Mannanase for formulations with pH 5-12

Abstract

A method for removing stains containing mannans by contacting the stains containing at least one mannan with a mannanase that is at least 80% identical to SEQ ID NO:1.

Description

[Technical field]

[0001] The present invention relates to a method for removing stains containing mannan. [Background technology]

[0002] The main 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 occur 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. Unlike unsubstituted mannans, galactomannans are water soluble. Due to the complex structural composition of plant cell walls, microorganisms growing on decaying plant material must possess several different enzymes capable of hydrolyzing these highly polymeric and almost insoluble materials. The two main endo-acting enzymes involved in the degradation of hemicellulose are beta-mannanase and beta-xylanase. In addition, the exo-acting enzymes beta-mannosidase, alpha-galactosidase and beta-glucosidase are required for the complete degradation of galactoglucomannans.

[0004] The main enzyme type involved in the degradation of the mannan backbone is endo-1,4-beta-mannanase (EC 3.2.1.78), which hydrolyzes internal glycosidic bonds in the mannan backbone. Endo-1,4-β-mannanase (EC 3.2.1.78) is a mannan degrading enzyme, also referred to herein as endo-β-1,4-D-mannanase, β-mannanase or mannanase. Since 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: For monogastric animals such as poultry and pigs, the use of mannanase enzymes as a feed additive has been shown to provide several beneficial effects since mannan is a poorly digestible feed ingredient that acts as an antinutritional factor. In the food industry, mannanase enzymes have been described for use in the production of instant coffee, where the enzyme reduces the viscosity of the coffee extract 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 functions. In such applications, mannopolymers of plant origin are subjected to hydrolysis by mannanases. Detergent applications: Mannanases facilitate the removal of stains / soils from food and cosmetic products that 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. Pulp and paper: 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.

[0006] 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. Summary of the Invention

[0007] There is a continuing need for enzymes that function in the harsh environment of detergent formulations. Various classes of enzymes are known to be useful in detergent formulations, such as proteases, amylases, cellulases, lipases, mannanases, pectate lyases and nucleases. Mannanases are useful components of washing and / or cleaning formulations because they remove some of the stains that contain hemicellulose. Insufficient removal of these types of stains usually results in graying of the fabric.

[0008] In the present specification, a stain containing mannan comprises at least one mannan, at least one galactomannan and / or at least one glucomannan, and in one embodiment further components, such as cellulose and / or hemicellulose. In addition, such stains may contain proteinaceous materials, starches and / or sugars. Galactomannans usually consist of a mannose backbone with galactose side groups. In the present specification, galactomannans include galactomannans with the following mannose to galactose ratios: fenugreek gum (about 1:1), guar gum (about 2:1), tara gum (about 3:1), locust bean gum or carob gum (about 4:1), cassia gum (about 5:1), said ratio being mannose:galactose. Galactomannans are often used in food and cosmetic products to increase the viscosity of liquid products.

[0009] In one embodiment, the at least one mannan-containing stain is contained on a textile.

[0010] It was therefore an objective to find mannan degrading enzymes that are catalytically active in formulations 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, 7-9, 7-12, 8-12, 8-10 and 7.5-8.5. Preferably the mannan degrading enzymes exhibit cleaning performance when provided in detergent formulations.

[0011] In one aspect, the invention provides a mannanase that is at least 80% identical to SEQ ID NO: 1 provided in an enzyme preparation that allows for flexible formulation into liquid detergent or cleaning formulations containing one enzyme or a mixture of enzymes. By "formulated into" it is meant that the enzyme preparation is added to a liquid formulation.

[0012] 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 having at least 80% identity to SEQ ID NO: 1. The mannanase has mannan decomposition activity at a pH in the range of 5-12 or 6-11, more preferably at a pH in the range of 6-10 or 7-9 or 7-12 or 8-12 or 8-10, and most preferably at a pH in the range of 7.5-8.5. At said pH, the mannanase exhibits cleaning performance for mannan-containing stains. Preferably, the method is a method for removing mannan-containing stains at a temperature of 60°C or less, 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.

[0013] In one aspect, the invention provides, on the other hand, a formulation comprising the mannanase of the invention, preferably having a pH in the range of 5-12, preferably in the range of 6-11, more preferably in the range selected from 6-10, 7-9, 7-12, 8-12, 8-10 and 7.5-8.5. 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 washing or cleaning. In one embodiment, the formulation is a detergent formulation and the mannanase exhibits cleaning performance when provided in the detergent formulation.

[0014] In general, an "enzyme" is a catalytically active protein or polypeptide that acts on a substrate to convert it into a product. This reaction, also referred to herein as an enzymatic conversion, typically takes place 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.

[0015] The mannanase according to the invention has mannanolytic activity and is of the enzyme class EC 3.2.1.78. In one embodiment, mannanolytic activity refers 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 5:1.

[0016] Mannanolytic or mannanase activity can be tested according to standard test 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), a substrate for the assay of endo-1,4-beta-D-mannanase. Carob is available, for example, as I-AZGMA from Megazyme. Mannanolytic 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 is the detection of reducing sugars when incubated with a substrate (such as guar gum or locust bean gum) - see Miller, GL Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugars. Analytical Chemistry 1959; 31, 426-428.

[0017] Enzymes are polypeptides that are typically identified by a polypeptide sequence (also referred to herein as an amino acid sequence). Polypeptide sequences are typically identified by SEQ ID NO: In accordance with the World Intellectual Property Organization (WIPO) standard ST.25 (1998), amino acids are represented herein using their three-letter code with the first letter capitalized or their single-letter equivalents.

[0018] 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 result in 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.

[0019] The parent polypeptide of the mannanase of the present invention has a polypeptide sequence according to SEQ ID NO:1.

[0020] "Variant polypeptide" refers to an enzyme that differs in amino acid sequence from its parent.

[0021] Variant polypeptide sequences in one embodiment are 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.

[0022] Sequence identity is usually expressed as "% sequence identity" or "% identity." To calculate sequence identity, the first step must be to produce a sequence alignment.

[0023] According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 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 default parameters of the program (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62).

[0024] After alignment of the two sequences, in a second step an identity score is determined from the resulting alignment.

[0025] As used herein, % identity is calculated by dividing the number of identical residues by the length of the alignment region showing the two aligned sequences in their entirety and multiplying by 100: % identity = (identical residues / length of alignment region showing the two aligned sequences in their entirety) x 100.

[0026] In one embodiment, the mannanase of the invention is 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, preferably when compared to the full-length amino acid sequence of SEQ ID NO: 1. The mannanase 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; amino acid Y is similar to amino acids F, H and W.

[0027] The mannanase of the present invention is a "mature polypeptide," which refers to the final form of the enzyme, including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence deletion, etc. The mature polypeptide may vary depending on the expression system, vector, promoter, and / or production process.

[0028] Enzymes are usually produced as liquid concentrates, often derived from fermentation broths. 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 the physical appearance at 20° C. and 101.3 kPa.

[0029] The liquid enzyme concentrate may be produced by dissolving the solid enzyme in a solvent. In such a case, the solvent is preferably selected from water and organic solvents. The liquid enzyme concentrate produced by dissolving the solid enzyme in a solvent contains the enzyme in an amount up to the saturation concentration.

[0030] As used herein, dissolution means that a solid compound is liquefied by contact with at least one solvent. Dissolution means complete dissolution of the solid compound without phase separation until a saturation concentration is reached in the specified solvent.

[0031] In one aspect of the invention, the enzyme concentrate may be water-free, meaning that there is no significant amount of water. In this specification, an insignificant amount of water means that the enzyme concentrate contains less than 25%, less than 20%, less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2% water by weight, or no water at all, all 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 a significant amount of water and contains organic solvent in an amount of about 10% to 90%, 20% to 85%, 30% to 80%, 40% to 75%, 50% to 70% by weight, all based on the total weight of the enzyme concentrate.

[0032] A liquid enzyme concentrate containing water may 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.

[0033] Fermentation refers to the process of culturing recombinant cells expressing the desired enzyme in a suitable nutrient medium that allows the recombinant host cells to grow and express the desired protein. At the end of the fermentation, the fermentation broth is usually collected and further processed, the fermentation broth containing a liquid fraction and a solid fraction. Depending on whether the enzyme is secreted into the liquid fraction, the desired protein or enzyme is 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.

[0034] The liquid enzyme concentrate comprises an amount of enzymes ranging from 0.1% to 40%, or 0.5% to 30%, or 1% to 25%, or 3% to 25%, or 5% to 25% by weight, all based on the total weight of the enzyme concentrate. In one embodiment, the liquid enzyme concentrate is produced by fermentation and is aqueous.

[0035] The aqueous enzyme concentrate resulting from fermentation comprises 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, the aqueous enzyme concentrate resulting from fermentation comprises 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. The aqueous enzyme concentrate resulting from fermentation may comprise residual components, such as salts from the fermentation medium, cell debris from the production host cells, and metabolic products produced by the production host cells during fermentation. In one embodiment, the residual components are present in the liquid enzyme concentrate 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.

[0036] Enzymes tend to lose enzymatic activity if left in an aqueous environment, and therefore it is conventional to convert the environment to an anhydrous form: 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, are usually utilized to temporarily inhibit enzyme activity until the enzyme inhibitor is released.

[0037] 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.

[0038] The enzyme preparation of the present invention includes a liquid enzyme concentrate comprising at least one mannanase of the present invention. The enzyme preparation of the present invention comprises only ingredients 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.

[0039] The liquid composition of the present invention is preferably free of surfactants, which means that the liquid composition contains less than about 10% by weight, less than about 7% by weight, less than about 5% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight of surfactants, based on the total weight of the liquid composition.

[0040] The liquid composition of the present invention is preferably free of complexing agents, which means that the liquid composition contains less than about 10% by weight, less than about 7% by weight, less than about 5% by weight, less than about 3% by weight, less than about 2% by weight, or less than about 1% by weight of a complexing agent, preferably an aminocarboxylate, based on the total weight of the liquid composition.

[0041] In one embodiment, the liquid composition of the present invention is surfactant-free and complexing agent-free.

[0042] Stabilization of enzymes relates to stability over time (e.g. storage stability), thermal stability, pH stability and chemical stability. In this specification, 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, which means that the enzyme reduces or avoids loss of enzyme activity over time.

[0043] In one embodiment, the at least one enzyme, preferably the at least one mannanase of 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.

[0044] The polyol includes polyols containing 2 to 6 hydroxyl groups. Suitable examples include glycol, 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, ethylene glycol, hexylene glycol, glycerol, sorbitol, mannitol, erythriol, glucose, fructose and lactose.

[0045] In one embodiment, the water-soluble salts are selected from salts such as NaCl or KCl and the alkali salts of lactic acid and formic acid.

[0046] In one embodiment of the invention, the at least one water-soluble salt is selected from water-soluble sources of zinc(II), calcium(II) and / or magnesium(II) ions in the finished composition that provide zinc(II), calcium(II) and / or magnesium(II) ions to the enzyme, 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)). Preferably, the water-soluble salt is selected from CaCl2 and MgCl2.

[0047] In one embodiment of the invention, 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 lanuginose lipase. In this context, the enzyme stabilizer is preferably selected from boron-containing compounds, polyols, peptide aldehydes, other stabilizers and mixtures thereof.

[0048] The boron-containing compound is 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 is also referred to as orthoboric acid.

[0049] In one embodiment, the at least one boron-containing compound is selected from the group consisting of arylboronic acids and their derivatives. In 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.

[0050] 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).

[0051] Other suitable derivatives include: 2-thienylboronic acid, 3-thienylboronic acid, (2-acetamidophenyl)boronic acid, 2-benzofuranylboronic acid, 1-naphthylboronic acid, 2-naphthylboronic acid, 2-FPBA, 3-FBPA, 1-thianthrenylboronic acid, 4-dibenzofuranboronic acid, 5-methyl-2-thienylboronic acid, 1-benzothiophene-2-boronic acid, 2-furanylboronic acid, 3-furanylboronic acid, 4,4-biphenyl-diboronic acid, 6-hydroxy-2-naphthaleneboronic acid, 4-(methylthio)phenylboronic acid, 4-(trimethylsilyl)phenylboronic acid, 3-bromothiopheneboronic acid, 4-methylthiopheneboronic acid, 2-naphthylboronic acid, 5-bromothiopheneboronic acid, 5-chlorothiopheneboronic acid, dimethylthio Opheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiopheneboronic acid, p-methyl-phenylethylboronic acid, 2-thianthrenylboronic acid, dibenzothiopheneboronic acid, 9-anthraceneboronic acid, 3,5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-bis-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, 4-methoxyphenylboronic acid and mixtures thereof.

[0052] In one embodiment, the at least one enzyme stabilizer is selected from peptide aldehydes, which are di-, tri- or tetrapeptide aldehydes and aldehyde analogs (B1-BO-R, where R is H, CH3, CX3, CHX2 or CH2X (X=halogen), BO is a single amino acid residue (in one embodiment with an optionally substituted aliphatic or aromatic side chain), and B1 consists of one or more amino acid residues (in one embodiment, one, two or three), optionally including an N-terminal protection group, or as described in WO09 / 118375 and WO98 / 13459, or protein-type protease inhibitors, such as RASI, BASI, WASI (rice, barley and wheat bifunctional alpha-amylase / subtilisin inhibitor) or CI2 or SSI. Preferably, the at least one peptide aldehyde is a tripeptide aldehyde.

[0053] By a compound that stabilizes the liquid enzyme preparation itself is meant 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.

[0054] Storage stability in the context of liquid formulations to those skilled in the art typically includes the aspects of product appearance and dosage uniformity.

[0055] The appearance of the product is influenced by the pH of the product and the presence of compounds such as preservatives, antioxidants, viscosity modifiers, emulsifiers, and the like.

[0056] Uniformity of dosage is usually related to homogeneity of the product.

[0057] The enzyme preparation of the present invention is alkaline or exhibits a neutral or weakly acidic pH value. The enzyme preparation may have a pH in the range of 5 to 12 or 6 to 11, more preferably in the range of 6 to 10 or 7 to 9 or 7 to 12 or 8 to 12 or 8 to 10, most preferably in the range of 7.5 to 8.5.

[0058] In one embodiment, the liquid enzyme preparation of the present invention comprises at least one preservative, which is added in an amount effective to prevent microbial contamination of the liquid enzyme preparation, preferably the aqueous enzyme preparation.

[0059] Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids and formaldehyde releasers.Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chloride, polyhexamethylene biguanide (PHMB), didecyldimethylammonium chloride (DDAC) and N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (Diamine).Non-limiting examples of suitable isothiazolinones include 1,2-benzisothiazolin-3-one (BIT), 2-methyl-2H-isothiazol-3-one (MIT), 5-chloro-2-methyl-2H-isothiazol-3-one (CIT), 2-octyl-2H-isothiazol-3-one (OIT) and 2-butyl-benzo[d]isothiazol-3-one (BBIT). Non-limiting examples of suitable organic acids include benzoic acid, sorbic acid, L-(+) lactic acid, formic acid, and salicylic acid. Non-limiting examples of suitable formaldehyde releasers include N,N'-methylene bismorpholine (MBM), 2,2',2"-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol (HHT), (ethylenedioxy)dimethanol, α,α',α"-trimethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-triethanol (HPT), 3,3'-methylenebis[5-methyloxazolidine] (MBO), and cis-1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride (CTAC).

[0060] Further useful preservatives include iodopropynyl butylcarbamate (IPBC), halogen releasing compounds such as dichloro-dimethyl-hydantoin (DCDMH), bromo-chloro-dimethyl-hydantoin (BCDMH) and dibromo-dimethyl-hydantoin (DBDMH); bromonitro compounds such as bronopol (2-bromo-2-nitropropane-1,3-diol), 2,2-dibromo-2-cyanoacetamide (DBNPA); aldehydes such as glutaraldehyde; phenoxyethanol; biphenyl-2-ol; and zinc or sodium pyrithione.

[0061] The enzyme preparation of the present invention preferably comprises at least one preservative 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, and 4,4'-dichloro-2-hydroxydiphenyl ether. Usually, the enzyme preparation of the present invention comprises at least one preservative in an amount ranging from 2 ppm to 5% by weight, based on the total weight of the liquid enzyme preparation. More preferably, the liquid enzyme preparation is free of preservatives, meaning that the preservative is contained in an amount of less than 1 ppm.

[0062] In one embodiment, the enzyme preparation of the invention is aqueous and comprises water in an amount in the range of 5% to 95% by weight, in the range of 5% to 30% by weight, in the range of 5% to 25% by weight, in the range of 30% to 80% by weight, or in the range of 20% to 70% by weight, all based on the total weight of the enzyme preparation.

[0063] In one embodiment, the enzyme preparation of the 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.Furthermore, the enzyme preparation of the invention may comprise at least one organic solvent selected from compounds such as 2-butoxyethanol, isopropyl alcohol and d-limonene.

[0064] In a preferred embodiment, the enzyme preparation of the invention comprises at least one water-miscible organic solvent. Water-miscible in this context means the property 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.

[0065] In one embodiment, the enzyme preparation 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% by weight to 60% by weight or in an amount ranging from 45% by weight to 55% by weight, all relative to the total weight of the enzyme preparation; Includes.

[0066] In one embodiment, the enzyme preparation 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 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.

[0067] 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 solvent.

[0068] The present invention provides a method for removing mannan-containing stains by contacting at least one mannan-containing stain with a mannanase having at least 80% identity to SEQ ID NO: 1. The mannanase has mannan-degrading activity at a pH in the range of 5-12 or 6-11, more preferably at a pH in the range of 6-10 or 7-9 or 7-12 or 8-12 or 8-10, and most preferably at a pH in the range of 7.5-8.5. At said pH, the mannanase exhibits cleaning performance on mannan-containing stains.

[0069] The mannanase disclosed herein has mannan decomposition activity at a temperature selected from below 60° C., below 40° C., and below 25° C. Thus, preferably, the method is a method for removing stains containing mannan at a temperature below 60° C., 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. Preferably, the temperature is a washing or cleaning temperature.

[0070] In one aspect, the present invention provides a method for removing stains containing mannan 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., (a) providing a liquid formulation comprising at least one mannanase that is at least 80% identical to SEQ ID NO:1; (b) contacting a stain containing mannan with the liquid formulation of (a); (c) allowing the enzyme to exert its catalytic 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 comprising the steps of:

[0071] 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. The liquid formulation is preferably a liquid detergent formulation.

[0072] In one aspect, the present invention relates to a formulation, preferably having a pH in the range of 5 to 12, comprising at least one mannanase at least 80% identical to SEQ ID NO: 1, which has increased washing or cleaning performance on stains comprising mannan, preferably stains comprising at least one galactomannan, more preferably stains comprising locust bean gum and / or guar gum. By increased washing or cleaning performance on stains comprising mannan, we mean increased washing or cleaning performance compared to a formulation lacking the mannanase of the present invention or compared to a formulation lacking any mannanase.

[0073] In one embodiment the formulation has a pH in the range of 6 to 11, more preferably in the range selected from 6 to 10, 7 to 9, 7 to 12, 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.

[0074] In one aspect, the invention relates to the use of a liquid enzyme preparation of the invention for formulating into detergent formulations, such as I&I and home care formulations for laundry and hard surface cleaning, where components (a) and (b) are mixed with one or more detergent ingredients in one or more steps, not in a specific order. By "formulating into" is meant adding the enzyme preparation to the liquid formulation.

[0075] The formulations according to the invention comprise one or more detergent ingredients. The ingredients selected depend on the desired washing or cleaning application and / or the physical form of the formulation, also referred to herein as a detergent formulation.

[0076] 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 effectively aids in its processing (maintains physical properties during processing, storage and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0077] Typically, detergent formulations are complex formulations of three or more detergent ingredients.

[0078] Since a detergent ingredient may have more than one function in the end use of the detergent formulation, 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.

[0079] The term "effective amount" includes the amounts of individual components that provide effective stain removal and effective cleaning conditions (e.g., pH, amount of sudsing), the amount of a particular component that effectively provides an optical benefit (e.g., optical brightening, dye transfer inhibition), and the amount of a particular component that effectively aids in processing (maintaining physical properties during processing, storage and use; e.g., rheology modifiers, hydrotropes, drying agents).

[0080] In one embodiment, the detergent formulation according to the present invention is a blend of three or more detergent components, where at least one component is effective in removing stains, at least one component is effective in providing optimal cleaning conditions, and at least one component is effective in maintaining the physical properties of the detergent.

[0081] 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 the skilled person may be "Detergent Formulations Encyclopedia", Solverchem Publications, 2016.

[0082] Detergent ingredients vary in type and / or amount in detergent formulations 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 local customs, which themselves relate to aspects such as the washing temperature used, washing machine configuration (vertical or horizontal axis washing machine), water consumption per wash cycle, etc., and geographical characteristics such as average water hardness.

[0083] 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 laundry formulations having from about 800 ppm to about 2,000 ppm of detergent components in the wash water, and high detergent concentration systems include laundry formulations having more than about 2,000 ppm of detergent components in the wash water.

[0084] The numerical ranges recited for the individual detergent ingredients indicate the amounts that will be included in the detergent formulation. Such ranges should be understood to be inclusive of the numbers defining the range, including each integer within the defined range.

[0085] 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 the weight of that substance)÷(total weight of the formulation)×100.

[0086] In one embodiment, the detergent formulation according to the present invention comprises one or more surfactants. By "surfactant" (used interchangeably herein as "surface active agent") is meant an organic chemical that when added to a liquid changes the properties of that liquid at an interface. Depending on its ionic charge, surfactants are called nonionic surfactants, anionic surfactants, cationic surfactants or amphoteric surfactants.

[0087] 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 previous editions of the same publications known to those skilled in the art.

[0088] In one embodiment, the detergent according to the present invention comprises a total amount of anionic surfactant 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 based on the total weight of the detergent formulation. In one embodiment, the detergent formulation according to the present invention comprises a total amount of anionic surfactant of about 11 wt.%, based on the total weight of the detergent formulation.

[0089] In one embodiment, the detergent composition according to the present invention comprises a compound of general formula (I):

[0090] [ka] The composition contains at least one anionic surfactant selected from the compounds of the formula:

[0091] The variables in general formula (I) are defined as follows:

[0092] 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 H 31 , nC 17 H 35 , i-C9H 19 ,I C 12 H 25 It is.

[0093] R 2 is H, C1~C 20 -Alkyl and C2-C 20 -alkenyl, where alkyl and / or alkenyl are linear or branched.

[0094] R 3 and R 4 are independent of each other, C1~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.

[0095] A - Ha-RCOO - , -SO3 - and RSO3 - R is selected from linear or branched C1-C8-alkyl and C1-C4-hydroxyalkyl, where alkyl is. - SO3 - (Fatty) alcohol / alkyl(ethoxy / ether) sulfate [(F)A(E)S], and A - Ga-RCOO - In some cases, they are called (fatty) alcohol / alkyl (ethoxy / ether) carboxylates [(F)A(E)C].

[0096] M + is selected from H and salt-forming cations. The salt-forming cations may be monovalent or polyvalent, and thus M + is 1 / v M v+ Examples include, but are not limited to, the sodium, potassium, magnesium, calcium, ammonium and ammonium salts of mono-, di- and triethanolamine.

[0097] The integers in the general formula (I) are defined as follows:

[0098] 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.

[0099] The anionic surfactants of general formula (I) can be of any structure, block or random copolymers.

[0100] Further suitable anionic surfactants include C 12 ~C 18 -sulfofatty acid alkyl esters (e.g., C 12 ~C 18 -sulfo fatty acid methyl esters), C 10 ~C 18 -Alkylarylsulfonic acids (e.g., nC 10 ~C 18 -Alkylbenzenesulfonic acid) and C 10 ~C 18 -Alkyl alkoxycarboxylate salts (M + ) are mentioned.

[0101] M + is in each case selected from the salt-forming cations. The salt-forming cations may be monovalent or polyvalent, so that M + is 1 / v M v+ Examples include, but are not limited to, the sodium, potassium, magnesium, calcium, ammonium and ammonium salts of mono-, di- and triethanolamine.

[0102] In one embodiment, the detergent formulation comprises at least two anionic surfactants selected from compounds of general formula (I), one of said anionic surfactants being R 1 C 11 , R 2is H, m is 2, n and o = 0, A - SO3 - , M + Na + The other surfactant is R 1 C 13 , R 2 is H, m is 2, n and o = 0, A - SO3 - , M + Na + It is characterized in that:

[0103] In one embodiment, the detergent formulation comprises a compound represented by the general formula (II):

[0104] [ka] [In formula (II), R 1 is C 10 ~C 13 -alkyl] In one embodiment, the detergent formulation comprises at least two anionic surfactants selected from compounds of formula (II), one of which is selected from the compounds of formula (II): 1 C 10 The other surfactant is R 1 C 13 Such compounds are also referred to herein as LAS (linear alkylbenzene sulfonates).

[0105] In one embodiment, the detergent formulation of the present invention comprises 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 of about 5.5% by weight, all based on the total weight of the detergent formulation.

[0106] In one embodiment, the detergent formulation according to the present invention has the general formula (III):

[0107] [ka] The composition comprises at least one nonionic surfactant.

[0108] The variables in formula (III) are defined as follows:

[0109] 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 It is.

[0110] R 2 is H, C1~C 20 -Alkyl and C2-C 20 -alkenyl, where alkyl and / or alkenyl are linear or branched.

[0111] R 3 and R 4 are independent of each other, C1~C 16 alkyl, which is straight 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.

[0112] R 5 H and C1-C18 The alkyl is selected from alkyl, which may be straight or branched.

[0113] The integers in the general formula (III) are defined as follows:

[0114] 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.

[0115] The nonionic surfactant of general formula (III) may be of any structure, either block or random, and is not limited to the sequence of formula (III) shown.

[0116] The compounds according to formula (III) are also referred to herein as alkyl polyethylene glycol ethers (AEOs).

[0117] In one embodiment, the detergent formulation comprises a compound represented by general formula (III): 1 is C 12 ~C 14 and R 5 In one embodiment, the detergent formulation comprises at least two nonionic surfactants selected from compounds of general formula (III), one of which is represented by R 1 C 12 , R 5 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.

[0118] In one embodiment, the detergent formulation according to the invention comprises 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 are also referred to herein as "builders" or "building agents", without meaning to limit such compounds to this function in the end use of the detergent formulation. In one embodiment, the detergent formulation of the present invention comprises at least one builder selected from non-phosphate based builders, such as sodium gluconate, citrates, silicates, carbonates, phosphonates, aminocarboxylates, polycarboxylates, polysulfonates, and polyphosphonates.

[0119] 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 mono-alkali metal salts, preferably trisodium salt, ammonium or substituted ammonium salts of citric acid, and citric acid itself. The citrates can be used as anhydrous compounds or as hydrates, e.g., sodium citrate dihydrate. In one embodiment, the citrates are 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 citrates ranging from about 1 to 3% by weight, all based on the total weight of the detergent formulation.

[0120] The detergent formulation of the present invention may comprise one or more hydrotropes. In one embodiment, the detergent formulation comprises one or more hydrotropes selected from organic solvents such as, but not limited to, ethanol, isopropanol, ethylene glycol, 1,2-propylene glycol, and further organic solvents known in the art that are water-miscible under normal conditions. In one embodiment, the detergent formulation of the present invention comprises 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.

[0121] 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.

[0122] In one embodiment, the detergent formulation of the present invention comprises, in addition to the mannanase of the present invention, at least one further enzyme selected from proteases, amylases, lipases, cellulases, mannanases and any other enzymes known in the art to be useful in detergent formulations.

[0123] The at least one enzyme in the detergent formulation of the present invention, in addition to the mannanase, may itself be stabilized by an enzyme stabilizer. In one embodiment, the at least one enzyme stabilizer is selected from boron-containing compounds, such as boric acid or its derivatives and boronic acid or its derivatives, their salts, and mixtures thereof, all as disclosed herein. In one embodiment, the at least one enzyme stabilizer is selected from the peptide aldehydes disclosed herein.

[0124] In one embodiment, the at least one enzyme stabilizer is selected from polyols containing 2 to 6 hydroxyl groups that stabilize proteases, suitable examples include glycol, 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol, ethylene glycol, hexylene glycol, glycerol, sorbitol, mannitol, erythritol, glucose, fructose and lactose.

[0125] In one embodiment, the detergent formulation of the present invention is a laundry detergent.

[0126] The term "laundry" refers to the process of treating textiles with a solution comprising the detergent formulation of the present invention, both in domestic and industrial laundry. In one embodiment, the laundry process is carried out by using a technical device, such as a domestic or industrial washing machine. A washing machine is also referred to herein as a washing machine. Alternatively, the laundry process may be carried out manually.

[0127] The term "textile" means any textile material including yarns (threads made of 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), cloth, and other articles.

[0128] The term "fibers" includes natural fibers, synthetic fibers and mixtures thereof. Examples of natural fibers are those of vegetable origin (e.g. flax, jute and cotton) or those of animal origin that contain proteins such as collagen, keratin and fibroin (e.g. silk, wool, angora, mohair, cashmere). Examples of fibers of synthetic origin are polyurethane fibers, e.g. Spandex® or Lycra®, polyester fibers, polyolefins, e.g. elastofin, or polyamide fibers, e.g. nylon. By fiber is meant a single fiber or a part of a textile, e.g. knitwear, woven or nonwoven fabric.

[0129] The present invention relates to 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 that is at least 80% identical to SEQ ID NO:1; (b) at least one detergent ingredient selected from surfactants, builders, and hydrotropes, present in an amount effective for cleaning and / or maintaining the physical properties of the detergent; The method includes the step of mixing the

[0130] In one embodiment, the present invention provides a method for providing a liquid mannanase-containing formulation, comprising one or more steps, in any order, (a) an enzyme preparation of the invention comprising at least one mannanase that is at least 80% identical to SEQ ID NO:1; (b) at least one detergent ingredient selected from surfactants, builders, and hydrotropes, present in an amount effective for cleaning and / or maintaining the physical properties of the liquid formulation; The method includes the step of mixing the

[0131] In one embodiment the formulation has a pH in the range of 5 to 12 or 6 to 11, more preferably in the range selected from 6 to 10, 7 to 9, 7 to 12, 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.

[0132] The laundry detergent formulation of the present invention exhibits cleaning performance evaluated under relevant washing conditions. In this specification, 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 stains containing mannans; preferably, the mannan-containing stains are selected from those containing galactomannans and glucomannans. In one embodiment, the cleaning performance relates to the removal of stains containing galactomannans, more preferably locust bean gum and / or guar gum.

[0133] The present invention relates to the use of at least one mannanase at least 80% identical to SEQ ID NO: 1 to increase the washing or cleaning performance of a detergent formulation on mannan-containing stains, preferably on stains containing at least one galactomannan, more preferably on stains containing locust bean gum and / or guar gum.

[0134] In one embodiment the detergent formulation has a pH in the range of 5 to 12 or 6 to 11, more preferably in the range selected from 6 to 10, 7 to 9, 7 to 12, 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.

[0135] 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.

[0136] 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). The present invention relates to a method comprising the steps of:

[0137] Preferably, the mannan containing stain comprises at least one galactomannan, more preferably locust bean gum and / or guar gum.

[0138] In one embodiment, in step (a), a textile is provided that includes a stain that includes mannan.

[0139] In one embodiment, the mannanase contained in the detergent according to the present invention removes mannan-containing stains from textile (a) by a step (c) of contacting the mannan-containing stains with the detergent (b). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0140] The present invention includes the following embodiments.

[0141] 1. A method for removing stains containing mannan by contacting at least one mannan-containing stain with a mannanase that is at least 80% identical to SEQ ID NO:1.

[0142] 2. A liquid enzyme preparation comprising a mannanase that is at least 80% identical to SEQ ID NO:1, at least one compound that stabilizes the liquid enzyme preparation itself, at least one solvent, and optionally at least one enzyme stabilizer.

[0143] 3. An enzyme preparation according to embodiment 2, having a pH in the range of 5 to 12 and preferably being liquid.

[0144] 4. A formulation having increased washing or cleaning performance against stains containing mannan, preferably against stains containing at least one galactomannan, preferably locust bean gum and / or guar gum, comprising a mannanase that is at least 80% identical to SEQ ID NO:1.

[0145] 5. The formulation of embodiment 3, having a pH in the range of 5 to 12 and preferably being liquid.

[0146] 6. The formulation of embodiments 4 and 5, comprising at least one component selected from surfactants, builders and hydrotropes in an amount effective to maintain the physical properties of the formulation and / or in an amount effective for cleaning.

[0147] 7. The formulation according to embodiments 4-6, which is preferably a detergent formulation, more preferably a laundry detergent formulation, more preferably a liquid laundry detergent.

[0148] 8. A method for providing a liquid detergent effective against mannan-containing stains, comprising one or more steps: (a) at least one mannanase having at least 80% identity to SEQ ID NO:1, preferably provided in an enzyme preparation as described in embodiments 2 and 3; (b) at least one component selected from surfactants, builders, and hydrotropes, present in an amount effective for cleaning and / or maintaining the physical properties of the detergent; The method of claim 1, further comprising the step of:

[0149] 9. A method of washing or cleaning comprising the steps of: (a) providing at least one mannan-containing stain, preferably the mannan-containing stain comprises at least one galactomannan, more preferably the mannan-containing stain comprises locust bean gum and / or guar gum; (b) providing a formulation as described in any one of embodiments 4 to 6; (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:

[0150] 10. The method of embodiment 8, wherein a mannan-containing stain is contained on the textile (a) and the polypeptide contained in the detergent (b) removes the mannan-containing stain from the textile (a).

[0151] 11. Use of at least one mannanase at least 80% identical to SEQ ID NO: 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. EXAMPLES

[0152] [Example 1] Evaluation of mannanase during Rotawash. The mannanase enzymes Man01, Man02 and Man03: Man01: mannanase according to SEQ ID NO:1 Man02: mannanase according to SEQ ID NO:2 Man03: mannanase according to SEQ ID NO:3 were tested in liquid detergents ES1_C (pH 8) and Persil non-Bio (pH 7.2) at wash temperatures of 40°C or 25°C.

[0153] [Table 1]

[0154] CFT CS-43 / Guar Gum Stain Monitor or CFT CS-73 / Locust Bean Gum Stain Monitor (CFT, Vlaardingen, NL) were washed together with cotton ballast fabric and steel balls in a wash solution using base formulation ES1_C under the following washing conditions in a Rotawash (Rotawash M228, SDL Atlas Inc., USA):

[0155] [Table 2]

[0156] After washing, the fabric was rinsed and dried in air. The cleaning performance for single stains was determined by measuring the average color intensity of the soiled fabric after washing using an Epson flatbed color image scanner (Expression 11000XL). In general, the higher the average intensity value, the better the performance. The results are also summarized in the following tables Ex1a, Ex1b and Ex1c.

[0157] [Table 3]

[0158] [Table 4]

[0159] [Table 5]

[0160] [Table 6]

[0161] [Table 7]

Claims

1. A method for removing stains comprising mannans by contacting at least one mannan-containing stain with a mannanase that is at least 90% identical to SEQ ID NO:1 at a temperature in the range of 5-60°C.

2. The method of claim 1 , wherein the stain comprising mannan is on a textile.

3. A liquid enzyme preparation comprising a mannanase that is at least 90% identical to SEQ ID NO:1, at least one compound that stabilizes the liquid enzyme preparation itself, and at least one solvent.

4. 4. The liquid enzyme preparation of claim 3, having a pH in the range of 5 to 12.

5. A laundry detergent formulation having increased washing or cleaning performance on mannan-containing stains, the laundry detergent formulation comprising a mannanase that is at least 90% identical to SEQ ID NO:1 and having a pH in the range of 5 to 12.

6. 6. The formulation of claim 5, comprising at least one component selected from surfactants, builders and hydrotropes in an amount effective to maintain the physical properties of the formulation and / or in an amount effective for cleaning.

7. 1. A method for providing a liquid laundry detergent that is effective against mannan-containing stains, comprising one or more steps of: (a) at least one mannanase that is at least 90% identical to SEQ ID NO:1; (b) at least one detergent ingredient selected from surfactants, builders, and hydrotropes, present in an amount effective for cleaning and / or maintaining the physical properties of the detergent; The method of claim 1, further comprising the step of:

8. 1. A method of washing or cleaning comprising the steps of: (a) providing a stain comprising at least one mannan; (b) providing a formulation according to claim 5 or 6; (c) contacting the mannan-containing stain (a) with the formulation of (b). A method comprising:

9. The method according to claim 8, wherein a stain containing mannan is formed on the textile (a), and a polypeptide contained in the detergent (b) removes the stain containing mannan from the textile (a).

10. 10. The method of claim 9, wherein the mannan-containing stain comprises at least one galactomannan.

11. Use of at least one mannanase at least 90% identical to SEQ ID NO: 1 to increase the cleaning performance of a detergent formulation on mannan-containing stains.

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