Mannanase variant

Abstract

To provide a variant of mannanase enzyme.SOLUTION: The prevent invention relates to mannanase at least 75% identical to SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, a poly-nucleotide encoding mannanase, an expression construct comprising the polynucleotide, and a host cell comprising the polynucleotide or the expression construct.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to variants of mannanase enzymes, which are useful in industrial applications where mannan degradation or modification is desirable, such as laundry and cleaning applications, feed, food, paper and pulp, and petroleum industries. The present invention also provides useful mannanase enzymes, polynucleotides encoding these enzymes, enzyme compositions, and methods for their production and use. [Background technology]

[0002] The primary role of hemicelluloses and galactomannans is to function as structural polysaccharides and / or energy reserves. In addition to amylose and amylopectin, which are the most widespread storage polysaccharides in plants, a diverse group of mannan-based polysaccharides is present in the seeds, roots, bulbs, and tubers of various plants. These include mannans, galactomannans, and glucomannans.

[0003] Mannans are polysaccharides with a backbone of β-1,4-linked D-mannopyranosyl residues. In most cases, mannans are highly insoluble in water but have a high water-binding capacity. Unlike unsubstituted mannans, galactomannans are water-soluble. Because of the complex structural composition of plant cell walls, microorganisms growing on decaying plant material must possess several different enzymes capable of hydrolyzing these highly polymerized, nearly insoluble materials. The two main endo-acting enzymes involved in the degradation of hemicellulose are β-mannanase and β-xylanase. Additionally, the complete degradation of galactoglucomannan requires the exo-acting enzymes β-mannosidase, α-galactosidase, and β-glucosidase.

[0004] The main enzyme type involved in mannan backbone degradation is endo-1,4-beta-mannanase (EC 3.2.1.78), which hydrolyzes internal glycosidic bonds in the mannan backbone. Endo-1,4-beta-mannanase (EC 3.2.1.78) is a mannan-degrading enzyme, sometimes referred to herein as endo-beta-1,4-D-mannanase, beta-mannanase, or mannanase. Because endo-1,4-beta-mannanase (EC 3.2.1.78) degrades the mannan backbone, mannan degradation includes the degradation of mannans, galactomannans, and / or glucomannans.

[0005] The uses of mannanase enzymes are widespread in food and feed applications, detergents and the paper and pulp industry: Since mannans are a contributing factor to the viscosity of the intestinal contents, thereby adversely affecting feed digestibility and animal growth rate, the use of mannanase enzymes as a feed additive has been shown to provide several beneficial effects. In the food industry, mannanase enzymes have been described for use in the production of instant coffee, where they reduce the viscosity of coffee extracts by hydrolysis of coffee mannans. Furthermore, mannanases are used to produce certain manno-oligomers that are of interest as functional food ingredients, e.g., manno-oligomers with prebiotic properties. In such applications, plant-derived mannopolymers are subjected to hydrolysis by mannanases.

[0006] · The use of mannanases in fruit juice processing and production is common because mannanases reduce viscosity, improve filtration rate, stability and help extract fruit components. Detergent applications: Mannanases facilitate the removal of stains / soil from food and cosmetic products, which often contain mannan-containing additives such as stabilizers, emulsifiers, and thickeners. In a more specific cleaning application, mannanases are applied to remove biofilms from surfaces or pipes that are required to be free of microorganisms, such as pharmaceutical equipment. In this application, mannanases are often used in combination with detergents and other enzymes such as carbohydrases and proteases. Paper and pulp: Mannanases are used in the enzyme-assisted bleaching of paper pulp. They are said to complement the action of xylanases. Mannanase is applied in the process of oil and gas well stimulation by hydraulic fracturing. Mannanase reduces the viscosity of the guar solution applied in this process. Mannanases are used for the controlled release of drugs or other materials from matrices composed of cross-linked galactomannans. Summary of the Invention

[0007] Activity under application conditions is an important parameter for many industrially applied enzymes, since these enzymes often tend to be insufficiently active under application conditions. Therefore, the object of the present invention was to find mannanase variants with improved performance, especially after storage for a certain period of time.

[0008] The mannanase variants of the present invention are advantageous in that they have good stability and mannanase activity, and can provide improved yields during production and better performance during use.

[0009] There is a continuing need for enzymes that function in the harsh environment of detergent formulations. Mannanases are useful components of washing and / or cleaning formulations because they remove some of the stains that contain hemicellulose.

[0010] As used herein, mannan-containing stains or mannan-containing stains comprise at least one mannan, at least one galactomannan, and / or at least one glucomannan, and in one embodiment, additional components, such as cellulose and / or hemicellulose. Furthermore, such stains may contain proteinaceous materials, starch, and / or sugars. Galactomannans typically consist of a mannose backbone with galactose side groups. As used herein, galactomannans include galactomannans with the following mannose-to-galactose ratios: fenugreek gum (approximately 1:1), guar gum (approximately 2:1), tara gum (approximately 3:1), locust bean gum or carob gum (approximately 4:1), and cassia gum (approximately 5:1), where the ratio is mannose:galactose. Galactomannans are often used in food and cosmetic products to increase the viscosity of liquid products.

[0011] Insufficient removal of these types of stains usually results in graying of the fabric. Therefore, a further object of the present invention was to find a catalytically active mannan-degrading enzyme in a formulation containing a surfactant and having a pH in the range of 5-12, preferably in the range of 6-11, more preferably selected from the ranges of 6-10, 7-12, 7-9, 8-12, 8-10, and 7.5-8.5.

[0012] The present invention provides mannanase variants with improved stability in detergents.

[0013] In one aspect, the present invention provides a mannanase having mannan-degrading activity at a pH in the range of 5 to 12 or 6 to 11, more preferably at a pH in the range of 6 to 10, 7 to 12, 7 to 9, 8 to 12, or 8 to 10, and most preferably at a pH in the range of 7.5 to 8.5. The mannanase of the present invention is at least 75% identical to SEQ ID NO: 2, and preferably at least 75% identical to the sequence of positions 31 to 490 of SEQ ID NO: 2. The sequence of positions 31 to 490 of SEQ ID NO: 2 is equivalent to SEQ ID NO: 3. In one embodiment, the mannanase of the present invention is at least 75% identical to the sequence of positions 31 to 327 of SEQ ID NO: 2. The sequence of positions 31 to 327 of SEQ ID NO: 2 is equivalent to SEQ ID NO: 4.

[0014] In one aspect, the present invention provides a polynucleotide sequence encoding a mannanase variant according to the present invention.

[0015] In one aspect, the present invention provides a vector comprising a polynucleotide sequence of the present invention.

[0016] In one aspect, the present invention provides a recombinant host cell comprising a polynucleotide of the present invention that enables the host cell to express at least one recombinant mannanase variant according to the present invention.

[0017] In one aspect, the invention provides a method of expressing a polynucleotide, comprising: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide. The present invention provides a method comprising:

[0018] In one aspect of the invention, the mannanase variants according to the invention are provided in an enzyme preparation that allows for flexible incorporation into liquid formulations containing one enzyme or a mixture of enzymes, such as liquid detergent formulations. By "incorporating into" is meant adding the enzyme preparation to the liquid formulation.

[0019] The enzyme preparation may further comprise other enzyme(s) selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases, cutinases, esterases, phytases, DNAses, pectinases, pectate lyases, pectinolytic enzymes, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases, together with suitable additives selected from the group consisting of compounds that stabilize the included enzymes, e.g., enzyme stabilizers, and compounds that stabilize the preparation, e.g., preservatives.

[0020] In one aspect, the present invention provides a formulation comprising a mannanase variant according to the present invention, preferably having a pH in the range of 5 to 12, preferably in the range of 6 to 11, more preferably in the range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10, and 7.5 to 8.5. Preferably, the formulation is a liquid formulation comprising at least one component selected from surfactants, builders, and hydrotropes, present in an amount effective to maintain the physical properties of the liquid formulation and / or in an amount effective for cleaning. In one embodiment, the formulation is a detergent formulation.

[0021] Detergent formulations comprising at least one mannanase variant according to the present invention are advantageous in terms of removing stains containing mannans.

[0022] array The sequences used herein are as follows: SEQ ID NO: 1 Polynucleotide sequence encoding the parent mannanase according to SEQ ID NO: 2:

number

number

number

number

[0023] Detailed description of the invention: It should be understood that as used herein and in the claims, "a" or "an" can mean one or more (in the sense of "at least one"), depending on the context in which it is used.

[0024] Furthermore, the term "at least" is understood to mean that the item or parameter to which it refers is limited in one direction, but open-ended in one or more other directions.

[0025] As used below, the terms "have", "comprise", "contain" or "include" or any grammatical variants thereof are used in a non-exclusive manner, and therefore these terms can refer both to a situation in which no further features are present in the entity described in this context other than the features introduced by these terms, and to a situation in which one or more further features are present.

[0026] Features introduced by "in one embodiment" or similar language are intended to be additional or alternative features and do not limit alternative embodiments of the invention, do not limit the scope of the invention, and do not limit the possibility of combining features introduced in this way with other additional or alternative or non-additional or alternative features of the invention. The term "may" is used herein to preferably encompass embodiments.

[0027] The term "about" as used herein means that for any number listed after said term, there is an interval precision that can achieve the technical effect. Thus, "about" as referred to herein preferably refers to the exact number or a range of ±15%, preferably ±10%, more preferably ±5% or even more preferably ±3% around said exact number.

[0028] In general, an "enzyme" is a catalytically active protein or polypeptide that acts on a substrate to convert the substrate into a product. This reaction, also referred to herein as an enzymatic conversion, typically occurs at the "active site" of the enzyme. An enzyme that performs an enzymatic conversion is enzymatically active or has enzymatic activity. Any polypeptide referred to herein as an "enzyme" refers to a catalytically active polypeptide.

[0029] The mannanase variants according to the present invention have mannan-degrading activity and are of the enzyme class EC 3.2.1.78. In one embodiment, the mannan-degrading activity corresponds to the degradation of at least one galactomannan. Preferably, the at least one galactomannan is characterized by a mannose:galactose ratio of about 1:1, about 2:1, about 3:1, about 4:1, and / or about 5:1.

[0030] Mannan-degrading activity or mannanase activity can be tested according to standard testing procedures known in the art. For example, the mannanase to be tested can be applied to a 4 mm diameter hole drilled in an agar plate containing 0.2% AZCL galactomannan (carob), i.e., a substrate for assaying endo-1,4-beta-D-mannanase. Carob is available, for example, from Megazyme, Inc. as I-AZGMA. (Megazyme's internet address: http: / / www.megazyme.com / Purchase / index.html). Mannan-degrading activity can be tested using carob galactomannan stained with Remazol Brilliant Blue in a liquid assay, as disclosed in McCleary, BV (1978). Carbohydrate Research, 67(1), 213-221. Another method of testing mannan-degrading activity uses the detection of reducing sugars when incubated with a substrate, e.g., guar gum or locust bean gum - for review see Miller, GL Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Analytical Chemistry 1959; 31: 426-428.

[0031] Enzymes are typically polypeptides identified by their polypeptide sequences (also referred to herein as amino acid sequences) as provided in the sequence listing accompanying this disclosure in accordance with World Intellectual Property Organization (WIPO) standard ST.25 (1998), which means that amino acids herein are represented using either their three-letter code with the first letter capitalized or their single-letter equivalents.

[0032] Polypeptides are typically encoded by polynucleotides, which are typically identified by polynucleotide sequences and SEQ ID NOs, provided in accordance with World Intellectual Property Organization (WIPO) standard ST.25 (1998), in the sequence listing accompanying this disclosure.

[0033] A "parent" polypeptide amino acid sequence is a starting sequence for introducing mutations into the sequence (e.g., by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) to produce a "variant" of the parent polypeptide amino acid sequence. Parents include wild-type or synthetically produced polypeptide amino acid sequences that are used as starting sequences for the introduction of (further) changes.

[0034] The parent polypeptide of the mannanase variant of the invention may have a polypeptide sequence according to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In one embodiment of the invention, the parent polypeptide has a sequence according to positions 31 to 490 of SEQ ID NO: 2. The sequence according to positions 31 to 490 of SEQ ID NO: 2 is equivalent to SEQ ID NO: 3. In another embodiment of the invention, the parent polypeptide has a sequence according to positions 31 to 327 of SEQ ID NO: 2. The sequence according to positions 31 to 327 of SEQ ID NO: 2 is equivalent to SEQ ID NO: 4.

[0035] "Variant polypeptide" refers to an enzyme that differs in amino acid sequence from its parent.

[0036] Variant polypeptide sequences may be defined by their "sequence identity" when compared to a parent sequence. An enzyme or polypeptide "at least x% identical to SEQ ID NO:X" means an enzyme or polypeptide having a polypeptide sequence that is x% identical when compared to a polypeptide sequence according to SEQ ID NO:X, where SEQ ID NO:X refers to a sequence according to the invention. In one embodiment, SEQ ID NO:X is selected from SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0037] A polynucleotide that is "at least y% identical to SEQ ID NO:Y" means a polynucleotide that has a polynucleotide sequence that is y% identical when compared to a polynucleotide sequence according to SEQ ID NO:Y, which corresponds to SEQ ID NO:1 in this specification.

[0038] Sequence identity is usually expressed as "% sequence identity" or "% identity." To calculate sequence identity, the first step is to produce a sequence alignment.

[0039] According to the present invention, alignments are generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, pp. 443-45). Preferably, the program "NEEDLE" (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the present invention, using the program's default parameters (polynucleotides: gap open=10.0, gap extend=0.5, and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5, and matrix=EBLOSUM62).

[0040] After the two sequences have been aligned, in a second step an identity score is determined from the resulting alignment.

[0041] In one embodiment, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing each sequence of the invention in its entirety and multiplying by 100: % identity = (identical residues / length of alignment region showing each sequence of the invention in its entirety) x 100.

[0042] Polypeptides according to the present invention The polypeptide of the present invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity. In one embodiment of the present invention, the mannanase variant of the present invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of positions 31 to 490 of SEQ ID NO: 2 or the sequence of SEQ ID NO: 3, and the polypeptide has mannan-degrading activity.

[0043] In one embodiment, the mannanase variant according to the present invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the sequence according to positions 31 to 327 of SEQ ID NO: 2 or the sequence according to SEQ ID NO: 4, and the polypeptide has mannan-degrading activity.

[0044] Mannanase variants may further include one or more conservative substitutions, meaning that one amino acid is replaced with a similar amino acid. Similar amino acids according to the present invention are defined as follows: amino acid A is similar to amino acid S; amino acid D is similar to amino acids E and N; amino acid E is similar to amino acids D, K, and Q; amino acid F is similar to amino acids W and Y; amino acid H is similar to amino acids N and Y; amino acid I is similar to amino acids L, M, and V; amino acid K is similar to amino acids E, Q, and R; amino acid L is similar to amino acids I, M, and V; amino acid M is similar to amino acids I, L, and V; amino acid N is similar to amino acids D, H, and S; amino acid Q is similar to amino acids E, K, and R; amino acid R is similar to amino acids K and Q; amino acid S is similar to amino acids A, N, and T; amino acid T is similar to amino acid S; amino acid V is similar to amino acids I, L, and M; amino acid W is similar to amino acids F and Y; and amino acid Y is similar to amino acids F, H, and W.

[0045] The mannanase variants according to the present invention are "mature polypeptides," which refer to the final form of the enzyme, including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, and signal sequence deletions. The mature polypeptide may vary depending on the expression system, vector, promoter, and / or production process. The mature mannanase variants according to the present invention may be at least 75% identical to the sequence according to positions 31-490 of SEQ ID NO: 2, or at least 75% identical to the sequence according to SEQ ID NO: 3. The mature mannanase variants according to the present invention may be at least 75% identical to the sequence according to positions 31-327 of SEQ ID NO: 2, or at least 75% identical to the sequence according to SEQ ID NO: 4.

[0046] In one aspect, the present invention provides a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, comprising one or more amino acid substitutions at amino acid positions selected from D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317 and A319, wherein the numbering is according to SEQ ID NO: 2.

[0047] Preferably, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 contains one or more amino acid substitutions at amino acid positions selected from D86, Q89, N96, L101, A112, A119, A124, S127, N129, S235, S281, G286, W289, N312 and A319, where the numbering is according to SEQ ID NO:2.

[0048] In one aspect, the present invention provides D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E , S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, Provided is a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, comprising one or more amino acid substitutions at amino acid positions selected from N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T and A319D / E, wherein the numbering is according to SEQ ID NO:2.

[0049] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 contains one or more amino acid substitutions selected from D86N, Q89V / L, N96D, L101V, S103Y, A112N, A119Y / H / T, N122S, A124D / E / C, S126E, S127A, N129M / L / F, S231Q / K / L, S235Y / H / R, D244N, E264Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296Y / H, D301T / E / C, T309L, N312Y / F and A319E, where numbering is according to SEQ ID NO:2.

[0050] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 contains one or more amino acid substitutions selected from D86N, Q89V, N96D, L101V, S103Y, A112N, A119Y / H / T, A124D / C, S126E, S127A, N129M / L / F, S235Y / H / R, D244N, S281L, G286E / L / Q / A, W289F / M / H, N296H, D301T / E, N312Y and A319E, where numbering is according to SEQ ID NO:2.

[0051] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 contains one or more amino acid substitutions selected from D86N, Q89V, N96D, L101V, A112N, A119Y, A124D / C, S127A, N129M / L / F, S235H, S281L, G286E / L / Q / A, W289F / M / H, N312Y and A319E, where numbering is according to SEQ ID NO:2.

[0052] In one embodiment, a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 contains at least one amino acid substitution selected from D328, G329, G330, D331, Y344, M359, Y374, L416 and W432, preferably selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, where numbering is according to SEQ ID NO:2, and wherein the polypeptide has mannan-degrading activity. More preferably, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 contains at least one amino acid substitution selected from D328I / Q, G329T, G330P, Y344Q / F, M359R / Y, Y374G / V / A7 and W432N / L / R / S.

[0053] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, and A319E; (b) at least one amino acid substitution selected from Q89V / L, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T, and A319D; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0054] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, N122S, A124D, S126E, S231Q / K / L, S235Y, D244N, W289M, S290A, N296Y, D301T, N312Y, and A319E; (b) at least one amino acid substitution selected from Q89V / L, S103Y, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R, E264Q, S281L, G286E / L / Q / A, W289F / H, N296H, D301E / C, T309L, and N312F; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0055] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, A124D, S126E, S235Y, D244N, W289M, D301T, N312Y, and A319E; (b) at least one amino acid substitution selected from Q89V, S103Y, A112N, A119Y / H / T, A124C, S127A, N129M / L / F, S235H / R, S281L, G286E / L / Q / A, W289F / H, N296H, and D301E; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0056] In one embodiment, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, A124D, W289M, N312Y, and A319E; (b) at least one amino acid substitution selected from Q89V, A112N, A119Y, A124C, S127A, N129M / L / F, S235H, S281L, G286E / L / Q / A, and W289F / H; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0057] In one embodiment, a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 contains at least one amino acid substitution selected from D328, G329, G330, D331, Y344, M359, Y374, L416 and W432, preferably selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, where numbering is according to SEQ ID NO:2, and wherein the polypeptide has mannan-degrading activity. More preferably, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 (additionally) comprises at least one amino acid substitution selected from D328I / Q, G329T, G330P, Y344Q / F, M359R / Y, Y374G / V / A7 and W432N / L / R / S. In one embodiment, at least one of these amino acid substitutions is present in addition to those disclosed above.

[0058] The present invention relates to D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317, A319 , D328, G329, G330, D331, N341, Y344, F346, T348, E349, S352, G356, M359, Y374, D379, L416 and W432, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

[0059] In one embodiment, the polypeptide at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 is selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A , N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R , E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N 296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, N341F, Y344Q / F / T, F346T, T348S / R / N / M / G, E349T / S / D / G, S35 2N / G, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V, L416W and W432P / N / L / R / S / T / G / H / I, numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0060] In one embodiment, the polypeptide at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 is selected from the group consisting of Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / R ... and one or more amino acid substitutions selected from C, T309L, N312F, L317T, A319D, D328I / Q / V, G329L / S / V, G330P / T, D331A / Q, Y344Q / F / T, F346T, T348R / N / M, E349T / D, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V and W432P / N / L / R / S / T / G / H / I, where numbering is according to SEQ ID NO: 2, wherein the polypeptide has mannan degrading activity.

[0061] In one embodiment, the polypeptide at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, A319E, G329T, G330D, D331A / Q, N341F, T348S / G, E349S / G, S352N / G, and L416W; (b)Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301 at least one amino acid substitution selected from E / C, T309L, N312F, L317T, A319D, D328I / Q / V, G329L / S / V, G330P / T, Y344Q / F / T, F346T, T348R / N / M, E349T / D, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V, W432P / N / L / R / S / T / G / H / I; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0062] In one embodiment, the present invention provides a polypeptide at least 75% identical to the sequence according to SEQ ID NO: 4, wherein the numbering is according to SEQ ID NO: 2, comprising one or more amino acid substitutions selected from D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317, A319 and D328I, and wherein the polypeptide has mannan degrading activity.

[0063] In one embodiment, the polypeptide at least 75% identical to the sequence according to SEQ ID NO: 4 is selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254 and one or more amino acid substitutions selected from W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, and A319D / E, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

[0064] In one embodiment, a polypeptide at least 75% identical to a sequence according to SEQ ID NO: 4 contains one or more amino acid substitutions selected from Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T and A319D, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

[0065] In one embodiment, the polypeptide at least 75% identical to the sequence according to SEQ ID NO: 4 is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, and A319E; (b) at least one amino acid substitution selected from Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T, and A319D; in combination, wherein an amino acid substitution as defined in (b) is not present when a substitution is present at the corresponding position as defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0066] A polypeptide molecule that is at least 75% identical to the sequence according to positions 31 to 490 or 31 to 327 of SEQ ID NO: 2, containing the substitutions disclosed above, is referred to as a "mannanase variant according to the invention."

[0067] In one aspect of the invention, the mannanase variant according to the invention comprises one or more conservative amino acid substitutions at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, the numbering being according to SEQ ID NO: 2. Mannanase variants according to the present invention may comprise a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D, V358I, S370A, Q423K, D459N, N461S, and V482L.Mannanase variants according to the present invention may comprise a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, the mannanase variant according to the present invention comprises one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, wherein the numbering is according to SEQ ID NO: 2.

[0068] In one embodiment, the mannanase variant according to the present invention comprises one or more amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, H332D, G335D, A360G, A365V, D372G, Q381N, S391Y, F398L, K433T, E438G, I449T, K475N, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, and the numbering is according to SEQ ID NO: 2. Mannanase variants according to the present invention may comprise a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D, H332D, G335D, A365V, D372G, Q381N, S391Y, E438G, and K475N. Mannanase variants according to the present invention may comprise a combination of amino acid substitutions selected from N39T, T45N, D64Q, S133D, E140S, and S168D. Preferably, mannanase variants according to the present invention comprise one or more amino acid substitutions selected from A360G and I449T, and the mannanase variant is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, where the numbering is according to SEQ ID NO: 2.

[0069] In one embodiment, the mannanase variant according to the present invention comprises one or more "conserved amino acid regions" within its polypeptide sequence. A "conserved amino acid region" herein is characterized by a sequence of several consecutive amino acids that are not mutated, and the number of consecutive amino acids may be 3 to 10, 4 to 10, 5 to 10, 6, 7, 8, 9, or 10. The one or more conserved amino acid regions may include G76-A77-N78-T79, R81-V83-L84, E115-V116-H117-D118, Y134-W135-I136, A154-N155-E156-W157, A191-G192-W193-G194-Q195, F218-S219-I220-H221-M222, and the like. -Y223-E224-Y225-A226-G227, N236-I237-D238, I249-G250-E251-F252-G253, G259-D260-V261-D262-E263 and G276-W277-L278-A279-W280, where the numbering is according to SEQ ID NO: 2.

[0070] In one embodiment, the mannanase variant according to the present invention has mannan-degrading activity in a formulation having a pH in the range of 5 to 12, preferably in the range of 6 to 11, more preferably in the range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10 and 7.5 to 8.5.

[0071] In one aspect of the present invention, the mannanase variant has improved stability in detergents when compared to the parent enzyme.

[0072] Improved detergent stability means increased residual mannan-degrading activity after a period of time at a particular temperature, as compared to the parent enzyme. "A period of time" can mean up to 6 days or even longer. "A particular temperature" can mean 37°C. Detergent stability can be determined in the presence of at least one anionic surfactant and / or at least one nonionic surfactant.

[0073] In one embodiment, the at least one anionic surfactant is selected from a compound according to general formula (I) disclosed herein and a compound according to general formula (II) disclosed herein. In one embodiment, the at least one nonionic surfactant is selected from a compound according to general formula (III) disclosed herein.

[0074] Mannanase variants according to the present invention include D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, I24, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314 The mannanase variant may be a polypeptide at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, having one or more amino acid substitutions at positions selected from P, L317, A319, D328, G329, G330, D331, Y344, M359, Y374, L416 and W432, where the numbering is according to SEQ ID NO: 2, and the mannanase variant has improved "stability in detergents" compared to the parent enzyme.

[0075] In one embodiment, the mannanase variants with improved detergent stability are selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273 T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y A polypeptide at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3, having one or more amino acid substitutions selected from A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein numbering is according to SEQ ID NO:2.

[0076] In one embodiment, mannanase variants with improved detergent stability include D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / A polypeptide at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3, having two or more amino acid substitutions selected from L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein numbering is according to SEQ ID NO:2.

[0077] In one embodiment, the mannanase variant with improved detergent stability comprises: (a) one or more amino acid substitutions selected from Q89V, N96D, L101V, A112N, A119Y, A124D, S127A, N129M / L / F, S281L, G286E / L / Q / A, W289F / M / H, N312Y, A319E, and W432P; (b)D86N, Q89V, L101T, S103Y / E / A, K107N, N108G, N109Q / A, A119H / T, N122S, A124E / C, S126E, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S29 at least one amino acid substitution selected from: 0A, N296H / F / Y, D301E / C / T, T309L, N312F, A314P, L317T, A319E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W, and W432N / L / R / S / T / G / H / I; A polypeptide at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3, in combination, wherein the amino acid substitutions defined in (b) are not present when there are substitutions at the corresponding positions defined in (a), and the numbering is according to SEQ ID NO:2.

[0078] In one embodiment of the present invention, the mannanase variants with improved detergent stability having the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 disclosed above contain at least one additional amino acid substitution selected from N341F, F346T, T348S / R / N / M / G, E349T / S / G / D, S352N / G, G356Y / V / T / Q / H / C and D379V. Such mannanase variants may have the advantageous property of being stable against degradation during their production process.

[0079] In this specification, being stable against degradation during production means that the fermentation stability of the mannanase variants of the present invention is at least 1.5 times, at least 1.6 times, or at least 2 times greater than that of the parent enzyme.

[0080] In one embodiment, stable to degradation during production refers to stability when expressed in a bacterial host cell, preferably a Bacillus host cell, more preferably a Bacillus subtilis host cell.

[0081] In one embodiment, stable to degradation during production means stability at fermentation temperatures in the range of 35°C to 45°C, preferably at a temperature of 37°C.

[0082] In one aspect of the present invention, the mannanase variant disclosed above having a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 with improved detergent stability comprises at least one conservative amino acid substitution at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V and V482L, wherein the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and the numbering is according to SEQ ID NO: 2. Mannanase variants according to the present invention having improved stability in detergents may contain a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D, V358I, S370A, Q423K, D459N, N461S, and V482L.Mannanase variants according to the present invention having improved stability in detergents may contain a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, the mannanase variants according to the present invention having improved stability in detergents comprise one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variants are at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and the numbering is according to SEQ ID NO: 2.

[0083] In one embodiment, the mannanase variant according to the present invention having improved stability in detergents comprises one or more amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, H332D, G335D, A360G, A365V, D372G, Q381N, S391Y, F398L, K433T, E438G, I449T, K475N, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and the numbering is according to SEQ ID NO: 2. Mannanase variants according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D, H332D, G335D, A365V, D372G, Q381N, S391Y, E438G, and K475N. Mannanase variants according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from N39T, T45N, D64Q, S133D, E140S, and S168D. Preferably, mannanase variants according to the present invention with improved detergent stability comprise one or more amino acid substitutions selected from A360G and I449T, and the mannanase variant is at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, where the numbering is according to SEQ ID NO: 2.

[0084] In one embodiment, the mannanase variants with improved detergent stability are selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K A polypeptide at least 75% identical to SEQ ID NO:4, wherein the numbering is according to SEQ ID NO:2, and wherein the polypeptide has one or more amino acid substitutions selected from 255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T and A319D / E.

[0085] In one embodiment, the mannanase variants with improved detergent stability are selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K A polypeptide at least 75% identical to SEQ ID NO:4, having two or more amino acid substitutions selected from 255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, where numbering is according to SEQ ID NO:2.

[0086] In one embodiment, the mannanase variant with improved detergent stability comprises: (a) one or more amino acid substitutions selected from Q89V, N96D, L101V, A112N, A119Y, A124D, S127A, N129M / L / F, S281L, G286E / L / Q / A, W289F / M / H, N312Y, and A319E; (b) at least one amino acid substitution selected from D86N, Q89V, L101T, S103Y / E / A, K107N, N108G, N109Q / A, A119H / T, N122S, A124E / C, S126E, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S290A, N296H / F / Y, D301E / C / T, T309L, N312F, A314P, L317T, and A319E; A polypeptide at least 75% identical to SEQ ID NO:4, having in combination, an amino acid substitution as defined in (b) not present if a substitution is present at the corresponding position as defined in (a), and numbering is according to SEQ ID NO:2.

[0087] In one embodiment of the present invention, the mannanase variant disclosed above having a sequence according to SEQ ID NO: 4 with improved detergent stability comprises at least one conservative amino acid substitution at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, wherein the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 4, the numbering being according to SEQ ID NO: 2. The mannanase variant according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D. Mannanase variants according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, mannanase variants according to the present invention with improved detergent stability comprise one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, and I323L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 4, where the numbering is according to SEQ ID NO: 2.

[0088] In one embodiment, a mannanase variant according to the present invention with improved detergent stability comprises one or more amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, wherein the mannanase variant is at least 75% identical to a sequence according to SEQ ID NO: 4, with numbering according to SEQ ID NO: 2. A mannanase variant according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D. A mannanase variant according to the present invention with improved detergent stability may comprise a combination of amino acid substitutions selected from N39T, T45N, D64Q, S133D, E140S, S168D.

[0089] In one embodiment, the mannanase variant according to the present invention with improved detergent stability comprises one or more conserved amino acid regions within its polypeptide sequence. A conserved amino acid region herein is characterized by a sequence of several consecutive amino acids that are not mutated, and the number of consecutive amino acids may be 3 to 10, 4 to 10, 5 to 10, 6, 7, 8, 9, or 10. The one or more conserved amino acid regions may be G76-A77-N78-T79, R81-V83-L84, E115-V116-H117-D118, Y134-W135-I136, A154-N155-E156-W157, A191-G192-W193-G194-Q195, F218-S219-I220-H221-M222, or the like. -Y223-E224-Y225-A226-G227, N236-I237-D238, I249-G250-E251-F252-G253, G259-D260-V261-D262-E263 and G276-W277-L278-A279-W280, where the numbering is according to SEQ ID NO: 2.

[0090] Polynucleotides The present invention relates to polynucleotides encoding mannanase variants according to the invention. The polynucleotides of the present invention encode polypeptides that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In one embodiment of the present invention, the polynucleotides encode polypeptides that are at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence according to SEQ ID NO: 3 or SEQ ID NO: 4. Polynucleotides of the invention may have a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:1.

[0091] In one aspect, the present invention provides a polynucleotide encoding a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and comprising one or more amino acid substitutions at amino acid positions selected from D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317 and A319, wherein the numbering is according to SEQ ID NO:2.

[0092] Preferably, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and containing one or more amino acid substitutions at amino acid positions selected from D86, Q89, N96, L101, A112, A119, A124, S127, N129, S235, S281, G286, W289, N312 and A319, where numbering is according to SEQ ID NO:2.

[0093] In one embodiment, the polynucleotide has an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and is selected from the group consisting of D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D and A319D / E, wherein the numbering is according to SEQ ID NO: 2.

[0094] In one embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and containing one or more amino acid substitutions selected from D86N, Q89V / L, N96D, L101V, S103Y, A112N, A119Y / H / T, N122S, A124D / E / C, S126E, S127A, N129M / L / F, S231Q / K / L, S235Y / H / R, D244N, E264Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296Y / H, D301T / E / C, T309L, N312Y / F and A319E, where numbering is according to SEQ ID NO:2.

[0095] In one embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and containing one or more amino acid substitutions selected from D86N, Q89V, N96D, L101V, S103Y, A112N, A119Y / H / T, A124D / C, S126E, S127A, N129M / L / F, S235Y / H / R, D244N, S281L, G286E / L / Q / A, W289F / M / H, N296H, D301T / E, N312Y and A319E, where numbering is according to SEQ ID NO:2.

[0096] In one embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 and containing one or more amino acid substitutions selected from D86N, Q89V, N96D, L101V, A112N, A119Y, A124D / C, S127A, N129M / L / F, S235H, S281L, G286E / L / Q / A, W289F / M / H, N312Y and A319E, where numbering is according to SEQ ID NO:2.

[0097] In one embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 and containing at least one amino acid substitution selected from D328, G329, G330, D331, Y344, M359, Y374, L416 and W432, preferably selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, where numbering is according to SEQ ID NO:2, and wherein the polypeptide has mannan-degrading activity. More preferably, the polypeptide having an amino acid sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 (additionally) contains at least one amino acid substitution selected from D328I / Q, G329T, G330P, Y344Q / F, M359R / Y, Y374G / V / A7 and W432N / L / R / S.

[0098] In one embodiment, the polynucleotide has an amino acid sequence at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, and A319E; (b) at least one amino acid substitution selected from Q89V / L, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T, and A319D; and (b) encoding a polypeptide comprising the amino acid substitutions defined in (b) in combination, wherein the amino acid substitutions defined in (b) are absent when substitutions are present at the corresponding positions defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0099] In one embodiment, the polynucleotide has an amino acid sequence that is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, (a) one or more amino acid substitutions selected from D86N, N96D, L101V, N122S, A124D, S126E, S231Q / K / L, S235Y, D244N, W289M, S290A, N296Y, D301T, N312Y, and A319E; (b) at least one amino acid substitution selected from Q89V / L, S103Y, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R, E264Q, S281L, G286E / L / Q / A, W289F / H, N296H, D301E / C, T309L, and N312F; and (b) encoding a polypeptide comprising the amino acid substitutions defined in (b) in combination, wherein the amino acid substitutions defined in (b) are absent when substitutions are present at the corresponding positions defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0100] In one embodiment, the polynucleotide has an amino acid sequence that is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, (a) one or more amino acid substitutions selected from D86N, N96D, L101V, A124D, S126E, S235Y, D244N, W289M, D301T, N312Y, and A319E (b) at least one amino acid substitution selected from Q89V, S103Y, A112N, A119Y / H / T, A124C, S127A, N129M / L / F, S235H / R, S281L, G286E / L / Q / A, W289F / H, N296H, and D301E; and (b) encoding a polypeptide comprising the amino acid substitutions defined in (b) in combination, wherein the amino acid substitutions defined in (b) are absent when substitutions are present at the corresponding positions defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0101] In one embodiment, the polynucleotide has an amino acid sequence that is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, (a) one or more amino acid substitutions selected from D86N, N96D, L101V, A124D, W289M, N312Y, and A319E (b) at least one amino acid substitution selected from Q89V, A112N, A119Y, A124C, S127A, N129M / L / F, S235H, S281L, G286E / L / Q / A, and W289F / H; and (b) encoding a polypeptide comprising the amino acid substitutions defined in (b) in combination, wherein the amino acid substitutions defined in (b) are absent when substitutions are present at the corresponding positions defined in (a), the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

[0102] In one embodiment, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, wherein the numbering is according to SEQ ID NO: 2, comprising at least one amino acid substitution selected from D328, G329, G330, D331, Y344, M359, Y374, L416 and W432, preferably selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, and wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity. More preferably, the polynucleotide encodes a polypeptide having an amino acid sequence at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, wherein the numbering is according to SEQ ID NO: 2, and wherein the sequence contains at least one amino acid substitution selected from D328I / Q, G329T, G330P, Y344Q / F, M359R / Y, Y374G / V / A7 and W432N / L / R / S.

[0103] Polynucleotides according to the present invention include D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317, A31 9, D328I, G329, G330, D331, N341, Y344, F346, T348, E349, S352, G356, M359, Y374, D379, L416 and W432, wherein the numbering is according to SEQ ID NO: 2, and the polypeptide encodes a mannanase variant according to the present invention that is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, and comprises one or more amino acid substitutions selected from:

[0104] Polynucleotides according to the invention are at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 and include any of the following: D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N12 9M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E26 4Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, N341F, Y344Q / F / T, F346T, T348S / R / N / M / G, E349T / S / D / G, S352N / G, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V, L416W and W432P / N / L / R / S / T / G / H / I, and the numbering is according to SEQ ID NO: 2.

[0105] In one embodiment, the polynucleotide is at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 and is selected from the group consisting of Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E and / or W432P / N / L / R / S / T / G / H / I, wherein the numbering is according to SEQ ID NO: 2.

[0106] In one embodiment, the polynucleotide is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, A319E, G329T, G330D, D331A / Q, N341F, T348S / G, E349S / G, S352N / G, and L416W; (b)Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E at least one amino acid substitution selected from the group consisting of / C, T309L, N312F, L317T, A319D, D328I / Q / V, G329L / S / V, G330P / T, Y344Q / F / T, F346T, T348R / N / M, E349T / D, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V and W432P / N / L / R / S / T / G / H / I; The mannanase variants according to the present invention encode mannanase variants that are at least 75% identical to the sequences according to SEQ ID NO: 2 or SEQ ID NO: 3, in combination, and the amino acid substitutions defined in (b) are not present if substitutions are present at the corresponding positions defined in (a), and the numbering is according to SEQ ID NO: 2.

[0107] In one embodiment, the present invention provides a polynucleotide encoding a mannanase variant according to the present invention that is at least 75% identical to the sequence according to SEQ ID NO: 4, comprising one or more amino acid substitutions selected from D86, Q89, N96, L101, S103, K107, N108, N109, A112, A119, N122, A124, S126, S127, N129, S231, I233, S235, D244, H254, K255, E264, S270, Q272, K273, N274, S281, G286, W289, S290, N296, D301, T309, N312, A314, L317 and A319, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

[0108] Polynucleotides according to the invention are at least 75% identical to SEQ ID NO: 4 and include any of the following: D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K The mannanase variants according to the present invention encode mannanase variants according to the present invention, which comprise one or more amino acid substitutions selected from 255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T and A319D / E, and the numbering is according to SEQ ID NO: 2.

[0109] In one embodiment, the polynucleotide is at least 75% identical to a sequence according to SEQ ID NO: 4 and is selected from the group consisting of Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, The present invention encodes a mannanase variant according to the present invention, comprising one or more amino acid substitutions selected from E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T and A319D, wherein the numbering is according to SEQ ID NO: 2.

[0110] In one embodiment, the polynucleotide is (a) one or more amino acid substitutions selected from D86N, N96D, L101V, S103E / A, N122S, A124D, S126E, S231Q / K / L / P / Y, I233V, S235Y, D244N, K255R, S270T, K273T, N274Q, W289M, S290A, N296Y, D301T, N312Y, A314P, and A319E; (b) at least one amino acid substitution selected from Q89V, L101T, S103Y, K107N, N108G, N109Q / A, A112N, A119Y / H / T, A124E / C, S127A, N129M / L / F, S235H / R / L / Q / N, D244I / V, H254W, K255Y / H, E264Q / V, Q272I, K273T, N274E / C, S281L, G286E / L / Q / A, W289F / H, N296H / F, D301E / C, T309L, N312F, L317T, and A319D; In combination, the mannanase variants according to the present invention encode mannanase variants that are at least 75% identical to the sequence according to SEQ ID NO: 4, wherein the amino acid substitutions defined in (b) are not present if substitutions are present at the corresponding positions defined in (a), and the numbering is according to SEQ ID NO: 2.

[0111] In one aspect of the invention, the polynucleotide encoding the mannanase variant according to the invention encodes one or more conservative amino acid substitutions at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, the numbering being according to SEQ ID NO: 2. Polynucleotides encoding mannanase variants according to the present invention may encode a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D, V358I, S370A, Q423K, D459N, N461S, and V482L.Polynucleotides encoding mannanase variants according to the present invention may comprise a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, the polynucleotide encoding the mannanase variant according to the present invention encodes one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, the numbering being according to SEQ ID NO: 2.

[0112] In one embodiment, the polynucleotide encoding the mannanase variant according to the present invention encodes one or more amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, H332D, G335D, A360G, A365V, D372G, Q381N, S391Y, F398L, K433T, E438G, I449T, K475N, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, where the numbering is according to SEQ ID NO: 2. Polynucleotides encoding mannanase variants according to the invention may encode a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D, H332D, G335D, A365V, D372G, Q381N, S391Y, E438G, and K475N. Polynucleotides encoding mannanase variants according to the invention may encode a combination of amino acid substitutions selected from N39T, T45N, D64Q, S133D, E140S, and S168D. Preferably, polynucleotides encoding mannanase variants according to the invention encode one or more amino acid substitutions selected from A360G and I449T, and the mannanase variants are at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, where the numbering is according to SEQ ID NO: 2.

[0113] In one embodiment, a polynucleotide encoding a mannanase variant according to the present invention encodes one or more conserved amino acid regions within its polypeptide sequence. A conserved amino acid region herein is characterized by a sequence of several consecutive amino acids that are not mutated, and the number of consecutive amino acids may be 3 to 10, 4 to 10, 5 to 10, 6, 7, 8, 9, or 10. The one or more conserved amino acid regions may be G76-A77-N78-T79, R81-V83-L84, E115-V116-H117-D118, Y134-W135-I136, A154-N155-E156-W157, A191-G192-W193-G194-Q195, F218-S219-I220-H221-M222, or -Y223-E224-Y225-A226-G227, N236-I237-D238, I249-G250-E251-F252-G253, G259-D260-V261-D262-E263 and G276-W277-L278-A279-W280, where the numbering is according to SEQ ID NO: 2.

[0114] In one embodiment, the polynucleotide encodes a mannanase variant with improved detergent stability, wherein the polypeptide is at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 and is selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein numbering is according to SEQ ID NO: 2.

[0115] In one embodiment, the polynucleotide encodes a mannanase variant with improved detergent stability, wherein the mannanase is at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 and is selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein numbering is according to SEQ ID NO: 2.

[0116] In one embodiment, the polynucleotide encodes a mannanase variant having improved detergent stability, the mannanase comprising: (a) one or more amino acid substitutions selected from Q89V, N96D, L101V, A112N, A119Y, A124D, S127A, N129M / L / F, S281L, G286E / L / Q / A, W289F / M / H, N312Y, A319E, and W432P; (b)D86N, Q89V, L101T, S103Y / E / A, K107N, N108G, N109Q / A, A119H / T, N122S, A124E / C, S126E, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S29 at least one amino acid substitution selected from: 0A, N296H / F / Y, D301E / C / T, T309L, N312F, A314P, L317T, A319E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W, and W432N / L / R / S / T / G / H / I; A polypeptide at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3, including in combination, wherein the amino acid substitutions defined in (b) are not present when there is a substitution at the corresponding position defined in (a), and the numbering is according to SEQ ID NO:2.

[0117] In one aspect of the present invention, the polynucleotide encoding a mannanase variant with improved stability in detergents, having a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 disclosed above, encodes at least one additional amino acid substitution selected from N341F, F346T, T348S / R / N / M / G, E349T / S / G / D, S352N / G, G356Y / V / T / Q / H / C and D379V.

[0118] In one embodiment, the polynucleotide encoding the mannanase variant according to the invention with improved detergent stability further encodes one or more conservative amino acid substitutions at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, wherein the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, the numbering being according to SEQ ID NO: 2. Polynucleotides encoding mannanase variants according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D, V358I, S370A, Q423K, D459N, N461S, and V482L.Polynucleotides encoding mannanase variants according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, the polynucleotide encoding the mannanase variant according to the present invention having improved stability in detergents further encodes one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, I323L, N345D, V358I, S370A, N383S, N384S, Q423K, F435Y, D459N, N461S, I463V, V482L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, the numbering being according to SEQ ID NO: 2.

[0119] In one embodiment, the polynucleotide encoding the mannanase variant according to the present invention having improved stability in detergents encodes one or more additional amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, H332D, G335D, A360G, A365V, D372G, Q381N, S391Y, F398L, K433T, E438G, I449T, K475N, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, the numbering being according to SEQ ID NO: 2. Polynucleotides encoding mannanase variants according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D, H332D, G335D, A365V, D372G, Q381N, S391Y, E438G, and K475N. Polynucleotides encoding mannanase variants according to the present invention with improved detergent stability may encode a combination of substitutions selected from N39T, T45N, D64Q, S133D, E140S, and S168D. Preferably, polynucleotides encoding mannanase variants according to the present invention with improved detergent stability encode one or two amino acid substitutions selected from A360G and I449T, and the mannanase variants are at least 75% identical to a sequence according to SEQ ID NO: 2 or SEQ ID NO: 3, with the numbering according to SEQ ID NO: 2.

[0120] In one embodiment, the polynucleotide encodes a mannanase variant according to the invention having improved stability in detergent, wherein the mannanase is at least 75% identical to SEQ ID NO: 4 and has the following amino acids: D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I and one or more amino acid substitutions selected from 233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T and A319D / E, where numbering is according to SEQ ID NO: 2.

[0121] In one embodiment, the polynucleotide encodes a mannanase variant with improved detergent stability, wherein the mannanase is at least 75% identical to SEQ ID NO: 4 and is selected from the group consisting of D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233 V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T and A319D / E, and numbering is according to SEQ ID NO: 2.

[0122] In one embodiment, the polynucleotide encodes a mannanase variant having improved detergent stability, wherein the mannanase is at least 75% identical to SEQ ID NO: 4; (a) one or more amino acid substitutions selected from Q89V, N96D, L101V, A112N, A119Y, A124D, S127A, N129M / L / F, S281L, G286E / L / Q / A, W289F / M / H, N312Y, and A319E; (b) at least one amino acid substitution selected from D86N, Q89V, L101T, S103Y / E / A, K107N, N108G, N109Q / A, A119H / T, N122S, A124E / C, S126E, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S290A, N296H / F / Y, D301E / C / T, T309L, N312F, A314P, L317T, and A319E; In combination, an amino acid substitution as defined in (b) is not present if a substitution is present at the corresponding position as defined in (a), and the numbering is according to SEQ ID NO:2.

[0123] In one embodiment, a polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability further encodes one or more conservative amino acid substitutions at the following positions: T32S, N37S, F61Y, I80V, Y90W, T91S, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, N258D, I323L, wherein the mannanase variant is at least 75% identical to a sequence according to SEQ ID NO: 4, the numbering is according to SEQ ID NO: 2. A polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from T32S, T91S, K99R, S100N, V125I, D179N, S183N, Y196F, D206E, N258D. A polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from T32S, N37S, F61Y, I80V, Y90W, K99R, S100N, V125I, L150I, D179N, S183N, Y196F, D206E, D229N, and I323L. Preferably, a polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability further encodes one or more conservative amino acid substitutions selected from N37S, F61Y, I80V, Y90W, T91S, L150I, D229N, N258D, and I323L, and the mannanase variant is at least 75% identical to the sequence according to SEQ ID NO: 4, where the numbering is according to SEQ ID NO: 2.

[0124] In one embodiment, a polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability encodes one or more additional amino acid substitutions at the following positions: N39T, T45N, D64Q, S133D, E140S, S168D, A173V, Q202N, S305D, wherein the mannanase variant is at least 75% identical to a sequence according to SEQ ID NO: 4, with numbering according to SEQ ID NO: 2. A polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability may encode a combination of amino acid substitutions selected from S133D, S168D, A173V, Q202N, S305D. A polynucleotide encoding a mannanase variant according to the present invention with improved detergent stability may encode a combination of substitutions selected from N39T, T45N, D64Q, S133D, E140S, S168D.

[0125] In one embodiment, the polynucleotide encodes a mannanase variant according to the present invention having improved stability in detergents, comprising one or more contiguous conserved amino acid regions characterized in that consecutive amino acids are not mutated, and the number of contiguous amino acids is 3 to 10, 4 to 10, 5 to 10, 6, 7, 8, 9, or 10. The one or more conserved amino acid regions include G76-A77-N78-T79, R81-V83-L84, E115-V116-H117-D118, Y134-W135-I136, A154-N155-E156-W157, A191-G192-W193-G194-Q195, F218-S219-I220-H221-M222 -Y223-E224-Y225-A226-G227, N236-I237-D238, I249-G250-E251-F252-G253, G259-D260-V261-D262-E263 and G276-W277-L278-A279-W280, where the numbering is according to SEQ ID NO: 2.

[0126] Methods for improving stability in surfactants In one aspect, the present invention provides D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S1 27A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P / S, L317T and A319D / E, wherein the numbering is according to SEQ ID NO: 2. The present invention relates to a method for increasing the stability in a detergent of a mannanase that is at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 by introducing one or more amino acid substitutions at amino acid positions selected from among: SEQ ID NO: 2, SEQ ID NO: 3, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P / S, L317T and A319D / E, wherein the numbering is according to SEQ ID NO: 2.

[0127] The increased detergent stability of the mannanase variants of the present invention relates to increased storage stability after storage at 37°C for up to 6 days when compared to the detergent stability of the respective parent enzymes.

[0128] In one embodiment, the mannanase variant of the present invention has a polypeptide sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, containing at least one amino acid substitution selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I.

[0129] In one aspect, the present invention relates to D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q2 72I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D30 1E / C / T, T309L, N312F / Y, A314P / S, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein the numbering is according to SEQ ID NO: 2.

[0130] In one aspect, the present invention provides D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E , S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, The present invention relates to a method for increasing the detergent stability of a mannanase that is at least 75% identical to SEQ ID NO: 4 by introducing one or more amino acid substitutions at amino acid positions selected from N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P / S, L317T and A319D / E, wherein the numbering is according to SEQ ID NO: 2.

[0131] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is selected from the group consisting of D86N, Q89V / L, N96D, L101T / V, S103Y / E, K107N, N108G, N109Q, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / Y, by introducing one or more amino acid substitutions at amino acid positions selected from D244N, H254W, K255Y / H, E264Q / V, S270T, K273T, N274Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P / S, L317T and A319D / E, where numbering is according to SEQ ID NO: 2. In one embodiment, detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W, and W432P / N / L / R / S / T / G / H / I within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, numbering according to SEQ ID NO: 2. Preferably, detergent stability of the mannanase variant is increased by at least about 10% when compared to the respective parent enzyme.

[0132] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is determined by the following sequence: D86N, Q89V / L, N96D, L101T / V, S103Y / E, N108G, N109Q, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S23 and A319D / E, numbering is according to SEQ ID NO: 2. In one embodiment, detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I / Q / V, G329L / S / T, G330P / D / T, D331A, Y344Q / F, M359R / Y / C / , Y374G / V / A / R / N, L416W and W432P / N / L / R / S / T / G / H within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, numbering according to SEQ ID NO: 2. Preferably, detergent stability of the mannanase variant is increased by at least about 20% when compared to the respective parent enzyme.

[0133] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is selected from the group consisting of D86N, Q89V / L, N96D, L101V, S103Y / E, N108G, N109Q, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P, I233V, I246V, I247V, I248V, I249V, I250V, I251V, I252V, I253V, I254V, I255V, I256V, I257V, I258V, I259V, I260V, I261V, I262V, I263V, I264V, I265V, I266V, I267V, I268V, I269V, I270V, I271V, I272V, I273V, I274V, I275V, I276V, I277V, I278V, I279V, I280V, I281V, I282V, I283V, I284V, I285V, I286V, I287V, I288V, I289V, I290V, I291V, I292V, I293V, I294V, I295V, I296V, I297V, I298V, I299V, I300V, I301V, I302V, I303V, I The detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from S235H / R / L / Y, D244N, K255Y, E264Q / V, K273T, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / Y, D301E / C / T, T309L, N312F / Y and A319D / E, where numbering is according to SEQ ID NO: 2. In one embodiment, detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I / Q, G329T, G330P, Y344Q / F, M359R / Y, Y374G / V / A and W432N / L / R / S, where numbering is according to SEQ ID NO: 2, within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, the detergent stability of the mannanase variant is increased by at least about 30% when compared to the respective parent enzyme.

[0134] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is increased by introducing one or more amino acid substitutions at amino acid positions selected from D86N, Q89V / L, N96D, L101V, S103Y, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L, S235H / R / Y, D244N, E264Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / Y, D301E / C / T, T309L, N312F / Y and A319E, where numbering is according to SEQ ID NO:2. In one embodiment, the detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I, G329T, Y374G and W432N / L / R within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, numbering according to SEQ ID NO: 2. Preferably, the detergent stability of the mannanase variant is increased by at least about 40% when compared to the respective parent enzyme.

[0135] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 is increased by introducing one or more amino acid substitutions at amino acid positions selected from D86N, Q89V, N96D, L101V, S103Y, A112N, A119Y / H / T, A124E / C / D, S126E, S127A, N129M / L / F, S231Q, S235H / R / Y, D244N, S281L, G286E / L / Q / A, W289F / M / H, N296H, D301E / C / T, N312F / Y and A319E, where numbering is according to SEQ ID NO: 2. In one embodiment, the detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I, G329T, Y374G and W432R within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, numbering according to SEQ ID NO: 2. Preferably, the detergent stability of the mannanase variant is increased by at least about 50% when compared to the respective parent enzyme.

[0136] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 is increased by introducing one or more amino acid substitutions at amino acid positions selected from D86N, Q89V, N96D, L101V, S103Y, A112N, A119Y / H / T, A124C / D, S126E, S127A, N129M / L / F, S235H / R / Y, D244N, S281L, G286E / L / Q / A, W289F / M / H, N296H, D301E / T, N312F / Y and A319E, where numbering is according to SEQ ID NO: 2. In one embodiment, the detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I, Y374G and W432R within a sequence at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, numbering according to SEQ ID NO: 2. Preferably, the detergent stability of the mannanase variant is increased by at least about 60% when compared to the respective parent enzyme.

[0137] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 is increased by introducing one or more amino acid substitutions at amino acid positions selected from D86N, Q89V, N96D, L101V, A112N, A119Y / H, A124C / D, S127A, N129M / L / F, S235H / Y, D244N, S281L, G286E / L / Q / A, W289F / M / H, N296H, D301T, N312F / Y and A319E, where numbering is according to SEQ ID NO:2. In one embodiment, the detergent stability is increased by introducing one or more amino acid substitutions at amino acid positions selected from D328I and W432R within a sequence at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3, where numbering is according to SEQ ID NO:2. Preferably, the detergent stability of the mannanase variant is increased by at least about 70% when compared to the respective parent enzyme.

[0138] In one embodiment, the detergent stability of a mannanase at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 is increased by introducing one or more amino acid substitutions at amino acid positions selected from D86N, Q89V, N96D, L101V, A112N, A119Y, A124C / D, S127A, N129M / L / F, S235H, S281L, G286E / L / Q / A, W289F / M / H, N312Y and A319E, numbering according to SEQ ID NO: 2. Preferably, the detergent stability of the mannanase variant is increased by at least about 80% when compared to the respective parent enzyme.

[0139] Mannanase production The present invention also provides a method for producing a mannanase variant according to the invention, comprising the steps of: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide. The present invention relates to a method including:

[0140] The present invention also provides a method for expressing a mannanase variant according to the present invention, comprising the steps of: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide. The present invention relates to a method including:

[0141] A polynucleotide encoding a polypeptide can be "expressed." The term "expression" or "gene expression" refers to the transcription of one or more specific genes or specific nucleic acid constructs. The term "expression" or "gene expression" specifically refers to the transcription of one or more genes or gene constructs into structural RNA (e.g., rRNA, tRNA) or mRNA, with or without the subsequent translation of the latter into protein. This process includes transcription of DNA and processing of the resulting mRNA product.

[0142] As used herein, a nucleic acid construct refers to a nucleic acid molecule, either single-stranded or double-stranded, isolated from a naturally occurring gene or modified to contain a nucleic acid segment in a manner not otherwise found in nature, or of synthetic origin, that contains one or more regulatory sequences. The term "regulatory sequence" refers to a nucleic acid sequence necessary for expression of a polynucleotide encoding a mannanase of the present invention. Each regulatory sequence may be native or foreign to the polynucleotide encoding the variant, or may be native or foreign to each other. Such regulatory sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, regulatory sequences include a promoter and transcriptional and translational stop signals. Regulatory sequences may also include linkers for the purpose of introducing specific restriction sites to facilitate ligation of regulatory sequences with the coding region of a polynucleotide encoding a variant.

[0143] Industrial production of enzymes is usually carried out by using an expression system. "Expression system" may refer to a host microorganism, expression host, host cell, production organism, or production strain, and these terms may be used interchangeably. In one embodiment, the expression host is selected from the group consisting of a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system. The expression host can be a wild-type cell or a recombinant cell, and is preferably a recombinant cell. As used herein, "wild-type cell" refers to a cell prior to a particular modification. The term "recombinant cell" (also referred to herein as "genetically modified cell") refers to a cell that has been genetically altered, modified, or engineered to exhibit an altered, modified, or different genotype compared to the wild-type from which it was derived. A "recombinant cell" may contain an exogenous polynucleotide encoding a particular protein or enzyme and thus be capable of expressing said protein or enzyme.

[0144] In one embodiment, the present invention is directed to a recombinant host cell comprising a polynucleotide encoding the mannanase described herein. The host cell can be any cell useful in the recombinant production of variants, including prokaryotic and eukaryotic cells.

[0145] Examples of expression hosts include, but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, Fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably selected from Bacillus subtilis, Bacillus pumilus and Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris, pastoris (also known as Komagataella phaffii), Myceliopthora thermophila (C1), Thermothelomyces thermophilus, Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei, and Saccharomyces, preferably Saccharomyces cerevisiae. The mannanase variants according to the present invention can be produced using host cells derived from the microorganisms listed above.

[0146] In one embodiment, the bacterial expression system is selected from E. coli, Bacillus, Pseudomonas, and Streptomyces. In one embodiment, the yeast expression system is selected from Candida, Pichia, Saccharomyces, and Schizosaccharomyces. In one embodiment, the fungal expression system is selected from Penicillium, Aspergillus, Fusarium, Myceliopthora, Rhizomucor, Rhizopus, Thermomyces, and Trichoderma.

[0147] Preferably, the recombinant host cells of the present invention are Gram-positive bacteria, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. More preferably, the host cell is a Bacillus cell, more preferably selected from the group consisting of Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and Bacillus stearothermophilus. stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis. Most preferably, the Bacillus cell is selected from Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, and Bacillus lentus. In one embodiment, the Bacillus cell is a Bacillus subtilis cell.

[0148] The present invention provides a fermentation method for producing a fermentation product, comprising: a) providing a recombinant host cell according to the present invention; and b) culturing the recombinant host cell under conditions that allow expression of the polynucleotide encoding the mannanase of the invention; The present invention provides a method comprising:

[0149] The term "heterologous" (or exogenous or foreign or recombinant) in the context of polynucleotides and polypeptides is defined herein as follows: (a) is not native to the host cell; or (b) native to the host cell but contains structural modifications, e.g., deletions, substitutions, and / or insertions, resulting from manipulation of the host cell's DNA by recombinant DNA techniques to alter the native sequence; or (c) Native to the host cell, but expression is quantitatively altered or directed from a different genomic location than in the native host cell as a result of manipulation of the host cell's DNA by recombinant DNA techniques (e.g., a stronger promoter).

[0150] Preferably, "heterologous" herein means "not native to the host cell."

[0151] In one aspect, the present invention relates to a host cell, preferably a Bacillus host cell, that expresses a polynucleotide encoding a polypeptide at least 75% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, comprising the amino acid substitutions disclosed above.

[0152] In one embodiment, the host cell expresses a polynucleotide encoding a mannanase variant that is at least 75% identical to SEQ ID NO: 2 or SEQ ID NO: 3, having an amino acid substitution at one or more positions selected from N341F, F346T, T348S / R / N / M / G, E349T / S / G / D, S352N / G, G356Y / V / T / Q / H / C and D379V, where numbering is according to SEQ ID NO: 2.

[0153] In one embodiment, the present invention is directed to a genetic construct comprising a polynucleotide encoding a mannanase of the present invention. As used herein, a "genetic construct" or "expression cassette" or "expression construct" refers to a DNA molecule comprising at least one polynucleotide sequence of the present invention to be expressed, operably linked to one or more control sequences (at least a promoter) described herein. Typically, an expression cassette contains three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site.

[0154] Additional regulatory elements may include transcriptional and translational enhancers. Intron sequences may also be added to the 5' untranslated region (UTR) or within the coding sequence to increase the amount of mature message that accumulates in the cytosol. Expression cassettes may be part of a vector or may be integrated into the genome of the host cell and replicated along with the genome of the host cell. Expression cassettes are typically capable of increasing or decreasing expression.

[0155] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the invention in bacterial host cells include the alpha-amylase gene of Bacillus amyloliquefaciens (amyQ), the alpha-amylase gene of Bacillus licheniformis (amyl), the penicillase gene of Bacillus licheniformis (penicillase, penP), the maltogenic amylase gene of Bacillus stearothermophilus (amyM), the levansucrase gene of Bacillus subtilis (sacB), the xylA and xylB genes of Bacillus subtilis, the cryIIIA gene of Bacillus thuringiensis (Agaisse and Lereclus, 1994, Molecular Biology 13: 97-107), the lac operon of E. coli, and the trc promoter of E. coli (Egon et al., 1988, Gene 69: 301-315), the agarose gene (dagA) of Streptomyces coelicolor, and the prokaryotic beta-lactamase gene (Villa-Kamaroff et al. 1978, Proc. Natl. Acad. Sci USA 75: 3727-3731), as well as the tac promoter (DaBoer et al. 1983, Proc. Natl. Acad. Sci USA 80: 21-25). Additional useful promoters are described in "Useful proteins from recombinant bacteria," Gilbert et al. 1980, Scientific American 242: 74-94, and Sambrook, J. et al. 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0156] As used herein, the term "vector" includes any type of construct suitable for carrying an exogenous polynucleotide sequence for transfer into another cell or for stable or transient expression within a given cell. As used herein, the term "vector" encompasses any type of cloning vehicle, including, but not limited to, plasmids, phagemids, viral vectors (e.g., phages), bacteriophages, baculoviruses, cosmids, fosmids, artificial chromosomes, or any other vector specific to a particular host of interest. Low-copy number and high-copy number vectors are also included. The exogenous polynucleotide sequence typically includes a coding sequence, which may be referred to herein as a "gene of interest." The gene of interest may include introns and exons, depending on the type of origin or destination of the host cell.

[0157] As used herein, a vector may provide segments for the transcription and translation of an exogenous polynucleotide upon transformation into a host cell or host cell organelle. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication necessary for its maintenance and / or replication in a particular cell type, one or more selectable markers, a polyadenylation signal, a suitable site for insertion of an exogenous coding sequence, e.g., a multiple cloning site, etc. One example is when the vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g., a plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the f1-ori and colE1. A vector can replicate without integrating into the host cell genome, e.g., as a plasmid in a bacterial host cell, or it can integrate some or all of its DNA into the host cell genome, resulting in replication and expression of that DNA.

[0158] Exogenous nucleic acids can be introduced into vectors by cloning. Cloning can mean that cleavage of the vector (e.g., within a multiple cloning site) and the exogenous polynucleotide by suitable means and methods (e.g., restriction enzymes) can create compatible structures within the individual nucleic acids that allow for controlled fusion of the exogenous nucleic acid with the vector. Once introduced into a vector, the exogenous nucleic acid, including the coding sequence, can be introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelle. A cloning vector suitable for expression of the exogenous polynucleotide sequence in the host cell or host cell organelle can be selected.

[0159] The terms "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer. That is, the term "transformation" as used herein is independent of vectors, shuttle systems, or host cells, and refers not only to polynucleotide transfer methods of transformation known in the art (see, e.g., Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but also encompasses any additional type of polynucleotide transfer method, such as, but not limited to, transduction or transfection. Plant tissue capable of subsequent clonal propagation, either by organogenesis or embryogenesis, can be transformed with a genetic construct and whole plants can be regenerated therefrom. The particular tissue selected will depend on the clonal propagation system available for and best suited to the particular species being transformed. In one embodiment of the present invention, a vector is used to transform a host cell.

[0160] The polynucleotides of the present invention can be transiently or stably introduced into a host cell, preferably a Bacillus host cell, and can be maintained in a non-integrated state, for example, as a plasmid. "Stable transformation" means that the transformed cell or cellular organelle passes on the nucleic acid containing the exogenous coding sequence to the cells or cellular organelles of subsequent generations. Typically, stable transformation occurs when the nucleic acid containing the exogenous coding sequence is integrated into a chromosome or as an episome (a separate element of nuclear DNA). "Transient transformation" means that once transformed, the cell or cellular organelle expresses the exogenous nucleic acid sequence for a period of time, usually within one generation. Typically, transient transformation occurs when the nucleic acid containing the exogenous nucleic acid sequence is not integrated into a chromosome or as an episome; alternatively, it is integrated into the host genome.

[0161] Enzymes are typically produced as liquid concentrates, often derived from fermentation broth. As used herein, "liquid enzyme concentrate" refers to any liquid enzyme-containing product that contains at least one enzyme. In the context of enzyme concentrates, "liquid" refers to their physical appearance at 20°C and 101.3 kPa.

[0162] A liquid enzyme concentrate can be produced by dissolving a solid enzyme in a solvent. The solvent can be selected from water and organic solvents. The liquid enzyme concentrate produced by dissolving a solid enzyme in a solvent can contain an amount of enzyme up to a saturation concentration.

[0163] As used herein, dissolution means that a solid compound is liquefied by contact with at least one solvent. Dissolution refers to the complete dissolution of the solid compound without phase separation until a saturation concentration is reached in the specified solvent.

[0164] In one aspect of the invention, the enzyme concentrate may be essentially water-free, meaning that no significant amount of water is present. As used herein, an insignificant amount of water means that the enzyme concentrate according to the invention contains less than 25%, 20%, 15%, 10%, 7%, 5%, 4%, 3%, 2% or no water, all by weight, based on the total weight of the enzyme concentrate. In one embodiment, a water-free enzyme concentrate means that the enzyme concentrate does not contain significant amounts of water and contains organic solvent in an amount of about 10%-90%, 20%-85%, 30%-80%, 40%-75%, or 50%-70% by weight, based on the total weight of the enzyme concentrate.

[0165] A liquid enzyme concentrate containing water may also be referred to as an "aqueous enzyme concentrate." In one embodiment, an "aqueous enzyme concentrate" is an enzyme-containing solution in which a solid enzyme product is dissolved in water. In one embodiment, "aqueous enzyme concentrate" refers to an enzyme-containing product resulting from enzyme production by fermentation.

[0166] Fermentation refers to the process of culturing recombinant cells expressing a desired enzyme in a suitable nutrient medium that allows the recombinant host cells to grow and express the desired protein. At the end of fermentation, the fermentation broth is usually collected and further processed, and the fermentation broth contains a liquid fraction and a solid fraction. Depending on whether the enzyme is secreted into the liquid fraction, the desired protein or enzyme can be recovered from the liquid fraction of the fermentation broth or from the cell lysate. The recovery of the desired enzyme uses methods known to those skilled in the art. Suitable methods for recovering the protein or enzyme from the fermentation broth include, but are not limited to, collection, centrifugation, filtration, extraction, and precipitation.

[0167] The aqueous enzyme concentrate resulting from fermentation may contain enzymes in an amount ranging from 0.1% to 40% by weight, or from 0.5% to 30% by weight, or from 1% to 25% by weight, or from 3% to 25% by weight, or from 5% to 25% by weight, all based on the total weight of the enzyme concentrate.

[0168] Aqueous enzyme concentrates resulting from fermentation typically contain water in an amount greater than about 50 wt. %, greater than about 60 wt. %, greater than about 70 wt. %, or greater than about 80 wt. %, based on the total weight of the enzyme concentrate. In one embodiment, aqueous enzyme concentrates resulting from fermentation contain water in an amount ranging from about 50 wt. % to 80 wt. % or from about 60 wt. % to 70 wt. %, based on the total weight of the enzyme concentrate. Aqueous enzyme concentrates resulting from fermentation may contain residual components, such as salts from the fermentation medium, cellular debris from the production host cells, and metabolic products produced by the production host cells during fermentation. In one embodiment, residual components may be present in the liquid (aqueous) enzyme concentrate (derived from fermentation) in an amount less than 30 wt. %, less than 20 wt. %, less than 10 wt. %, or less than 5 wt. %, based on the total weight of the aqueous enzyme concentrate.

[0169] Enzymes tend to lose enzymatic activity when left in an aqueous environment, and therefore it is conventional practice to convert them to anhydrous forms: aqueous concentrates may be freeze-dried or spray-dried, for example, in the presence of a carrier material, to form aggregates. Solid enzyme products usually need to be "dissolved" before use. To stabilize enzymes in liquid products, enzyme inhibitors, preferably reversible enzyme inhibitors, may be utilized to temporarily inhibit enzyme activity until the enzyme inhibitor is released.

[0170] Enzyme preparations The enzyme preparations of the present invention are preferably liquid. In the context of an enzyme preparation, "liquid" refers to its physical appearance at 20°C and 101.3 kPa.

[0171] The enzyme preparation of the present invention includes a liquid enzyme concentrate containing at least one mannanase variant according to the present invention, and contains only components effective for stabilizing the enzyme preparation or the enzyme contained in the enzyme preparation, such as at least one enzyme stabilizer, at least one compound that stabilizes the liquid enzyme preparation itself, and at least one solvent.

[0172] In one aspect, the present invention provides a liquid enzyme preparation comprising a mannanase variant according to the present invention that is at least 75% identical to SEQ ID NO: 2, preferably a mannanase that is at least 75% identical to the sequence according to SEQ ID NO: 3, at least one compound that stabilizes the liquid enzyme preparation itself, at least one solvent, and optionally at least one enzyme stabilizer.

[0173] The liquid enzyme preparations of the present invention are preferably surfactant-free. By surfactant-free, we mean that the enzyme preparation contains less than about 15% by weight of surfactant, based on the total weight of the enzyme preparation. In this specification, surfactant in the context of the enzyme preparation refers to the total amount of surfactant.

[0174] Preferably, surfactant-free means that the enzyme preparation contains less than about 10 wt.%, less than about 7 wt.%, less than about 5 wt.%, less than about 3 wt.%, less than about 2 wt.%, or less than about 1 wt.% surfactant, based on the total weight of the enzyme preparation.

[0175] In one embodiment, surfactant-free means that no actively added surfactants are included in the enzyme preparation of the present invention, which means that the enzyme preparation of the present invention may contain surfactants (as by-products) that result from the fermentation process from which the enzyme concentrate is derived.

[0176] The liquid enzyme preparation of the present invention preferably does not contain a complexing agent. By "complexing agent-free," we mean that the liquid enzyme preparation contains less than about 15% by weight of a complexing agent, based on the total weight of the liquid enzyme preparation. In the context of the liquid enzyme preparation of the present invention, "complexing agent" refers to the total amount of complexing agent.

[0177] Preferably, free of complexing agents means that the enzyme preparation contains less than about 10 wt. %, less than about 7 wt. %, less than about 5 wt. %, less than about 3 wt. %, less than about 2 wt. %, or less than about 1 wt. % complexing agents, based on the total weight of the liquid enzyme preparation.

[0178] In one embodiment, complexing agent-free means that the enzyme preparation of the present invention does not contain actively added complexing agents, which means that the enzyme preparation of the present invention may contain complexing agents (as by-products) that result from the fermentation process from which the enzyme concentrate is derived.

[0179] In one embodiment, the liquid enzyme preparation of the present invention is surfactant-free and complexing agent-free.

[0180] Compounds that stabilize the liquid enzyme preparation itself A compound that stabilizes the liquid enzyme preparation itself means any compound other than an enzyme stabilizer that is required to establish the storage stability of the liquid preparation in an amount effective to ensure storage stability.

[0181] Storage stability in the context of liquid formulations to those skilled in the art typically includes the aspects of product appearance and dosage uniformity.

[0182] Product appearance is influenced by the pH of the product and the presence of compounds such as preservatives, antioxidants, viscosity modifiers, emulsifiers, and the like.

[0183] Uniformity of dosage is usually related to homogeneity of the product.

[0184] The enzyme preparation of the present invention may be alkaline or may exhibit a neutral or slightly acidic pH value, and may have a pH in the range of 5 to 12, preferably in the range of 6 to 11, and more preferably in the range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10, and 7.5 to 8.5.

[0185] The liquid enzyme preparation of the present invention may comprise at least one preservative. The preservative is added in an amount effective to prevent microbial contamination of the liquid enzyme preparation, preferably the aqueous enzyme preparation. The at least one preservative may be selected from: Benzylhemiformal, also known as (benzyloxy)methanol (CAS No. 14548-60-8); (Ethylenedioxy)dimethanol, also known as Dascocide 9; (Ethylenedioxy)dimethanol (reaction product of ethylene glycol and paraformaldehyde (EGForm)) (CAS. No. 3586-55-8); α,α',α"-Trimethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-triethanol, also known as: tris(N-hydroxypropyl)hexahydrotriazine, hexahydro-1,3,5-tris(2-hydroxypropyl)-s-triazine (HPT, CAS No. 25254-50-6); 2,2-Dibromo-2-cyanoacetamide (DBNPA, CAS No. 10222-01-2); 2,2'-dithiobis[N-methylbenzamide] (DTBMA, CAS No. 2527-58-4); 2-Bromo-2-(bromomethyl)pentanedinitrile (DBDCB, CAS No. 35691-65-7); 2-Butanone, peroxide, also known as: 2-butanone-peroxide (CAS No. 1338-23-4); 2-butyl-benzo[d]isothiazol-3-one (BBIT, CAS No. 4299-07-4); 2-methyl-2H-isothiazol-3-one (MIT, CAS No. 2682-20-4); 2-octyl-2H-isothiazol-3-one (OIT, CAS No. 26530-20-1); 5-chloro-2-methyl-2H-isothiazol-3-one (CIT, CMIT, CAS No. 26172-55-4); a mixture of 5-chloro-2-methyl-2H-isothiazol-3-one (CMIT, EINECS 247-500-7) and 2-methyl-2H-isothiazol-3-one (MIT, EINECS 220-239-6) (CMIT / MIT mixture, CAS No. 55965-84-9); 1,2-benzisothiazol-3(2H)-one (BIT, CAS No. 2634-33-5); 3,3'-methylenebis[5-methyloxazolidine] (oxazolidine / MBO, CAS No. 66204-44-2); 4,4-dimethyloxazolidine (CAS No. 51200-87-4); 7a-Ethyldihydro-1H,3H,5H-oxazolo[3,4-c]oxazole (EDHO, CAS No. 7747-35-5); Benzyl alcohol (CAS No. 100-51-6); Biphenyl-2-ol (CAS No. 90-43-7); Biphenyl-2-ol and its salts, o-phenylphenol, MEA-o-phenylphenate, potassium phenylphenate, sodium phenylphenate; Sodium 2-biphenylate (CAS No. 132-27-4); cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (cis CTAC, CAS No. 51229-78-8); Didecyldimethylammonium chloride (DDAC, CAS No. 68424-95-3 and CAS No. 7173-51-5); Dodecylguanidine monohydrochloride (CAS No. 13590-97-1); Ethanol (CAS. No. 64-17-5); n-propanol (1-propanol, CAS No. 71-23-8); Hexa-2,4-dienoic acid (sorbic acid, CAS No. 110-44-1) and its salts (e.g., calcium sorbate, sodium sorbate); Potassium (E,E)-hexa-2,4-dienoate (potassium sorbate, CAS No. 24634-61-5); Hydrogen peroxide (CAS No. 7722-84-1); Lactic acid and its salts; L-(+)lactic acid (CAS No.79-33-4); 2-methyl-1,2-benzothiazol-3(2H)-one (MBIT, CAS No. 2527-66-4); Methenamine 3-chloroallylochloride (CTAC, CAS No. 4080-31-3); Monochloroamine produced from ammonium carbamate and a chlorine source N,N'-methylenebismorpholine (MBM, CAS No. 5625-90-1); N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (diamine, CAS No. 2372-82-9); N-(trichloromethylthio)phthalimide (Folpet, CAS No. 133-07-3); p-[(diiodomethyl)sulfonyl]toluene (CAS No. 20018-09-1); Peracetic acid (CAS No. 79-21-0); Polyhexamethylene biguanide hydrochloride (PHMB, CAS No. 1802181-67-4), polyhexamethylene biguanide hydrochloride (PHMB, CAS No. 27083-27-8), such as poly(iminoimidocarbonyl)iminohexamethylene hydrochloride, poly(iminocarbonimidoyliminocarbonimidoylimino-1,6-hexanediyl), polyaminopropyl biguanide; Pyridine-2-thiol 1-oxide, sodium salt (sodium pyrithione, CAS No. 3811-73-2); Zinc pyrithione (zinc pyrithione, CAS No. 13463-41-7); Silver chloride (CAS No. 7783-90-6), a reaction product of titanium dioxide and silver chloride; Sodium azide (CAS No. 26628-22-8); tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione (TMAD, CAS No. 5395-50-6); tetrakis(hydroxymethyl)phosphonium sulfate (2:1) (THPS, CAS No. 55566-30-8); Salts of benzoic acid, such as ammonium benzoate, calcium benzoate, magnesium benzoate, MEA benzoate, potassium benzoate; Esters of benzoic acid, for example, butyl benzoate, ethyl benzoate, isobutyl benzoate, isopropyl benzoate, methyl benzoate, phenyl benzoate, propyl benzoate; Benzoic acid and its sodium salt (CAS No. 65-85-0, CAS No. 532-32-1); Propionic acid and its salts, such as ammonium propionate, calcium propionate, magnesium propionate, potassium propionate, sodium propionate; Salicylic acid and its salts, such as calcium salicylate, magnesium salicylate, MEA salicylate, sodium salicylate, potassium salicylate, TEA salicylate; Inorganic sulfites and bisulfites, for example, sodium sulfite, ammonium sulfite, ammonium bisulfite, potassium sulfite, potassium bisulfite, sodium bisulfite, sodium metasulfite, potassium metasulfite, potassium metabisulfite; Chlorobutanol (CAS No. 57-15-8); Butyl 4-hydroxybenzoate and its salts, such as butylparaben, sodium butylparaben, potassium butylparaben; Propyl 4-hydroxybenzoate and its salts, such as propylparaben, sodium propylparaben, potassium propylparaben; Isopropyl-4-hydroxybenzoic acid and its salts and esters; Isobutyl-4-hydroxybenzoic acid and its salts and esters; Benzyl-4-hydroxybenzoic acid and its salts and esters; Pentyl-4-hydroxybenzoic acid and its salts and esters; 4-hydroxybenzoic acid and its salts and esters, for example, methylparaben, ethylparaben, potassium ethylparaben, potassium paraben, potassium methylparaben, sodium methylparaben, sodium ethylparaben, sodium paraben, calcium paraben, calcium methylparaben, calcium ethylparaben; 3-acetyl-6-methylpyran-2,4(3H)-dione and its salts, such as dehydroacetic acid, sodium dehydroacetate (Cas No. 520-45-6, 4418-26-2, 16807-48-0); 3,3'-Dibromo-4,4'-hexamethylenedioxydibenzamidine and its salts (including isethionates), such as dibromohexamidine isethionate (CAS No. 93856-83-8); Thiomersal (CAS No. 54-64-8); Phenylmercury salts (including borates), for example, phenylmercury acetate, phenylmercury benzoate (CAS Nos. 62-38-4 and 94-43-9); Undec-10-enoic acid and its salts, such as undecylenic acid, potassium undecylenate, sodium undecylenate, calcium undecylenate, MEA undecylenate, TEA undecylenate; 5-Pyrimidineamines, 1,3-bis(2-ethylhexyl)hexahydro-5-methyl-, for example, hexetidine (CAS No. 141-94-6); 1-(4-chlorophenyl)-3-(3,4-dichlorophenyl)urea, for example, triclocarban (CAS No. 101-20-2); Chlorocresols, such as p-chloro-m-cresol (CAS No. 59-50-7); Chloroxylenol (CAS No. 88-04-0, 1321-23-9); N,N"-Methylenebis[N'-[3-(hydroxymethyl)-2,5-dioxoimidazolidin-4-yl]urea], also known as: Imidazolidinyl urea (CAS No. 39236-46-9); Methenamine (CAS No. 100-97-0); Methenamine 3-chloroallyl chloride, also known as Quaternium-15 (CAS No. 4080-31-3); 1-(4-chlorophenoxy)-1-(imidazol-1-yl)-3,3-dimethylbutan-2-one, also known as climbazole (CAS No. 38083-17-9); 1,3-Bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, also known as DMDM hydantoin (CAS No. 6440-58-0); 1-hydroxy-4-methyl-6-(2,4,4-trimethylpentyl)-2 pyridone and its monoethanolamine salts, such as 1-hydroxy-4-methyl-6-(2,4,4-trimethylpentenyl)-2 pyridone, piroctone olamine (CAS No. 50650-76-5, 68890-66-4); 2,2'-methylenebis(6-bromo-4-chlorophenol), also known as: bromochlorophene (CAS No. 15435-29-7); 4-Isopropyl-m-cresol, also known as: o-cymen-5-ol (CAS No. 3228-02-2); 2-benzyl-4-chlorophenol, also known as: chlorophene (CAS No. 120-32-1); 2-Chloroacetamide (CAS No. 79-07-2); N,N'-bis(4-chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediamidine and its digluconates, diacetates and dihydrochlorides, for example, chlorhexidine, chlorhexidine digluconate, chlorhexidine diacetate, chlorhexidine dihydrochloride (CAS Nos. 55-56-1, 56-95-1, 18472-51-0, 3697-42-5); Alkyl (C12-C22)trimethylammonium bromides and chlorides, such as behentrimonium chloride, cetrimonium bromide, cetrimonium chloride, lautrimonium bromide, lautrimonium chloride, steartrimonium bromide, steartrimonium chloride (CAS Nos. 17301-53-0, 57-09-0, 112-02-7, 1119-94-4, 112-00-5, 1120-02-1, 112-03-8); 4,4-dimethyl-1,3-oxazolidine (CAS No. 51200-87-4); N-(hydroxymethyl)-N-(dihydroxymethyl-1,3-dioxo-2,5-imidazolidinyl-4)-N'-(hydroxymethyl)urea, also known as diazolidinylurea (CAS No. 78491-02-8); Benzenecarboximidamide, 4,4'-(1,6-hexanediylbis(oxy))bis-, and its salts (including isothionates and p-hydroxybenzoates), such as hexamidine, hexamidine diisethionate, hexamidine paraben (CAS Nos. 3811-75-4, 659-40-5, 93841-83-9); 5-Ethyl-3,7-dioxa-1-azabicyclo[3.3.0]octane, also known as: 7-ethylbicyclooxazolidine (CAS No. 7747-35-5); 3-(p-chlorophenoxy)-propane-1,2-diol, also known as: chlorophenesin (CAS No. 104-29-0); Sodium hydroxymethylaminoacetate, also known as: sodium N-(hydroxymethyl)glycinate, sodium hydroxymethylglycinate (CAS No. 70161-44-3); Benzenemethanaminium, N,N-dimethyl-N-[2-[2-[4-(1,1,3,3,-tetramethylbutyl)phenoxy]ethoxy]ethyl]-, chloride, also known as: benzethonium chloride (CAS No. 121-54-0); Benzalkonium chlorides, bromides and saccharinates, such as benzalkonium chloride, benzalkonium bromide, benzalkonium saccharinate (CAS Nos. 8001-54-5, 63449-41-2, 91080-29-4, 68989-01-5, 68424-85-1, 68391-01-5, 61789-71-7, 85409-22-9); Methanol, (phenylmethoxy), also known as: benzylhemiformal (CAS. No. 14548-60-8); 3-iodo-2-propynyl butylcarbamate (IPBC, CAS No. 55406-53-6); Ethyl lauroyl arginate HCl (CAS No. 60372-77-2); 1,2,3-propanetricarboxylic acid, 2-hydroxy-, monohydrate and 1,2,3-propanetricarboxylic acid, 2-hydroxy-silver(1+) salt, monohydrate, INCI: citrate (and) silver citrate; Tetrahydro-3,5-dimethyl-1,3,5-thiadiazine-2-thione (also known as 3,5-dimethyl-1,3,5-thiadiazinan-2-thione, Protectol® DZ, Protectol® DZ P, Dazomet, (CAS No. 533-74-4); 2,4-Dichlorobenzyl alcohol (CAS No. 1777-82-8, also known as dichlorobenzyl alcohol, 2,4-dichloro-benzenemethanol, (2,4-dichlorophenyl)methanol, DCBA, Protectol® DA); 1-propanol (CAS No. 71-23-8, also known as n-propanol, propan-1-ol, n-propyl alcohol, Protectol® NP S); 5-Bromo-5-nitro-1,3-dioxane (CAS No. 30007-47-7, also known as 5-bromo-5-nitro-m-dioxane, Bronidox®); 2-Bromo-2-nitropropane-1,3-diol (CAS No. 52-51-7, also known as 2-bromo-2-nitro-1,3-propanediol, Bronopol®, Protectol® BN, Myacide AS); Glutaraldehyde (CAS No. 111-30-8, also known as: 1-5-pentanedial, pentane-1,5-dial, glutaral, glutaric dialdehyde, Protectol® GA, Protectol® GA 50, Myacide® GA); Glyoxal (CAS No. 107-22-2; synonyms: ethanedial, oxylaldehyde, 1,2-ethanedial, Protectol® GL); 2,4,4'-trichloro-2'-hydroxydiphenyl ether (CAS No. 3380-34-5, also known as triclosan, Irgasan® DP 300, Irgacare® MP, TCS); 4,4'-Dichloro-2-hydroxydiphenyl ether (CAS No. 3380-30-1), also known as 5-chloro-2-(4-chlorophenoxy)phenol, Diclosan, DCPP (commercially available under the trade name Tinosan® HP 100 (BASF) as a 30 wt% solution of 4,4'-dichloro-2-hydroxydiphenyl ether in 1,2-propylene glycol); 2-phenoxyethanol (CAS No. 122-99-6, also known as: phenoxyethanol, methylphenyl glycol, Phenoxetol, ethylene glycol phenyl ether, ethylene glycol monophenyl ether, Protectol® PE); Phenoxypropanol (CAS No. 770-35-4, CAS No. 4169-04-4, propylene glycol phenyl ether, phenoxyisopropanol, 1-phenoxy-2-propanol, 2-phenoxy-1-propanol); Glucoprotamine (CAS No. 164907-72-6, chemical description: reaction product of glutamic acid and alkylpropylenediamine, alias: Glucoprotamine 50); Cyclohexylhydroxyldiazenium-1-oxide, potassium salt (CAS No. 66603-10-9, also known as N-cyclohexyl-diazenium dioxide, potassium HDO, Xyligene, Protectol® KD); Formic acid (CAS No. 64-18-6, also known as methanoic acid, Protectol® FM, Protectol® FM 75, Protectol® FM 85, Protectol® FM 99, Lutensol® FM) and its salts, such as sodium formate (CAS No. 141-53-7); performic acid and its salts; Inorganic silver complexes, such as silver zeolites and silver glass compounds (e.g. Irgaguard® B5000, Irgaguard® B6000, Irgaguard® B7000) and others described in WO-A-99 / 18790 (EP 1 041 879 B1); 1,3,5-tris-(2-hydroxyethyl)-hexahydro-1,3,5-triazine (CAS No. 4719-04-4, also known as hexyhydrotriazine, tris(hydroethyl)hexyhydrotriazine, hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine, 2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol, Protectol® HT).

[0186] One or more preservatives disclosed above may be added to the enzyme preparation at a concentration of 0.0001 to 10% based on the total weight of the enzyme preparation.

[0187] Preferably, the enzyme preparation comprises 2-phenoxyethanol at a concentration of 0.01% to 5%, more preferably 0.1% to 2%, and / or 2-bromo-2-nitropropane-1,3-diol at a concentration of 5 ppm to 5000 ppm, more preferably 20 ppm to 1000 ppm, and / or glutaraldehyde at a concentration of 2 ppm to 5000 ppm, more preferably 10 ppm to 2000 ppm, and / or formic acid (as the acid or its salts) at a concentration of 0.01% to 3%, more preferably 0.05% to 0.5%, and / or 4,4'-dichloro-2-hydroxydiphenyl ether at a concentration of 0.001% to 1%, more preferably 0.002% to 0.6% (in each case relative to the total weight of the enzyme preparation).

[0188] In a preferred embodiment, the enzyme preparation is an aqueous enzyme preparation comprising 2-phenoxyethanol, 2-bromo-2-nitropropane-1,3-diol, glutaraldehyde and / or formic acid (as the acid or its salts) in the amounts indicated above.

[0189] In one embodiment, the liquid enzyme preparations of the present invention are preservative-free, meaning that they contain less than 1 ppm of preservatives. In one embodiment, "preservative-free" means that no actively added preservatives are present in the enzyme preparations of the present invention. This means that the enzyme preparations may contain preservatives (as by-products) resulting from the fermentation process from which the enzyme concentrate is derived.

[0190] In one aspect, the present invention relates to a method for preserving an aqueous enzyme preparation according to the present invention against microbial contamination or growth, the method comprising the step of adding an antimicrobial agent selected from the group consisting of 2-phenoxyethanol, glutaraldehyde, 2-bromo-2-nitropropane-1,3-diol, and formic acid in acid form or as a salt thereof to an aqueous enzyme concentrate comprising a mannanase variant according to the present invention.

[0191] solvent In one embodiment, the enzyme preparation of the present invention is aqueous and comprises water in an amount ranging from 5% to 95% by weight, from 5% to 30% by weight, from 5% to 25% by weight, from 30% to 80% by weight, or from 20% to 70% by weight, all based on the total weight of the enzyme preparation.

[0192] In one embodiment, the enzyme preparation of the present invention comprises at least one organic solvent selected from ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, ethylene glycol, propylene glycol, 1,3-propanediol, butanediol, glycerol, diglycol, propyl diglycol, butyl diglycol, hexylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, and phenoxyethanol, with ethanol, isopropanol, or propylene glycol being preferred. Additionally, the enzyme preparation of the present invention may comprise at least one organic solvent selected from compounds such as 2-butoxyethanol, isopropyl alcohol, and d-limonene.

[0193] In a preferred embodiment, the enzyme preparation of the present invention comprises at least one water-miscible organic solvent. Water-miscible in this context refers to the ability of an organic solvent to mix in water in all proportions to form a homogeneous solution. Preferably, the at least one water-miscible solvent is selected from ethanol, isopropanol, or 1,2-propylene glycol.

[0194] In one embodiment, the enzyme preparation of the present invention comprises: (a) water in an amount ranging from about 20% to 50%; and (b) at least one organic solvent in an amount ranging from 30% to 60% by weight or from 45% to 55% by weight, all relative to the total weight of the enzyme preparation; Includes.

[0195] In one embodiment, the enzyme preparation of the present invention comprises an organic solvent in an amount ranging from 0% to 20% by weight, based on the total weight of the enzyme preparation. Preferably, the enzyme preparation of the present invention comprises water in an amount ranging from about 30% to 80% by weight and at least one organic solvent in an amount of less than 10%, less than 5%, or less than 1% by weight, all based on the total weight of the enzyme preparation.

[0196] In one embodiment, the enzyme preparation contains water in an amount ranging from 5% to 15% by weight, based on the total weight of the enzyme preparation, and does not contain significant amounts of organic solvents, for example, 1% or less by weight of organic solvents.

[0197] Enzyme Stabilizers Stabilization of enzymes relates to stability over time (e.g., storage stability), thermal stability, pH stability, and chemical stability. As used herein, the term "enzyme stability" preferably relates to the retention of enzyme activity as a function of time, e.g., during storage or handling. Enzyme stabilizers stabilize enzymes in a liquid, preferably aqueous, environment, meaning that they reduce or prevent loss of enzyme activity over time.

[0198] In one embodiment, the at least one enzyme, preferably the at least one mannanase variant according to the invention, is stabilized by the presence of a water-soluble source of calcium and / or magnesium ions in the enzyme preparation. In one embodiment, the at least one enzyme stabilizer is selected from a polyol or a water-soluble salt.

[0199] The polyol may be selected from polyols containing 2 to 6 hydroxyl groups. Suitable examples include glycol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,6-hexanediol, hexylene glycol, glycerol, sorbitol, mannitol, erythriol, glucose, fructose and lactose.

[0200] The water-soluble salts may be selected from salts such as NaCl or KCl and alkali salts of lactic and formic acid.

[0201] In one embodiment of the present invention, the water-soluble salt may be selected from water-soluble sources of zinc(II), calcium(II), and / or magnesium(II) ions, as well as water-soluble sources of other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)) in the finished composition to provide zinc(II), calcium(II), and / or magnesium(II) ions for the enzyme. Preferably, the water-soluble salt is selected from CaCl and MgCl.

[0202] The enzyme preparation may comprise one or more enzymes other than the mannanase variant according to the present invention selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases different from the mannanase variant according to the present invention, cutinases, esterases, phytases, DNAses, pectinases, pectate lyases, pectinolytic enzymes, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases.

[0203] In one embodiment, the enzyme preparation further comprises a protease, preferably a serine protease (EC 3.4.21), more preferably a subtilisin EC 3.4.21.62, and / or a lipase, preferably a triacylglycerol lipase (EC 3.1.1.3), more preferably a Thermomyces lanuginosa lipase. Preferably, the enzyme preparation comprises at least one enzyme stabilizer selected from boron-containing compounds, polyols, peptide aldehydes, other stabilizers, and mixtures thereof.

[0204] The boron-containing compound may be selected from boric acid or a derivative thereof, and boronic acid or a derivative thereof, such as arylboronic acid or a derivative thereof, salts thereof, and mixtures thereof. Herein, boric acid may also be referred to as orthoboric acid.

[0205] In one embodiment, the boron-containing compound is selected from the group consisting of arylboronic acids and their derivatives, hi one embodiment, the boron-containing compound is selected from the group consisting of benzeneboronic acid (BBA), also known as phenylboronic acid (PBA), its derivatives, and mixtures thereof.

[0206] In one embodiment, the phenylboronic acid derivative is selected from the group consisting of 4-formylphenylboronic acid (4-FPBA), 4-carboxyphenylboronic acid (4-CPBA), 4-(hydroxymethyl)phenylboronic acid (4-HMPBA), and p-tolylboronic acid (p-TBA).

[0207] In one embodiment, the enzyme preparation, preferably an enzyme preparation additionally comprising a subtilisin protease, comprises about 0.1-2 wt. % of at least one boron-containing compound, based on the total weight of the enzyme preparation. Preferably, the enzyme preparation comprises about 0.15-1 wt. %, or 0.2-0.5 wt. %, or about 0.3 wt. % of at least one boron-containing compound, based on the total weight of the enzyme preparation. More preferably, the enzyme preparation comprises about 0.3 wt. % of 4-FPBA, based on the total weight of the enzyme preparation.

[0208] In one embodiment, the at least one enzyme stabilizer may be selected from (tri)peptide stabilizers. The at least one peptide stabilizer has formula (D):

[0209] [ka] The compound may be selected from the following compounds:

[0210] R in formula (D) 1 , R 2 , R 3 , R4 , R 5 and Z is defined as follows: R 1 , R 2 and R 3 each independently represents hydrogen, optionally substituted C 1-8 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 1-8 alkoxy, optionally substituted 3- to 12-membered cycloalkyl, and optionally substituted 6- to 10-membered aryl; or each R 1 , R 2 and R 3 is independently selected as -(CH2)3- and also bonds to the nitrogen atom of -NH-C(H)-, thereby forming -NC(H)R 1、2又は3 - forms a 5-membered heterocyclic ring; R 4 and R 5 each independently represents hydrogen, optionally substituted C 1-8 Alkyl, optionally substituted C 2-6 Alkenyl, optionally substituted C 1-8 Alkoxy, optionally substituted C 1-4 Acyl, optionally substituted C 1-8 selected from the group consisting of alkylphenyl (e.g., benzyl) and optionally substituted 6-10 membered aryl; or R 4 and R 5 taken together form an optionally substituted 5- or 6-membered ring; Z is selected from hydrogen, an N-terminal protecting group, and one or more amino acid residues optionally containing an N-terminal protecting group.

[0211] In one preferred embodiment, the peptide stabilizer according to formula (D) is characterized by:

[0212] R 1 is NH-CHR 1 -CO is a group such that Val is an L- or D-amino acid residue, and R 2 is NH-CHR2 -CO is a group such that the L- or D-amino acid residue of Ala is present, and R 3 is NH-CHR 3 -CO is a group such that the L- or D-amino acid residue of Leu is present; and The N-terminal protecting group Z is selected from benzyloxycarbonyl (Cbz), p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), formyl, acetyl (Ac), methyloxy, alkoxycarbonyl, methoxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc) or tert-butyloxycarbonyl (Boc); preferably, the N-terminal protecting group Z is benzyloxycarbonyl (Cbz).

[0213] In one embodiment, the enzyme preparation, preferably an enzyme preparation additionally comprising a subtilisin protease, comprises about 0.1-2 wt. % of at least one peptide stabilizer, based on the total weight of the enzyme preparation. Preferably, the enzyme preparation comprises about 0.15-1 wt. %, or 0.2-0.5 wt. %, or about 0.3 wt. % of at least one peptide stabilizer, based on the total weight of the enzyme preparation.

[0214] Uses of mannanase In one aspect, the present invention relates to a formulation comprising at least one mannanase variant according to the present invention, preferably having a pH in the range of 5-12.

[0215] In one aspect, the present invention relates to the use of a mannanase variant according to the present invention for incorporating into a detergent formulation, such as an I&I and home care formulation for laundry and hard surface cleaning, wherein at least one mannanase variant according to the present invention and at least one detergent component are mixed with one or more detergent components in one or more steps in an unspecified order. In one embodiment, the mannanase variant according to the present invention is comprised in a liquid enzyme preparation.

[0216] In one embodiment the formulation has a pH in the range of 6 to 11, more preferably in a range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10 and 7.5 to 8.5. In one embodiment the formulation is a detergent formulation, preferably a liquid detergent formulation.

[0217] Detergent formulations according to the present invention comprise at least one mannanase of the present invention and one or more detergent ingredients, the ingredients selected depending on the desired washing or cleaning application and / or physical form of the detergent formulation.

[0218] The term "detergent ingredient" is defined herein to mean any type of ingredient suitable for detergent formulations, e.g., surfactants, building agents, polymers, bleaching systems. Any ingredient known in the art that recognizes known properties is a suitable detergent ingredient according to the present invention. In one embodiment, detergent ingredient refers to an ingredient that, when present in an effective amount, provides cleaning or cleaning performance or an ingredient that effectively aids processing (maintains physical properties during processing, storage, and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0219] Typically, detergent formulations are complex blends of three or more detergent ingredients.

[0220] A detergent ingredient may have more than one function in the end use of the detergent formulation, so any detergent ingredient referred to herein in the context of a particular function may also have another function in the end use of the detergent formulation. The function of a particular detergent ingredient in the end use of the detergent formulation is usually determined by its amount in the detergent formulation, i.e., the effective amount of the detergent ingredient.

[0221] The term "effective amount" includes the amount of individual ingredients that provide effective stain removal and effective cleaning conditions (e.g., pH, amount of sudsing), the amount of certain ingredients that effectively provide optical benefits (e.g., optical brightening, dye transfer inhibition), and the amount of certain ingredients that effectively aid processing (maintaining physical properties during processing, storage, and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0222] In one embodiment, a detergent formulation according to the present invention is a blend of three or more detergent ingredients, at least one ingredient effective in stain removal, at least one ingredient effective in providing optimal cleaning conditions, and at least one ingredient effective in maintaining the physical properties of the detergent.

[0223] Individual detergent ingredients and their use in detergent formulations are known to those skilled in the art. Suitable detergent ingredients include, inter alia, surfactants, builders, polymers, alkaline bleaching systems, optical brighteners, foam suppressors and stabilizers, hydrotropes, and corrosion inhibitors. Further examples are described, for example, in "Complete Technology Book on Detergents with Formulations (Detergent Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray Dried Washing Powder)", Engineers India Research Institute (EIRI), 6th edition (2015). Another reference book for those skilled in the art can be "Detergent Formulations Encyclopedia", Solverchem Publications, 2016.

[0224] Detergent ingredients vary in type and / or amount in a detergent formulation depending on the desired application, e.g., washing white textiles, colored textiles, and wool. The ingredients selected are further determined by the physical form of the detergent formulation (liquid, solid, gel, etc., provided in pouches or tablets). For example, the ingredients selected for laundry formulations are further determined by regional practices, which themselves relate to aspects such as the wash temperature used, washing machine configuration (vertical axis or horizontal axis), water consumption per wash cycle, etc., and geographic characteristics such as average water hardness.

[0225] For example, low detergent concentration systems include laundry formulations having less than about 800 ppm of detergent components in the wash water, medium detergent concentration systems include those having from about 800 ppm to about 2,000 ppm of detergent components in the wash water, and high detergent concentration systems include those having more than about 2,000 ppm of detergent components in the wash water.

[0226] The numerical ranges recited for individual detergent ingredients provide the amounts to be included in a detergent formulation. Such ranges should be understood to be inclusive of the numbers defining the range and to include each integer within the defined range.

[0227] Unless otherwise stated, "% by weight" or "% w / w" is meant to relate to the total detergent formulation, in which case "% by weight" or "% w / w" is calculated as follows: concentration of a substance as (weight of the substance) ÷ (total weight of the formulation) × 100.

[0228] In one embodiment, the detergent formulation according to the present invention comprises one or more surfactants. "Surfactant" (used interchangeably herein as "surface active agent") means an organic chemical that, when added to a liquid, changes the properties of that liquid at an interface. Depending on their ionic charge, surfactants are called nonionic surfactants, anionic surfactants, cationic surfactants, or amphoteric surfactants.

[0229] Non-limiting examples of surfactants are disclosed in McCutcheon's 2016 Detergents and Emulsifiers and McCutcheon's 2016 Functional Materials (both North American and International Edition, MC Publishing Co, 2016 edition). Further useful examples are disclosed in earlier editions of the same publications known to those skilled in the art.

[0230] In one embodiment, detergents according to the present invention comprise a total amount of anionic surfactants in the range of 1 wt.% to 30 wt.%, in the range of 3 wt.% to 25 wt.%, in the range of 5 wt.% to 20 wt.%, or in the range of 8 wt.% to 15 wt.%, all relative to the total weight of the detergent formulation. In one embodiment, detergent formulations according to the present invention comprise a total amount of anionic surfactants of about 11 wt.%, relative to the total weight of the detergent formulation.

[0231] In one embodiment, the detergent formulation according to the present invention comprises a compound of general formula (I):

[0232] [ka] The composition contains at least one anionic surfactant selected from the compounds of the formula:

[0233] The variables in general formula (I) are defined as follows:

[0234] R 1 is C1~C 23 -Alkyl (e.g., 1-, 2-, 3-, 4-C1-C 23 -alkyl) and C2-C 23 -alkenyl, where alkyl and / or alkenyl are linear or branched, 2-, 3-, or 4-alkyl; examples are n-CH 15 , n-C9H 19 , nC 11 H 23 , nC 13 H 27 , nC 15 H31 , nC 17 H 35 , i-C9H 19 ,I C 12 H 25 is.

[0235] R 2 is H, C1~C 20 -Alkyl and C2-C 20 -alkenyl, wherein alkyl and / or alkenyl are linear or branched.

[0236] R 3 and R 4 are independently C1 to C 16 -alkyl, where alkyl is straight or branched chain, examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl.

[0237] A - Ha-RCOO - , -SO3 - and RSO3 - and R is selected from linear or branched C1-C8 alkyl and C1-C4 hydroxyalkyl, where alkyl is. - is SO3 - (Fatty) alcohol / alkyl (ethoxy / ether) sulfate [(F)A(E)S], and A - Ga-RCOO - In this case, it is sometimes called (fatty) alcohol / alkyl (ethoxy / ether) carboxylate [(F)A(E)C].

[0238] M + is selected from H and salt-forming cations. The salt-forming cations may be monovalent or polyvalent, and therefore M + is 1 / v Mv+ Examples include, but are not limited to, sodium, potassium, magnesium, calcium, ammonium and ammonium salts of mono-, di- and triethanolamine.

[0239] The integers in the general formula (I) are defined as follows:

[0240] m is in the range of 0 to 200, preferably 1 to 80, more preferably 3 to 20; n and o are each independently in the range of 0 to 100; n is preferably in the range of 1 to 10, more preferably 1 to 6; and o is preferably in the range of 1 to 50, more preferably 4 to 25. The sum of m, n, and o is at least 1, and preferably the sum of m, n, and o is in the range of 5 to 100, more preferably 9 to 50.

[0241] The anionic surfactants of general formula (I) can be of any structure, block copolymers or random copolymers.

[0242] Further suitable anionic surfactants include C 12 ~C 18 -sulfofatty acid alkyl esters (e.g., C 12 ~C 18 -sulfofatty acid methyl ester), C 10 ~C 18 - alkylarylsulfonic acids (e.g., nC 10 ~C 18 -alkylbenzenesulfonic acid) and C 10 ~C 18 -Alkyl alkoxy carboxylate salts (M + ) are listed.

[0243] M + is in each case selected from salt-forming cations. The salt-forming cations may be monovalent or polyvalent, and therefore M + is 1 / v M v+Examples include, but are not limited to, sodium, potassium, magnesium, calcium, ammonium and ammonium salts of mono-, di- and triethanolamine.

[0244] The detergent formulation may comprise at least two anionic surfactants selected from compounds of general formula (I), one of said anionic surfactants being R 1 C 11 , R 2 is H, m is 2, n and o = 0, A - is SO3 - , M + Na + The other surfactant is R 1 C 13 , R 2 is H, m is 2, n and o = 0, A - is SO3 - , M + Na + It is characterized in that:

[0245] In one embodiment, the detergent formulation comprises a compound of general formula (II):

[0246] [ka] [In formula (II), R 1 is C 10 ~C 13 -alkyl] The composition contains at least one anionic surfactant selected from the compounds of the formula:

[0247] The detergent formulation may comprise at least two anionic surfactants selected from compounds of general formula (II), one of said anionic surfactants being R 1 C 10 The other surfactant is R 1 C 13 Such compounds may be referred to herein as LAS (linear alkyl benzene sulfonates).

[0248] The detergent formulations of the present invention may comprise a total amount of nonionic surfactants in the range of about 1% to about 15% by weight, in the range of about 3% to about 12% by weight, or in the range of about 4% to about 8% by weight, all based on the total weight of the detergent formulation. In one embodiment, the detergent formulation of the present invention comprises a total amount of nonionic surfactants in the range of about 5.5% by weight, based on the total weight of the detergent formulation.

[0249] In one embodiment, the detergent formulation according to the present invention comprises a compound of general formula (III):

[0250] [ka] The composition contains at least one nonionic surfactant.

[0251] The variables in general formula (III) are defined as follows:

[0252] R 1 is C1~C 23 -Alkyl and C2-C 23 -alkenyl, where alkyl and / or alkenyl are linear or branched; examples are n-CH 15 , n-C9H 19 , nC 11 H 23 , nC 13 H 27 , nC 15 H 31 , nC 17 H 35 , i-C9H 19 ,I C 12 H 25 is.

[0253] R 2 is H, C1~C 20 -Alkyl and C2-C 20 -alkenyl, wherein alkyl and / or alkenyl are linear or branched.

[0254] R 3 and R 4 are independently C1 to C16 alkyl, which is straight-chain or branched; examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, neopentyl, 1,2-dimethylpropyl, isoamyl, n-hexyl, isohexyl, sec-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, isodecyl.

[0255] R 5 is H and C1~C 18 The alkyl is selected from alkyl, which is straight or branched chain.

[0256] The integers in the general formula (III) are defined as follows:

[0257] m is in the range of 0 to 200, preferably 1 to 80, more preferably 3 to 20; n and o are each independently in the range of 0 to 100; n is preferably in the range of 1 to 10, more preferably 1 to 6; and o is preferably in the range of 1 to 50, more preferably 4 to 25. The sum of m, n and o is at least 1, and preferably the sum of m, n and o is in the range of 5 to 100, more preferably 9 to 50.

[0258] The nonionic surfactant of general formula (III) may have any structure, such as a block structure or a random structure, and is not limited to the arrangement of formula (III) shown.

[0259] Compounds according to formula (III) may be referred to herein as alkyl polyethylene glycol ethers (AEOs).

[0260] In one embodiment, the detergent formulation comprises a compound represented by general formula (III): wherein m is in the range of 3 to 11, preferably 7 or less; n and o are 0; and R 1 is C 12 ~C 14 and R 5is H. The detergent formulation may comprise at least two nonionic surfactants selected from compounds of general formula (III), one of which is 1 C 12 , R 5 is characterized in that: is H, m is 7, n and o=0, and other surfactants are R 1 C 14 , R 5 is H, m is 7, n and o=0.

[0261] The detergent formulations according to the present invention may comprise one or more compounds selected from complexing agents (chelating agents, sequestrating agents), precipitating agents, and ion exchange compounds capable of forming water-soluble complexes with calcium and magnesium. Such compounds may also be referred to herein as "builders" or "building agents," although this is not meant to limit such compounds to this function in the end-use detergent formulation. In one embodiment, the detergent formulations of the present invention comprise at least one builder selected from non-phosphate builders, such as sodium gluconate, citrate, silicate, carbonate, phosphonate, aminocarboxylate, polycarboxylate, polysulfonate, and polyphosphonate.

[0262] In one embodiment, the detergent formulation of the present invention comprises at least one "citrate" selected from mono- and dialkali metal salts of citric acid, particularly the mono-alkali metal salt, preferably the trisodium salt; ammonium or substituted ammonium salts of citric acid; and citric acid itself. The citrate can be used as an anhydrous compound or as a hydrate, e.g., sodium citrate dihydrate. The citrate can be present in a total amount ranging from 0% to about 20% by weight, from about 0.5% to about 10% by weight, or from 1 to 5% by weight, all based on the total weight of the detergent formulation. In one embodiment, the detergent formulation of the present invention comprises a total amount of citrate ranging from about 1 to 3% by weight, based on the total weight of the detergent formulation.

[0263] The detergent formulations of the present invention may include one or more hydrotropes. The one or more hydrotropes may be selected from organic solvents such as, but not limited to, ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and additional organic solvents known in the art that are water-miscible under normal conditions. In one embodiment, the detergent formulations of the present invention include 1,2-propylene glycol in a total amount ranging from 5 to 10% by weight, preferably about 6% by weight, all based on the total weight of the detergent formulation.

[0264] In one embodiment, the detergent formulation of the present invention does not comprise any further enzymes other than the mannanase according to the present invention.

[0265] In one embodiment, the detergent formulation of the present invention comprises, in addition to the mannanase variant according to the present invention, at least one further enzyme selected from proteases, amylases, lipases, cellulases, mannanases and any other enzyme known in the art to be useful in detergent formulations.

[0266] The detergent formulation may comprise one or more enzymes other than the mannanase variants according to the present invention selected from the group consisting of proteases, amylases, cellulases, lipases, xylanases, mannanases different from the mannanase variants according to the present invention, cutinases, esterases, phytases, DNAses, pectinases, pectate lyases, pectinolytic enzymes, carbohydrases, arabinases, galactanases, xanthanases, xyloglucanases, laccases, peroxidases and oxidases.

[0267] The detergent formulation may also include a water-soluble source of calcium and / or magnesium ions. In one embodiment, the detergent formulation includes at least one enzyme stabilizer selected from a polyol and a water-soluble salt.

[0268] The polyol may be selected from polyols containing 2 to 6 hydroxyl groups. Suitable examples include glycol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,4-butanediol, 1,2-pentanediol, 1,6-hexanediol, hexylene glycol, glycerol, sorbitol, mannitol, erythriol, glucose, fructose and lactose.

[0269] The water-soluble salts may be selected from salts such as NaCl or KCl and alkali salts of lactic and formic acid.

[0270] In one embodiment of the present invention, the water-soluble salt may be selected from water-soluble sources of zinc(II), calcium(II), and / or magnesium(II) ions, as well as water-soluble sources of other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and oxovanadium(IV)) in the finished composition to provide zinc(II), calcium(II), and / or magnesium(II) ions for the enzyme. Preferably, the water-soluble salt is selected from CaCl and MgCl.

[0271] The detergent formulation may include at least one enzyme stabilizer selected from the boron-containing compounds and peptide stabilizers disclosed above.

[0272] The mannanase variant according to the present invention preferably exhibits mannan-degrading activity at a temperature selected from below 60° C., below 40° C. and below 25° C. Preferably, the temperature is a washing or cleaning temperature. Mannan-degrading activity in the context of washing or cleaning herein relates to its ability to remove stains containing mannan.

[0273] In one embodiment, the detergent formulation of the present invention is a laundry detergent.

[0274] The term "laundry" refers to the process of treating textiles with a solution comprising the detergent formulation of the present invention, both in relation to domestic and industrial laundering. The laundering process may be carried out by using a technical device, for example, a domestic or industrial washing machine. A washing machine is also referred to herein as a washing machine. Alternatively, the laundering process may be carried out manually.

[0275] The term "textile" means any textile material, including yarns (threads made from natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and fabrics (textiles made by weaving, knitting, or felting fibers) made from these materials, such as garments (any article of clothing made from textiles), fabrics, and other articles.

[0276] The term "fiber" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are those of plant origin (e.g., flax, jute and cotton) or those of animal origin containing proteins such as collagen, keratin and fibroin (e.g., silk, wool, angora, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers, such as Spandex® or Lycra®, polyester fibers, polyolefins, such as elastofin, or polyamide fibers, such as nylon. A fiber can be a single fiber or part of a textile, for example, knitwear, woven fabric or nonwoven fabric.

[0277] The present invention provides a method for providing a liquid mannanase-containing formulation, preferably a liquid detergent formulation, more preferably a liquid laundry detergent formulation, comprising in one or more steps: (a) at least one mannanase variant according to the present invention, preferably provided in an enzyme preparation of the present invention; (b) at least one detergent ingredient selected from surfactants, builders, and hydrotropes, present in an effective cleaning amount and / or in an amount effective to maintain the physical properties of the detergent; The method relates to a method comprising the step of mixing

[0278] In one embodiment the formulation has a pH in the range of 5 to 12 or 6 to 11, more preferably in a range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10 and 7.5 to 8.5. In one embodiment the formulation is a detergent formulation, preferably a liquid detergent formulation, more preferably a liquid laundry detergent formulation.

[0279] The laundry detergent formulations of the present invention exhibit cleaning performance under relevant washing conditions. As used herein, the term "relevant washing / cleaning conditions" refers to the conditions actually used in a washing machine or in a manual washing process, in particular temperature, time, cleaning mechanism, foam concentration, detergent type, and water hardness. In one embodiment, the cleaning performance herein relates to the removal of mannan-containing stains; preferably, the mannan-containing stains are selected from those containing galactomannan and glucomannan. In one embodiment, the cleaning performance relates to the removal of locust bean gum and / or guar gum stains.

[0280] In one aspect, the present invention provides a method for removing mannan-containing stains by contacting at least one mannan-containing stain with a mannanase of the present invention. The mannanase has mannan-degrading activity at a pH in the range of 5 to 12 or 6 to 11, more preferably 6 to 10, 7 to 9, 7 to 12, 8 to 12, or 8 to 10, and most preferably 7.5 to 8.5. At these pH levels, the mannanase exhibits cleaning performance for mannan-containing stains. Preferably, this method removes mannan-containing stains at a temperature of 60°C or less, preferably about 5 to 60°C, preferably about 5 to 40°C, and more preferably about 10 to 40°C.

[0281] In one aspect, the present invention provides a method for producing a method for manufacturing a semiconductor device comprising the steps of: (a) providing a liquid formulation comprising at least one mannanase according to the present invention; (b) contacting a mannan-containing stain with the liquid formulation of (a); (c) allowing the enzyme to exert its enzymatic activity on the stain for a period of time ranging from about 10 to 90 minutes, preferably 20 to 80 minutes, more preferably 30 to 70 minutes, or even more preferably 40 to 60 minutes. The present invention relates to a method for removing stains containing mannan by the above process at a temperature of 60°C or less, preferably in the range of about 5 to 60°C, preferably in the range of about 5 to 40°C, more preferably in the range of about 10 to 40°C.

[0282] In one embodiment, the method for removing mannan-containing stains is characterized in that it is a washing method preferably carried out under mechanical agitation in a washing machine, and the liquid formulation is preferably a liquid detergent formulation.

[0283] The present invention relates to the use of at least one mannanase variant according to the invention for increasing the washing or cleaning performance of a detergent formulation on stains containing mannan, preferably stains containing locust bean gum and / or guar gum.

[0284] In one embodiment, the detergent formulation has a pH in the range of 5 to 12 or 6 to 11, more preferably in a range selected from 6 to 10, 7 to 12, 7 to 9, 8 to 12, 8 to 10 and 7.5 to 8.5. In one embodiment, the formulation is a liquid detergent formulation, preferably a liquid laundry detergent formulation.

[0285] In one aspect, the present invention relates to a formulation comprising at least one mannanase according to the present invention, preferably having a pH in the range of 5 to 12, which formulation has increased washing or cleaning performance on mannan-containing stains, more preferably on stains comprising locust bean gum and / or guar gum. Increased washing or cleaning performance on mannan-containing stains means increased washing or cleaning performance compared to a formulation lacking the mannanase of the present invention or compared to a formulation lacking any mannanase. The washing or cleaning performance preferably relates to the removal of mannan-containing stains, preferably stains comprising locust bean gum and / or guar gum.

[0286] In one embodiment, the washing or cleaning performance is increased at washing or cleaning temperatures below 60°C, preferably in the range of about 5-60°C, preferably in the range of about 5-40°C, more preferably in the range of about 10-40°C.

[0287] In one aspect, the present invention provides a method of washing or cleaning comprising the steps of: (a) providing a stain comprising at least one mannan; (b) providing a detergent formulation according to the present invention; (c) contacting the mannan-containing stain with the detergent of (b) preferably at a wash temperature in the range of about 10 to 40°C. The present invention relates to a method, comprising:

[0288] In one embodiment, step (a) includes providing a textile, the textile including a stain comprising mannan.

[0289] In one embodiment, step (c) is carried out in a washer under mechanical agitation.

[0290] In one embodiment, at least one mannanase of the present invention contained in a detergent according to the present invention removes mannan-containing stains from textile (a) by (c) contacting the mannan-containing stains with the detergent (b).

[0291] Further embodiments of the present invention: 1. D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122 S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244 I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K273T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, D3 28I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, N341F, Y344Q / F / T, F346T, T348S / R / N / M / G, E349T / S / D / G, S352N / G, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V, L416W and W432P / N / L / R / S / T / G / H / I, and having at least one amino acid substitution selected from the group consisting of 28I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, N341F, Y344Q / F / T, F346T, T348S / R / N / M / G, E349T / S / D / G, S352N / G, G356Y / V / T / Q / H / C, M359R / Y / C / Q, Y374G / V / A / R / N / P, D379V, L416W and W432P / N / L / R / S / T / G / H / I, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

[0292] 2. A polynucleotide encoding the mannanase described in embodiment 1, which is at least 75% identical to SEQ ID NO:1.

[0293] 3. An expression construct comprising the polynucleotide of embodiment 2.

[0294] 4. A host cell comprising the polynucleotide of claim 2 or the expression construct of embodiment 3.

[0295] 5. A method for expressing the mannanase variant described in embodiment 1, comprising: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide. A method comprising:

[0296] 6. A liquid enzyme preparation comprising the mannanase described in embodiment 1, at least one compound that stabilizes the liquid enzyme preparation itself, such as a preservative, at least one solvent, such as glycerol, and optionally at least one enzyme stabilizer.

[0297] 7.D86N, Q89V, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A11 9Y / H / T, N122S, A124E / C / D, S126E, S127A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, E264Q / V, S270T, Q272I, K27 3T, N274E / C / Q, S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / A method for increasing the stability in a detergent of a mannanase that is at least 75% identical to SEQ ID NO: 3 or SEQ ID NO: 4 by introducing at least one amino acid substitution at an amino acid position selected from T, T309L, N312F / Y, A314P, L317T, A319D / E, D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I, wherein the numbering is according to SEQ ID NO: 2.

[0298] 8. A formulation according to embodiment 1, preferably a liquid formulation, preferably having a pH in the range of 5 to 12.

[0299] 9. A method for providing a detergent formulation, comprising, in one or more steps: (a) at least one mannanase according to embodiment 1, optionally provided in an enzyme preparation according to claim 6; (b) at least one component selected from surfactants, builders, and hydrotropes, all in an amount effective for cleaning performance or to maintain the physical properties of the detergent; The method of claim 1, further comprising the step of:

[0300] 10. A method of washing or cleaning comprising: (a) providing a stain comprising at least one mannan; (b) providing a formulation according to embodiment 8; (c) contacting the mannan-containing stain (a) with the formulation of (b) at a washing or cleaning temperature preferably in the range of 5 to 60°C. A method comprising:

[0301] 11. The method of embodiment 10, wherein a stain comprising mannan is provided on textile (a), and the polypeptide contained in detergent (b) removes the stain comprising mannan from textile (a).

[0302] 12. Use of at least one mannanase according to embodiment 1 for increasing the cleaning performance of a detergent formulation on mannan-containing stains, preferably at a washing or cleaning temperature in the range of 5 to 60°C.

[0303] 13. The use according to embodiment 12, wherein the stain comprising at least one mannan comprises locust bean gum and / or guar gum. [Example]

[0304] [Example 1] Expression and purification of mannanase variants The gene was synthesized by GenScript (New Jersey, USA) and cloned into a Bacillus expression vector. The construct was received from GenScript as sequence-verified plasmid DNA and transformed into Bacillus subtilis. Five microliters of plasmid DNA (20–200 ng / μL) was added to 500 μL of freshly prepared competent B. subtilis cells and incubated at 37°C for 3.5 hours. The cells were then plated onto LB + 50 μg / mL kanamycin agar plates and grown overnight at 37°C. To confirm the expression of mannanase in B. subtilis, the resulting colonies were screened by colony PCR and sequencing. Prior to PCR, each colony was lysed in a buffer containing 20 mM DTT and 0.5 mg / mL proteinase K at 55°C for 5 minutes, followed by 95°C for 6 minutes. A 20 μL PCR reaction was performed using 1 μL of lysed cells and TaKara Ex Taq (TaKaRa Cat#RR001) polymerase as follows: initial denaturation at 98°C for 3 minutes, followed by 30 cycles of denaturation, annealing, and extension at 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 2.5 minutes. The PCR reaction was completed by a final extension at 72°C for 5 minutes. Mannanase expression was performed, for example, in a microtiter plate format. Fermentation was carried out at 30°C under 1000 rpm agitation for approximately 48 hours. The final fermentation broth was centrifuged at 2500 × g for 15 minutes at 4°C to obtain a cell-free supernatant. Protein quantification was estimated using an automated capillary gel electrophoresis system: LabChip® GX II (PerkinElmer, USA) equipped with the HT Protein Express LabChip® and HT Protein Express Reagent Kit. Protein purity determination and quantification was performed using the Regular Sensitivity HT Protein Express 200 assay and analysis was performed using LabChip® GX Reviewer 5.3 software.Molecular weight determinations were performed by the LabChip software using a bracketing ladder of protein standards (as part of the HT Protein Express Reagent Kit) to assign and quantify the MW of the peaks, or by using known protein standards and the "Titer" function within the instrument analysis software, or by generating a standard curve using peak areas obtained from the LabChip of a series of protein standards with known concentrations, resulting in quantification of the protein of interest.

[0305] [Example 2] Mannanase expression Expression of mannanase was carried out in 384-well deep-well plates. Fermentation was carried out at 37°C under 1000 rpm agitation for approximately 48 hours. The final fermentation broth was centrifuged at 2500 x g for 15 minutes at 4°C to obtain a cell-free supernatant.

[0306] Protein quantification was estimated using an automated capillary gel electrophoresis system: LabChip® GX II (PerkinElmer, USA) equipped with the HT Protein Express LabChip® and HT Protein Express Reagent Kit. Protein purity determination and quantification were performed using the Regular Sensitivity HT Protein Express 200 assay, and analysis was performed using LabChip® GX Reviewer 5.3 software. Molecular weight determination was performed by the LabChip software using a bracketing ladder of protein standards (as part of the HT Protein Express Reagent Kit) to assign and quantify peak MWs, or by using known protein standards and the "Titer" function within the instrument analysis software, or by generating a standard curve using peak areas obtained from the LabChip of a series of protein standards with known concentrations, resulting in quantification of the protein of interest.

[0307] The degradation stability of mannanase variants was determined by calculating the amount of full-length enzyme and the percent full-length mannanase (amount of full-length ÷ (sum of full-length mannanase and observed degradation products) × 100).

[0308] [Table 1]

[0309] [Example 3] Mannanase activity after storage Shelf-life stability was determined for ES1 formulations adjusted to pH 8 at 37°C for 6 days.

[0310] [Table 2]

[0311] 900 μl of the buffer / detergent formulation was supplemented with 15.2 mg / mL of mannanase enzyme. The probe was stored at 37°C for 6 days. At specific time points, 50 μl of sample was taken from the original probe and diluted with 800 μl of HEPES buffer. The sample was incubated at 37°C for 5 minutes. 200 μl of this sample was mixed with 200 μl of substrate (1% Azo Carob Galactomannan) and incubated for an additional 60 minutes. 200 μl of the reaction mixture was then added to 350 μl of 95% ethanol on ice and incubated for 10 minutes. The reaction mixture was then centrifuged at 4000 × g for 10 minutes. The absorbance of 250 μl of the resulting supernatant was determined at 590 nm. The mannanase activity measured after storage at the time points indicated in the table below is presented as a percentage improvement compared to the parent enzyme, which is set to 1.0.

[0312] [Table 3-1] [Table 3-2]

Claims

1. A mannanase variant that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4, comprising the amino acid substitution E264Q / V, wherein the numbering is according to SEQ ID NO: 2, and the polypeptide has mannan-degrading activity.

2. The variants are D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S12 7A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, S270T, Q272I, K273T, N274E / C / Q, S28 1L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, wherein the numbering is according to SEQ ID NO: 2, and wherein the polypeptide has mannan-degrading activity.

3. 3. The mannanase variant of claim 1, wherein the variant has a polypeptide sequence at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 3, and includes at least one amino acid substitution selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I.

4. A polynucleotide encoding the mannanase of any one of claims 1 to 3, which preferably is at least 90% identical to SEQ ID NO:

1.

5. An expression construct comprising the polynucleotide of claim 4.

6. 6. A host cell comprising the polynucleotide of claim 4 or the expression construct of claim 5.

7. A method for expressing the mannanase variant of any one of claims 1 to 3, comprising: (a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention by introducing into the host cell a nucleic acid construct comprising a polynucleotide encoding a mannanase variant according to the invention; (b) culturing the recombinant host cell of step (a) under conditions conducive to expression of the polynucleotide; and (c) optionally recovering the protein of interest encoded by the polynucleotide. A method comprising:

8. A liquid enzyme preparation comprising a mannanase described in any one of claims 1 to 3, at least one compound that stabilizes the liquid enzyme preparation, such as a preservative, at least one solvent, such as glycerol, and optionally at least one enzyme stabilizer.

9. A method for increasing the stability in a detergent of a mannanase that is at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 by introducing the amino acid substitution E264Q / V, wherein the numbering is according to SEQ ID NO:

2.

10. Variants are D86N, Q89V / L, N96D, L101T / V, S103Y / E / A, K107N, N108G, N109Q / A, A112N, A119Y / H / T, N122S, A124E / C / D, S126E, S1 27A, N129M / L / F, S231Q / K / L / P / Y, I233V, S235H / R / L / Q / N / Y, D244I / V / N, H254W, K255Y / H / R, S270T, Q272I, K273T, N274E / C / Q 10. The method of claim 9, wherein the polypeptide has a polypeptide sequence at least 90% identical to SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4, comprising at least one amino acid substitution selected from: S281L, G286E / L / Q / A, W289F / M / H, S290A, N296H / F / Y, D301E / C / T, T309L, N312F / Y, A314P, L317T, A319D / E, wherein the numbering is according to SEQ ID NO:2, and wherein the polypeptide has mannan-degrading activity.

11. The method of claim 9 or 10, wherein the stability in detergent of a mannanase at least 90% identical to SEQ ID NO: 2 or SEQ ID NO: 3 is increased by introducing at least one amino acid substitution at an amino acid position selected from D328I / Q / V, G329L / S / V / T, G330P / D / T, D331A / Q, Y344Q / F / T, M359R / Y / C / Q, Y374G / V / A / R / N / P, L416W and W432P / N / L / R / S / T / G / H / I.

12. A formulation according to any one of claims 1 to 3, preferably a liquid formulation, preferably having a pH in the range of 5 to 12.

13. 1. A method for providing a detergent formulation, comprising, in one or more steps: (a) at least one mannanase according to any one of claims 1 to 3, optionally provided in an enzyme preparation according to claim 7; (b) at least one component selected from surfactants, builders, and hydrotropes, all in an amount effective for cleaning performance or to maintain the physical properties of the detergent; The method of claim 1, further comprising the step of:

14. 1. A method of washing or cleaning comprising: (a) providing a stain comprising at least one mannan; (b) providing the formulation of claim 12; (c) contacting the mannan-containing stain (a) with the formulation of (b) at a washing or cleaning temperature preferably in the range of 5 to 60°C. A method comprising:

15. 15. The method of claim 14, wherein a stain containing mannan is provided on textile (a), and the polypeptide contained in detergent (b) removes the stain containing mannan from textile (a).

16. 4. Use of at least one mannanase according to any one of claims 1 to 3 for increasing the cleaning performance of a detergent formulation on mannan-containing stains, preferably at washing or cleaning temperatures in the range of 5 to 60°C.

17. 17. Use according to claim 16, wherein the at least one mannan-containing stain comprises locust bean gum and / or guar gum.

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