DESCRIPTION

Different classes of enzymes are known to be useful in detergent formulations such as prote ase, amylase, cellulase, lipase, mannanase, pectate lyase, and nuclease. Mannanases are use ful components of washing and/or cleaning formulations since mannanases remove part of hemicellulose comprising stains. Insufficient removal of these types of stains may e.g. result in fabric graying.

The major constituents of hemicellulose are hetero-1 ,4-D-xylans and herto-1 ,4-beta-mannans. Mannans are polysaccharides with a backbone of b-1 ,4-linked D-mannopyranosyl residues, which may comprise galactose or acetyl substitutions and may have glucose residues in the backbone. The main enzyme type participating in the degradation of mannans are endo-1 ,4- beta-mannanases (EC 3.2.1 .78), which hydrolyze the internal glycoside bonds in the mannan backbone. Endo-1 ,4^-mannanases (EC 3.2.1 .78) are mannan-degrading enzymes which may be called endo-b-1 ,4-D-mannanase, b-mannanase, or mannanase herein.

There is a need for mannanase enzymes that perform in the harsh environment of detergent formulations as well as under harsh washing conditions. Hence, it was the objective to find a mannanase suitable for use in formulations having a pH in the range of 5-1 1 , preferably in the range of 5.5-10, more preferably in the range of 6-9, most preferably in the range of 6.5-8, pref erably detergent formulations.

The present invention provides a mannanase comprising a catalytic domain and optionally a carbohydrate binding domain (CBD) which is catalytically active and storage stable at pH 5-1 1 , preferably 5.5-10, more preferably 6-9, most preferably 6.5-8. In one embodiment, the catalytic domain of said mannanase originates from T choderma reesei mannanase according to SEQ ID NO: 1 of WO 2008/009673, wherein the catalytic domain of the mannanase enzyme is at least 90% similar or identical to said SEQ ID NO:1 and comprises at least one amino acid sub stitution at position selected from 3, 31 , 66, 97, 1 13, 173, 181 , 201 , 207, 215, 259, 274, 280, 282, and 331 within SEQ ID NO: 1 of WO 2008/009673. The optional CBD is located at the C- terminus of the catalytic domain, preferably without any linking domain, wherein the CBD may have a sequence that is at least 90% similar or identical to SEQ ID NO: 10 of WO 201 1/085747. The mannanases as disclosed in WO 2008/009673 and WO 201 1/085747 are described to have increased stability when compared to the wild-type T reesei mannanase against low pH conditions e.g. pH 2.3.

The invention at hand provides a formulation having a pH in the range of 5-1 1 , preferably in the range of 5.5-10, more preferably in the range of 6-9, most preferably in the range of 6-8, com prising a T. reesei mannanase variant as disclosed herein, wherein said mannanase variant is

(a) active at the pH of the formulation, and

(b) is storage-stable when stored at 37°C for up to 22 days.

Preferably, the formulation is a liquid formulation comprising at least one component selected from surfactants, builders, and hydrotropes is present in amounts effective in cleaning perfor mance or effective in maintaining the physical characteristics of the formulation.

Generally,“enzymes” are catalytically active proteins acting on substrates and converting these into products. This reaction may be called enzymatic conversion herein which typically takes place at the“active site” of an enzyme. Enzymes exerting enzymatic conversion are enzymati cally active or have enzymatic activity. Any polypeptide called“enzyme” herein means polypep tides being catalytically active.

The mannanases according to the invention have mannan degrading activity. Mannan degrad ing activity or mannanase activity may be tested according to standard test procedures known in the art. For example: a mannanase to be tested may be applied to 4 mm diameter holes punched out in agar plates comprising 0.2% AZCL galactomannan (carob), i.e. substrate for the assay of endo-1 ,4-beta-D-mannanase available as CatNo. I-AZGMA from the company

Megazyme (Megazyme's Internet address: http://www. megazyme. com/Purchase/index. html).

Enzymes such as a mannanase of the invention are polypeptides which are usually identified by polypeptide sequences (also called amino acid sequences herein). Polypeptide sequences may be identified by a SEQ ID NO. According to the World Intellectual Property Office (WIPO) Standard ST.25 (1998) the amino acids herein are represented using three-letter code with the first letter as a capital or the corresponding one letter.

The terms polypeptide and polypeptide sequence are used synonymously with mannanase of the invention. A mannanase of the invention is a variant of a parent mannanase having a parent polypeptide amino acid sequence. A“parent” polypeptide amino acid sequence is the starting sequence for introduction of muta tions (e.g. by introducing one or more amino acid substitutions, insertions, deletions, or a com bination thereof) to the sequence, resulting in“variants” of the parent polypeptide amino acid sequences. A parent includes: A wild-type polypeptide amino acid sequence or synthetically generated polypeptide amino acid sequence that is used as starting sequence for introduction of (further) changes.

In one embodiment, the parent polypeptide for the catalytic domain of the mannanase in this invention is originating from a T choderma reesei mannanase according to SEQ ID NO: 1 of WO 2008/009673 which optionally comprises a CBD located at the C-terminus of the catalytic domain, preferably without any linking domain, wherein the CBD preferably has a sequence according to SEQ ID NO: 10 of WO 201 1/085747.

A“variant polypeptide” refers to an enzyme that differs from its parent in its amino acid se quence. While the definition below describes variants in the context of amino acid changes, nu cleic acids may be similarly modified, e.g. by substitutions.

A variant polypeptide of the invention herein means mannanases of the invention as disclosed herein which are originating from a parent Trichoderma reesei mannanase according to SEQ ID NO: 1 of WO 2008/009673 which optionally comprises a CBD located at the C-terminus of the catalytic domain, preferably without any linking domain, wherein the CBD preferably has a se quence according to SEQ ID NO: 10 of WO 201 1/085747.

“Substitutions” are described by providing the original amino acid followed by the number of the position within the amino acid sequence, followed by the substituted amino acid. For example, the substitution of histidine at position 120 with alanine is designated as“His120Ala” or“H120A”

In one embodiment, the variant polypeptide for the catalytic domain of the mannanase of the invention comprises at least one substitution at a position selected from 3, 66, 1 13, 181 , 207, 215, and 274 within the sequence as provided in SEQ ID NO: 1 of WO 2008/009673 and has mannan degrading activity. Preferably, the variant polypeptide for catalytic domain of the man nanase of the invention comprises at least one substitution selected from S3R, S66P, N1 13Y,

V181 H, L207F, A215T, and F274L within the sequence of SEQ ID NO: 1 of WO 2008/009673.

The substitution of one amino acid with a similar amino acid may be called“conservative muta tion” herein. Similar amino acids according to the invention are defined as follows: amino acid A is similar to amino acids S; amino acid D is similar to amino acids E and N; amino acid E is simi lar to amino acids D, K, and Q; amino acid F is similar to amino acids W and Y; amino acid FI 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 acids 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, FI, and W.

Conservative amino acid substitutions may occur everywhere within the full length of the se quence of a polypeptide sequence of a functional protein such as an enzyme. Usually, con servative mutations do not change the catalytic activity of an enzyme.

In one embodiment, such mutations are not pertaining to the functional domains of an enzyme such as the mannanase of the invention. In one embodiment, conservative mutations are not pertaining to the catalytic centers of an enzyme such as the mannanase of the invention.

In one embodiment, the variant polypeptide for catalytic domain of the mannanase of the inven tion comprises at least one conservative substitution at a position selected from 31 , 97, 173, 201 , 259, 280, 282, and 331 within the sequence as provided in SEQ ID NO: 1 according to SEQ ID NO: 1 of WO 2008/009673 and has mannan degrading activity. Preferably, the variant polypeptide for catalytic domain of the mannanase comprises at least one conservative substi tution selected from F31Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S within the sequence according to SEQ ID NO: 1 of WO 2008/009673.

Variant polypeptides may be defined by their“sequence similarity” when compared to a parent sequence. Sequence similarity usually is provided as“% sequence similarity” or“%-similarity”.

% sequence similarity takes into account that defined sets of amino acids share similar proper ties, e.g. by their size, by their hydrophobicity, by their charge, or by other characteristics. Simi lar amino acids are amino acids that substitute similar amino acids and are called“conservative mutation” herein.

Variant polypeptides of the invention or mannanases of the invention“being x% similar to a mannanase comprising substitutions at positions as disclosed within the sequence according to e.g. SEQ ID NO: 1 of WO 2008/009673” means that“the variant polypeptide has a polypeptide sequence which is at least x% similar to a polypeptide sequence according to e.g. SEQ ID NO: 1 of WO 2008/009673 comprising at least one of said substitutions”. For calculation of sequence similarity, in a first step, a sequence alignment has to be produced. According to this invention, a pairwise global alignment has to be produced, meaning that two sequences have to be aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.

According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program“NEEDLE” (The Europe an Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap ex- tend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and ma- trix=EBLOSUM62).

After aligning two sequences, in a second step, a similarity value is determined from the align ment produced.

In one embodiment, the %-similarity is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length multiplied with 100: %-similarity = [ (identical residues + similar residues) / length of the alignment region which is showing the respective sequence of this invention over its complete length] ^* 100.

In a preferred embodiment, the %-similarity is calculated by dividing the number of identical res idues plus the number of similar residues by the length of the alignment region which is showing the two aligned sequences over their complete length multiplied with 100: %-similarity = [ (iden tical residues + similar residues) / length of the alignment region which is showing the two aligned sequences over their complete length] ^* 100.

In one embodiment, the variant polypeptide for catalytic domain of the mannanase of the inven tion is at least 85% similar to SEQ ID NO: 1 of WO 2008/009673. The mannanase of the inven tion may be 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%, at least 99% similar to SEQ ID NO: 1 of WO

2008/009673.

In one embodiment, the variant polypeptide for catalytic domain of the mannanase of the inven tion which is at least 85%, preferably at least 95%, more preferably at least 98% similar to SEQ ID NO: of WO 2008/009673 comprises at least one substitution at a position selected from 3,

66, 1 13, 181 , 207, 215, and 274 within the sequence as provided in SEQ ID NO: 1 of

W02008/009673, and has mannan degrading activity. Preferably, at least one substitution is selected from S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L. Variant polypeptide sequences may be defined by their“sequence identity” when compared to a parent sequence. Sequence identity usually is provided as“% sequence identity” or“% identity”. For calculation of sequence identities, in a first step a sequence alignment has to be produced as disclosed above.

Variant polypeptides of the invention or mannanases of the invention“being x% identical to a mannanase comprising substitutions at positions as disclosed within the sequence according to e.g. SEQ ID NO: 1 of WO 2008/009673” means that“the variant polypeptide has a polypeptide sequence which is at least x% identical to a polypeptide sequence according to e.g. SEQ ID NO: 1 of WO 2008/009673 comprising at least one of said substitutions”.

In one embodiment, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of this invention over its complete length multiplied with 100: %-identity = (identical residues / length of the alignment region which is showing the respective sequence of this invention over its complete length) ^* 100.

In a preferred embodiment, the %-identity is calculated by dividing the number of identical resi dues by the length of the alignment region which is showing the two aligned sequences over their complete length multiplied with 100: %-identity = (identical residues / length of the align ment region which is showing the two aligned sequences over their complete length) ^* 100.

In one embodiment, the variant polypeptide for catalytic domain of the mannanase of the inven tion is at least 85% similar or identical to SEQ ID NO: 1 of WO 2008/009673. The variant poly peptide for catalytic domain of the mannanase of the invention may be 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%, at least 99% similar or identical to SEQ ID NO: 1 of WO 2008/009673.

In one embodiment, the variant polypeptide for catalytic domain of the mannanase of the inven tion which is at least 85%, preferably at least 90%, more preferably at least 95% similar or iden tical to SEQ ID NO: 1 of WO 2008/009673 comprises within the sequence of SEQ ID NO: 1 of WO 2008/009673 at least one substitution selected from S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L. Preferably, the variant polypeptide for catalytic domain further comprises at least one conservative substitution selected from F31Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S. In one embodiment, the variant polypeptide for catalytic domain of the mannanase which is at least 85%, preferably at least 90%, more preferably at least 95% similar or identical to SEQ ID NO: 1 of WO 2008/009673 comprises within the sequence of SEQ ID NO:1 of WO 2008/009673 the substitutions S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L and at least one con servative substitution selected from F31 Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S.

In one embodiment, the catalytic domain of the mannanase according to the invention compris es a sequence according to SEQ ID NO: 6 of WO 201 1/085747.

The mannanase of the invention may comprise a polypeptide sequence according to SEQ ID NO: 6 of WO 201 1/085747 and at its C-terminus a polypeptide sequence according to SEQ ID NO: 10 of WO 201 1 /085747.

The mannanase of the invention (comprising a CBD or not) is a“mature polypeptide” meaning an enzyme in its final form including any post-translational modifications, glycosylation, phos phorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence de letion. A mature polypeptide can vary depending upon the expression system, vector, promoter, and/or production process.

Enzymatic activity may change during storage or operational use of the enzyme. Parameters influencing the enzymatic activity of an enzyme and/or storage stability and/or operational stabil ity are for example pH, temperature, and presence of oxidative substances.

A mannanase according to the invention is active at a pH in the range of 5-1 1 , preferably in the range of 5.5-10, more preferably in the range of 6-9, most preferably in the range of 6-8.

The term“enzyme stability” relates to the retention of enzymatic activity as a function of time during storage or operation. The term“storage” herein means to indicate the fact of products or formulations or formulations being stored from the time of being manufactured to the point in time of being used in final application. Retention of enzymatic activity as a function of time dur ing storage in detergent may be called“storage stability” herein.

To determine and quantify changes in catalytic activity of enzymes stored or used under certain conditions over time, the“initial enzymatic activity” is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, a potential loss of enzymatic activity can be determined in its extent. The extent of enzymatic ac tivity loss determines an enzymes stability or non-stability. A mannanase of the invention is storage stable at pH in the range of 5-1 1 , preferably in the range of 5.5-10, more preferably in the range of 6-9, most preferably in the range of 6-8.

In one embodiment storage stable means, that a mannanase of the invention retains its man- nan-degrading activity at a pH as disclosed over a time period of up to 22 days of storage at 37°C. According to the invention, the mannanase retains its mannan-degrading activity, when the extent of enzymatic activity loss after storage is less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5%.

In one embodiment, a mannanase of the invention is storage-stable in a liquid formulation at the pH and storage conditions as disclosed.“Liquid” herein is related to the physical appearance at 20°C and 101.3 kPa.

In one embodiment, a mannanase of the invention is storage-stable in a liquid detergent formu lation at the pH and storage conditions as disclosed.

In one aspect, the invention relates to a formulation having a pH in the range of 5-1 1 , preferably in the range of 5.5-10, more preferably in the range of 6-9, most preferably in the range of 6-8, and even more preferably 7-9 comprising at least one mannanases as disclosed herein.

In one aspect, the invention relates to a formulation having a pH in the range of 6-8 comprising at least one mannanase being at least 90% similar to a mannanase comprising

(a) a catalytic domain comprising at least one amino acid substitution at position selected from 3, 31 , 66, 97, 1 13, 173, 181 , 201 , 207, 215, 259, 274, 280, 282, and 331 within SEQ ID NO: 1 of WO 2008/009673, and

(b) optionally a carbohydrate binding domain (CBD) according to SEQ ID NO: 10 of

WO 201 1/085747 at the C-terminus of the catalytic domain, preferably without any linking domain in between.

In one embodiment, the formulation comprises a variant polypeptide for catalytic domain of the mannanase which is at least 90%, preferably at least 95% similar or identical to SEQ ID NO: 1 of WO 2008/009673 and comprises within the sequence of SEQ ID NO:1 of WO 2008/009673 the substitutions S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L and at least one con servative substitution selected from F31 Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S.

In one embodiment, said formulation is liquid. In one embodiment, said liquid formulation is a storage-stable detergent formulation, preferably wherein storage-stability is characterized by the loss of mannan-degrading activity of the man- nanase at a pH of 6-8 is less than 15% after storage at 37°C for up to 22 days.

A detergent formulation according to the invention comprises one or more detergent compo nents). The component(s) chosen depend on the desired wash or cleaning application and/or physical form of the detergent formulation.

The term“detergent component” is defined herein to mean any types of ingredient, which is suitable for detergent formulation, such as surfactants, building agents, polymers, bleaching systems. Any component(s) known in the art acknowledging their known characteristics are suitable detergent component(s) according to the invention. Detergent components in one em bodiment means components which provide washing or cleaning performance, or which effec tively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants) when present in effective amounts.

Usually, a detergent formulation is a complex formulation of more than two detergent compo nents.

Detergent components may have more than one function in the final application of a detergent formulation, therefore any detergent component mentioned in the context of a specific function herein, may also have another function in the final application of a detergent formulation. The function of a specific detergent component in the final application of a detergent formulation usually depends on its amount within the detergent formulation, i.e. the effective amount of a detergent component.

The term“effective amount” includes amounts of individual components to provide effective stain removal and effective cleaning conditions (e.g. pH, quantity of foaming), amounts of cer tain components to effectively provide optical benefits (e.g. optical brightening, dye transfer in hibition), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desic cants).

In one embodiment, the detergent formulation according to the invention is a formulation of more than two detergent components, wherein at least one component is effective in stain- removal, at least one component is effective in providing the optimal cleaning conditions, and at least one component is effective in maintaining the physical characteristics of the detergent. Individual detergent components and usage in detergent formulation are known to those skilled in the art. Suitable detergent components comprise inter alia surfactants, builders, polymers, alkaline, bleaching systems, fluorescent whitening agents, suds suppressors and stabilizers, hydrotropes, and corrosion inhibitors. Further examples are described e.g. in“complete Tech nology 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 may be“Detergent Formulations Encyclopedia”, Solverchem Publications,

2016.

Detergent components vary in type and/or amount in a detergent formulation depending on the desired application such as laundering white textiles, colored textiles, and wool. The compo nents) chosen further depend on physical form of a detergent formulation (liquid, solid, gel, provided in pouches or as a tablet, etc.). The component(s) chosen e.g. for laundering formula tions further depend on regional conventions which themselves are related to aspects like washing temperatures used, mechanics of laundry machine (vertical vs. horizontal axis ma chines), water consumption per wash cycle etc. and geographical characteristics like average hardness of water.

For example: A low detergent concentration system includes laundering formulations where less than about 800 ppm of detergent components are present in the wash water. A medium deter gent concentration includes laundering formulations where between about 800 ppm and about 2,000 ppm of detergent components are present in the wash water. A high detergent concentra tion includes laundering formulations where more than about 2,000 ppm of detergent compo nents are present in the wash water.

The numeric ranges recited for the individual detergent components provide amounts com prised in detergent formulations. Such ranges have to be understood to be inclusive of the numbers defining the range and include each integer within the defined range.

If not described otherwise,“% by weight” or“% w/w” is meant to be related to total detergent formulation. In this case“% by weight” or“% w/w” is calculated as follows: concentration of a substance as the weight of that substance divided by the total weight of the formulation, multi plied by 100.

In one embodiment, the detergent formulation according to the invention comprises one or more surfactant(s). "Surfactant" (synonymously used herein with“surface active agent”) means an organic chemical that, when added to a liquid, changes the properties of that liquid at an inter- face. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or ampho teric.

Non-limiting examples of surfactants are disclosed McCutcheon's 2016 Detergents and Emulsi fiers, and McCutcheon's 2016 Functional Materials, both North American and International Edi- tion, MC Publishing Co, 2016 edition. Further useful examples are disclosed in earlier editions of the same publications which are known to those skilled in the art.

In one embodiment, the detergent according to the invention comprises a total amount of anion ic surfactant which in the range of 1 % to 30%by weight, in the range of 3% to 25% by weight, in the range of 5% to 20% by weight, or in the range of 8% to 15% by weight, all relative to the total weight of the detergent formulation. In one embodiment, the detergent formulation of the invention comprises a total amount of anionic surfactant of about 1 1% by weight relative to the total weight of the detergent formulation.

In one embodiment, the detergent composition according to the invention comprises at least one anionic surfactant selected from compounds of general formula (I):

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

R ¹ is selected from Ci-C23-alkyl (such as 1 -, 2-, 3-, 4- Ci-C23-alkyl) and C2-C23-alkenyl, wherein alkyl and/or alkenyl are linear or branched, and wherein 2-, 3-, or 4-alkyl; examples are n-C Hi ₅ , n-CgHig, n-Cii H23, n-Ci3H27, n-Ci ₅ H _3i , n-Ci7H35, 1-C ₉ H1 ₉ , i-Ci2H25· R ² is selected from H, CrC2o-alkyl and C2-C2o-alkenyl, wherein alkyl and/or alkenyl are linear or branched.

R ³ and R ⁴ , each independently selected from Ci-Ci ₆ -alkyl, wherein alkyl is linear 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. A is selected from -RCOO , -SO ₃ and RSO ₃ , wherein R is selected from linear or branched Ci- Cs-alkyl, and C ₁ -C ₄ hydroxyalkyl, wherein alkyl is. Compounds might be called (fatty) alco hol/alkyl (ethoxy/ether) sulfates [(F)A(E)S] when A- is SO ₃ , (fatty) alcohol/alkyl (ethoxy/ether) carboxylate [(F)A(E)C] when A- is -RCOO .

M ⁺ is selected from FI and salt forming cations. Salt forming cations may be monovalent or mul tivalent; hence M ⁺ equals 1/v M ^v+ . Examples include but are not limited to sodium, potassium, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanolamine.

The integers of the general formula (I) are defined as follows: m is in the range of zero to 200, preferably 1 -80, more preferably 3-20; n and 0, each inde pendently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; 0 preferably is in the range of 1 to 50, more preferably 4 to 25. The sum of m, n and 0 is at least one, preferably the sum of m, n and 0 is in the range of 5 to 100, more preferably in the range of from 9 to 50.

Anionic surfactants of the general formula (I) may be of any structure, block copolymers or ran dom copolymers.

Further suitable anionic surfactants include salts (M ⁺ ) of C12-C18 sulfo fatty acid alkyl esters (such as C12-C18 sulfo fatty acid methyl esters), Cio-Ci ₈ -alkylarylsulfonic acids (such as n-Cio- Cis-alkylbenzene sulfonic acids) and C10-C18 alkyl alkoxy carboxylates.

M ⁺ in all cases is selected from salt forming cations. Salt forming cations may be monovalent or multivalent; hence M ⁺ equals 1/v M ^v+ . Examples include but are not limited to sodium, potassi um, magnesium, calcium, ammonium, and the ammonium salt of mono-, di, and triethanola mine.

The detergent formulation may comprise at least two anionic surfactants, selected from com pounds of general formula (I), wherein one of said anionic surfactants is characterized in R ¹ being Cn, R ² being FI, m being 2, n and 0 = 0, A being SO ₃ , M ⁺ being Na ⁺ and the other surfac tant is characterized in R ¹ being C ₁₃ , R ² being FI, m being 2, n and 0 = 0, A being SO ₃ , M ⁺ be ing Na ⁺ .

In one embodiment, the detergent formulation comprises at least one anionic surfactant select ed from compounds of general formula (II): wherein R ¹ in formula (II) is C10-C13 alkyl. The detergent formulation may comprise at least two anionic surfactants, selected from compounds of general formula (II), wherein one of said anion ic surfactants is characterized in R ¹ being C10, and the other surfactant is characterized in R ¹ being C13. Compounds like this may be called LAS (linear alkylbenzene sulfonates) herein.

The detergent formulation of the invention may comprise a total amount of non-ionic 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 relative to the total weight of the detergent formulation. In one embodiment, the detergent formulation of the invention comprises a total amount of non-ionic surfactants of about 5.5% by weight relative to the total weight of the detergent formulation.

In one embodiment, the detergent formulation according to the invention comprises at least one non-ionic surfactant according to general formula (III):

The variables of the general formula (III) are defined as follows:

R ¹ is selected from C ₁ -C ₂₃ alkyl and C ₂ -C ₂₃ alkenyl, wherein alkyl and/or alkenyl are linear or branched; examples are n-C ₇ Hi ₅ , n-CgHig, n-Cn H ₂ 3, n-Ci3H ₂ 7, n-Ci ₅ H _3i , n-Ci ₇ H ₃ 5, i-CgHig, i- C12H25·

R ² is selected from H, C ₁ -C ₂₀ alkyl and C ₂ -C ₂₀ alkenyl, wherein alkyl and/or alkenyl are linear or branched.

R ³ and R ⁴ , each independently selected from C1-C16 alkyl, wherein alkyl is linear 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.

R ⁵ is selected from H and C1-C18 alkyl, wherein alkyl is linear or branched.

The integers of the general formula (III) are defined as follows: m is in the range of zero to 200, preferably 1 -80, more preferably 3-20; n and 0, each inde pendently in the range of zero to 100; n preferably is in the range of 1 to 10, more preferably 1 to 6; 0 preferably is in the range of 1 to 50, more preferably 4 to 25. The sum of m, n and 0 is at least one, preferably the sum of m, n and 0 is in the range of 5 to 100, more preferably in the range of from 9 to 50.

The non-ionic surfactants of the general formula (III) may be of any structure, is it block or ran dom structure, and is not limited to the displayed sequence of formula (III).

Compounds according to formula (III) may be called alkyl polyethyleneglycol ether (AEO) here in.

In one embodiment, the detergent formulation comprises at least one non-ionic surfactant se lected from general formula (III), wherein m is in the range of 3 to 1 1 , preferably not more than 7; n and 0 is 0, R ¹ is C12-C14, R ⁵ is H. The detergent formulation may comprise at least two non ionic surfactants, selected from compounds of general formula (III), wherein one of said non ionic surfactants is characterized in R ¹ being C12, R ⁵ being H, m is 7, n and 0 = 0, and the other surfactant is characterized in R ¹ being C14, R ⁵ being H, m being 7, n and 0 = 0.

The detergent formulation according to the invention may comprise one or more compounds selected from complexing agents (chelating agents, sequestrating agents), precipitating agents, and ion exchange compounds which may form water-soluble complexes with calcium and mag nesium. Such compounds may be called“builders” or“building agents” herein, without meaning to limit such compounds to this function in the final application of a detergent formulation. In one embodiment, the detergent formulation of the invention comprises at least one builder selected from non-phosphate based builders such as sodium gluconate, citrate(s), silicate(s), car bonate^), phosphonate(s), amino carboxylate(s), polycarboxylate(s), polysulfonate(s), and pol- yphosphonate(s).

In one embodiment, the detergent formulation of the invention comprises at least one“citrate” selected from the mono- and the dialkali metal salts and in particular the mono- and preferably the trisodium salt of citric acid, ammonium or substituted ammonium salts of citric acid as well as citric acid as such. Citrate can be used as the anhydrous compound or as the hydrate, for example as sodium citrate dihydrate. The citrate may be comprised in a total amount in the range of 0% to about 20% by weight, in the range of about 0.5% to about 10% by weight, or in the range of 1 -5% by weight, all relative to the total weight of the detergent formulation. In one embodiment, the detergent formulation of the invention comprises a total amount of citrate in the range of about 1 -3% relative to the total weight of the detergent formulation

The detergent formulation of the invention may comprise one or more hydrotropes. One or more hydrotropes may be selected from organic solvents such as ethanol, isopropanol, ethylene gly col, 1 ,2-propylene glycol, and further organic solvents known in the art that are water-miscible under normal conditions without limitation. In one embodiment, the detergent formulation of the invention comprises 1 ,2-propylene glycol in a total amount in the range of 5-10% by weight, preferably of about 6% by weight, all relative to the total weight of the detergent formulation.

In one embodiment, the detergent formulation of the invention does not comprise any further enzyme besides the mannanase according to the invention.

In one embodiment, the detergent formulation of the invention comprises at least one further enzyme besides the mannanase of the invention, selected from proteases, amylases, lipases, cellulases, mannanases and any other enzymes known in the art to be useful in detergent for mulations.

At least one enzyme which is additionally to the mannanase comprised in the detergent formu lation of the invention may itself be stabilized by an enzyme stabilizer. Enzyme stabilizers may be selected from boron-comprising compounds such as boric acid or its derivatives and boronic acid or its derivatives, from salts thereof, and from mixtures thereof.

In one embodiment, at least one enzyme stabilizer is selected from polyols comprising from 2 to 6 hydroxyl groups to stabilize protease. Suitable examples include glycol, propylene glycol, 1 ,2-propane diol, 1 ,2-butane diol, ethylene glycol, hexylene glycol, glycerol, sorbitol, mannitol, erythriol, glucose, fructose, and lactose, and erythritan.

In one embodiment, the detergent formulation of the invention is a laundering detergent.

The term“laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution comprising a detergent formulation of the present invention. The laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand. The term“textile” means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics (a textile made by weaving, knitting or felting fibers) made of these materials such as garments (any article of clothing made of textile), cloths and other articles.

The term“fibers” includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheep wool, angora, mohair, cashmere). Examples for fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, wovens, or nonwovens.

In one aspect, the invention relates to a method to provide a liquid storage-stable mannanase- detergent comprising the steps of mixing in one or more steps

(a) at least one mannanase being at least 90% similar or identical to a mannanase as dis closed herein, and

(b) at least one detergent component selected from surfactants, builders, and hydrotropes present in amounts effective in cleaning performance or effective in maintaining the physi cal characteristics of the detergent.

The invention relates to a method to provide a liquid storage-stable mannanase-comprising de tergent, comprising the steps of mixing in one or more steps

(a) at least one mannanase being at least 90% similar or identical to a mannanase compris ing i. a catalytic domain comprising at least one amino acid substitution at position select ed from 3, 31 , 66, 97, 1 13, 173, 181 , 201 , 207, 215, 259, 274, 280, 282, and 331 within SEQ ID NO: 1 of WO 2008/009673, and

ii. optionally a carbohydrate binding domain (CBD) according to SEQ ID NO: 10 of WO 201 1/085747 at the C-terminus of the catalytic domain, preferably without any linking domain in between;

(b) at least one detergent component selected from surfactants, builders, and hydrotropes present in amounts effective in cleaning performance or effective in maintaining the physi cal characteristics of the detergent. In one embodiment, at least one mannanase is a variant polypeptide for catalytic domain of the mannanase which is at least 90%, preferably at least 95% similar or identical to SEQ ID NO: 1 of WO 2008/009673 and comprises within the sequence of SEQ ID NO:1 of WO 2008/009673 the substitutions S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L and at least one con servative substitution selected from F31 Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S.

The laundering detergent of the invention exerts wash performance which is evaluated under relevant wash conditions. The term "relevant wash/cleaning conditions" herein refers to the conditions, particularly temperature, time, cleaning mechanics, suds concentration, type of de tergent and water hardness, actually used in laundry machines, or in manual washing process es. In one embodiment, wash performance herein is related towards removal of mannan- comprising stains; preferably mannan-comprising stains are selected from those comprising galactomannans and glucomannans. In one embodiment, wash performance relates to removal of stains comprising locust bean and/or guar gum.

Increase in wash performance herein is related to increase in wash-performance towards man- nan-comprising stains. Increase is usually determined by comparison of a detergent formulation comprising a mannanase of the invention with a detergent formulation lacking mannanase, es pecially the mannanase of the invention.

In one aspect, the invention relates to the use of at least one mannanase as disclosed herein to increase wash performance of a laundry detergent towards at least one mannan-comprising stain when compared to laundry detergents not comprising said mannanase.

The invention relates to the use of at least one mannanase being at least 90% similar or identi cal to a mannanase comprising

(a) a catalytic domain comprising at least one amino acid substitution at position selected from 3, 31 , 66, 97, 1 13, 173, 181 , 201 , 207, 215, 259, 274, 280, 282, and 331 within SEQ ID NO: 1 of WO 2008/009673, and

(b) optionally a carbohydrate binding domain (CBD) according to SEQ ID NO: 10 of

WO 201 1/085747 at the C-terminus of the catalytic domain, preferably without any linking domain in between; to increase wash performance of a laundry detergent towards mannan-comprising stains, pref erably stains comprising locust bean and/or guar gum. In one embodiment, at least one mannanase is used which is a variant polypeptide for catalytic domain of the mannanase which is at least 90%, preferably at least 95% similar or identical to SEQ ID NO: 1 of WO 2008/009673 and comprises within the sequence of SEQ ID NO:1 of WO 2008/009673 the substitutions S3R, S66P, N1 13Y, V181 H, L207F, A215T, and F274L and at least one conservative substitution selected from F31 Y, Q97R, N173H, T201 S, Q259R, Q280R, N282D, and N331 S.

In one embodiment, the wash performance is increased at laundering temperatures £40°C, preferably in the range of about 5-40°C, preferably in the range of about 10-40°C, more prefer ably in the range of about 20-40°C.

In one aspect, the invention relates to a method of laundering, comprising the steps of

(a) providing a textile to be cleaned from mannan-comprising stains;

(b) providing a detergent formulation according to the invention

(c) contacting the textile to be cleaned (a) with the detergent of (b).

In one embodiment, the textile in step (a) comprises mannan-comprising stains.

In one embodiment, the mannanase comprised in the detergent according to the invention re moves the mannan-comprising stains from the textile (a).

Removal of stains herein corresponds to DE as a measure of the achieved stain removal effect or washing effect. DE may be determined by measuring reflectance values of the stains on the monitors using a sphere reflectance spectrometer (SF 500 type from Datacolor, USA, wave length range 360-700nm, optical geometry d/8°) with a UV cutoff filter at 460 nm. For example, with the aid of the CIE-Lab color space classification, the brightness L ^* , the value a ^* on the red - green color axis and the b ^* value on the yellow - blue color axis, can be measured before and after washing. DE is calculated according to the following formula:

According to the invention, DE is increased towards mannan-comprising stains, when compar ing the washing performance of a detergent comprising a mannanase of the invention with a detergent lacking said mannanase. In one embodiment, DE is increased by at least 2. DE may be increased by at least 2.5, by at least 3, or by at least 3.5. In one embodiment, DE is in creased by at least 2.5 at a pH of 8, preferably at a washing temperature of 20°C and/or 40°C.

Example 1 - Mannanase activity/storage-stability at various pH

The activity of the variant mannanase enzymes was determined at pH 6-8. For doing this, the mannanase comprising the catalytic domain according to SEQ ID NO: 6 of WO 201 1/085747 and the CBD according to SEQ ID NO: 10 of WO 201 1/085747, was tested in HEPES (pH 8.0) buffer or Persil Non-Bio formulation at various pH values before storage and after storage 3days and 22 days at 37°C.

900mI of HEPES (pH 8.0) buffer or Persil NonBio formulation was supplemented with 0.1 mg/ml_ mannanase enzyme. The probes were stored at 37°C for several days. 50mI of a sample was taken from the original probe at a certain point in time and diluted in 700mI HEPES buffer. The sample was incubated for 5 min at room temperature. 200mI of this sample was mixed with 200mI of substrate (1% Azo Carob Galactomannan) and further incubated for 60 min. 200mI of the reaction mix was then added to 350mI 95% ethanol on ice and incubated for 10 min. After wards, the reaction mix was centrifuged at 4000 x g for 10 min. The absorption of 250 mI of the resulting supernatant was determined at 590nm. The results of the individual time points are provided in Table Ex1 below. Mannanase activity measured after storage at points in time as indicated in Table Ex1 are provided in relative measures to 100% before storage (time = 0).

Table Ex1 : mannanase activity at various pH before storage and after storage at 37°C; data normalized to activity before storage (Od)

Example 2 - Mannanase performance at various pH

The performance of the variant mannanase enzyme comprising the catalytic domain according to SEQ ID NO: 6 of WO 201 1/085747 and the CBD according to SEQ ID NO: 10 of WO 201 1/085747, was determined at different pH values. The mannanase was tested in 4 g/L liquid detergent (ES1_M formulation). ES1 M:

Tested stain: CFT CS73 (locust bean gum with pigment, CFT, Vlaardingen, NL)

The unwashed soiled swatches were put into open cylindric [stainless steel] vessels (ratio height to diameter 1 :1.5) of a size < 2000ml_, filling rate <80% comprising one of the following types of washing liquor:

Sample (a): water (hardness 2.5 mmol/L; Ca ²⁺ : Mg ²⁺ : HCO3 = 4:1 :8)

Sample (b): water (hardness 2.5 mmol/L; Ca ²⁺ : Mg ²⁺ : HCO3 = 4:1 :8) + 0.4 ppm mannanase

Control sample (a) and mannanase comprising sample (b) were evaluated at different pH val ues: The soiled swatches were shaken in the washing liquor at a fabric/liquor ration of 1 :38 on a shaking table device at 375 rpm and at 40°C for 20 min.

Rinse step: After washing the soiled swatches remained in the vessel and were rinsed under continuous tap water (12-21 °dH) flow (2-6 l/min) for < 5 min.

Drying Step: The rinsed swatches were dried under continuous air stream and stored in a dark closed room under ambient condition until they were measured. The brightness L ^* , the value a ^* on the red - green color axis and the b ^* value on the yellow - blue color axis, were measured by using MACH 5 multi area color measurement (Center for Testmaterials (CFT) BV, NL-3130 AC Vlaardingen). The change of the color value (DE) value, defined and calculated automatically by the evaluation color tools on the following equation: DE = root [D Delta a ^* 2 + D Delta b ^* 2 + D Delta L ^* 2]

DE is a measure of the achieved stain removal effect or washing effect. All measurements were repeated three times to yield an average number. Note that higher DE values represent better washing effects when compared to lower DE values. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units. Table Ex2: mannanase wash performance at 40°C on stain CFT CS73 at different pH values in ES1_M detergent expressed in DE values

Example 3: Evaluation of Mannanases in LOM

The mannanase enzymes were tested in 4 g/L liquid detergent (ES1_C) at 25°C or 40°C wash ing temperature.

Selected stain monitors (CFT CS73/ locust bean gum with pigment and CFT CS43/guar gum, both from CFT, Vlaardingen, NL) were washed together with cotton ballast fabric and steel balls in wash liquor using base formulation ES1_C (formulation as described in example 1 , having pH 8-8.5) in the launder-o-meter (LOM, LP2 Typ, SDL Atlas Inc., USA) under the following washing conditions:

Test conditions

After the washing, the fabrics were rinsed, spin-dried and dried in the air. The wash perfor mance for the single stains was determined by measuring the remission value of the soiled fab ric after wash with the spectrophotometer from Fa. Datacolor (Elrepho 2000) at 460 nm. In gen- eral, the higher the remission value, the better the performance. The results are also outlined below in Table Ex3.

Table Ex3: Wash performance of mannanase variant on mannan-comprising stains in ES1_C detergent formulation; values in R% (460 nm)

Example 4: Evaluation of Mannanases in LOM

The mannanase enzyme was tested in 4 g/L liquid detergent (ES1_C).

The soiled swatches were washed together with ballast fabric (3.5 kg) and one soiled ballast sheet wfk SBL 2004 in a Miele Household washing machine (Softtronic W1935 WTL) under the following washing conditions:

Drying step: After the washing and spinning, the soiled swatches are removed from the washing machine and line dried in a dark closed room for 24h.

The brightness L ^* , the value a ^* on the red - green color axis and the b ^* value on the yellow - blue color axis, were measured by using MACH 5 multi area color measurement (Center for Testmaterials (CFT) BV, NL-3130 AC Vlaardingen) - see example 2.

Tested stains:

^* Warwick Equest Limited, Unit 55, Derwentside Business Centre, County Durham, DH8 6BN, UK Table Ex4: Wash performance of mannanase variants on mannan-comprising stains in ES1_C detergent formulation. The performance is shown as DE; washing temperature 20°C and 40°C