate). In some embodiments, the inorganic perhydrate salt is an alkali metal salt. In some embodiments, it is included as a crystalline solid but not additionally protected inorganic perhydrate salt, but in some other embodiments, the salt is coated. Suitable salts include, for example, those described in EP 2100949. Bleach activators are typically organic peracid precursors that enhance bleaching during cleaning at temperatures of 60°C and below. Bleach activators suitable for use herein include compounds and / or optionally substituted peroxybenzoic acid that give, under perhydrolysis conditions, preferably aliphatic peroxycarboxylic acids having from about 1 to about 10 carbon atoms, particularly from about 2 to about 4 carbon atoms. Suitable bleach activators include, for example, those described in EP 2100949. Bleach catalysts typically include, for example, manganese triazacyclononane and related complexes, and cobalt, copper, manganese, and iron complexes, and those described in US Pat. Nos. 4,246,612, 5,227,084; 4,810,410; and WO 99 / 06521 and EP 2100949.

[0192] In certain embodiments, cleaning compositions described herein further comprise one or more catalytic metal complexes. In certain embodiments, metal-containing bleaching catalysts can be used. In other embodiments, metal bleaching catalysts comprise a catalytic system, which includes: transition metal cations (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cations) with limited bleaching catalytic activity, auxiliary metal cations (e.g., zinc or aluminum cations) with little or no bleaching catalytic activity, and chelates with limited stability constants for catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetrakis (methylenephosphonic acid) and water-soluble salts thereof (see, e.g., US 4,430,243). In certain embodiments, cleaning compositions described herein are catalyzed by manganese compounds. Such compounds and usage levels are well known in the art (see, e.g., US 5,576,282). In other embodiments, cobalt bleaching catalysts can be used in cleaning compositions described herein. Various cobalt bleach catalysts are known in the art (see, for example, US Pat. Nos. 5,597,936 and 5,595,967) and are readily prepared by known procedures.

[0193] In some additional embodiments, the cleaning compositions described herein further comprise a transition metal complex of a macropolycyclic rigid ligand (MRL). As a practical matter and not by way of limitation, in some embodiments, the compositions and cleaning methods provided herein are adjusted to provide at least one part per hundred million of active MRL species in an aqueous washing medium, and in other embodiments, provide from about 0.005 ppm to about 25 ppm, from about 0.05 ppm to about 10 ppm, or from about 0.1 ppm to about 5 ppm of MRL in the wash liquor.

[0194] In some embodiments, the transition metal in the instantaneous transition metal bleach catalyst includes, but is not limited to, manganese, iron, and chromium. In other embodiments, MRLs include, but are not limited to, special ultra-rigid ligands (e.g., 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane) that form cross-linked bridges. Suitable transition metal MRLs are readily prepared by known procedures (see, e.g., WO 2000 / 32601 and US 6,225,464).

[0195] In some embodiments, the cleaning compositions described herein further comprise a metal care agent. The metal care agent is used to prevent and / or reduce the rust, corrosion and / or oxidation of metals, including aluminum, stainless steel and non-ferrous metals (e.g., silver and copper). Suitable metal care agents include those described in EP 2100949, WO 94 / 26860 and WO 94 / 26859. In some embodiments, the metal care agent is a zinc salt. In some further embodiments, the cleaning compositions described herein comprise from about 0.1% to about 5% by weight of one or more metal care agents.

[0196] Cleaning compositions as described herein can be used for cleaning surfaces, tableware, or fabrics. Typically, at least a portion of a surface, tableware, or fabric is contacted with (i) at least one variant as described herein or (ii) at least one cleaning composition as described herein, and then optionally washed and / or rinsed. For purposes of this disclosure, "washing" includes but is not limited to scrubbing and mechanical agitation. In certain embodiments, cleaning compositions are typically used in a solution at a concentration of about 500ppm to about 15,000ppm. When the washing solvent is water, the water temperature is typically in the range of from about 5°C to about 90°C, and when fabric is involved, the mass ratio of water to fabric is typically from about 1:1 to about 30:1.

[0197] Some embodiments relate to a cleaning method comprising contacting an effective amount of (i) a mannanase variant described herein or (ii) a cleaning composition described herein with an item or surface containing a soil or stain containing mannan to hydrolyze the mannan contained in the soil or stain.

[0198] In some embodiments, one or more mannanase variants described herein are used to prevent, reduce and / or remove biofilm on one or more articles selected from textiles and fabrics.

[0199] One or more mannanase variants described herein hydrolyze polysaccharide chains containing mannose units, including but not limited to mannan, galactomannan and glucomannan, so that such polypeptides are particularly suitable for carrying out mannan hydrolysis reactions involving polysaccharide substrates containing 1,4-β-D-mannosidic bonds. In general, under conditions suitable for carrying out mannan hydrolysis reactions, donor molecules are incubated in the presence of mannanase variants described herein, and then the products are optionally separated from the reaction. Alternatively, in the context of food, the product can become a component of food without separation. In certain embodiments, the donor molecule is a polysaccharide chain comprising mannose units, including but not limited to mannan, glucomannan, galactomannan and galactoglucomannan.

[0200] In one embodiment, one or more mannanase variants described herein are used in a method for extracting palm kernel oil. Another embodiment relates to a method for extracting palm kernel oil from palm kernel or palm kernel meal, the method comprising providing palm kernel and / or palm kernel meal and treating the seeds or cakes with one or more mannanase variants described herein.

[0201] In one embodiment, the compositions comprising the mannanase variants described herein are used to process and / or manufacture animal feed or food for humans. In yet another embodiment, the mannanase variants described herein can be an additive for feeding non-human animals. In another embodiment, the mannanase variants described herein can be used for human food, such as, for example, as an additive for human food.

[0202] Several nutritional factors may limit the amount of inexpensive plant material that can be used to prepare animal feed or food for humans. For example, plant materials containing oligomannans (such as mannans, galactomannans, glucomannans, and galactoglucomannans) can reduce the ability of animals to digest and absorb nutritional compounds (such as minerals, vitamins, sugars, and fats). Specifically, these negative effects are due to the high viscosity of the mannan-containing polymers and the ability of the mannan-containing polymers to absorb nutritional compounds. These effects can be reduced by including in the feed an enzyme that degrades the mannan-containing polymer (the endo-β-mannanase described herein), thereby making the proportion of mannan-containing polymers typically found in the inexpensive plant material that would be included in the feed higher, which ultimately reduces the cost of the feed. In addition, the mannanase variants described herein can break down the mannan-containing polymers into simpler sugars that can be more easily assimilated to provide additional energy.

[0203] In further embodiments, an animal feed containing plant material is incubated in the presence of a mannanase variant described herein under conditions suitable for breaking down mannan-containing polymers.

[0204] In another embodiment, a bread improver composition comprises a mannanase variant as described herein, optionally in combination with mannan or glucomannan or galactomannan, and further optionally in combination with one or more other enzymes.

[0205] The term "non-human animal" includes all non-ruminants and ruminants. In a specific embodiment, the non-ruminant is selected from the group consisting of, but not limited to: horses and monogastric animals, such as, but not limited to, pigs, poultry, swine, and fish. In other embodiments, pigs can be, but not limited to, piglets, growing pigs, and sows; poultry can be, but not limited to, turkeys, ducks, and chickens, which include, but are not limited to, broilers and laying hens; and fish can be, but are not limited to, salmon, trout, tilapia, catfish, and carp; and crustaceans, including, but not limited to, shrimps and prawns. In other embodiments, ruminants are selected from the group consisting of, but not limited to, cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns, and wildebeests.

[0206] In some embodiments, the mannanase variants described herein are used to pretreat feed rather than as a feed additive. In some embodiments, the mannanase variants described herein are added or used to pretreat feed for weaned pigs, nursery pigs, piglets, finishing pigs, growing pigs, finishing pigs, laying hens, broilers, and turkeys.

[0207] In another embodiment, the mannanase variants described herein are added or used to pretreat feed from plant materials such as palm kernel, coconut, konjac, locust bean gum, guar gum, soybean, barley, oats, flax, wheat, corn, linseed, citrus pulp, cottonseed, peanuts, rapeseed, sunflower, peas and lupins.

[0208] The mannanase variants described herein are thermostable, and thus the mannanase variants described herein can be used in a method of producing a pelleted feed, wherein heat is applied to the feed mixture prior to the pelleting step. In another embodiment, the mannanase variants described herein are added to other feed ingredients prior to the pelleting step or after the pelleting step (i.e., to an already formed feed pellet).

[0209] In yet another embodiment, the food processing or feed supplement composition containing the mannanase variants described herein may optionally further contain other substitutes selected from colorants, aroma compounds, stabilizers, vitamins, minerals and other feed or food enhancing enzymes. This is particularly applicable to so-called premixes.

[0210] In still further embodiments, the food additives according to the present invention may be combined in appropriate amounts with other food components, such as, for example, grains or plant proteins, to form processed food products.

[0211] In one embodiment, the animal feed composition and / or animal feed additive composition and / or pet food comprises a polypeptide described herein.

[0212] Another embodiment relates to a method for preparing an animal feed composition and / or an animal feed additive composition and / or a pet food, the method comprising mixing a mannanase variant described herein with one or more animal feed ingredients and / or animal feed additive ingredients and / or pet food ingredients.

[0213] Additional embodiments relate to the use of the mannanase variants described herein in the preparation of animal feed compositions and / or animal feed additive compositions and / or pet foods. The phrase "pet food" means food for domestic animals, such as, but not limited to, dogs; cats; gerbils; hamsters; chinchillas; fancy rats; guinea pigs; avian pets, such as canaries, parakeets and parrots; reptile pets, such as turtles, lizards and snakes; and aquatic pets, such as tropical fish and frogs.

[0214] The terms "animal feed composition", "feed" and "feedstock" are used interchangeably and may comprise one or more feed ingredients selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and / or large grains (e.g., maize or sorghum); b) by-products from cereals, such as corn gluten meal, Distillers Dried Grain Solubles (DDGS), particularly corn-based Distillers Dried Grain Solubles (cDDGS), wheat bran, semolina, wheat flour, rice bran, rice hulls, oat hulls, palm kernels and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupin, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, and sesame; d) oils and fats obtained from plant and animal sources; and e) minerals and vitamins.

[0215] In one aspect, the food composition or additive can be liquid or solid.In one aspect of the invention, the food composition is a beverage, including but not limited to fermented beverages (such as beer and wine).

[0216] In the context of the present invention, the term "fermented beverage" is intended to include any beverage produced by a process including a fermentation process, such as microbial fermentation (such as bacterial and / or yeast fermentation).

[0217] In one aspect of the invention, the fermented beverage is beer. The term "beer" is intended to include any fermented wort produced by fermenting / brewing starchy plant material. Typically, beer is produced from malt or as an adjunct ingredient of starchy plant material, or any combination of malt and adjunct ingredients. As used herein, the term "malt" is understood to be any germinated cereal grain, such as germinated barley or wheat.

[0218] As used herein, the term "adjunct ingredient" refers to any starch and / or sugar-containing plant material other than malt (e.g., barley or wheat malt). Examples of adjunct ingredients include, for example, common corn grits, refined corn grits, ground yeast for brewing, rice, sorghum, refined corn starch, barley, barley starch, hulled barley, wheat, wheat starch, baked cereals, cereal flakes, rye, oats, potatoes, cassava, tapioca, and syrups, such as corn syrup, cane syrup, invert syrup, barley and / or wheat syrup, etc., which can be used as a source of starch.

[0219] As used herein, the term "mash" refers to an aqueous slurry of any starch and / or sugar-containing plant material such as grist (e.g., including crushed barley malt, crushed barley) and / or other adjunct ingredients, or combinations thereof, mixed with water and subsequently separated into wort and spent grains.

[0220] As used herein, the term "wort" refers to the unfermented liquid run-off after grist is extracted during mashing.

[0221] In another aspect, the present invention relates to a method for preparing a fermented beverage (such as beer), which method comprises mixing the mannanase variant described herein with malt and / or adjunct ingredients.

[0222] Examples of beer include: all-malt beer, beer brewed according to the "Reinheitsgebot", ale, IPA, lager, bitter beer, sparkling wine (second beer), third beer, dry beer, thin beer, pale beer, low-alcohol beer, low-calorie beer, porter, dark beer, tout, malt liquor, non-alcoholic beer, non-alcoholic malt liquor, etc., and alternative cereal and malt beverages, such as fruit-flavored malt beverages, for example citrus-flavored malt beverages, such as lemon, orange, lime or berry-flavored malt beverages; alcohol-flavored malt beverages, such as vodka, rum or tequila-flavored malt liquor, or coffee-flavored malt beverages, such as coffee-flavored malt liquor, etc.

[0223] One aspect of the invention relates to the use of the mannanase variants described herein in the production of a fermented beverage, such as beer.

[0224] Another aspect relates to a method of providing a fermented beverage, the method comprising the step of contacting mash and / or wort with a mannanase variant as described herein.

[0225] A further aspect relates to a method for providing a fermented beverage, the method comprising the steps of: (a) preparing a mash, (b) filtering the mash to obtain wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer, wherein the mannanase variant described herein is added to: (i) the mash of step (a) and / or (ii) the wort of step (b) and / or (iii) the wort of step (c).

[0226] According to yet another aspect, a fermented beverage (such as beer) is produced or provided by a method comprising one or more of the following steps: (1) contacting mash and / or wort with a mannanase variant described herein; and / or (2) (a) preparing mash, (b) filtering the mash to obtain wort, and (c) fermenting the wort to obtain a fermented beverage, such as beer, wherein the mannanase variant described herein is added to: (i) the mash of step (a) and / or (ii) the wort of step (b) and / or (iii) the wort of step (c).

[0227] Generally, the coffee extract is incubated in the presence of the mannanase variants described herein under conditions suitable for hydrolyzing galactomannans present in the liquid coffee extract.

[0228] In another aspect, the present invention relates to a method for preparing a baked product, the method comprising adding the mannanase variants described herein to a dough, followed by baking the dough. Examples of baked products are well known to those skilled in the art, and include bread, rolls, puff pastry, sweet yeast dough, buns, cakes, crackers, biscuits, rolls, waffles, pancakes, tortillas, breakfast cereals, extruded products, and the like.

[0229] Mannanase variants as described herein can be added to dough as part of a bread improver composition. Bread improvers are compositions containing multiple ingredients that improve the quality of dough properties and baked products (e.g., bread and cakes). Bread improvers are usually added to industrial baking processes due to their beneficial effects (e.g., dough stability and bread texture and volume). Bread improvers usually contain fats and oils and additives, such as emulsifiers, enzymes, antioxidants, oxidants, stabilizers, and reducing agents. In addition to any polypeptide of the present invention, other enzymes that may also be present in bread improvers or that may be used together with any polypeptide of the present invention include amylases, hemicellulases, amylolytic complexes, lipases, proteases, xylanases, pectinases, pullulanases, non-starch polysaccharide degrading enzymes, and oxidoreductases (e.g., glucose oxidases), lipoxygenases, or ascorbic acid oxidases.

[0230] In one embodiment, the mannanase variants described herein can be added to the dough as part of a bread improver composition that also includes a source of glucomannan and / or galactomannan, such as konjac gum, guar gum, locust bean gum (Ceratonia siliqua), copra meal, ivory nut mannan (Phytelephas macrocarpa), seaweed mannan extract, coconut meal, and brewer's yeast cell walls (which can be dried or used in the form of brewer's yeast extract). Other acceptable mannan derivatives for use in the present invention include unbranched β-1,4-linked mannan homopolymers and mannooligosaccharides (mannobiose, mannotriose, mannotetrose, and mannopentose). The mannanase variants described herein can further be used alone or in combination with glucomannan and / or galactomannan and / or galactoglucomannan to improve dough tolerance; dough flexibility and / or dough stickiness; and / or crumb structure, and to delay bread staling. In another aspect, the mannanase hydrolysate is used as a soluble prebiotic (such as manno-oligosaccharides (MOS)), which promotes the growth of lactic acid bacteria, which are generally associated with good health, when found in favorable population densities in the colon.

[0231] In one aspect, the dough to which any of the polypeptides of the invention are added comprises bran or oat, rice, millet, maize or pulse flour in addition to or in place of pure wheat flour (ie, not a pure white flour dough).

[0232] In another embodiment, the mannanase variants described herein can be added to milk or any other dairy product to which glucomannan and / or galactomannan have been added. Typical sources of glucomannan and / or galactomannan are listed above in terms of baking, and include guar gum or konjac gum. The combination of the mannanase variants described herein with glucomannan and / or galactomannan releases mannanase hydrolysates (manno-oligosaccharides) that act as soluble prebiotics by promoting the selective growth and proliferation of probiotics (particularly Bifidobacteria and Lactobacillus lactic acid bacteria), which are generally associated with good health when found in the large intestine or colon at favorable population densities.

[0233] Another aspect relates to a method for preparing milk or a milk product, the method comprising adding a mannanase variant as described herein and any glucomannan or galactomannan or galactoglucomannan.

[0234] In another aspect, the mannanase variants described herein are used in combination with any glucomannan or galactomannan before or after addition to a dairy-based food to produce a dairy-based food containing a prebiotic mannan hydrolysate. In a further aspect, the manno-oligosaccharide-containing dairy product so produced can increase the beneficial human intestinal microflora population, and in yet another aspect, a dairy-based food may contain the mannanase variants described herein together with any source of glucomannan and / or galactomannan and / or galactoglucomannan, and in a dosage sufficient for inoculation with at least one bacterial strain known to be beneficial in the human large intestine (such as bifidobacteria or lactobacilli). In one aspect, the dairy-based food is a yogurt or a milk drink.

[0235] The mannanase variants described herein can further be used for enzyme-assisted bleaching of pulp (e.g., chemical pulp, semi-chemical pulp, kraft pulp, mechanical pulp, and pulp prepared by the sulfite process). Generally, the pulp is incubated with one or more mannanase variants described herein under conditions suitable for bleaching the pulp.

[0236] In some embodiments, the pulp is chlorine-free pulp, and the pulp is bleached with oxygen, ozone, hydrogen peroxide or peroxyacid. In some embodiments, one or more mannanase variants described herein are used for enzyme-assisted bleaching of pulp exhibiting low lignin content, and the pulp is produced by a modified pulping process or a continuous pulping process. In some other embodiments, one or more mannanase variants described herein are used alone or preferably in combination with xylanase and / or endoglucanase and / or alpha-galactosidase and / or cellobiohydrolase.

[0237] Galactomannans (such as guar gum and locust bean gum) are widely used as thickeners in foods (e.g., ice cream) and for printing pastes for textile printing (e.g., printing on T-shirts). Therefore, the mannanase variants described herein can also be used to reduce the consistency or viscosity of mannan-containing substrates. In some embodiments, one or more mannanase variants described herein are used to hydrolyze galactomannans in waste streams of food (e.g., ice cream) manufacturing. In certain embodiments, the mannanase variants described herein are used to reduce the viscosity of residual food in processing equipment, thereby promoting cleaning after processing. In certain other embodiments, the mannanase variants described herein are used to reduce the viscosity of printing pastes, thereby helping to wash off excess printing pastes after textile printing. In general, a substrate containing mannan is incubated with a mannanase variant described herein under conditions suitable for reducing the viscosity of the substrate containing mannan.

[0238] In yet further embodiments, one or more of the mannanase variants described herein can be used in the oil and gas industry for, e.g., controlling the viscosity of drilling fluids; increasing the rate at which fluids used in hydraulic fracturing create subsurface fractures extending from a borehole into the rock; cleaning borehole filter cakes; and combinations thereof.

[0239] Other aspects and embodiments of the compositions and methods of the present invention will be apparent from the foregoing description and the following examples. Various alternative embodiments beyond the embodiments described herein may be employed in practicing the present invention without departing from the spirit and scope of the present invention. Therefore, the claims (rather than the specific embodiments described herein) define the scope of the present invention, and therefore the scope of the present invention covers methods and structures within the scope of the claims and their equivalents.

[0240] Non-limiting examples of the compositions and methods disclosed herein are as follows:

[0241] 1. A mannanase variant comprising an amino acid substitution at a position selected from the group consisting of 32, 72, 161 and 172 compared to a parent mannanase, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of the parent enzyme of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0242] 2. A mannanase variant comprising amino acid substitutions, wherein the variant comprises substitutions selected from the group consisting of: 19D, 32Y, 34D, 72V, 93Q, 131S, 136P, 139R, 161G, 172F, 225N / Q, 259D, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0243] 2b A mannanase variant comprising amino acid substitutions, wherein the variant comprises substitutions selected from the group consisting of: P019D, F032Y, N034D, I072V, K093Q, T131S, A136P, D139R, A161G, V172F, G225N / Q, G259D, N261D / E and F276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0244] 2c A mannanase variant comprising amino acid substitutions, wherein the variant comprises substitutions selected from the group consisting of: E019D, F032Y, N034D, I072V, K093Q, T131S, A136P, D139R, A161G, V172F, C225N / Q, G259D, R261D / E and F276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:2, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO:2.

[0245] 3. A mannanase variant comprising two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of: 19D, 32Y, 72V, 93Q, 131S, 136P, 139R, 161G, 168T, 172F, 225N / Q, 259D / E, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0246] 3a. A mannanase variant comprising two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of: P019D, F032Y, I072V, K093Q, T131S, A136P, D139R, A161G, P168T, V172F, G225N / Q, G259D / E, N261D / E and F276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0247] 3b. A mannanase variant comprising two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of: E019D, F032Y, I072V, K093Q, T131S, A136P, D139R, A161G, P168T, V172F, C225N / Q, G259D / E, R261D / E and F276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO:2, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO:2.

[0248] 4. The mannanase variant of embodiment 3, wherein the variant comprises an amino acid substitution selected from the group consisting of 19D-276W, 32Y-259D, 93Q-276W, 131S-276W, 136P-276W, 139R-276W, 161G-276W, 225N / Q-276W, 259D / E-276W and 261D / E-276W.

[0249] 4a. The mannanase variant of embodiment 3b, wherein the variant comprises an amino acid substitution selected from the group consisting of E019D-F276W, F032Y-G259D, K093Q-F276W, T131S-F276W, A136P-F276W, D139R-F276W, A161G-F276W, C225N-F276W, C225Q-F276W, G259D-F276W, G259E-F276W, R261D-F276W and R261E-F276W.

[0250] 5. The mannanase variant of embodiment 3, wherein the variant comprises an amino acid substitution selected from the group consisting of 19D-131S-276W, 32Y-261D-276W, 32Y-259D-276W, 32Y-172F-259D, 168T-259D-276W, 259D-261E-276W, and 259Q-261E-276W.

[0251] 5b. The mannanase variant of embodiment 3b, wherein the variant comprises an amino acid substitution selected from the group consisting of E019D-A068S-F276W, E019D-T131S-F276W, F032Y-R261D-F276W, F032Y-G259D-F276W, F032Y-T062E-G259D, F032Y-V172F-G259D, P168A-G259D-F276W, P168T-G259D-F276W, G259D-R261E-F276W and G259Q-R261E-F276W.

[0252] 6. A mannanase variant comprising an amino acid substitution selected from the group consisting of: Y061W-G259D-R261E-F276W, Y061W-T062E-G259D-R261E-F276W, Y061W-V228T-G259D-R261E-F276W, V228T-G259D-R261E-F276W, F032Y-G259D-R261E-F276W, F032Y-Y061W-Y167F-P168S-G259D-R261E-F276W, F032Y-Y061W-G259D-R261E-F276W , F032Y-Y061W-T062E-G259D-R261E-F276W, F032Y-T062E-R261D-F276W , F032Y-T062E-G259D-F276W, T062E-G259D-R261E-F276W, V059S-Q060L -G259D-R261E-F276W, V059S-Q060L-Y061W-G259D-R261E-F276W, V059S -Q060L-T062E-G259D-R261E-F276W, T062E-V228T-G259D-R261E-F276W , F032Y-T062E-G259D-R261E-F276W, F032Y-T062E-G259D-R261E-F276W -T284E, E019D-A068S-T131S-F276W, F032Y-T062E-G259D-F276W-T284E , F032Y-T062E-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-G259D -F276W-T284E, F032Y-T062E-A068S-G259D-F276W-T284E, F032Y-Y061W -T062E-G259D-F276W-T284E, F032Y-V059S-Q060L-T062E-G259D-F276W -T284E, N010T-F032Y-T062E-G259D-F276W-T284E, F032Y-T062E-Y167F -P168S-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-V228T-G259D -F276W-T284E, F032Y-T062E-I072V-Y167F-P168S-G259D-F276W-T284E,F032Y-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-V228T-G259D-F276W-T284E、F032Y-V059S、Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-G259D-F276W-T284E、F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E and N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E, F032Y-Y061W-T062E-G259D-R261E-F276W-T284E, F032Y-T062E-A068S-G259D-R261E-F276W-T284 E. F032Y-T062E-T131S-G259D-R261E-F276W-T284E, F032Y-T062E-N150D -G259D-R261E-F276W-T284E, F032Y-T062E-P168S-G259D-R261E-F276W- T284E, F032Y-T062E-L235K-G259D-R261E-F276W-T284E, N010T-F032Y-Y 061W-T062E-P168A-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T06 2E-Y167F-P168S-V228T-G259E-F276W-T284E, F032Y-Y061W-T062E-T131 S-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T062E -A136P-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W- T062E-K093Q-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y 061W-T062E-Y167F-P168S-V228T-L235K-G259D-F276W-T284E, N010T-F0 32Y-Y061W-T062E-D139R-Y167F-P168S-V228T-G259D-F276W-T284E, N01 0T-F032Y-Y061W-T062E-N150D-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-Y167F-P168A-V228T-G259D-F276W-T284E,N010T-F032Y-Y061W-T062E-T131S-Y167F-P168S-V228T-G259D-F276W-T284E , N010T-F032Y-Y061W-T062E-P168S-V228T-G259D-F276W-T284E, N010T-F032 Y-Y061W-T062E-A068S-Y167F-P168A-G259D-F276W-T284E, and N010T-F032Y-Y061W-T062E-A068S-Y167F-P168A-G259E-F276W-T284E, wherein the amino acid positions of the variants are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variants have at least 80% identity with the amino acid sequence of SEQ ID NO: 1.

[0253] 7. The mannanase variant of embodiments 1-3, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0254] 7a. The mannanase variant of any preceding embodiment, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0255] 8. The mannanase variant of embodiments 1-3, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:1.

[0256] 8a. The mannanase variant of any preceding embodiment, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6.

[0257] 9. The mannanase variant of embodiments 1-3, wherein the variant has improved stability when compared to a parent mannanase or a reference mannanase.

[0258] 9a. The mannanase variant of any preceding embodiment, wherein the variant has improved stability when compared to a parent mannanase or a reference mannanase.

[0259] 10. The mannanase of embodiment 9, wherein the variant has equal or improved cleaning performance in detergent when compared to a parent mannanase or a reference mannanase.

[0260] 10. The mannanase of embodiment 9, wherein the variant has equal or improved cleaning performance in detergent when compared to a parent mannanase or a reference mannanase.

[0261] 10b. The mannanase of any preceding embodiment, wherein the variant has equal or improved cleaning performance in detergent when compared to a parent mannanase or a reference mannanase.

[0262] 11. The mannanase variant of embodiments 1-3, wherein the mannanase variant has mannanase activity.

[0263] 11a. The mannanase variant of any preceding embodiment, wherein the mannanase variant has mannanase activity.

[0264] 11b. The mannanase variant of any preceding embodiment, wherein the mannanase activity is in the presence of a surfactant and / or a protease.

[0265] 12. A polynucleotide comprising a nucleic acid sequence encoding a variant as described in any one of embodiments 1-3, wherein the polynucleotide is optionally isolated.

[0266] 12a. A polynucleotide comprising a nucleic acid sequence encoding a variant as described in any preceding embodiment, wherein the polynucleotide is optionally isolated.

[0267] 12b. The polynucleotide of embodiment 12 or 12a, wherein the nucleic acid sequence is operably linked to a promoter.

[0268] 12c. An expression vector or expression cassette comprising the polynucleotide of embodiment 12 or 12a or 12b.

[0269] 12d. A recombinant host cell comprising the polynucleotide of embodiment 12 or 12a or 12b or the vector or cassette of embodiment 12d.

[0270] 13. An enzyme composition comprising one or more mannanase variants as described in embodiments 1-3.

[0271] 13a. An enzyme composition comprising one or more mannanase variants according to any preceding embodiment.

[0272] 14. The enzyme composition of embodiment 13 or 13a, wherein the composition is an enzyme granule.

[0273] 15. The enzyme composition of any one of embodiments 13, 13a or 14, further comprising one or more additional enzymes selected from the group consisting of acyltransferases, amylases, α-amylases, β-amylases, α-galactosidases, arabinanases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, DNAse (also known as nuclease or dispersin), endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase 、 Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, PETase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthan lyase, xanthanase, xylan acetylesterase, xylanase, xylosidase, and combinations or mixtures thereof.

[0274] 16. A cleaning composition comprising the mannanase variant of any one of embodiments 1-3.

[0275] 16a. A cleaning composition comprising the mannanase variant of any one of embodiments 1-11.

[0276] 16b. The cleaning composition of embodiment 16, further comprising: at least one surfactant; at least one ion selected from calcium and zinc; at least one auxiliary ingredient; at least one stabilizer; from about 0.001 wt. % to about 1.0 wt. % of the mannanase variant of any one of embodiments 1-11; at least one bleaching agent; and / or at least one enzyme or enzyme derivative selected from the group consisting of: acyltransferase, amylase, α-amylase, β-amylase, α-galactosidase, arabinanase, arabinosidase, arylesterase, β-galactosidase, β-glucanase, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, DNA enzyme (also known as nuclease or dispersin), endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase 、 Hyaluronidase, keratinase, laccase, lactase, ligninase, lipase, lipolytic enzyme, lipoxygenase, lysozyme, mannanase, metalloproteinase, nuclease, oxidase, oxidoreductase, pectate lyase, pectin acetylesterase, pectinase, pentosanase, perhydrolase, peroxidase, PETase, phenoloxidase, phosphatase, phospholipase, phosphodiesterase, phytase, polyesterase, polygalacturonase, protease, pullulanase, reductase, rhamnogalacturonase, tannase, transglutaminase, xanthan lyase, xanthanase, xylan acetylesterase, xylanase, xylosidase, and combinations or mixtures thereof.

[0277] 17. The cleaning composition of embodiment 16, wherein the cleaning composition is a detergent composition selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, a medical device cleaning detergent, and a hard surface cleaning detergent.

[0278] 17a. The cleaning composition of any of embodiments 16-17, wherein the cleaning composition is in a form selected from the group consisting of a liquid, a powder, a granular solid, a tablet, a flake, and a unit dose.

[0279] 17b. The cleaning composition of any one of embodiments 16-17 or embodiment 17a, wherein the composition contains phosphate or is free of phosphate and / or contains boron or is free of boron.

[0280] 18. A method of cleaning, comprising contacting a surface or item in need of cleaning with an effective amount of a mannanase variant as described in any one of Examples 1-3 or an enzyme composition as described in any one of Examples 13-15; and optionally further comprising the step of rinsing the surface or item after contacting the surface or item with the variant or enzyme composition.

[0281] 19. The method of embodiment 18, wherein the article is a medical device, tableware, or fabric.

[0282] 20. A method for producing the mannanase variant of any one of embodiments 1-3, the method comprising: (a) stably transforming the host cell of embodiment 12c with the expression vector of embodiment 12b; (b) culturing the transformed host cell under conditions suitable for the host cell to produce the mannanase variant or recombinant polypeptide or an active fragment thereof; and (c) recovering the mannanase variant or recombinant polypeptide or an active fragment thereof.

[0283] 21. A food or feed composition and / or a food additive, comprising the mannanase variant or recombinant polypeptide according to any one of embodiments 1-3.

[0284] 22. Use of the mannanase variant or recombinant polypeptide according to any one of embodiments 1 to 3 in the preparation of a food or feed composition and / or a food or feed additive and / or a food or feed raw material and / or a pet food.

[0285] 23. A mannanase variant comprising an amino acid substitution selected from the group consisting of: X0136P-X019D, X0136P-X0225N, X0136P-X032Y, X0136P-X072V, X0136P-X093Q, X0136P-X131S, X0136P-X139R, X0136P-X161G, X0136P-X168T, X0136P-X172F, X0136P-X225Q, X0136P-X259D, X0136P-X259E, X0136P-X261D, X0136P-X261E, X0136P-X276W, X0136P-X277 019D-X0225N, X019D-X032Y, X019D-X072V, X019D-X093Q, X019D-X131S, 19D-X139R, X019D-X161G, X019D-X168T, X019D-X172F, X019D-X225Q, D-X259D, X019D-X259E, X019D-X261D, X019D-X261E, X019D-X276W, -X032Y, X0225N-X072V, X0225N-X093Q, X0225N-X131S, 25N-X161G, X0225N-X168T, X0225N-X172F, X0225N-X261D, X0225N-X261E, X0225N-X276W, X032Y-X072V, X032Y-X093Q , X032Y-X131S, X032Y-X139R, X032Y-X161G, 032Y-X225Q, X032Y-X259D, X032Y-X259E, X032Y-X261D, X032Y-X261E, 2Y-X276W, X072V-X093Q, X072V-X131S, X072V-X139R, X072V-X161G, -X168T, X072V-X172F, X072V-X225Q, X072V-X259D, X072V-X259E, 261D, X072V-X261E, X072V-X276W, X093Q-X131S, X093Q-X139R, 1G, X093Q-X168T, X093Q-X172F, X093Q-X225Q,X093Q-X261D, X093Q-X261E, X093Q-X276W, X131S-X139R, X131S-X225Q, X131S-X259D, X131S-X259E, X131S-X261D, X139R-X168T, X139R-X172F, X139R-X225Q, X139R-X259D, X139R-X276W, X161G-X168T, X161G-X172F, X161G-X225Q, X161G-X261E, X161G-X276W, X168T-X172F, X168T-X225Q, X168T-X261E, X168T-X276W, X172F-X225Q, X172F-X259D, X172F-X276W, X225Q-X259D, X225Q-X259E, X225Q-X261D, X225Q-X261E, X225Q-X276W, X259D-X261D, X259D-X261E, X259D-X276W, X259E-X261D, X259E-X261E, X259E-X276W, X261D-X276W and X261E-X276W.

[0286] 24. A mannanase variant comprising an amino acid substitution selected from the group consisting of: X0136P-X019D-X0225N, X0136P-X019D-X032Y, X0136P-X019D-X072V, X0136P-X019D-X093Q, X0136P-X019D-X131S, X0136P-X019D-X139R, X0136P-X019D-X161G, X0136P-X019D-X168T, X0136P-X019D-X172F, X0136P-X019D-X225Q, X0136P-X019D-X25 9D, X0136P-X019D-X259E, X0136P-X019D-X261D, 0136P-X019D-X276W, X0136P-X0225N-X032Y, X0136P-X0225N-X072V, X013 6P-X0225N-X093Q, X0136P-X0225N-X131S, X0136P-X0225N-X139R, X0136 P-X0225N-X161G, X0136P-X0225N-X168T, X0136P-X0225N-X172F, X0225N-X259D, X0136P-X0225N-X259E, X0136P-X0225N-X261D, X0136P-X 0225N-X261E, X0136P-X0225N-X276W, X0136P-X032Y-X072V, Y-X093Q, X0136P-X032Y-X131S, X0136P-X032Y-X139R, X0136P-X032Y-X1 61G, X0136P-X032Y-X168T, X0136P-X032Y-X172F, 0136P-X032Y-X259D, X0136P-X032Y-X259E, X0136P-X032Y-X261D, X0136 P-X032Y-X261E, X0136P-X032Y-X276W, X0136P-X072V-X093Q, X0136P-X07 2V-X131S, X0136P-X072V-X139R, X0136P-X072V-X161G, 168T, X0136P-X072V-X172F,X0136P-X072V-X259E、X0136P-X072V-X261D、X0136P-X072V-X261E、X0136P-X072V-X276W、X0136P-X093Q-X131S、X0136P-X093Q-X139R、X0136P-X093Q-X161G、X0136P-X093Q-X168T、X0136P-X093Q-X172F、X0136P-X093Q-X225Q、X0136P-X093Q-X259D、X0136P-X093Q-X259E、X0136P-X093Q-X261D、X0136P-X093Q-X261E、X0136P-X093Q-X276W、X0136P-X131S-X139R、X0136P-X131S-X161G、X0136P-X131S-X168T、X0136P-X131S-X172F、X0136P-X131S-X225Q、X0136P-X131S-X259D、X0136P-X131S-X259E、X0136P-X131S-X261D、X0136P-X131S-X261E、X0136P-X131S-X276W、X0136P-X139R-X161G、X0136P-X139R-X168T、X0136P-X139R-X172F、X0136P-X139R-X225Q、X0136P-X139R-X259D、X0136P-X139R-X259E、X0136P-X139R-X261D、X0136P-X139R-X261E、X0136P-X139R-X276W、X0136P-X161G-X168T、X0136P-X161G-X172F、X0136P-X161G-X225Q、X0136P-X161G-X259D、X0136P-X161G-X259E、X0136P-X161G-X261D、X0136P-X161G-X261E、X0136P-X161G-X276W、X0136P-X168T-X172F、X0136P-X168T-X225Q、X0136P-X168T-X259D、X0136P-X168T-X259E、X0136P-X168T-X261D、X0136P-X168T-X261E、X0136P-X168T-X276W、X0136P-X172F-X225Q、X0136P-X172F-X259D、X0136P-X172F-X259E、X0136P-X172F-X261D、X0136P-X172F-X261E、X0136P-X172F-X276W、X0136P-X225Q-X259D、X0136P-X225Q-X259E、X0136P-X225Q-X261D、X0136P-X225Q-X261E、X0136P-X225Q-X276W、X0136P-X259D-X261D、X0136P-X259D-X261E、X0136P-X259D-X276W、X0136P-X259E-X261D、X0136P-X259E-X261E、X0136P-X259E-X276W、X0136P-X261D-X276W、X0136P-X261E-X276W、X019D-X0225N-X032Y、X019D-X0225N-X072V、X019D-X0225N-X093Q、X019D-X0225N-X131S、X019D-X0225N-X139R、X019D-X0225N-X161G、X019D-X0225N-X168T、X019D-X0225N-X172F、X019D-X0225N-X259D、X019D-X0225N-X259E、X019D-X0225N-X261D、X019D-X0225N-X261E、X019D-X0225N-X276W、X019D-X032Y-X072V、X019D-X032Y-X093Q、X019D-X032Y-X131S、X019D-X032Y-X139R、X019D-X032Y-X161G、X019D-X032Y-X168T、X019D-X032Y-X172F、X019D-X032Y-X225Q、X019D-X032Y-X259D、X019D-X032Y-X259E、X019D-X032Y-X261D、X019D-X032Y-X261E、X019D-X032Y-X276W、X019D-X072V-X093Q、X019D-X072V-X131S、X019D-X072V-X139R、X019D-X072V-X161G、X019D-X072V-X168T、X019D-X072V-X172F、X019D-X072V-X225Q、X019D-X072V-X259D、X019D-X072V-X259E、X019D-X072V-X261D、X019D-X072V-X261E、X019D-X072V-X276W、X019D-X093Q-X131S、X019D-X093Q-X139R、X019D-X093Q-X161G、X019D-X093Q-X168T、X019D-X093Q-X172F、X019D-X093Q-X225Q、X019D-X093Q-X259D、X019D-X093Q-X259E、X019D-X093Q-X261D、X019D-X093Q-X261E、X019D-X093Q-X276W、X019D-X131S-X139R、X019D-X131S-X161G、X019D-X131S-X168T、X019D-X131S-X172F、X019D-X131S-X225Q、X019D-X131S-X259D、X019D-X131S-X259E、X019D-X131S-X261D、X019D-X131S-X261E、X019D-X131S-X276W、X019D-X139R-X161G、X019D-X139R-X168T、X019D-X139R-X172F、X019D-X139R-X225Q、X019D-X139R-X259D、X019D-X139R-X259E、X019D-X139R-X261D、X019D-X139R-X261E、X019D-X139R-X276W、X019D-X161G-X168T、X019D-X161G-X172F、X019D-X161G-X225Q、X019D-X161G-X259D、X019D-X161G-X259E、X019D-X161G-X261D、X019D-X161G-X261E、X019D-X161G-X276W、X019D-X168T-X172F、X019D-X168T-X225Q、X019D-X168T-X259D、X019D-X168T-X259E、X019D-X168T-X261D、X019D-X168T-X261E、X019D-X168T-X276W、X019D-X172F-X225Q、X019D-X172F-X259D、X019D-X172F-X259E、X019D-X172F-X261D、X019D-X172F-X261E、X019D-X172F-X276W、X019D-X225Q-X259D、X019D-X225Q-X259E、X019D-X225Q-X261D、X019D-X225Q-X261E、X019D-X225Q-X276W、X019D-X259D-X261D、X019D-X259D-X261E、X019D-X259D-X276W、X019D-X259E-X261D、X019D-X259E-X261E、X019D-X259E-X276W、X019D-X261D-X276W、X019D-X261E-X276W、X0225N-X032Y-X072V、X0225N-X032Y-X093Q、X0225N-X032Y-X131S、X0225N-X032Y-X139R、X0225N-X032Y-X161G、X0225N-X032Y-X168T、X0225N-X032Y-X172F、X0225N-X032Y-X259D、X0225N-X032Y-X259E、X0225N-X032Y-X261D、X0225N-X032Y-X261E、X0225N-X032Y-X276W、X0225N-X072V-X093Q、X0225N-X072V-X131S、X0225N-X072V-X139R、X0225N-X072V-X161G、X0225N-X072V-X168T、X0225N-X072V-X172F、X0225N-X072V-X259D、X0225N-X072V-X259E、X0225N-X072V-X261D、X0225N-X072V-X261E、X0225N-X072V-X276W、X0225N-X093Q-X131S、X0225N-X093Q-X139R、X0225N-X093Q-X161G、X0225N-X093Q-X168T、X0225N-X093Q-X172F、X0225N-X093Q-X259D、X0225N-X093Q-X259E、X0225N-X093Q-X261D、X0225N-X093Q-X261E、X0225N-X093Q-X276W、X0225N-X131S-X139R、X0225N-X131S-X161G、X0225N-X131S-X168T、X0225N-X131S-X172F、X0225N-X131S-X259D、X0225N-X131S-X259E、X0225N-X131S-X261D、X0225N-X131S-X261E、X0225N-X131S-X276W、X0225N-X139R-X161G、X0225N-X139R-X168T、X0225N-X139R-X172F、X0225N-X139R-X259D、X0225N-X139R-X259E、X0225N-X139R-X261D、X0225N-X139R-X261E、X0225N-X139R-X276W、X0225N-X161G-X168T、X0225N-X161G-X172F、X0225N-X161G-X259D、X0225N-X161G-X259E、X0225N-X161G-X261D、X0225N-X161G-X261E、X0225N-X161G-X276W、X0225N-X168T-X172F、X0225N-X168T-X259D、X0225N-X168T-X259E、X0225N-X168T-X261D、X0225N-X168T-X261E、X0225N-X168T-X276W、X0225N-X172F-X259D、X0225N-X172F-X259E、X0225N-X172F-X261D、X0225N-X172F-X261E、X0225N-X172F-X276W、X0225N-X259D-X261D、X0225N-X259D-X261E、X0225N-X259D-X276W、X0225N-X259E-X261D、X0225N-X259E-X261E、X0225N-X259E-X276W、X0225N-X261D-X276W、X0225N-X261E-X276W、X032Y-X072V-X093Q、X032Y-X072V-X131S、X032Y-X072V-X139R、X032Y-X072V-X161G、X032Y-X072V-X168T、X032Y-X072V-X172F、X032Y-X072V-X225Q、X032Y-X072V-X259D、X032Y-X072V-X259E、X032Y-X072V-X261D、X032Y-X072V-X261E、X032Y-X072V-X276W、X032Y-X093Q-X131S、X032Y-X093Q-X139R、X032Y-X093Q-X161G、X032Y-X093Q-X168T、X032Y-X093Q-X172F、X032Y-X093Q-X225Q、X032Y-X093Q-X259D、X032Y-X093Q-X259E、X032Y-X093Q-X261D、X032Y-X093Q-X261E、X032Y-X093Q-X276W、X032Y-X131S-X139R、X032Y-X131S-X161G、X032Y-X131S-X168T、X032Y-X131S-X172F、X032Y-X131S-X225Q、X032Y-X131S-X259D、X032Y-X131S-X259E、X032Y-X131S-X261D、X032Y-X131S-X261E、X032Y-X131S-X276W、X032Y-X139R-X161G、X032Y-X139R-X168T、X032Y-X139R-X172F、X032Y-X139R-X225Q、X032Y-X139R-X259D、X032Y-X139R-X259E、X032Y-X139R-X261D、X032Y-X139R-X261E、X032Y-X139R-X276W、X032Y-X161G-X168T、X032Y-X161G-X172F、X032Y-X161G-X225Q、X032Y-X161G-X259D、X032Y-X161G-X259E、X032Y-X161G-X261D、X032Y-X161G-X261E、X032Y-X161G-X276W、X032Y-X168T-X172F、X032Y-X168T-X225Q、X032Y-X168T-X259D、X032Y-X168T-X259E、X032Y-X168T-X261D、X032Y-X168T-X261E、X032Y-X168T-X276W、X032Y-X172F-X225Q、X032Y-X172F-X259D、X032Y-X172F-X259E、X032Y-X172F-X261D、X032Y-X172F-X261E、X032Y-X172F-X276W、X032Y-X225Q-X259D、X032Y-X225Q-X259E、X032Y-X225Q-X261D、X032Y-X225Q-X261E、X032Y-X225Q-X276W、X032Y-X259D-X261D、X032Y-X259D-X261E、X032Y-X259D-X276W、X032Y-X259E-X261D、X032Y-X259E-X261E、X032Y-X259E-X276W、X032Y-X261D-X276W、X032Y-X261E-X276W、X072V-X093Q-X131S、X072V-X093Q-X139R、X072V-X093Q-X161G、X072V-X093Q-X168T、X072V-X093Q-X172F、X072V-X093Q-X225Q、X072V-X093Q-X259D、X072V-X093Q-X259E、X072V-X093Q-X261D、X072V-X093Q-X261E、X072V-X093Q-X276W、X072V-X131S-X139R、X072V-X131S-X161G、X072V-X131S-X168T、X072V-X131S-X172F、X072V-X131S-X225Q、X072V-X131S-X259D、X072V-X131S-X259E、X072V-X131S-X261D、X072V-X131S-X261E、X072V-X131S-X276W、X072V-X139R-X161G、X072V-X139R-X168T、X072V-X139R-X172F、X072V-X139R-X225Q、X072V-X139R-X259D、X072V-X139R-X259E、X072V-X139R-X261D、X072V-X139R-X261E、X072V-X139R-X276W、X072V-X161G-X168T、X072V-X161G-X172F、X072V-X161G-X225Q、X072V-X161G-X259D、X072V-X161G-X259E、X072V-X161G-X261D、X072V-X161G-X261E、X072V-X161G-X276W、X072V-X168T-X172F、X072V-X168T-X225Q、X072V-X168T-X259D、X072V-X168T-X259E、X072V-X168T-X261D、X072V-X168T-X261E、X072V-X168T-X276W、X072V-X172F-X225Q、X072V-X172F-X259D、X072V-X172F-X259E、X072V-X172F-X261D、X072V-X172F-X261E、X072V-X172F-X276W、X072V-X225Q-X259D、X072V-X225Q-X259E、X072V-X225Q-X261D、X072V-X225Q-X261E、X072V-X225Q-X276W、X072V-X259D-X261D、X072V-X259D-X261E、X072V-X259D-X276W、X072V-X259E-X261D、X072V-X259E-X261E、X072V-X259E-X276W、X072V-X261D-X276W、X072V-X261E-X276W、X093Q-X131S-X139R、X093Q-X131S-X161G、X093Q-X131S-X168T、X093Q-X131S-X172F、X093Q-X131S-X225Q、X093Q-X131S-X259D、X093Q-X131S-X259E、X093Q-X131S-X261D、X093Q-X131S-X261E、X093Q-X131S-X276W、X093Q-X139R-X161G、X093Q-X139R-X168T、X093Q-X139R-X172F、X093Q-X139R-X225Q、X093Q-X139R-X259D、X093Q-X139R-X259E、X093Q-X139R-X261D、X093Q-X139R-X261E、X093Q-X139R-X276W、X093Q-X161G-X168T、X093Q-X161G-X172F、X093Q-X161G-X225Q、X093Q-X161G-X259D、X093Q-X161G-X259E、X093Q-X161G-X261D、X093Q-X161G-X261E、X093Q-X161G-X276W、X093Q-X168T-X172F、X093Q-X168T-X225Q、X093Q-X168T-X259D、X093Q-X168T-X259E、X093Q-X168T-X261D、X093Q-X168T-X261E、X093Q-X168T-X276W、X093Q-X172F-X225Q、X093Q-X172F-X259D、X093Q-X172F-X259E、X093Q-X172F-X261D、X093Q-X172F-X261E、X093Q-X172F-X276W、X093Q-X225Q-X259D、X093Q-X225Q-X259E、X093Q-X225Q-X261D、X093Q-X225Q-X261E、X093Q-X225Q-X276W、X093Q-X259D-X259E、X093Q-X259D-X261D、X093Q-X259D-X261E、X093Q-X259D-X276W、X093Q-X259E-X261D、X093Q-X259E-X261E、X093Q-X259E-X276W、X093Q-X261D-X276W、X093Q-X261E-X276W、X131S-X139R-X161G、X131S-X139R-X168T、X131S-X139R-X172F、X131S-X139R-X225Q、X131S-X139R-X259D、X131S-X139R-X259E、X131S-X139R-X261D、X131S-X139R-X261E、X131S-X139R-X276W、X131S-X161G-X168T、X131S-X161G-X172F、X131S-X161G-X225Q、X131S-X161G-X259D、X131S-X161G-X259E、X131S-X161G-X261D、X131S-X161G-X261E、X131S-X161G-X276W、X131S-X168T-X172F、X131S-X168T-X225Q、X131S-X168T-X259D、X131S-X168T-X259E、X131S-X168T-X261D、X131S-X168T-X261E、X131S-X168T-X276W、X131S-X172F-X225Q、X131S-X172F-X259D、X131S-X172F-X259E、X131S-X172F-X261D、X131S-X172F-X261E、X131S-X172F-X276W、X131S-X225Q-X259D、X131S-X225Q-X259E、X131S-X225Q-X261D、X131S-X225Q-X261E、X131S-X225Q-X276W、X131S-X259D-X261D、X131S-X259D-X261E、X131S-X259D-X276W、X131S-X259E-X261D、X131S-X259E-X261E、X131S-X259E-X276W、X131S-X261D-X276W、X131S-X261E-X276W、X139R-X161G-X168T、X139R-X161G-X172F、X139R-X161G-X225Q、X139R-X161G-X259D、X139R-X161G-X259E、X139R-X161G-X261D、X139R-X161G-X261E、X139R-X161G-X276W、X139R-X168T-X172F、X139R-X168T-X225Q、X139R-X168T-X259D、X139R-X168T-X259E、X139R-X168T-X261D、X139R-X168T-X261E、X139R-X168T-X276W、X139R-X172F-X225Q、X139R-X172F-X259D、X139R-X172F-X259E、X139R-X172F-X261D、X139R-X172F-X261E、X139R-X172F-X276W、X139R-X225Q-X259D、X139R-X225Q-X259E、X139R-X225Q-X261D、X139R-X225Q-X261E、X139R-X225Q-X276W、X139R-X259D-X261D、X139R-X259D-X261E、X139R-X259D-X276W、X139R-X259E-X261D、X139R-X259E-X261E、X139R-X259E-X276W、X139R-X261D-X276W、X139R-X261E-X276W、X161G-X168T-X172F、X161G-X168T-X225Q、X161G-X168T-X259D、X161G-X168T-X259E、X161G-X168T-X261D、X161G-X168T-X261E、X161G-X168T-X276W、X161G-X172F-X225Q、X161G-X172F-X259D、X161G-X172F-X259E、X161G-X172F-X261D、X161G-X172F-X261E、X161G-X172F-X276W、X161G-X225Q-X259D、X161G-X225Q-X259E、X161G-X225Q-X261D、X161G-X225Q-X261E、X161G-X225Q-X276W、X161G-X259D-X261D、X161G-X259D-X261E、X161G-X259D-X276W、X161G-X259E-X261D、X161G-X259E-X261E、X161G-X259E-X276W、X161G-X261D-X276W、X161G-X261E-X276W, X168T-X172F-X225Q, X168T-X172F-X259D, X168T-X172F-X259E, X168T-X172F-X261D, X168T-X172F-X261E, X168T-X172F-X276W, X168T-X225Q-X259D, X168T-X225Q-X259E, X168T-X225Q-X261D, X168T-X225Q-X261E, X168T-X225Q-X276W, X168T-X259D-X261D, X168T-X259D-X261E, X168T-X259D-X276W, X168T-X259E-X261D, X168T-X259E-X261E, X168T-X259E-X276W, X168T-X261D-X276W, X168T-X261E-X276W, X172F-X225Q-X259D, X172F-X225Q-X259E, X172F-X225Q-X261D, X172F-X225Q-X261E, X172F-X225Q-X276W, X172F-X259D-X261D, X172F-X259D-X261E, X172F-X259D-X276W, X172F-X259E-X261D, X172F-X259E-X261E, X172F-X259E-X276W, X172F-X261D-X276W, X172F-X261E-X276W, X225Q-X259D-X261D, X225Q-X259D-X261E, X225Q-X259D-X276W, X225Q-X259E-X261D, X225Q-X259E-X261E, X225Q-X259E-X276W, X225Q-X261D-X276W, X225Q-X261E-X276W, X259D-X261D-X276W, X259D-X261E-X276W, X259E-X261D-X276W and X259E-X261E-X276W.

[0287] 25. A mannanase variant comprising an amino acid substitution selected from the group consisting of: X0136P-X019D-X0225N-X032Y, X0136P-X019D-X0225N-X072V, X0136P-X019D-X0225N-X093Q, X0136P-X019D-X0225N-X131S, X0136P-X019D-X0225N-X139R, X0136P-X019D-X0225N-X161G, X0136P-X019D-X0225N-X168T, X0136P-X019D-X0225N-X172F, X0136P-X019D-X0225N-X173 6P-X019D-X0225N-X259D, X0136P-X019D-X0225N-X259E, X0136P-X019D-X 0225N-X261D, X0136P-X019D-X0225N-X261E, ,X0136P-X019D-X032Y-X072V, D-X032Y-X131S, X0136P-X019D-X032Y-X139R, X0136P-X019D-X032Y-X161G ,X0136P-X019D-X032Y-X168T, D-X032Y-X225Q, X0136P-X019D-X032Y-X259D, X0136P-X019D-X032Y-X259E ,X0136P-X019D-X032Y-X261D, D-X032Y-X276W, X0136P-X019D-X072V-X093Q, X0136P-X019D-X072V-X131S ,X0136P-X019D-X072V-X139R, D-X072V-X168T, X0136P-X019D-X072V-X172F, X0136P-X019D-X072V-X225Q ,X0136P-X019D-X072V-X259D, -X072V-X261D, 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276W、X139R-X168T-X261E-X276W、X139R-X172F-X225Q-X259D、X139R-X172F-X225Q-X259E、X139R-X172F-X225Q-X261D、X139R-X172F-X225Q-X261E、X139R-X172F-X225Q-X276W、X139R-X172F-X259D-X261D、X139R-X172F-X259D-X261E、X139R-X172F-X259D-X276W、X139R-X172F-X259E-X261D、X139R-X172F-X259E-X261E、X139R-X172F-X259E-X276W、X139R-X172F-X261D-X276W、X139R-X172F-X261E-X276W、X139R-X225Q-X259D-X261D、X139R-X225Q-X259D-X261E、X139R-X225Q-X259D-X276W、X139R-X225Q-X259E-X261D、X139R-X225Q-X259E-X261E、X139R-X225Q-X259E-X276W、X139R-X225Q-X261D-X276W、X139R-X225Q-X261E-X276W、X139R-X259D-X261D-X276W、X139R-X259D-X261E-X276W、X139R-X259E-X261D-X276W、X139R-X259E-X261E-X276W、X161G-X168T-X172F-X225Q、X161G-X168T-X172F-X259D、X161G-X168T-X172F-X259E、X161G-X168T-X172F-X261D、X161G-X168T-X172F-X261E、X161G-X168T-X172F-X276W、X161G-X168T-X225Q-X259D、X161G-X168T-X225Q-X259E、X161G-X168T-X225Q-X261D、X161G-X168T-X225Q-X261E、X161G-X168T-X225Q-X276W、X161G-X168T-X259D-X261D、X161G-X168T-X259D-X261E、X161G-X168T-X259D-X276W、X161G-X168T-X259E-X261D、X161G-X168T-X259E-X261E、X161G-X168T-X259E-X276WX161G-X168T-X261D-X276W、X161G-X168T-X261E-X276W、X161G-X172F-X225Q-X259D、X161G-X172F-X225Q-X259E、X161G-X172F-X225Q-X261D、X161G-X172F-X225Q-X261E、X161G-X172F-X225Q-X276W、X161G-X172F-X259D-X261D、X161G-X172F-X259D-X261E、X161G-X172F-X259D-X276W、X161G-X172F-X259E-X261D、X161G-X172F-X259E-X261E、X161G-X172F-X259E-X276W、X161G-X172F-X261D-X276W、X161G-X172F-X261E-X276W、X161G-X225Q-X259D-X261D、X161G-X225Q-X259D-X261E、X161G-X225Q-X259D-X276W、X161G-X225Q-X259E-X261D、X161G-X225Q-X259E-X261E、X161G-X225Q-X259E-X276W、X161G-X225Q-X261D-X276W、X161G-X225Q-X261E-X276W、X161G-X259D-X261D-X276W、X161G-X259D-X261E-X276W、X161G-X259E-X261D-X276W、X161G-X259E-X261E-X276W、X168T-X172F-X225Q-X259D、X168T-X172F-X225Q-X259E、X168T-X172F-X225Q-X261D、X168T-X172F-X225Q-X261E、X168T-X172F-X225Q-X276W、X168T-X172F-X259D-X261D、X168T-X172F-X259D-X261E、X168T-X172F-X259D-X276W、X168T-X172F-X259E-X261D、X168T-X172F-X259E-X261E、X168T-X172F-X259E-X276W、X168T-X172F-X261D-X276W、X168T-X172F-X261E-X276W、X168T-X225Q-X259D-X261D、X168T-X225Q-X259D-X261E、X168T-X225Q-X259D-X276W、X168T-X225Q-X259E-X261D、X168T-X225Q-X259E-X261E、X168T-X225Q-X259E-X276W、X168T-X225Q-X261D-X276W、X168T-X225Q-X261E-X276WX168T-X259D-X261D-X276W、X168T-X259D-X261E-X276W、X168T-X259E-X261D-X276W、X168T-X259E-X261E-X276W、X172F-X225Q-X259D-X261D、X172F-X225Q-X259D-X261E、X172F-X225Q-X259D-X276W、X172F-X225Q-X259E-X261D、X172F-X225Q-X259E-X261E、X172F-X225Q-X259E-X276W、X172F-X225Q-X261D-X276W、X172F-X225Q-X261E-X276W、X172F-X259D-X261D-X276W、X172F-X259D-X261E-X276W, X172F-X259E-X261D-X276W, X172F-X259E-X261E-X276W, X225Q-X259D-X261D-X276W, X225Q-X259D-X261E-X276W, X225Q-X259E-X261D-X276W and X225Q-X259E-X261E-X276W.

[0288] Examples

[0289] It should be understood that although these examples indicate embodiments of the present disclosure, they are provided by way of illustration only. From the above discussion and these examples, those skilled in the art may make various changes and modifications to the present disclosure to adapt it to various uses and conditions. Such modifications are also intended to fall within the scope of the appended claims.

[0290] Example 1

[0291] Determination method

[0292] Protein determination assay Law

[0293] HPLC detection and quantification - Protein concentration was determined using a high performance liquid chromatography (HPLC) method that measures the integrated peak area. Samples were obtained from filtered culture supernatants and prepared as 3-fold dilutions in 10 mM HEPES buffer (pH 8). HPLC was performed on an Agilent 1200 series HPLC system equipped with a Poroshell 300SB-C8 column (2.1×75 mm) and a Poroshell 300SB-C8 guard column (2.1×12.5 mm) using a gradient eluent consisting of water and acetonitrile solvents (each supplemented with 0.1% TFA). The sample was eluted at a flow rate of 0.5 mL / min at 65°C. Proteins were detected by measuring the absorbance at 225 nm, and peaks were integrated using ChemStation OpenLabsoftware (Agilent Technologies). The protein concentration of the sample was determined based on a standard curve of the parent protein.

[0294] Bradford protein quantitation—using the Bradford assay (Thermo Scientific, Inc.

[0295] Protein concentration was measured using the Thermo Scientific Coomassie Protein Assay Kit #23200. Samples were obtained from filtered culture supernatants and prepared as 3-fold dilutions in 10 mM HEPES buffer (pH 8). The protein concentration of the samples was determined based on a standard curve of the parent protein. Background protein was subtracted using a Bacillus subtilis host that does not express recombinant mannanase.

[0296] Mannanase activity assay

[0297] Mannanase activity was determined by measuring the hydrolysis of locust bean gum (LBG) galactomannan (Sigma-Aldrich, #G0753) substrate (approximately 0.5% (w / v) LBG substrate) in solution. To prepare the working substrate solution, LBG powder was dissolved in a heated solution of 50 mM Tris-HCl buffer (pH 7.5) with stirring. After cooling to room temperature, the solution was centrifuged and the clear supernatant was used as the substrate solution. The enzyme was diluted in enzyme dilution buffer (100 mM HEPES (pH 8), 0.005% 80), and aliquots of the diluted enzyme solution are added to the holes of a microtiter plate (e.g., Corning 3358) containing LBG substrate solution. The plates are sealed and incubated at 40°C with 900rpm stirring for 10 minutes (e.g., in an iEMS incubator / oscillator, Thermo Fisher Scientific). After incubation, the reducing sugars released are quantified using the BCA reagent assay (Catalog No. 23225, Thermo Scientific Pierce). Specifically, aliquots from each well of the LBG assay plate are added to a PCR plate containing a BCA working reagent solution (prepared according to the manufacturer's instructions); the ratio of sample to working reagent is 1:9 (v / v). The plate is sealed and incubated at 95°C in a thermal cycler (e.g., Applied Biosystems GeneAmp PCR System 9700) for 2.5min. After the plate has cooled to 25°C, the reaction solution is transferred to a fresh flat-bottomed transparent polystyrene MTP (e.g., Falcon 353075) and the absorbance at 562 nm is measured in a microplate reader spectrophotometer (e.g., SpectraMax Plus 384, Molecular Devices). The absorbance value of the sample without mannanase (blank) is subtracted from the absorbance value of the sample containing mannanase. The resulting absorbance is taken as a measure of mannanase activity.

[0298] Detergents used in the assay

[0299] Two detergents were used in the following examples. Chinese National Standard HDL (CNS) was purchased from the China Cleaning Industry Association. The composition information of CNS is described in "Determination of detergency and cycle of washing property for laundry detergents", National Standard of the People's republic of China, ICS 71.100.40, Y43 (GB / T 13174-2021), www.chinesestandard.net / PDF / BOOK.aspx / GBT13174-2021, and is shown in

[0300] Table 2. The composition of HDL detergent formulation Model A is shown in Table 3.

[0301]

[0302]

[0303]

[0304] Stability determination Law

[0305] The stability of the mannanase variants was tested under the stress conditions described in the Examples and by measuring the residual mannanase activity of the samples after incubation under those conditions. To measure the initial (non-stress) activity, enzyme samples were diluted in 100 mM HEPES (pH 8), 0.005% 80 and added to the detergent conditions as described in the examples and then immediately stored at -80°C. To measure stress activity, enzyme samples were diluted in 100 mM HEPES (pH 8), 0.005% 80°C and added to detergent conditions and incubated as described in the Examples, followed by storage at -80°C. The samples were then assayed for activity on the LBG substrate using the assay described in the "Mannanase Activity Assay" section above. The residual activity percentage (%) was calculated by taking the ratio of the stressed activity to the non-stressed activity and multiplying by 100.

[0306] Cleaning performance of mannanase variants using the micro-swatch assay

[0307] The cleaning performance of mannanase samples was tested on locust bean gum (LBG) micro-swatches (CFT CS-73, Center for Testmaterials, Vlaardingen, The Netherlands), and the results of the mannanase variants are reported relative to the performance of the parent enzyme or a reference enzyme. The cleaning performance was measured using a high-throughput assay developed to measure the removal of galactomannan from industrial soils. The assay measures the release of LBG from industrial soils containing LBG. The BCA reaction using commercially available reagents (Cat. No. 23225, Thermo Scientific Pierce) was used to measure the reducing ends of oligosaccharides in solution in the presence of the enzyme compared to a blank control. This measurement correlates with the cleaning performance of the enzyme. When the mannanase hydrolyzes galactomannan, oligosaccharides of different lengths with reducing ends can be released from the cotton swatch. The bicinchoninic acid in the BCA reagent then allows for high sensitivity colorimetric detection of the oligosaccharides by reducing Cu 2+ Cu 1+ Two 5 mm diameter LBG microswatches were placed in each well of a flat-bottom 96-well assay plate. 80% buffer dilution enzyme. 90 μL micro-swatch assay buffer (6g / L HDL detergent formulation model A, 5mM HEPES pH 8.0, 200ppm 3: 1Ca2+ / Mg2+ water hardness) and 10 μL aliquots of the diluted enzyme are added to each well of a 96-well micro-swatch assay plate, with a combined volume of 100 μL. The plate is sealed and incubated at 25°C with 1150rpm stirring for 20min (e.g., in an iEMS incubator shaker, Thermo Fisher Scientific). After incubation, as described in the "Mannanase Activity Assay" section above, the BCA reagent assay is used to quantify the released reducing sugars. The resulting absorbance is used as a measure of cleaning performance.

[0308] The performance index (PI) of an enzyme compares the performance of a tested variant (measured value) with that of a parent or reference enzyme (theoretical value or measured value) at the same protein concentration. The theoretical value of the cleaning performance of the parent or reference enzyme at the relevant protein concentration is calculated using parameters extracted from the Langmuir fit of the measured values ​​of the standard curve of the parent or reference enzyme.

[0309] All mannanase variants described in this application show cleaning performance equal to or better than the parent enzyme or reference enzyme.

[0310] Example 2

[0311] Variant and strain construction and sample preparation

[0312] Design the synthetic gene encoding the mannanase variant based on Paenibacillus species A1 mannanase PspMan4 (SEQ ID NO:1) to express in Bacillus subtilis. Use methods known in the art to clone and express these genes in Bacillus subtilis. In order to generate samples of the mannanase variants for characterization, the Bacillus subtilis strain is grown in a culture medium (a semi-defined medium enriched based on MOPS buffer, urea as the main nitrogen source, glucose as the main carbon source, and supplemented with soy peptone) by standard methods, and the supernatant is filtered through a 0.2 μm filter plate (PALL). According to the assay method described in Example 1, the culture supernatant filtered is used to measure enzyme activity, stability and cleaning performance.

[0313] Example 3

[0314] Evaluation of the stability of mannanase variants in detergent formulations

[0315] The stability of the mannanase variants was tested under stress conditions described for the table below using the method described in Example 1 Stability Assay, where the results are reported as percent residual activity (%). As mentioned on the data sheet, the mutations of the mannanase variants are reported relative to the parent enzyme PspMan4 (SEQ ID NO: 1) or PspMan138 (SEQ ID NO: 2). PspMan138 is a variant of PspMan4 with the following mutations: P019E-S030T-T038E-S059V-L060Q-K063R-N067D-N097D-V103I-Y129M-F167Y-Q184L-G225C-T228V-Y235L-K244L-S258D-N261R-ins298Q.

[0316] The stability of the mannanase variants compared to the PspMan4 parent was tested using stress condition A (30°C for 5 minutes) in 25% (v / v) aqueous solution of HDL detergent formulation Model A described in Example 1. The stability results under these conditions are reported as residual activity using the calculations described in Example 1, and these values ​​are reported in Table 4.

[0317]

[0318] The stability of the mannanase variants compared to the PspMan138 parent was tested in 25% (v / v) aqueous solution of the HDL detergent formulation Model A described in Example 1 using either stress condition B (incubation at 55°C for 20 minutes) or stress condition C (incubation at 55°C for 60 minutes). The residual activity results are reported in Table 5.

[0319]

[0320]

[0321]

[0322]

[0323] ND – Not determined.

[0324] The following stress conditions were also used, with the Chinese National Standard (CNS) detergent (including 660 ppm The stability of the mannanase variants compared to the parent PspMan138 was tested in aqueous solutions of 1% (v / v) detergent and 37°C for 48 hours; Stress condition E: 50% (v / v) detergent and 50°C for 24 hours; Stress condition F: 50% (v / v) detergent and 55°C for 48 hours. The stability results under these conditions are reported as % residual activity using the calculations described in Example 1, and these values ​​are reported in Table 6.

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341] ND – Not determined.

Claims

1. A mannanase variant comprising an amino acid substitution at a position selected from the group consisting of 32, 72, 161 and 172, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

2. A mannanase variant comprising amino acid substitutions, wherein the variant comprises amino acid substitutions selected from the group consisting of: 19D, 32Y, 34D, 72V, 93Q, 131S, 136P, 139R, 161G, 172F, 225N / Q, 259D, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

3. A mannanase variant comprising two or more amino acid substitutions, wherein the two or more amino acid substitutions are selected from the group consisting of: 19D, 32Y, 72V, 93Q, 131S, 136P, 139R, 161G, 168T, 172F, 225N / Q, 259D / E, 261D / E and 276W, wherein the amino acid positions of the variant are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variant has at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

4. The mannanase variant of claim 3, wherein the variant comprises an amino acid substitution selected from the group consisting of 19D-276W, 32Y-259D, 93Q-276W, 131S-276W, 136P-276W, 139R-276W, 161G-276W, 225N / Q-276W, 259D / E-276W and 261D / E-276W.

5. The mannanase variant of claim 3, wherein the variant comprises an amino acid substitution selected from the group consisting of 19D-131S-276W, 32Y-261D-276W, 32Y-259D-276W, 32Y-172F-259D, 168T-259D-276W, 259D-261E-276W, and 259Q-261E-276W.

6. A mannanase variant comprising an amino acid substitution selected from the group consisting of: Y061W-G259D-R261E-F276W, Y061W-T062E-G259D-R261E-F276W, Y061W-V228T-G259D-R261E-F276W, V228T-G259D-R261E-F276W, F032Y-G259D-R261E-F276W, F032Y-Y061W-Y167F-P168S-G259D-R261E-F276W, F032Y-Y061W-G259D-R261E-F276W , F032Y-Y061W-T062E-G259D-R261E-F276W, F032Y-T062E-R261D-F276W , F032Y-T062E-G259D-F276W, T062E-G259D-R261E-F276W, V059S-Q060L -G259D-R261E-F276W, V059S-Q060L-Y061W-G259D-R261E-F276W, V059S -Q060L-T062E-G259D-R261E-F276W, T062E-V228T-G259D-R261E-F276W , F032Y-T062E-G259D-R261E-F276W, F032Y-T062E-G259D-R261E-F276W -T284E, E019D-A068S-T131S-F276W, F032Y-T062E-G259D-F276W-T284E , F032Y-T062E-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-G259D -F276W-T284E, F032Y-T062E-A068S-G259D-F276W-T284E, F032Y-Y061W -T062E-G259D-F276W-T284E, F032Y-V059S-Q060L-T062E-G259D-F276W -T284E, N010T-F032Y-T062E-G259D-F276W-T284E, F032Y-T062E-Y167F -P168S-V228T-G259D-F276W-T284E, F032Y-T062E-I072V-V228T-G259D -F276W-T284E, F032Y-T062E-I072V-Y167F-P168S-G259D-F276W-T284E,F032Y-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-V228T-G259D-F276W-T284E、F032Y-V059S、Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-G259D-F276W-T284E、F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-G259D-F276W-T284E、F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-G259D-F276W-T284E、F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-G259D-F276W-T284E、N010T-F032Y-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-I072V-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-Y167F-P168S-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-V228T-G259D-F276W-T284E、N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-G259D-F276W-T284E and N010T-F032Y-V059S-Q060L-Y061W-T062E-A068S-I072V-Y167F-P168S-V228T-G259D-F276W-T284E, F032Y-Y061W-T062E-G259D-R261E-F276W-T284E, F032Y-T062E-A068S-G259D-R261E-F276W-T284 E. F032Y-T062E-T131S-G259D-R261E-F276W-T284E, F032Y-T062E-N150D -G259D-R261E-F276W-T284E, F032Y-T062E-P168S-G259D-R261E-F276W- T284E, F032Y-T062E-L235K-G259D-R261E-F276W-T284E, N010T-F032Y-Y 061W-T062E-P168A-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T06 2E-Y167F-P168S-V228T-G259E-F276W-T284E, F032Y-Y061W-T062E-T131 S-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W-T062E -A136P-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y061W- T062E-K093Q-Y167F-P168S-V228T-G259D-F276W-T284E, N010T-F032Y-Y 061W-T062E-Y167F-P168S-V228T-L235K-G259D-F276W-T284E, N010T-F0 32Y-Y061W-T062E-D139R-Y167F-P168S-V228T-G259D-F276W-T284E, N01 0T-F032Y-Y061W-T062E-N150D-Y167F-P168S-V228T-G259D-F276W-T284 E. N010T-F032Y-Y061W-T062E-Y167F-P168A-V228T-G259D-F276W-T284E,N010T-F032Y-Y061W-T062E-T131S-Y167F-P168S-V228T-G259D-F276W-T284E , N010T-F032Y-Y061W-T062E-P168S-V228T-G259D-F276W-T284E, N010T-F032 Y-Y061W-T062E-A068S-Y167F-P168A-G259D-F276W-T284E, and N010T-F032Y-Y061W-T062E-A068S-Y167F-P168A-G259E-F276W-T284E, wherein the amino acid positions of the variants are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and wherein the variants have at least 80% identity with the amino acid sequence of SEQ ID NO:

1.

7. The mannanase variant of claim 1, wherein the variant comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:

1.

8. The mannanase variant of claim 1, wherein the variant is derived from a parent or reference polypeptide having 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:

1.

9. The mannanase variant of claim 1, wherein the variant has improved stability when compared to a parent mannanase or a reference mannanase.

10. The mannanase of claim 9, wherein the variant has equal or improved cleaning performance in a detergent when compared to a parent mannanase or a reference mannanase.

11. The mannanase variant of claim 1, wherein the mannanase variant has mannanase activity.

12. A polynucleotide comprising a nucleic acid sequence encoding the variant of claim 1, wherein the polynucleotide is optionally isolated.

13. An enzyme composition comprising one or more mannanase variants according to claim 1.

14. The enzyme composition of claim 13, wherein the composition is an enzyme granule.

15. The enzyme composition of claim 13, further comprising one or more other enzymes selected from the group consisting of acyltransferases, amylases, α-amylases, β-amylases, α-galactosidases, arabinanases, arabinosidases, arylesterases, β-galactosidases, β-glucanases, carrageenase, catalase, cellulase, chondroitinase, cutinase, dispersin, endo-glucanase, endo-β-mannanase, exo-β-mannanase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, hexosaminidase, esterase, exo-mannanase, galactanase, glucoamylase, hemicellulase, aminosuccinylcholine, hydroxyethyl esterase ... enzymes, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipolytic enzymes, lipoxygenases, lysozymes, mannanases, metalloproteinases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetylesterases, pectinases, pentosanases, perhydrolases, peroxidases, PETases, phenoloxidases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, subtilisins, tannases, transglutaminases, xylan acetylesterases, xylanases, and xylosidases; and combinations thereof.

16. A cleaning composition comprising the mannanase variant of claim 1.

17. The cleaning composition of claim 16, wherein the cleaning composition is a detergent composition selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, a medical device cleaning detergent, and a hard surface cleaning detergent.

18. A method of cleaning, comprising contacting a surface or item in need of cleaning with an effective amount of the mannanase variant of claim 1 or the enzyme composition of claim 13; and optionally further comprising the steps of: The surface or item is rinsed after contacting the surface or item with the variant or enzyme composition.

19. The method of claim 18, wherein the article is a medical device, tableware, or fabric.

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