The present invention is directed to an enzyme preparation, preferably a liquid enzyme prepara tion, comprising

component (a): at least one compound according to general formula (I)

(R ² ) ₃ N ⁺ -(CH ₂ ) _n C(R ³ )(R ⁴ )-(0-X) _m -0-C(0)-R ¹ A (I)

wherein

n is selected from 1 to 12,

m is selected from zero to 50,

R ¹ is selected from Ci-Cio-alkyl, linear or branched, linear or branched C2-C4 alkenyl, and C ₆ -Cio-aryl, wherein R ¹ may bear one or more hydroxyl or C=0 or COOH groups, partially or fully neutralized, if applicable,

R ² are same or different and selected from Ci-Cio-alkyl, phenyl,

R ³ and R ⁴ are same or different and selected from hydrogen and Ci-C4-alkyl,

X is C2-C4-alkylen, and

A- is an inorganic or organic counteranion,

component (b): at least one enzyme selected from the group of hydrolases (EC 3), preferably selected from amylases and proteases, more preferably selected from alpha- amylases (EC 3.2.1 .1 ) and subtilisin type proteases (EC 3.4.21 .62);

and optionally

component (c): at least one compound selected from solvents, enzyme stabilizers different from component (a), and compounds stabilizing the liquid enzyme preparation as such.

Enzymes are usually produced commercially as a liquid concentrate, frequently derived from a fermentation broth. The enzyme tends to lose enzymatic activity if it remains in an aqueous en vironment and so it is conventional practice to convert it to an anhydrous form: aqueous con centrates may be lyophilized or spray-dried e.g. in the presence of a carrier material to form aggregates. Usually, solid enzyme products need to be“dissolved” prior to use. To stabilize enzymes in liquid products enzyme inhibitors are usually employed, preferably reversible en zyme inhibitors, to inhibit enzyme activity temporarily until the enzyme inhibitor is released.

The problem to be solved for the current invention relates to providing a compound helping to reduce loss of enzymatic activity during storage of liquid enzyme containing products, even if the liquid enzyme containing product comprises complexing agents such as EDTA and/or DTPA and/or MGDA and/or GLDA. It was a further objective of the present invention to provide an enzyme preparation that allows to be flexibly formulated into liquid detergent formulations or cleaning formulations with either one type of enzymes or mixtures of enzymes.

The problem was solved by providing compounds according to general formula (I):

(R ² ) ₃ N ⁺ -(CH ₂ ) _n C(R ³ )(R ⁴ )-(0-X) _m -0-C(0)-R ¹ A (I)

wherein,

n is selected from 1 to 12,

m is selected from zero to 50,

R ¹ is selected from CrCio-alkyl, linear or branched, linear or branched C2-C4 alkenyl, and C ₆ -Cio-aryl, wherein R ¹ may bear one or more hydroxyl or C=0 or COOH groups, partially or fully neutralized, if applicable,

R ² are same or different and selected from CrCio-alkyl, phenyl,

R ³ and R ⁴ are same or different and selected from hydrogen and Ci-C4-alkyl,

X is C2-C4-alkylen, and

A- is an inorganic or organic counteranion; and

wherein said compound supports retention of enzymatic activity of at least one enzyme selected from the group of hydrolases (EC 3), preferably from the group of amylases; during storage of the same within liquid products.

Enzyme names are known to those skilled in the art based on the recommendations of the No menclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzyme names include: an EC (Enzyme Commission) number, recommended name, alternative names (if any), catalytic activity, and other factors.; see

http://www.sbcs.qmul.ac.uk/iubmb/enzyme/EC3/ in the version last updated on 28 June, 2018.

In one aspect, the invention provides an enzyme preparation containing component (a): at least one enzyme stabilizer selected from compounds according to general formula (I)

(R ² ) ₃ N ⁺ -(CH ₂ ) _n C(R ³ )(R ⁴ )-(0-X) _m -0-C(0)-R ¹ A (I)

wherein

n is selected from 1 to 12,

m is selected from zero to 50,

R ¹ is selected from Ci-Cio-alkyl, linear or branched, linear or branched C2-C4 alkenyl, and C ₆ -Cio-aryl, wherein R ¹ may bear one or more hydroxyl or C=0 or COOH groups, partially or fully neutralized, if applicable,

R ² are same or different and selected from Ci-Cio-alkyl, phenyl,

R ³ and R ⁴ are same or different and selected from hydrogen and Ci-C4-alkyl,

X is C2-C4-alkylen, and

A- is an inorganic or organic counteranion, and

component (b): at least one enzyme selected from the group of hydrolases (EC 3), preferably selected from amylases and proteases, more preferably selected from alpha- amylases (EC 3.2.1.1 ) and subtilisin type proteases (EC 3.4.21.62);

and optionally

component (c): at least one compound selected from solvents, enzyme stabilizers different from component (a), and compounds stabilizing the liquid enzyme preparation as such.

The enzyme preparation of the invention may be liquid at 20°C and 101 .3 kPa. Liquids include solutions, emulsions and dispersions, gels etc. as long as the liquid is fluid and pourable. In one embodiment of the present invention, liquid detergent formulations according to the present in vention have a dynamic viscosity in the range of about 500 to about 20,000 mPa ^* s, determined at 25°C according to Brookfield, for example spindle 3 at 20 rpm with a Brookfield viscosimeter LVT-II.

In one embodiment, liquid means that the enzyme preparation does not show visible precipitate formation or turbidity after storage of the liquid enzyme preparation, preferably after at least 20 days of storage at 37°C. Component (a)

Component (a) is a salt of an organic ester of choline or of a derivative of choline. The anion of salt - the counterion - may be inorganic or organic, organic being preferred. Examples of inor ganic counterions of salt (component (a)) are nitrate, hydroxide, sulphate, phosphate, hy- drogenphosphate, dihydrogenphosphate, carbonate, bicarbonate, and halide, for example bro mide or chloride. Preferred are halide, especially chloride, and sulphate, carbonate, and bicar bonate. Examples of organic counterions are lactate, acetate, tartrate, citrate, and CH3SO3 (methanesulfonate). In embodiments with divalent or trivalent counterions, the respective molar amounts cation is present.

The nitrogen atom in component (a) bears three methyl groups and a hydroxyethyl group. The term derivatives of choline as used in the context of the present invention refers to compounds that bear at least one alkyl group other than a methyl group, or a hydroxyalkyl group other than a 2-hydroxyethyl group, or further alkoxy groups.

More specifically, component (a) is a compound of general formula (I)

(R ² ) ₃ N ⁺ -(CH ₂ ) _n C(R ³ )(R ⁴ )-(0-X) _m -0-C(0)-R ¹ A (I)

wherein the variables in formula (I) are defined as follows:

n is selected from 1 to 12, for example 1 to 9, preferably 1 , 2, 3, or 4, and even more pref erably n is 1 ;

m is selected from zero to 50, for example 2 to 50, preferred is 10 to 25. Most preferably, however, m is zero;

R ¹ is selected from CrCio-alkyl, linear or branched, linear or branched C2-C4 alkenyl, and C ₆ -Cio-aryl, wherein R ¹ may bear one or more hydroxyl or C=0 or COOH groups, partially or fully neutralized, if applicable. In an embodiment, R ¹ is selected from linear or branched CrCio-alkyl, preferably linear or branched Ci-C ₆ -alkyl, more preferably linear or branched Ci-C4-alkyl. Preferred examples of R ¹ are non-substituted Ci-C ₆ -alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, n-hexyl, preferred non-substituted CrCio-alkyl are methyl and ethyl. In one embodiment, R ¹ is selected from linear or branched ethenyl and propenyl, preferably linear ethenyl. In one embodiment, R ¹ is C6-aryl (-C6H5).

In one embodiment R ¹ is furthermore selected from substituted CrCio-alkyl such as - CH(OH)-CH(OH)-COOH, -CH(OH)-CH ₃ , -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (£)- CH=CHCOOH, (Z)-CH=CHCOOH, para-HO-CeHU-, o,p-dihydroxyphenyl, and 3,4,5- trihydroxyphenyl. In a preferred embodiment, 0-C(0)-R ¹ together constitute a citrate.

Even more preferred, R ¹ is methyl; R ² are same or different and selected from phenyl and CrCio-alkyl, for example methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec.-butyl, tert.-butyl, n-pentyl, iso-pentyl, sec.-pentyl, neo-pentyl, 1 ,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec.-hexyl, n- heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl, 2-n-propyl-heptyl, or iso-decyl, preferred are linear CrCio-alkyl and more preferred are linear Ci-C4-alkyl, even more preferred at least two R ² groups are CH ₃ and the third R ² is selected from linear CrCio-alkyl, and most pre ferred, all R ² are the same and methyl;

R ³ and R ⁴ are same or different and selected from hydrogen and Ci-C4-alkyl, preferred are n- for example methyl, ethyl, n-propyl, and n-butyl, and even more preferred both R ³ and R ⁴ are hydrogen. In another embodiment, R ³ is Ci-C4-alkyl and R ⁴ is hydrogen, preferably R ³ is methyl and R ⁴ is hydrogen;

X is C -C ₄ -alkylen, for example -CH ₂ -CH ₂ -, -CH(CH ₃ )-CH ₂ -, -(CH ₂ ) ₃ -, CH ₂ -CH(CH ₃ )-, or - (CH ₂ ) -; and

A- is a counteranion, inorganic or organic. Examples of inorganic counterions of compo nent (a) are sulphate, phosphate, hydrogenphosphate, dihydrogenphosphate, carbonate, bicarbonate, and halide, for example bromide or chloride. Preferred are halide, especially chloride, and sulphate, carbonate, and bicarbonate. Examples of organic counterions are lactate, acetate, tartrate, citrate, and CH ₃ S0 ₃ ^_ (methanesulfonate). Preferred is me- thanesulfonate.

Component (a) is not a surfactant. In one embodiment, a solution of 5 g component (a) in 1000 g water has a dynamic surface tension of > 45 mN/m at 20°C and 101.3 kPa. A solution of 5 g component (a) in 1000 g water may have a dynamic surface tension of ³ 50 mN/m at 20°C and 101 .3 kPa.

It is to be noted that component (a) characterized in m=0 is not a surfactant since the molecule is lacking a hydrophobic side chain and therefore is lacking surfactant characteristics.

The dynamic surface tension may be measured with a bubble pressure tensiometer, wherein the maximum internal pressure of a gas bubble which is formed in a liquid by means of a capil lary is measured; the measured value usually corresponds to the surface tension at a certain surface age, the time from the start of the bubble formation to the occurrence of the pressure maximum. In one embodiment, the surface age during measuring the dynamic surface tension at 20°C and 101.3 kPa with a bubble pressure tensiometer is 50 ms.

Most preferred example of component (a) are salts of choline esters with tartrate or citrate as counterion, and salts of acetyl choline, choline citrate, and of choline tartrate, for example lac tates, acetates, tartrates, citrates. In embodiments wherein counteranion A- is - or may be - divalent such as sulphate, tartrate, carbonate, or polyvalent such as phosphate or citrate, the necessary positive charge may be furnished by another salt (component (a)) derived cation, or by alkali metal cations such as po tassium or preferably sodium, or by ammonium, non-substituted or substituted with Ci-C4-alkyl and/or with 2-hydroxyethyl.

In embodiments wherein R ¹ bears one or more carboxyl groups they may be free COOH groups or partially or fully neutralized with alkali, for example potassium or especially sodium, or they may be esterified, for example with (R ² ) ₃ N ⁺ -(CH ₂ ) _n -(0-X) _m -0H. Such embodiments result in the di- or triester, if applicable, of the respective di- or tricarboxylic acid. Mixtures of mono- and diesters of, e.g., tartaric acid or citric acid, and mixtures of di- and triesters of citric acid are fea sible as well.

In a preferred embodiment of the present invention, component (a) is a compound according to formula (II)

(CH ₃ ) ₃ N ⁺ -(CH ₂ ) ₂ -0-C(0)-R ⁵ (A ¹ ) (II)

wherein (A ¹ )- is selected from methanesulfonate, tartrate and citrate

and wherein R ⁵ is selected from -CH2-C(OH)(COOX ² )-CH2-COOX ² and

-CH(OH)-CH(OH)-COOX ¹

wherein X ¹ is selected from hydrogen, alkali metal - especially sodium - and (CH ₃ ) ₃ N ⁺ -(CH ₂ )2- and wherein X ² are same or different and selected from hydrogen, alkali metal - especially so dium - and (CH ₃ ) ₃ N ⁺ -(CH ₂ )2-. In a preferred embodiment, the ester group of 0-C(0)-R ⁵ and (A ¹ )- correspond to each other.

In one embodiment of the present invention, liquid enzyme preparations contain component (a) in amounts in the range of 0.1% to 30% by weight, relative to the total weight of the enzyme preparation. The enzyme preparation may contain component (a) in amounts in the range of 0.1 % to 15% by weight, 0.25% to 10% by weight, 0.5% to 10% by weight, 0.5% to 6% by weight, or 1% to 3% by weight, all relative to the total weight of the enzyme preparation.

In one embodiment of the present invention, component (a) contains as an impurity a com pound (a’) which is as follows:

(R ² ) ₃ N ⁺ -(CH ₂ ) _n C(R ³ )(R ⁴ )-(0-X) _m -0H R ¹ -COO (III)

wherein the variables in formula (III) are:

R ¹ , R ² , X, n and m are the same as in the corresponding salt according to formula (I) as dis closed above. Said impurity may amount to up to 50 mole-%, preferably 0.1 to 20 mole-%, even more preferably 1 to 10 mole-% of component (a). Although impurity compound (a’) may stem from the synthesis of component (a) and may be removed by purification methods it is not pre ferred to remove it.

Component (b)

In one aspect of the invention, at least one enzyme comprised in component (b) is part of a liq uid enzyme concentrate.“Liquid enzyme concentrate” herein means any liquid enzyme comprising product comprising at least one enzyme.“Liquid” in the context of enzyme concen trate is related to the physical appearance at 20°C and 101.3 kPa.

The liquid enzyme concentrate may result from dissolution of solid enzyme in solvent. The sol vent may be selected from water and an organic solvent. A liquid enzyme concentrate resulting from dissolution of solid enzyme in solvent may comprise amounts of enzyme up to the satura tion concentration.

Dissolution herein means, that solid compounds are liquified by contact with at least one sol vent. Dissolution means complete dissolution of a solid compound until the saturation concen tration is achieved in a specified solvent, wherein no phase-separation occurs.

In one aspect of the invention, component (b) of the resulting enzyme concentrate may be free of water, meaning that no significant amounts of water are present. Non-significant amounts of water herein means, that the enzyme preparation comprises 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% by weight water, all relative to the total weight of the enzyme concentrate, or no water. In one embodiment, enzyme concentrate free of water free of water means that the enzyme concen trate does not comprise significant amounts of water but does comprise organic solvents in amounts of 30-80% by weight, relative to the total weight of the enzyme concentrate.

Liquid enzyme concentrates comprising water may be called“aqueous enzyme concentrates”. Aqueous enzyme concentrates may be enzyme-comprising solutions, wherein solid enzyme product has been dissolved in water. In one embodiment“aqueous enzyme concentrate” means enzyme-comprising products resulting from enzyme production by fermentation.

Fermentation means the process of cultivating recombinant cells which express the desired enzyme in a suitable nutrient medium allowing the recombinant host cells to grow (this process may be called fermentation) and express the desired protein. At the end of the fermentation, fermentation broth usually is collected and further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction. Depending on whether the enzyme has been secreted into the liquid fraction or not, the desired protein or enzyme may be recovered from the liquid fraction of the fermentation broth or from cell lysates. Recovery of the desired enzyme uses methods known to those skilled in the art. Suitable methods for recovery of proteins or enzymes from fermentation broth include but are not limited to collection, centrifugation, filtra tion, extraction, and precipitation.

Liquid enzyme concentrates, may comprise amounts of enzyme in the range of 0.1 % to 40% by weight, or 0.5% to 30% by weight, or 1 % to 25% by weight, or 3% to 25% by weight, or 5% to 25% by weight, all relative to the total weight of the enzyme concentrate. In one embodiment, liquid enzyme concentrates are resulting from fermentation and are aqueous.

Aqueous enzyme concentrates resulting from fermentation may comprise water in amounts of more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme concen trate. Aqueous enzyme concentrates which result from fermentation, may comprise residual components such as salts originating from the fermentation medium, cell debris originating from the production host cells, metabolites produced by the production host cells during fermentation. In one embodiment, residual components may be comprised in liquid enzyme concentrates in amounts less than 30% by weight, less than 20% by weight less, than 10% by weight, or less than 5% by weight, all relative to the total weight of the aqueous enzyme concentrate.

At least one enzyme comprised in component (b) is selected from hydrolases (EC 3), hereinaf ter also referred to as enzyme (component (b)). Preferred enzymes are selected from the group of enzymes acting on ester bond (E.C. 3.1 ), glycosylases (E.C. 3.2), and peptidases (E.C. 3.4). Enzymes acting on ester bond (E.C. 3.1 ), are hereinafter also referred to as lipases. Glycosyl ases (E.C. 3.2) are hereinafter also referred to as either amylases, cellulases, or mannanases. Peptidases are hereinafter also referred to as proteases.

Hydrolases comprised in component (b) are identified by polypeptide sequences (also called amino acid sequences herein). The polypeptide sequence specifies the three-dimensional struc ture including the“active site” of an enzyme which in turn determines the catalytic activity of the same. Polypeptide sequences may be identified by a SEQ ID NO. According to the World Intel lectual 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.

Any enzyme comprised in component (b) according to the invention relates to parent enzymes and/or variant enzymes, both having enzymatic activity. Enzymes having enzymatic activity are enzymatically active or exert enzymatic conversion, meaning that enzymes act on substrates and convert these into products. The term“enzyme” herein excludes inactive variants of an en zyme. A“parent” sequence (of a parent protein or enzyme, also called“parent enzyme”) is the starting sequence for introduction of changes (e.g. by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) to the sequence, resulting in“variants” of the parent sequences. The term parent enzyme (or parent sequence) includes wild-type enzymes (sequences) and synthetically generated sequences (enzymes) which are used as starting se quences for introduction of (further) changes.

The term“enzyme variant” or“sequence variant” or“variant enzyme” refers to an enzyme that differs from its parent enzyme in its amino acid sequence to a certain extent. If not indicated otherwise, variant enzyme“having enzymatic activity” means that this variant enzyme has the same type of enzymatic activity as the respective parent enzyme.

In describing the variants of the present invention, the nomenclature described as follows is used:

Amino acid substitutions are described by providing the original amino acid of the parent en zyme followed by the number of the position within the amino acid sequence, followed by the substituted amino acid.

Amino acid deletions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by ^* .

Amino acid insertions are described by providing the original amino acid of the parent enzyme followed by the number of the position within the amino acid sequence, followed by the original amino acid and the additional amino acid. For example, an insertion at position 180 of lysine next to glycine is designated as“Gly180Glyl_ys” or“G180GK”.

In cases where a substitution and an insertion occur at the same position, this may be indicated as S99SD+S99A or in short S99AD. In cases where an amino acid residue identical to the exist ing amino acid residue is inserted, it is clear that degeneracy in the nomenclature arises. If for example a glycine is inserted after the glycine in the above example this would be indicated by G180GG.

Where different alterations can be introduced at a position, the different alterations are separat ed by a comma, e.g.“Arg170Tyr, Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid.

Enzyme variants may be defined by their sequence similarity and/or identity when compared to a parent enzyme. Sequence similarity and identity usually is provided as“% sequence similari ty” or“%-similarity” and“% sequence identity” or“% identity”, respectively. For calculation of sequence similarities and identities, 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 (gap open=10.0, gap extend=0.5 and ma- trix=EBLOSUM62).

% 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. Here in, the exchange of one amino acid with a similar amino acid may be called“conservative muta tion”. Amino acid similarity according to this invention: amino acid A is similar to amino acids S; amino acid D is similar to amino acids E and N; amino acid E is similar to amino acids D and 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 and M and V; amino acid K is similar to amino acids E and Q and R; amino acid L is similar to amino acids I and M and V; amino acid M is similar to amino acids I and L and V; amino acid N is similar to amino acids D and H and S; amino acid Q is similar to amino acids E and K and R; amino acid R is similar to amino acids K and Q; amino acid S is similar to amino acids A and N and T ; amino acid T is similar to amino acids S; amino acid V is similar to amino acids I and L and M; amino acid W is similar to amino acids F and Y; amino acid Y is similar to amino acids F and H and W.

Conservative amino acid substitutions may occur over the full-length of the sequence of a poly peptide sequence of a functional protein such as an enzyme. In one embodiment, such muta tions are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.

To take conservative mutations into account, a value for sequence similarity of two amino acid sequences may be calculated from the same alignment, which is used to calculate %-identity.

According to this invention, the following calculation of %-similarity applies: %-similarity = [ (identical residues + similar residues) / length of the alignment region which is showing the re spective sequence(s) of this invention over its complete length ] ^* 100.

According to this invention, enzyme variants may be described as an amino acid sequence which is at least m% similar to the respective parent sequences with“m” being an integer be tween 10 and 100. In one embodiment, variant enzymes are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similar when compared to the full-length polypeptide sequence of the parent enzyme, wherein the variant enzyme has enzy matic activity.

According to this invention, the following calculation of %-identity applies: %-identity = (identical residues / length of the alignment region which is showing the respective sequence of this in vention over its complete length) ^* 100.

According to this invention, enzyme variants may be described as an amino acid sequence which is at least n% identical to the amino acid sequence of the respective parent enzyme with “n” being an integer between 10 and 100. In one embodiment, variant enzymes are at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 when compared to the full-length amino acid sequence of the parent en zyme, wherein the enzyme variant has enzymatic activity.

“Enzymatic activity” means the catalytic effect exerted by an enzyme, which usually is ex pressed as units per milligram of enzyme (specific activity) which relates to molecules of sub strate transformed per minute per molecule of enzyme (molecular activity).

Variant enzymes may have enzymatic activity according to the present invention when said en zyme variants exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the enzymatic activity of the respective parent enzyme. In one embodiment, variant enzymes have increased enzymatic activity when com pared to the respective parent enzyme, meaning that the enzymatic activity of the variant en zyme is more then 100% when compared to the parent enzyme.

In one embodiment, component (b) comprises at least one hydrolase selected from the group of amylases.“Amylases” according to the invention (alpha and/or beta) include those of bacterial or fungal origin (EC 3.2.1.1 and 3.2.1.2, respectively). Preferably, component (b) comprises at least one enzyme selected from the group of alpha-amylases (EC 3.2.1.1 ). Chemically modified or protein engineered mutants are included.

Amylases according to the invention have“amylolytic activity” or“amylase activity” involving (endo)hydrolysis of glucosidic linkages in polysaccharides alpha-amylase activity may be de termined by assays for measurement of alpha-amylase activity which are known to those skilled in the art. Examples for assays measuring alpha-amylase activity are:

alpha-amylase activity can be determined by a method employing Phadebas tablets as sub strate (Phadebas Amylase Test, supplied by Magle Life Science). Starch is hydrolyzed by the alpha-amylase giving soluble blue fragments. The absorbance of the resulting blue solution, measured spectrophotometrically at 620 nm, is a function of the alpha-amylase activity. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha- amylase protein) of the alpha-amylase in question under the given set of conditions.

alpha-amylase activity can also be determined by a method employing the Ethyliden-4-nitro- phenyl-alpha-D-maltoheptaosid (EPS). D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase. Following the cleavage, the alpha-glucosidase included in the kit to digest the substrate to liberate a free PNP molecule which has a yellow color and thus can be measured by visible spectophotometry at 405nm. Kits containing EPS substrate and alpha-glucosidase is manufactured by Roche Costum Biotech (cat. No. 10880078103). The slope of the time dependent absorption-curve is directly proportional to the specific activity (ac tivity per mg enzyme) of the alpha-amylase in question under the given set of conditions.

Amylolytic activity may be provided in units per gram enzyme. For example, 1 unit alpha- amylase may liberate 1.0 mg of maltose from starch in 3 min at pH 6.9 at 20°C.

At least one amylase comprised in component (b) may be selected from the following:

• amylases from Bacillus licheniformis having SEQ ID NO:2 as described in WO 95/10603.

Suitable variants are described in WO 95/10603 comprising one or more substitutions in the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188,

190, 197, 201 , 202, 207, 208, 209, 21 1 , 243, 264, 304, 305, 391 , 408, and 444 which have amylolytic activity. Variants are described in WO 94/02597, WO 94/018314, WO 97/043424 and SEQ ID NO:4 of WO 99/019467.

• amylases from B. stearothermophilus having SEQ ID NO:6 as disclosed in WO 02/10355 or an amylase with optionally having a C-terminal truncation over the wildtype sequence. Suitable variants of SEQ ID NO:6 include those comprising a deletion in positions 181 and/or 182 and/or a substitution in position 193.

• amylases from Bacillus sp.707 having SEQ ID NO:6 as disclosed in WO 99/19467. Pre ferred variants of SEQ NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269.

• amylases from Bacillus halmapalus having SEQ ID NO:2 or SEQ ID NO:7 as described in WO 96/23872, also described herein as SP-722. Preferred variants are described in WO 97/3296, WO 99/194671 and WO 2013/001078.

• amylases from Bacillus sp. DSM 12649 having SEQ ID NO:4 as disclosed in WO

00/22103.

• amylases from Bacillus strain TS-23 having SEQ ID NO:2 as disclosed in WO

2009/061380. • amylases from Cytophaga sp. having SEQ ID NO:1 as disclosed in WO 2013/184577.

• amylases from Bacillus megaterium DSM 90 having SEQ ID NO:1 as disclosed in WO 2010/104675.

• amylases from Bacillus sp. comprising amino acids 1 to 485 of SEQ ID NO:2 as described in WO 00/60060.

amylases from Bacillus amyloliquefaciens or variants thereof, preferably selected from amylases according to SEQ ID NO: 3 as described in WO 2016/092009.

amylases having SEQ ID NO: 12 as described in WO 2006/002643 or amylase variants comprising the substitutions Y295F and M202LITV within said SEQ ID NO:12.

amylases having SEQ ID NO:6 as described in WO 201 1/098531 or amylase variants comprising a substitution at one or more positions selected from the group consisting of 193 [G,A,S,T or M], 195 [F,W,Y,L,I or V], 197 [F,W,Y,L,I or V], 198 [Q or N], 200

[F,W,Y,L,I or V], 203 [F,W,Y,L,I or V], 206 [F,W,Y,N,L,I,V,H,Q,D or E], 210 [F,W,Y,L,I or V], 212 [F,W,Y,L,I or V], 213 [G,A,S,T or M] and 243 [F,W,Y,L,I or V] within said SEQ ID NO:6.

• amylases having SEQ ID NO:1 as described in WO 2013/001078 or amylase variants comprising an alteration at two or more (several) positions corresponding to positions G304, W140, W189, D134, E260, F262, W284, W347, W439, W469, G476, and G477 within said SEQ ID NO:1 .

• amylases having SEQ ID NO:2 as described in WO 2013/001087 or amylase variants comprising a deletion of positions 181 +182, or 182+183, or 183+184, within said SEQ ID NO:2, optionally comprising one or two or more modifications in any of positions corre sponding to W140, W159, W167, Q169, W189, E194, N260, F262, W284, F289, G304, G305, R320, W347, W439, W469, G476 and G477 within said SEQ ID NO:2.

• amylases which are hybrid alpha-amylases from above mentioned amylases as for exam ple as described in WO 2006/066594;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy- brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

Preferably, at least one amylase is selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23,

SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO

2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

Suitable amylases comprised in component (b) include amylase variants of the amylases dis closed herein having amylase activity which are at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or identical when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. According to the present invention, component (b) may comprise a combination of at least two amylases as disclosed above.

In one embodiment, component (b) comprises a combination of at least one amylase as dis closed above and at least one further enzyme preferably selected from proteases, lipases, cel- lulases, and mannanases, preferably a combination of at least one amylase and at least one protease.

At least one enzyme comprised in component (b) may be selected from the group of proteases, preferably selected from the group of serine endopeptidases (EC 3.4.21 ), most preferably se lected from the group of subtilisin type proteases (EC 3.4.21 .62).

Proteases are members of class EC 3.4. Proteases include aminopeptidases (EC 3.4.1 1 ), di peptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl- dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metallocarboxypeptidas- es (EC 3.4.17), cysteine-type carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21 ), cysteine endopeptidases (EC 3.4.22), aspartic endopepti dases (EC 3.4.23), metallo-endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), or endopeptidases of unknown catalytic mechanism (EC 3.4.99).

In one embodiment, component (b) comprises at least one protease selected from serine prote ases (EC 3.4.21 ). Serine proteases or serine peptidases are characterized by having a serine in the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. Preferably, at least one serine protease (EC 3.4.21 ) is selected from the group of sub- tilopeptidase (EC 3.4.21 .62) - as proposed e.g. by Siezen et al. (1991 ), Protein Eng. 4:719-737 and Siezen et al. (1997), Protein Science 6:501 -523. Subtilopeptidases include the subtilisin family, thermitase family, the proteinase K family, the lantibiotic peptidase family, the kexin fami ly and the pyrolysin family.

Subtilisins may be serine proteases from the family S8 as defined by the MEROPS database (http://merops.sanger.ac.uk). Peptidase family S8 comprises the serine endopeptidase subtilisin and its homologues. The subtilisin related class of serine proteases shares a common amino acid sequence defining a catalytic triad which distinguishes them from the chymotrypsin related class of serine proteases. Subtilisins and chymotrypsin related serine proteases both have a catalytic triad comprising aspartate, histidine and serine. Examples include the subtilisins as described in WO 89/06276 and EP 0283075, WO 89/06279, WO 89/09830, WO 89/09819,

WO 91 /06637 and WO 91/02792. Proteases are active proteins exerting“protease activity” or“proteolytic activity”. Proteolytic ac tivity is related to the rate of degradation of protein by a protease or proteolytic enzyme in a de fined course of time.

The methods for analyzing proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381 -395). Proteolytic activity may be determined by using Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD405.

Proteolytic activity may be provided in units per gram enzyme. For example, 1 U protease may correspond to the amount of protease which sets free 1 pmol folin-positive amino acids and peptides (as tyrosine) per minute at pH 8.0 and 37°C (casein as substrate).

Proteases of the subtilisin type (EC 3.4.21 .62) may be bacterial proteases originating from a microorganism selected from Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces protease, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fuso- bacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

In one embodiment of the present invention, component (b) comprises at least one protease selected from the following: subtilisin from Bacillus amyloliquefaciens BRN' (described by Vasantha et al. (1984) J. Bacteriol. Volume 159, p. 81 1 -819 and JA Wells et al. (1983) in Nucle ic Acids Research, Volume 1 1 , p. 791 1 -7925); subtilisin from Bacillus licheniformis (subtilisin Carlsberg; disclosed in EL Smith et al. (1968) in J. Biol Chem, Volume 243, pp. 2184-2191 , and Jacobs et al. (1985) in Nucl. Acids Res, Vol 13, p. 8913-8926); subtilisin PB92 (original se quence of the alkaline protease PB92 is described in EP 283075 A2); subtilisin 147 and/or 309 (Esperase®, Savinase®, respectively) as disclosed in WO 89/06279; subtilisin from Bacillus lentus as disclosed in WO 91/02792, such as from Bacillus lentus DSM 5483 or the variants of Bacillus lentus DSM 5483 as described in WO 95/23221 ; subtilisin from Bacillus alcalophilus (DSM 1 1233) disclosed in DE 10064983; subtilisin from Bacillus gibsonii (DSM 14391 ) as dis closed in WO 2003/054184; subtilisin from Bacillus sp. (DSM 14390) disclosed in

WO 2003/056017; subtilisin from Bacillus sp. (DSM 14392) disclosed in WO 2003/055974; sub tilisin from Bacillus gibsonii (DSM 14393) disclosed in WO 2003/054184; subtilisin having SEQ ID NO: 4 as described in WO 2005/063974; subtilisin having SEQ ID NO: 4 as described in WO 2005/103244; subtilisin having SEQ ID NO: 7 as described in WO 2005/103244; and subtilisin having SEQ ID NO: 2 as described in application DE 102005028295.4. In one embodiment, component (b) comprises at least subtilisin 309 (which might be called Savinase herein) as disclosed as sequence a) in Table I of WO 89/06279 or a variant thereof which is at least 80% similar or identical thereto and has proteolytic activity.

Examples of useful proteases in accordance with the present invention comprise the variants described in: WO 92/19729, WO 95/23221 , WO 96/34946, WO 98/201 15, WO 98/201 16,

WO 99/1 1768, WO 01/44452, WO 02/088340, WO 03/006602, WO 2004/03186,

WO 2004/041979, WO 2007/006305, WO 201 1/036263, WO 201 1 /036264, and

WO 201 1 /072099. Suitable examples comprise especially variants of subtilisin protease derived from SEQ ID NO:22 as described in EP 1921 147 (which is the sequence of mature alkaline pro tease from Bacillus !entus DSM 5483) with amino acid substitutions in one or more of the follow ing positions: 3, 4, 9, 15, 24, 27, 33, 36, 57, 68, 76, 77, 87, 95, 96, 97, 98, 99, 100, 101 , 102,

103, 104, 106, 1 18, 120, 123, 128, 129, 130, 131 , 154, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 (according to the BPN' numbering), which have proteolytic activity. In one embodiment, such a protease is not mutated at positions Asp32, His64 and Ser221 (according to BPN’ numbering).

Component (b) may comprise a protease variant having proteolytic activity which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or identical when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above.

In one embodiment, at least one protease comprised in component (b) has SEQ ID NO:22 as described in EP 1921 147, or a protease which is at least 80% similar or identical thereto and has proteolytic activity. In one embodiment, said protease is characterized by having amino acid glutamic acid (E), or aspartic acid (D), or asparagine (N), or glutamine (Q), or alanine (A), or glycine (G), or serine (S) at position 101 (according to BPN’ numbering) and has proteolytic ac tivity. In one embodiment, said protease comprises one or more further substitutions: (a) threo nine at position 3 (3T), (b) isoleucine at position 4 (4I), (c) alanine, threonine or arginine at posi tion 63 (63A, 63T, or 63R), (d) aspartic acid or glutamic acid at position 156 (156D or 156E), (e) proline at position 194 (194P), (f) methionine at position 199 (199M), (g) isoleucine at position 205 (205I), (h) aspartic acid, glutamic acid or glycine at position 217 (217D, 217E or 217G), (i) combinations of two or more amino acids according to (a) to (h). At least one protease may be at least 80% similar or identical to SEQ ID NO:22 as described in EP 1921 147 and is character ized by comprising one amino acid (according to (a)-(h)) or combinations according to (i) to gether with the amino acid 101 E, 101 D, 101 N, 101 Q, 101 A, 101 G, or 101 S (according to BPN’ numbering) and having proteolytic activity. In one embodiment, said protease is characterized by comprising the mutation (according to BPN’ numbering) R101 E, or S3T + V4I + V205I, or R101 E and S3T, V4I, and V205I, or S3T + V4I + V199M + V205I + L217D, and having proteo lytic activity.

In one embodiment, protease according to SEQ ID NO:22 as described in EP 1921 147 is char acterized by comprising the mutation (according to BPN’ numbering) S3T + V4I + S9R + A15T + V68A + D99S + R101 S + A103S + 1104V + N218D, and having proteolytic activity.

According to the present invention, component (b) may comprise a combination of at least two proteases, preferably selected from the group of serine endopeptidases (EC 3.4.21 ), more pref erably selected from the group of subtilisin type proteases (EC 3.4.21.62) - all as disclosed above.

In one embodiment, component (b) comprises at least one protease selected from proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof having proteolytic activity, as disclosed above.

In one embodiment, component (b) comprises at least one protease selected from subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof having proteolytic activity, as dis closed above.

In one embodiment, component (b) comprises a combination of at least one alpha-amylase (EC 3.2.1.1 ) as disclosed above, preferably selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity;

and at least one protease as disclosed above, preferably selected from the group of subtilisin type proteases (EC 3.4.21 .62), more preferably selected from

• proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof as disclosed above; and

• subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof as disclosed above.

In one embodiment, component (b) comprises a combination of at least one alpha-amylase (EC 3.2.1.1 ) as disclosed above, and optionally one or more further enzymes, preferably selected from lipases, proteases, cellulases and mannan degrading enzymes (mannanases).

At least one enzyme comprised in component (b) may be selected from the group of lipases. “Lipases”,“lipolytic enzyme”,“lipid esterase”, all refer to an enzyme of EC class 3.1.1 (“carbox ylic ester hydrolase”). Lipase means active protein having lipase activity (or lipolytic activity; triacylglycerol lipase, EC 3.1.1 .3), cutinase activity (EC 3.1.1.74; enzymes having cutinase ac tivity may be called cutinase herein), sterol esterase activity (EC 3.1.1 .13) and/or wax-ester hy drolase activity (EC 3.1.1.50).

The methods for determining lipolytic activity are well-known in the literature (see e.g. Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, p. 63-71 ). E.g. the lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C:16) and releases pNP which is yellow and can be detected at 405 nm.

“Lipolytic activity” means the catalytic effect exerted by a lipase, which may be provided in lipo lytic units (LU). For example, 1 LU may correspond to the amount of lipase which produces 1 pmol of titratable fatty acid per minute in a pH stat. under the following conditions: temperature 30°C.; pH=9.0; substrate may be an emulsion of 3.3 wt.% of olive oil and 3.3% gum arabic, in the presence of 13 mmol/l Ca ²⁺ and 20 mmol/l NaCI in 5 mmol/l Tris-buffer. Lipases which may be comprised in component (b) include those of bacterial or fungal origin. In one aspect of the invention, a suitable lipase (component (b)) is selected from the following: lipases from Humicola (synonym Thermomyces), e.g. from H. lanuginosa {T. lanuginosus) as described in EP 258068, EP 305216, WO 92/05249 and WO 2009/109500 or from H. insolens as described in WO 96/13580; lipases derived from Rhizomucor miehei as described in WO 92/05249; lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. from P. alcaligenes or P. pseudoalcaligenes (EP 218272, WO 94/25578, WO 95/30744,

WO 95/35381 , WO 96/00292), P. cepacia (EP 331376), P. stutzeri (GB 1372034), P. fluo- rescens, Pseudomonas sp. strain SD705 (WO 95/06720 and WO 96/27002), P. wisconsinensis (WO 96/12012), Pseudomonas mendocina (WO 95/14783), P. glumae (WO 95/35381 , WO 96/00292); lipase from Streptomyces griseus (WO 201 1/150157) and S. pristinaespiralis (WO 2012/137147), GDSL-type Streptomyces lipases (WO 2010/065455); lipase from Thermobifida fusca as disclosed in WO 201 1/084412; lipase from Geobacillus stearothermophilus as dis closed in WO 201 1/084417; Bacillus lipases, e.g. as disclosed in WO 00/60063, lipases from B. subtilis as disclosed in Dartois et al. (1992), Biochemica et Biophysica Acta, 1 131 , 253-360 or WO 201 1 /084599, B. stearothermophilus (JP S64-074992) or B. pumilus (WO 91 /16422); lipase from Candida antarctica as disclosed in WO 94/01541 ; cutinase from Pseudomonas mendocina (US 5389536, WO 88/09367); cutinase from Magnaporthe grisea (WO 2010/107560); cutinase from Fusarum solani pisi as disclosed in WO 90/09446, WO 00/34450 and WO 01/92502; and cutinase from Humicola lanuginosa as disclosed in WO 00/34450 and WO 01/92502.

Suitable lipases also include those referred to as acyltransferases or perhydrolases, e.g. acyl- transferases with homology to Candida antarctica lipase A (WO 2010/1 1 1 143), acyltransferase from Mycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE7 family (WO 2009/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant (WO 2010/100028).

Component (b) may comprise lipase variants of the above described lipases which have lipolytic activity. Such suitable lipase variants are e.g. those which are developed by methods as dis closed in WO 95/22615, WO 97/04079, WO 97/07202, WO 00/60063, WO 2007/087508,

EP 407225 and EP 260105.

Component (b) may comprise lipase variants having lipolytic activity which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or iden tical when compared to the full-length polypeptide sequence of the parent enzyme as disclosed above. In one embodiment, component (b) comprises at least one lipase selected from fungal triacyl- glycerol lipase (EC class 3.1.1 .3). Fungal triacylglycerol lipase may be selected from Thermo- myces lanuginose lipase. In one embodiment, Thermomyces lanuginosa lipase is selected from triacylglycerol lipase according to amino acids 1 -269 of SEQ ID NO:2 of US 5869438 and vari ants thereof having lipolytic activity. Triacylglycerol lipase according to amino acids 1 -269 of SEQ ID NO:2 of US 5869438 may be called Lipolase herein.

Thermomyces lanuginosa lipase may be selected from variants having lipolytic activity which are 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% similar or identical when compared to the full- length polypeptide sequence of amino acids 1 -269 of SEQ ID NO:2 of US 5869438. Preferably, said Thermomyces lanuginosa lipase may be characterized by having amino acid T231 R and N233R. Said Thermomyces lanuginosa lipase may further comprise one or more of the follow ing amino acid exchanges: Q4V, V60S, A150G, L227G, P256K.

According to the present invention, component (b) may comprise a combination of at least two lipases, preferably selected from triacylglycerol lipase according to amino acids 1 -269 of SEQ ID NO:2 of US 5869438 and variants thereof having lipolytic activity as disclosed above.

At least one enzyme comprised in component (b) may be selected from the group of cellulases. At least one cellulase may be selected from cellobiohydrolase (1 ,4-P-D-glucan cellobiohydro- lase, EC 3.2.1.91 ), endo-ss-1 ,4-glucanase (endo-1 ,4-P-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and ss-glucosidase (EC 3.2.1.21 ). Preferably, component (b) comprises at least one cellulase of the glycosyl hydrolase family 7 (GH7, pfam00840), preferably selected from en- doglucanases (EC 3.2.1 .4).

"Cellulases",“cellulase enzymes” or“cellulolytic enzymes” (component (b)) are enzymes in volved in hydrolysis of cellulose. Assays for measurement of“cellulase activity” or“cellulolytic activity” are known to those skilled in the art. For example, cellulolytic activity may be deter mined by virtue of the fact that cellulase hydrolyses carboxymethyl cellulose to reducing carbo hydrates, the reducing ability of which is determined colorimetrically by means of the ferricya- nide reaction, according to Hoffman, W. S., J. Biol. Chem. 120, 51 (1937).

Cellulolytic activity may be provided in units per gram enzyme. For example, 1 unit may liberate 1.0 pmole of glucose from cellulose in one hour at pH 5.0 at 37 °C (2 hour incubation time).

Cellulases according to the invention include those of bacterial or fungal origin. In one embodi ment, at least one cellulase is selected from cellulases comprising a cellulose binding domain.

In one embodiment, at least one cellulase is selected from cellulases comprising a catalytic do main only, meaning that the cellulase lacks cellulose binding domain. At least one enzyme comprised in component (b) may be selected from the group of mannan degrading enzymes. At least one mannan degrading enzyme may be selected from b-manno- sidase (EC 3.2.1.25), endo-1 ,4^-mannosidase (EC 3.2.1.78), and 1 ,4^-mannobiosidase (EC 3.2.1.100). Preferably, at least one mannan degrading enzyme is selected from the group of endo-1 ,4^-mannosidase (EC 3.2.1.78), a group of enzymes which may be called endo-b-1 ,4-D- mannanase, b-mannanase, or mannanase herein.

A polypeptide having mannanase activity may be tested for mannanase activity according to standard test procedures known in the art, such as by applying a solution to be tested to 4 mm diameter holes punched out in agar plates containing 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).

Component (b) may comprise at least one mannanase selected from alkaline mannanase of Family 5 or 26. The term“alkaline mannanase” is meant to encompass mannanases having an enzymatic activity of at least 40% of its maximum activity at a given pH ranging from 7 to 12, preferably 7.5 to10.5.

At least one mannanase comprised in component (b) may be selected from mannanases origi nating from Bacillus organisms, such as described in JP-0304706 [beta-mannanase from Bacil lus sp.], JP-63056289 [alkaline, thermostable beta-mannanase], JP-63036774 [Bacillus micro organism FERM P-8856 producing beta-mannanase and beta-mannosidase at an alkaline pH], JP-08051975 [alkaline beta-mannanases from alkalophilic Bacillus sp. AM-001], WO 97/1 1 164 [mannanase from Bacillus amyloliquefaciens ], WO 91/18974 [mannanase active at an extreme pH and temperature], WO 97/1 1 164 [mannanase from Bacillus amyloliquefaciens ],

WO 2014/100018 [endo-(3-mannanase1 cloned from a Bacillus circulans or Bacillus lentus strain CMG1240 (Blemanl ; see U.S. 5,476,775)]. Suitable mannanases are described in WO 99/064619]

At least one mannanase comprised in component (b) may be selected from mannanases origi nating from Trichoderma organisms, such as disclosed in WO 93/24622 or in WO 201 1/085747.

Component (b) may comprise mannanase variants having mannanase activity which are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar or identical when compared to the full-length polypeptide sequence of the corresponding parent enzyme as disclosed above. In one embodiment, component (b) comprises a combination of at least one alpha-amylase (EC 3.2.1.1 ) as disclosed above, preferably selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity;

and at least one alkaline mannanase, preferably selected from the group of endo-1 ,4-b- mannosidase (EC 3.2.1.78) as disclosed above; and

optionally one or more further enzymes, preferably selected from

• protease as disclosed above, preferably selected from the group of serine endopeptidases (EC 3.4.21 ), more preferably selected from the group of subtilisin type proteases (EC 3.4.21.62). • lipase as disclosed above, preferably selected from triacylglycerol lipase according to amino acids 1 -269 of SEQ ID NO:2 of US 5869438 and variants thereof having lipolytic activity; and

• cellulase as disclosed above, preferably of the GH7 family, more preferably selected from endoglucanases (EC 3.2.1 .4) as disclosed above.

Component (c)

In one embodiment, enzyme preparations and/or other enzyme containing products of the in vention comprises component (c). Component (c) comprises at least one compound selected from enzyme stabilizers different from component (a), compounds stabilizing the enzyme prepa ration as such, and solvents.

In one embodiment, the enzyme preparation of the invention does not comprise any enzyme stabilizer different from component (a). In another embodiment, the enzyme preparation of the invention does comprise at least one enzyme stabilizer different from component (a). Prefera bly, the enzyme preparation of the invention comprising at least one enzyme stabilizer different from component (a) is liquid.

At least one enzyme stabilizer different from component (a) may be selected from boron- containing compounds, polyols, peptide stabilizers such as peptide aldehydes, other enzyme stabilizers, and mixtures thereof.

Boron-containing compounds (component (c)) may be selected from boric acid or its derivatives and from boronic acid or its derivatives such as aryl boronic acids or its derivatives, from salts thereof, and from mixtures thereof. Boric acid herein may be called orthoboric acid.

In one embodiment, boron-containing compound (component (c)) is selected from the group consisting of aryl boronic acids and its derivatives. In one embodiment, boron-containing com pound is selected from the group consisting of benzene boronic acid (BBA) which is also called phenyl boronic acid (PBA), derivatives thereof, and mixtures thereof.

In one embodiment phenyl-boronic acid derivatives (component (c)) are selected from the group consisting of 4-formyl phenyl boronic acid (4-FPBA), 4-carboxy phenyl boronic acid (4-CPBA), 4-(hydroxymethyl) phenyl boronic acid (4-HMPBA), and p-tolylboronic acid (p-TBA).

Other suitable derivatives (component (c)) include: 2-thienyl boronic acid, 3-thienyl boronic acid, (2-acetamidophenyl) boronic acid, 2-benzofuranyl boronic acid, 1 -naphthyl boronic acid, 2- naphthyl boronic acid, 2-FPBA, 3-FBPA, 1 -thianthrenyl boronic acid, 4-dibenzofuran boronic acid, 5-methyl-2-thienyl boronic acid, 1 -benzothiophene-2 boronic acid, 2-furanyl boronic acid, 3-furanyl boronic acid, 4,4 biphenyl-diboronic acid, 6-hydroxy-2-naphthaleneboronic acid, 4- (methylthio) phenyl boronic acid, 4-(trimethylsilyl) phenyl boronic acid, 3-bromothiophene bo- ronic acid, 4-methylthiophene boronic acid, 2-naphthyl boronic acid, 5-bromothiophene boronic acid, 5-chlorothiophene boronic acid, dimethylthiophene boronic acid, 2-bromophenyl boronic acid, 3-chlorophenyl boronic acid, 3-methoxy-2-thiophene boronic acid, p-methyl-phenylethyl boronic acid, 2-thianthrenyl boronic acid, di-benzothiophene boronic acid, 9-anthracene boronic acid, 3,5 dichlorophenyl boronic, acid, diphenyl boronic acid anhydride, o-chlorophenyl boronic acid, p-chlorophenyl boronic acid, m-bromophenyl boronic acid, p-bromophenyl boronic acid, p- fluorophenyl boronic acid, octyl boronic acid, 1 ,3,5 trimethylphenyl boronic acid, 3-chloro-4- fluorophenyl boronic acid, 3-aminophenyl boronic acid, 3,5-bis-(trifluoromethyl) phenyl boronic acid, 2,4 dichlorophenyl boronic acid, 4-methoxyphenyl boronic acid, and mixtures thereof.

Polyols (component (c)) may be selected from polyols containing from 2 to 6 hydroxyl groups. Suitable examples include glycol, propylene glycol, 1 ,2-propane diol, 1 ,2-butane diol, 1 ,2- pentane diol, ethylene glycol, hexylene glycol, glycerol, sorbitol, mannitol, erythriol, glucose, fructose, and lactose.

At least one polyol may be selected from diols containing from 4 to 10 C-atoms. In one aspect of the invention the -OH groups in the diols are vicinally positioned, as e.g. in 1 ,2-pentane diol.

In another aspect of the invention, the -OH groups are localized terminally, as e.g. 1 ,6-hexane diol.

In one embodiment, the diol having vicinally positioned -OH groups contains 4 to 10 C-atoms, preferably 4 to 8 C-atoms, more preferably 4 to 6 C-atoms, most preferably 4 to 5 C-atoms. The diol may be selected from 1 ,2-butandiol and 1 ,2-pentandiol. The diol having vicinally positioned -OH groups may be comprised in the enzyme preparation in amounts in the range of 1% to 5% by weight, or in amounts of about 4% by weight, all relative to the total weight of the enzyme preparation.

In one embodiment, the diol having terminal -OH groups contains 3 to 10 C-atoms, preferably 4 to 8 C-atoms. The diol may be selected from 1 ,4-butanediol, 1 ,6-hexanediol and 1 ,8-octanediol. The diol having terminal -OH groups may be comprised in the enzyme preparation in amounts in the range of 10% to 30% by weight, or 12% to 27% by weight, all relative to the total weight of the enzyme preparation.

In one embodiment, diol means a combination of at least two diols, wherein at least one of the diols is selected from diols having terminal -OH groups containing 3 to 10 C-atoms, preferably 4 to 8 C-atoms. More preferably, the diol having terminal -OH groups is selected from 1 ,4- butanediol, 1 ,6-hexanediol and 1 ,8-octanediol.

In one embodiment, polyol comprises at least two diols, wherein • the first diol is selected from diols having vicinally positioned -OH groups containing 4 to 10 C-atoms, preferably 4 to 8 C-atoms, more preferably 4 to 6 C-atoms, most preferably 4 to 5 C-atoms; said diol may be selected from 1 ,2-butandiol and 1 ,2-pentandiol; and

• the second diol is selected from diols having terminal -OH groups containing 3 to 10 C- atoms, preferably 4 to 8 C-atoms; said diol may be selected from 1 ,4-butanediol, 1 ,6- hexanediol and 1 ,8-octanediol.

In one embodiment, polyol comprises a mixture of diols having vicinally positioned -OH groups containing 4 to 10 C-atoms and diols is selected from diols having terminal -OH groups contain ing 3 to 10 C-atoms in a mixing ration of 1 :10, 1 :9, 1 :8, 1 :7, or 1 :6. The mixing ratio is preferably 1 :6.75.

In one embodiment, the enzyme preparation comprises about 2-5% by weight 1 ,2-butandiol in combination with about 10-30% by weight of at least one diol selected from 1 ,4-butanediol, 1 ,6- hexanediol and 1 ,8-octanediol; % by weight relative to the total weight of the enzyme prepara tion.

In one embodiment, the enzyme preparation comprises about 2-5% by weight 1 ,2-pentandiol in combination with about 10-30% by weight of at least one diol selected from 1 ,4-butanediol, 1 ,6- hexanediol and 1 ,8-octanediol; % by weight relative to the total weight of the enzyme prepara tion.

Component (c) may comprise at least one peptide stabilizer. Peptide stabilizers may be select ed from di-, tri- or tetrapeptide aldehydes and aldehyde analogues (either of the form B1 -BO-R wherein, R is H, CH ₃ , CX ₃ , CHX ₂ , or CH ₂ X (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 compris ing an N-terminal protection group, or as described in WO 09/1 18375 and WO 98/13459, or a protease inhibitor of the protein type such as RASI, BASI, WASI (bifunctional alpha- amylase/subtilisin inhibitors of rice, barley and wheat) or Cl ₂ or SSI.

At least one peptide stabilizer may be selected from a compound of formula (Da) or a salt thereof or from a compound of formula (Db):

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ and Z within formulae (Da) and (Db) are defined as follows:

R ¹ , R ² and R ³ are each independently selected from the group consisting of hydrogen, optionally substituted Ci-s alkyl, optionally substituted C2-6 alkenyl, optionally substituted Ci-s alkoxy, op tionally substituted 3- to 12-membered cycloalkyl, and optionally substituted 6- to 10-membered aryl; or wherein each R ¹ , R ² and R ³ is independently selected as -(CH ₂ )3- which is also attached to the nitrogen atom of -NH-C(H)- so that -N-C(H)R ^{1 2 or 3} - forms a 5-membered heterocyclic ring;

R ⁴ and R ⁵ are each independently selected from the group consisting of hydrogen, optionally substituted Ci-s alkyl, optionally substituted C2-6 alkenyl, optionally substituted Ci-s alkoxy, op tionally substituted Ci-4 acyl, optionally substituted Ci-s alkyl phenyl (e.g. benzyl), and optionally substituted 6- to 10-membered aryl; or wherein R ⁴ and R ⁵ are joined to form an optionally sub stituted 5- or 6-membered ring;

Z is selected from hydrogen, an N-terminal protection group, and one or more amino acid resi dues optionally comprising an N-terminal protection group.

Preferably, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Gly, Ala,

Val, Leu, lie, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gin, Tyr, Cys, Lys, Arg, His, Asn, Glu, m- tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle. More preferably, R ¹ is a group such that NH- CHR ¹ -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, Phe, lie, His or Thr. Even more preferably, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, lie or His.

Preferably, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Gly, Ala,

Val, Leu, lie, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gin, Tyr, Cys, Lys, Arg, His, Asn, Glu, m- tyrosine, 3,4-dihydroxyphenylalanine, Nva, or Nle. More preferably, R ² is a group such that NH- CHR ² -CO is an L or D-amino acid residue of Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva or Nle. Even more preferably, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, lie or His and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D- amino acid residue of Ala, Val, Gly, Arg, Leu, lie or His and R ² is a group such that NH-CHR ² - CO is an L or D-amino acid residue of Gly. In one embodiment R ¹ is a group such that NH- CHR ¹ -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, lie or His and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Pro. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Ala, Val, Gly, Arg, Leu, lie or His and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Val.

In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Ala and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid resi due of Gly and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala,

Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Arg and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D- amino acid residue of Leu and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of lie and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val. In one embodiment R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of His and R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, Gly, Pro or Val.

In one embodiment, R ³ is a group selected from optionally substituted Ci-s alkyl, such as CH ₂ Si(CH ₃ )3, Ci-s alkylphosphates such as (CH ₂ ) _n PO(OR)2, Ci-s alkylnitriles such as CH ₂ CN, Ci-s alkylsulfones such as CH ₂ S0 ₂ R, Ci-s alkylethers such as (CH ₂ ) _n OR, Ci-s alkylesters such as CH ₂ C0 ₂ R, and Ci-s alkylamides; optionally substituted Ci-s alkoxy, optionally substituted 3- to 12-membered cycloalkyl, such as cyclohexylmethyl; and optionally substituted 6- to 10- membered aryl, wherein R is independently selected from the group consisting of hydrogen, optionally substituted Ci-s alkyl, optionally substituted Ci-s alkoxy, optionally substituted 3- to 12- membered cycloalkyl, optionally substituted 6- to 10-membered aryl, and optionally substituted 6- to 10-membered heteroaryl and n is an integer from 1 to 8, i.e. 1 , 2, 3, 4, 5, 6, 7 or 8.

Preferably, R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Tyr, m- tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Ala, Met, Nva, Leu, lie or Nle or other non- natural amino acids carrying alkyl groups. More preferably, R ³ is a group such that NH-CHR ³ - CO is an L or D-amino acid residue of Tyr, Phe, Val, Ala or Leu.

In one embodiment, R ¹ , R ² and R ³ is a group such that NH-CHR ¹ -CO, NH-CHR ² -CO and NH- CHR ³ -CO each is an L or D-amino acid residue of Gly, Ala, Val, Leu, lie, Met, Pro, Phe, Trp, Ser, Thr, Asp, Gin, Tyr, Cys, Lys, Arg, His, Asn, Glu, m-tyrosine, 3,4-dihydroxyphenylalanine, Nva or Nle.

In one embodiment, R ¹ and R ² is a group such that NH-CHR ¹ -CO and NH-CHR ² -CO each is an L or D-amino acid residue of Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva or Nle, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Tyr, m-tyrosine, 3,4-dihydroxyphe nylalanine, Phe, Val, Ala, Met, Nva, Leu, lie or Nle.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Gly or Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Tyr, Ala, or Leu.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Leu.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Gly, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Tyr.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Ala.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Norleucine.

In one embodiment, R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Norvaline.

In one embodiment, R ⁴ and R ⁵ are each independently selected from hydrogen, methyl, ethyl, i- propyl, n-propyl, i-butyl, s-butyl, n-butyl, i-pentyl, 2-pentyl, 3-pentyl, neopentyl, cyclopentyl, cy clohexyl, and benzyl.

R ⁴ and R ⁵ may each independently be selected from methyl, ethyl, isopropyl, 2-butyl or 3-pentyl. More preferably, R ⁴ and R ⁵ are both methyl, ethyl, isopropyl, 2-butyl or 3-pentyl. Z may be selected from hydrogen, an N-terminal protection group, and one or more amino acid residues optionally comprising an N-terminal protection group. Preferably, Z is an N-terminal protection group.

The N-terminal protection group may be selected from formyl, acetyl (Ac), benzoyl (Bz), tri- fluoroacetyl, fluorenylmethyloxycarbonyl (Fmoc), methoxysuccinyl, aromatic and aliphatic ure thane protecting groups, benzyloxycarbonyl (Cbz), ferf-butyloxycarbonyl (Boc), adaman- tyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p- methoxyphenyl (PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate, a me- thylamino carbonyl/methyl urea group, tityl (Trt), 3,5-dimethoxyphenylisoproxycarbonyl (Ddz), 2- (4-biphenyl)isopropoxycarbonyl (Bpoc), 2-nitrophenylsulfenyl (Nps), 2-(4-nitrophenylsulfonyl)- ethoxycarbonyl (Nsc), 1 ,1 -dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc), (1 ,1 -di- oxonaphtho[1 ,2-b]thiophene-2-yl)methyloxycarbonyl (a-Nsmoc), 1 -(4,4-dimethyl-2,6-dioxo- cyclohex-1 -ylidene)-3-methylbutyl (ivDde), 2,7-di-tert-butyl-Fmoc (Fmoc ^* ), 2-fluoro-Fmoc (Fmoc(2F)), 2-monoisooctyl-Fmoc (mio-Fmoc) and 2,7-diisooctyl-Fmoc (dio-Fmoc), tetrachlo- rophthaloyl (TCP), 2-phenyl(methyl)sulfonio)ethyloxycarbonyl tetrafluoroborate (Pms), ethane- sulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps), allyloxycarbonyl (Alloc), o-nitrobenzenesulfonyl (oNBS), 2,4-dinitrobenzenesulfonyl (dNBS), Benzothiazole-2-sul- fonyl (Bts), 2,2,2-trichloroethyloxycarbonyl (Troc), dithiasuccinoyl (Dts), p-nitrobenzyloxycarbo- nyl (pNZ), a-Azidoacids, Propargyloxycarbonyl (Poc), o-Nitrobenzyloxycarbonyl (oNZ), 4-Nitro- veratryloxycarbonyl (NVOC), 2-(2-Nitrophenyl)propyloxycarbonyl (NPPOC), 2-(3,4-Methylene- dioxy-6-nitrophenyl)propyloxycarbonyl (MNPPOC), 9-(4-Bromophenyl)-9-fluorenyl (BrPhF), Az- idomethyloxycarbonyl (Azoc), Hexafluoroacetone (FIFA), 2-Chlorobenzyloxycarbonyl (Cl-Z), Trifluoroacetyl (tfa), 2-(Methylsulfonyl)ethoxycarbonyl (Msc), Tetrachlorophthaloyl (TCP), Phe- nyldisulphanylethyloxycarbonyl (Phdec), 2-Pyridyldisulphanylethyloxycarbonyl (Pydec), or 4- Methyltrityl (Mtt).

If Z is one or more amino acid residue(s) comprising an N-terminal protection group, the N- terminal protection group is preferably a small aliphatic group, e.g., formyl, acetyl, fluorenylme thyloxycarbonyl (Fmoc), ferf-butyloxycarbonyl (Boc), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or a methylamino carbonyl/methyl urea group. In the case of a tripep tide, the N-terminal protection group is preferably a bulky aromatic group such as benzoyl (Bz), benzyloxycarbonyl (Cbz), p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP).

Further suitable N-terminal protection groups are described in Greene’s Protective Groups in Organic Synthesis, Fifth Edition by Peter G. M. Wuts, published in 2014 by John Wiley & Sons, Inc and in Isidro-Llobet et al., Amino Acid-Protecting Groups, Chem. Rev. 2009 109(6), 2455- 2504.

Preferably, the N-terminal protection group is selected from benzyloxycarbonyl (Cbz), p- methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p- methoxyphenyl (PMP), formyl, acetyl (Ac), methyloxy, alkoxycarbonyl, methoxycarbonyl, fluo- renylmethyloxycarbonyl (Fmoc), or ferf-butyloxycarbonyl (Boc). Most preferably, the N-terminal protection group is benzyloxycarbonyl (Cbz).

In a preferred embodiment, the peptide stabilizer is selected from compounds according to for mula (Db), wherein

• R ¹ and R ² is a group such that NH-CHR ¹ -CO and NH-CHR ² -CO each is an L or D-amino acid residue selected from Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva or Nle, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue selected from Tyr, m-tyrosine, 3,4-dihydroxyphe-nylalanine, Phe, Val, Ala, Met, Nva, Leu, lie or Nle;

and

• the N-terminal protection group Z is selected from benzyloxycarbonyl (Cbz), p- methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p- methoxyphenyl (PMP), formyl, acetyl (Ac), methyloxy, alkoxycarbonyl, methoxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc), or ferf-butyloxycarbonyl (Boc).

In a more preferred embodiment, the peptide stabilizer according to formula (Db) is character ized in

• R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Leu;

and

• the N-terminal protection group Z is selected from benzyloxycarbonyl (Cbz), p- methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p- methoxyphenyl (PMP), formyl, acetyl (Ac), methyloxy, alkoxycarbonyl, methoxycarbonyl, fluorenylmethyloxycarbonyl (Fmoc), or ferf-butyloxycarbonyl (Boc); preferably, the N- terminal protection group Z is benzyloxycarbonyl (Cbz).

In one embodiment, the enzyme preparation comprises about 0.1 -2% by weight relative to the total weight of the enzyme preparation of at least one peptide stabilizer. Preferably, the enzyme preparation comprises about 0.15-1%, or 0.2-0.5%, or about 0.3% by weight relative to the total weight of the enzyme preparation of at least one peptide stabilizer. More preferably, the enzyme preparation comprises about 0.3% by weight relative to the total weight of the enzyme prepara tion of a peptide stabilizer according to formula (Db) characterized in

• R ¹ is a group such that NH-CHR ¹ -CO is an L or D-amino acid residue of Val, R ² is a group such that NH-CHR ² -CO is an L or D-amino acid residue of Ala, and R ³ is a group such that NH-CHR ³ -CO is an L or D-amino acid residue of Leu;

and

• the N-terminal protection group Z is benzyloxycarbonyl (Cbz).

Other enzyme stabilizers (component (c)) may be selected from salts like NaCI or KCI, and alka li salts of lactic acid and formic acid.

Other enzyme stabilizers (component (c)) may be selected from water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g. barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (I I), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).

In one embodiment, the enzyme preparation of the invention does not comprise any compound stabilizing the enzyme preparation as such. In another embodiment, the enzyme preparation of the invention does comprise at least one compound stabilizing the liquid enzyme preparation as such. In one embodiment, the enzyme preparation of the invention comprising at least one compound stabilizing the enzyme preparation as such is liquid.

Compounds stabilizing the enzyme preparation as such means any compound except enzyme stabilizers needed to establish storage stability of an enzyme preparation, preferably a liquid enzyme preparation, in amounts effective to ensure storage stability.

Storage stability in the context of enzyme preparations usually includes aspects of appearance of the product and uniformity of dosage.

Appearance of an enzyme preparation, preferably liquid enzyme preparation, is influenced by the pH of the product and by the presence of compounds such as preservatives, antioxidants, viscosity modifiers, emulsifiers etc.

Uniformity of dosage is usually related to the homogeneity of a product.

Inventive enzyme preparations may be alkaline or exhibit a neutral or slightly acidic pH value, for example 6 to 14, 6.5 to 13, 8 to 10.5, or 8.5 to 9.0. The liquid enzyme preparation may comprise at least one preservative. Preservatives are add ed in amounts effective in preventing microbial contamination of the liquid enzyme preparation, preferably the aqueous enzyme preparation.

Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids, and formaldehyde releasing agents. Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chlorides, polyhexameth- ylene 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 releasing agent include N,N'-methylenebismorpholine (MBM), 2,2',2"-(hexahydro-1 ,3,5-triazine- 1 ,3,5- triyl)triethanol (HHT), (ethylenedioxy)dimethanol, .alpha...alpha.', .alpha. "-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).

Further useful preservatives include iodopropynyl butylcarbamate (IPBC), halogen releasing compounds such as dichloro-dimethyl-hydantoine (DCDMH), bromo-chloro-dimethyl-hydantoine (BCDMH), and dibromo-dimethyl-hydantoine (DBDMH); bromo-nitro compounds such as Bronopol (2-bromo-2-nitropropane-1 ,3-diol), 2,2-dibromo-2-cyanoacetamide (DBNPA); alde hydes such as glutaraldehyde; phenoxyethanol; Biphenyl-2-ol; and zinc or sodium pyrithione.

An enzyme preparation of the in invention, preferably a liquid enzyme preparation, may or may not comprise a solvent.

In one embodiment, the inventive enzyme preparation is aqueous, comprising water in amounts 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, or in the range of 20% to 70% by weight, all relative to the total weight of the enzyme preparation.

In one embodiment, the enzyme preparation of the invention comprises at least one organic solvent selected from ethanol, n-propanol, iso-propanol, n-butanol, iso-butanol, sec.-butanol, ethylene glycol, propylene glycol, 1 ,3-propane diol, butane diol, glycerol, diglycol, propyl di glycol, butyl diglycol, hexylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, and phenoxyethanol, preferred are ethanol, isopropanol or propyl ene glycol. Further, 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. Said enzyme preparation may comprise organic solvents in amounts in the range of 0% to 20% by weight relative to the total weight of the enzyme preparation. In one embodiment, the en zyme preparation comprises water in amounts in the range of 5% to 15% by weight and no sig nificant amounts of organic solvent, for example 1 % by weight or less, all relative to the total weight of the enzyme preparation.

In one embodiment, the enzyme preparation of the invention is liquid and comprises component (a): at least one enzyme stabilizer selected from compounds according to general formula (I) or (II) - component (a) as disclosed above

component (b): at least one enzyme selected from the group of hydrolases (EC 3), preferably selected from amylases and proteases, more preferably selected from alpha- amylases (EC 3.2.1.1 ) and subtilisin type proteases (EC 3.4.21.62);

and

component (c): at least one solvent as disclosed above, optionally at least one enzyme stabi lizer different from component (a) as disclosed above, preferably selected from polyols, peptide stabilizers such as peptide aldehydes and other stabi lizers as disclosed above; and further optionally at least one compound stabi lizing the liquid enzyme preparation preferably selected from preservatives and antioxidants.

wherein alpha-amylase may be selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009; • hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

and wherein subtilisin type proteases may be selected from

• proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof as disclosed above; and

• subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof as disclosed above.

In one embodiment, said enzyme preparation comprises component (a) according to formula (I), wherein R ¹ is selected from -CH(OH)-CH(OH)-COOH, -CH(OH)-CH ₃ , -CH ₂ -C(OH)(COOH)-CH ₂ - COOH, (E)-CH=CHCOOH, (Z)-CH=CHCOOH, and 3,4,5-trihydroxyphenyl. Preferably, compo nent (a) according to formula (I) is further characterized by R ² selected from methyl and butyl, R ³ is selected from H, methyl and propyl, R ⁴ is H, n=1 , and m=0.

Preparation of enzyme preparation:

The invention relates to a process for making an enzyme preparation, said process comprising the step of mixing at least component (a) as disclosed above and component (b) as disclosed above and optionally component (c) as disclosed above. Mixing may be done in one or more steps in any order.

In one embodiment component (c) comprises at least one solvent as disclosed above. At the time at least one solvent is present at least one enzyme stabilizer different from component (a) as disclosed above may be added to further stabilize the resulting liquid enzyme preparation.

Component (b) may be solid. Solid component (b) may be added to solid component (a) prior to contact of both with at least one solvent (component (c)). Contact with at least one solvent (component (c)) usually results in solubilizing of at least one molecule component (a) and at least one molecule component (b), resulting in stabilization of at least one molecule component (b). In one embodiment, solid components (a) and (b) are completely dissolved in at least one solvent (component (c)) without phase separation.

Component (b) may be comprised in a liquid, preferably aqueous enzyme concentrate. Usually this means that component (b) is already solubilized in a solvent, preferably water.

Solid component (a) may be dissolved in at least one solvent (component (c)) prior to mixing with solid component (b) or when contacted with a liquid enzyme concentrate already compris ing solvent. In one embodiment, component (a) is completely dissolved in at least one solvent (component (c)) prior to mixing with solid component (b) or a liquid, preferably aqueous enzyme concentrate comprising component (b).

Enzyme stabilization

The invention relates to a method of stabilizing at least one hydrolase comprised in an enzyme containing product by the step of adding at least one compound according to formula (I) or (II) (as disclosed as component (a) herein). In one embodiment, the method of stabilizing at least one hydrolase includes a step of adding at least one enzyme stabilizer different from component (a) (as disclosed as component (c) herein).

Therefore, the invention also relates to the use of at least one compound according to formula (I) or (II) (as disclosed as component (a) herein) to stabilize hydrolytic activity of at least one hydrolase comprised in an enzyme containing product.

An enzyme containing product according to the invention may be solid or liquid and comprises at least one hydrolase (as disclosed as component (b) herein). An enzyme containing product may be component (b) as disclosed above alone or in combination with other components such as solvents. An enzyme containing product may be an enzyme concentrate or any formulation comprising at least one enzyme which needs to be stabilized or stabilized further.

In one embodiment, the enzyme containing product comprises at least one enzyme according to component (b) and at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed below. Preferably, such an enzyme containing product further comprises at least one surfactant selected from non-ionic surfactants, amphoteric surfactants, anionic surfac tants, and cationic surfactants, all as described below.

The enzyme containing product preferably comprises at least one alpha-amylases (EC 3.2.1.1 ); and/or at least one subtilisin type proteases (EC 3.4.21.62) and

optionally at least one enzyme selected from lipases, cellulases and mannan degrading en zymes. At least one alpha-amylase may be selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

At least one subtilisin type proteases may be selected from

• proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof as disclosed above; and

• subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof as disclosed above.

At least one lipase may be selected from fungal triacylglycerol lipase (EC class 3.1.1 .3), prefer ably from Thermomyces lanuginose lipase; more preferably the lipase is selected from Ther- momyces lanuginosa lipase according to amino acids 1 -269 of SEQ ID NO:2 of US 5869438 and variants thereof as disclosed above.

In one embodiment, at least one compound according to formula (I) or (II) is used in combina tion with at least one enzyme stabilizer different from component (a), selected from boron- containing compounds, polyols, peptide stabilizers such as peptide aldehydes and other stabi lizers as disclosed above (component (c)). In one embodiment, a boron-containing product is added in amounts effective to reversibly inhibit the proteolytic activity of at least one protease comprised in the enzyme containing product.

The invention relates to the use of a compound according to formula (I) or (II) (component (a) as disclosed above) as additive for a solid enzyme containing product. In such a case, the enzy matic activity of the hydrolase is stabilized when the compound according to formula (I) or (II) and the hydrolase are contacted with at least one solvent [component (c)].

Contact with at least one solvent (component (c)) may result in solubilizing of at least one mole cule component (a) and at least one molecule component (b), resulting in stabilization of at least one molecule component (b). In one embodiment, solid components (a) and (b) are completely dissolved in at least one solvent (component (c)) without phase separation.

In one embodiment of the present invention, component (a) is added in amounts in the range of 0.1 % to 30% by weight, relative to the total weight of the enzyme preparation. The enzyme preparation may comprise component (a) in amounts in the range of 0.1 % to 15% by weight, 0.25% to 10% by weight, 0.5% to 10% by weight, 0.5% to 6% by weight, or 1 % to 3% by weight, all relative to the total weight of the enzyme preparation.

In one embodiment, at least one enzyme stabilizer different from component (a) is added in amounts effective to reversibly inhibit the proteolytic activity of at least one protease comprised in component (b).

Stabilization of an enzyme may relate to stability in the course of time (e.g. storage stability), thermal stability, pH stability, and chemical stability. The term“enzyme stability” herein prefera bly relates to the retention of enzymatic activity as a function of time e.g. during storage or op eration. The term“storage” herein means to indicate the fact of products or compositions 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 during storage is called“storage stability”. In one embodiment, storage means storage for at least 20 days at 37°C. Storage may mean storage for 3, 7, 14, 21 , 28 or 42 days at 37°C. To determine changes in enzymatic activity over time, the“initial enzymatic activity” of an en zyme may be measured under defined conditions at time zero (i.e. before storage) and the“en zymatic activity after storage” may be measured at a certain point in time later (i.e. after stor age).

The enzymatic activity after storage divided by the initial enzymatic activity multiplied by 100 gives the“residual enzymatic activity” (a%).

An enzyme is stable according to the invention, when its residual enzymatic activity is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% when compared to the initial enzymatic activity be fore storage.

Subtracting“residual enzymatic activity” a% from 100% gives the“loss of enzymatic activity during storage” when compared to the initial enzymatic activity before storage. In one embodi ment, an enzyme is stable according to the invention when essentially no loss of enzymatic ac tivity occurs during storage, i.e. loss in enzymatic activity equals 0% when compared to the ini tial enzymatic activity before storage. Essentially no loss of enzymatic activity within this inven tion may mean that the loss of enzymatic activity is less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% when compared to the initial enzymatic activity before storage.

In one aspect of the invention, a compound according to formula (I) or (II) (as disclosed as component (a) herein) stabilizes at least one amylase, preferably selected from alpha-amylases (EC 3.2.1 .1 ) (as disclosed as component (b) herein). In one embodiment, component (a) is used to stabilize amylase [component (b)] within a liquid enzyme preparation. In one embodi ment, component (a) is used to stabilize amylase [component (b)] within a liquid enzyme con taining product preferably comprising at least one surfactant, more preferably further comprising at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as disclosed be low. Stabilization in this context may mean stabilization during storage at 37°C for 3, 7, 14, 21 , 28 and/or 42 days.

In one embodiment, the addition of component (a) to component (b) stabilizes at least one al pha-amylase during storage, wherein stabilization is characterized by

(a) residual amylolytic activity after storage at 37°C for 21 days being ³65% when compared to the initial amylolytic activity before storage and/or (b) residual amylolytic activity after storage at 37°C for 28 days being ³55% when compared to the initial amylolytic activity before storage and/or

(c) residual amylolytic activity after storage at 37°C for 42 days being ³40 when compared to the initial amylolytic activity before storage;

wherein component (a) is preferably comprised in amounts in the range of 1% to 5% by weight, more preferably in the range of 1 .5% to 2% by weight, both relative to the total weight of the composition, and/or wherein amylase is preferably comprised in amounts in the range of 0.2% to 2% by weight, more preferably in about 0.5% by weight, both relative to the total weight of the composition, and/or wherein EDTA and/or DTPA and/or MGDA (methyl glycine diacetic acid) and/or GLDA (glutamic acid diacetic acid) may be comprised in amounts in the range of 10% to 30% by weight, preferably in the range of 15% to 25%, all relative to the total weight of the composition. MGDA and GLDA are known as sequestrants for alkaline earth metal ions such as Ca ²⁺ and Mg ²⁺ - see disclosure below.

In one embodiment, at least one alpha-amylase is stabilized by addition of component (a) ac cording to formula (I), wherein R ¹ is selected from -CH(OH)-CH(OH)-COOH, -CH(OH)-CH ₃ , -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl. Preferably, component (a) according to formula (I) is further characterized by R ² selected from methyl and butyl, R ³ is selected from H, methyl and propyl, R ⁴ is H, n=1 , and m=0.

In one embodiment, stabilization is characterized by

(a) loss of amylolytic activity during storage at 37°C for 21 days being £35% when com pared to the initial amylolytic activity before storage and/or

(b) loss of amylolytic activity during storage at 37°C for 28 days being £45% when com pared to the initial amylolytic activity before storage and/or

(c) loss of amylolytic activity during storage at 37°C for 42 days being £60% when com pared to the initial amylolytic activity before storage.

In one embodiment, alpha-amylase is selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity; • amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

In one aspect of the invention, a compound according to formula (I) or (II) (as disclosed as component (a) herein) stabilizes at least one protease, preferably selected from the group of serine endopeptidases (EC 3.4.21 ), more preferably selected from the group of subtilisin type proteases (EC 3.4.21 .62) (as disclosed as component (b) herein). In one embodiment, compo nent (a) is used to stabilize protease [component (b)] within a liquid enzyme preparation. In one embodiment, component (a) is used to stabilize protease [component (b)] within a liquid enzyme containing product preferably comprising at least one surfactant, more preferably further com prising at least one complexing agent selected from EDTA, DTPA, MGDA and GLDA as dis closed below. Stabilization in this context may mean stabilization during storage at 37°C for 3,

7, 14, 21 , 28 and/or 42 days.

In one embodiment, the addition of component (a) to component (b) stabilizes subtilisin type protease during storage, wherein stabilization is characterized by

(a) residual proteolytic activity after storage at 37°C for 21 days being ³73% when com

pared to the initial amylolytic activity before storage and/or

(b) residual proteolytic activity after storage at 37°C for 28 days being ³67% when com

pared to the initial amylolytic activity before storage and/or (c) residual proteolytic activity after storage at 37°C for 42 days being ³60 when compared to the initial amylolytic activity before storage;

wherein component (a) is preferably comprised in amounts in the range of 1% to 5% by weight, more preferably in the range of 1 .5% to 2% by weight, both relative to the total weight of the composition, and/or wherein protease is preferably comprised in amounts in the range of 0.2% to 2% by weight, more preferably in about 2.5% to 3% by weight, both relative to the total weight of the composition, and/or wherein EDTA and/or DTPA and/or MGDA (methyl glycine diacetic acid) and/or GLDA (glutamic acid diacetic acid) may be comprised in amounts in the range of 10% to 30% by weight, preferably in the range of 15% to 25%, all relative to the total weight of the composition. MGDA and GLDA are known as sequestrants for alkaline earth metal ions such as Ca ²⁺ and Mg ²⁺ - see disclosure below.

In one embodiment, at least one subtilisin type protease is stabilized by addition of component

(a) according to formula (I), wherein R ¹ is selected from -CH(OH)-CH(OH)-COOH,

-CH(OH)-CH ₃ , -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, (Z)-CH=CHCOOH, and 3,4,5-trihydroxyphenyl. Preferably, component (a) according to formula (I) is further character ized by R ² selected from methyl and butyl, R ³ is selected from H, methyl and propyl, R ⁴ is H, n=1 , and m=0.

In one embodiment, stabilization is characterized by

(a) loss of proteolytic activity during storage at 37°C for 21 days being £27% when com

pared to the initial proteolytic activity before storage and/or

(b) loss of proteolytic activity during storage at 37°C for 28 days being £33% when com

pared to the initial proteolytic activity before storage and/or

(c) loss of proteolytic activity during storage at 37°C for 42 days being £40% when com

pared to the initial proteolytic activity before storage.

In one embodiment, subtilisin type protease is selected from

• proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof as disclosed above; and

• subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof as disclosed above.

In one embodiment, the addition of component (a) to component (b) stabilizes alpha-amylase and/or subtilisin type protease during storage preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is character ized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (£)-CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl, R ² is -CH3, R ³ and R ⁴ are H, n=1 , and m=0.

In one embodiment, the addition of component (a) to component (b) stabilizes alpha-amylase and/or subtilisin type protease during storage preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is character ized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl, preferably -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, R ² and R ³ are -CH ₃ , R ⁴ is H, n=1 and m=0.

In one embodiment, the addition of component (a) to component (b) stabilizes alpha-amylase and/or subtilisin type protease during storage preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is character ized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl, preferably-CH ₂ -C(OH)(COOH)-CH ₂ -COOH, R ² is -CH ₃ , R ³ is propyl, R ⁴ is H, n=1 and m=0.

In one embodiment, the addition of component (a) to component (b) stabilizes alpha-amylase and/or subtilisin type protease during storage preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is character ized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl, preferably -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, and R ² is two times methyl and one time butyl, R ³ and R ⁴ are H, n=1 , and m=0.

The invention relates to a method of reducing loss of hydrolytic activity of a hydrolase com prised in an enzyme containing product by the step of adding at least one compound according to formula (I) or (II) (as disclosed as component (a) herein). In one embodiment, the method of stabilizing at least one hydrolase includes a step of adding at least one enzyme stabilizer differ ent from component (a) (as disclosed as component (c) herein).

The invention also relates to the use of at least one compound according to formula (I) or (II) (as disclosed as component (a) herein) to reduce the loss of hydrolytic activity of at least one hydro lase comprised in an enzyme containing product.

In one embodiment, the loss of hydrolytic activity is reduced during storage preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein component (a) is preferably comprised in amounts in the range of 1 % to 5% by weight, more preferably in the range of 1 .5% to 2% by weight, both relative to the total weight of the composition, and EDTA and/or DTPA are comprised in amounts up to 3% by weight, preferably up to 2.5% and/or MGDA and/or GLDA are comprised in amounts in the range of 10% to 30% by weight, preferably in the range of 15% to 25%, all relative to the total weight of the composi tion.

Calculation of % reduced loss of enzymatic activity is done as follows: (% loss of enzymatic ac tivity of stabilized enzyme) - (% loss of enzymatic activity of non-stabilized enzyme). The value for reduced loss indicates the reduced loss of enzymatic activity of at least one enzyme com prised in component (b) in the presence of component (a) when compared to the loss of enzy matic activity of the same enzyme(s) in the absence of component (a) at a certain point in time.

Reduced loss of enzymatic activity within this invention may mean that the loss of enzymatic activity is reduced in the presence of component (a) by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, when compared to the loss of enzymatic activity in the absence of component (a).

In one embodiment, reduced loss of alpha-amylase activity is characterized by

(a) reduced loss of amylolytic activity during storage at 37°C for 21 days being ³40% when compared to the loss of proteolytic activity in the absence of component (a) and/or

(b) reduced loss of amylolytic activity during storage at 37°C for 28 days being ³35%, when compared to the loss of proteolytic activity in the absence of component (a) and/or

(c) reduced loss of amylolytic activity during storage at 37°C for 42 days being ³30%, or ³10% when compared to the loss of proteolytic activity in the absence of component (a). wherein alpha-amylase is preferably comprised in amounts in the range of 0.2% to 2% by weight, more preferably in about 0.5% by weight, all relative to the total weight of the enzyme containing product; and wherein alpha-amylase is selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity;

• amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity; • amylase from Bacillus halmapalus or variants thereof having amylolytic activity, preferably selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO 2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amy lolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; preferably, the hy brid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity.

In one embodiment, reduced loss of subtilisin type protease activity is characterized by

(a) reduced loss of proteolytic activity during storage at 37°C for 21 days being ³5% when compared to the loss of proteolytic activity in the absence of component (a) and/or

(b) reduced loss of proteolytic activity during storage at 37°C for 28 days being ³9%when compared to the loss of proteolytic activity in the absence of component (a) and/or

(c) reduced loss of proteolytic activity during storage at 37°C for 42 days being ³10% when compared to the loss of proteolytic activity in the absence of component (a).

wherein subtilisin type protease is preferably comprised in amounts in the range of 0.2% to 2% by weight, more preferably in about 2.5% to 3% by weight, all relative to the total weight of the enzyme containing product. In embodiments, the subtilisin type protease (EC 3.4.21.62) of the above embodiments is be selected from

• proteases according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof as disclosed above; and

subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof as disclosed above. In one embodiment, the alpha-amylase and/or subtilisin-type protease is stabilized during stor age preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA by addition of a compound characterized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (£)- CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5-trihydroxyphenyl, R ² is -CH ₃ , R ³ and R ⁴ are H, n=1 , and m=0.

In one embodiment, the alpha-amylase and/or subtilisin-type protease is stabilized during stor age preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA by addition of a compound characterized by R ¹ in the compound according to formula is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (£)- CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5-trihydroxyphenyl, preferably -CH ₂ - C(OH)(COOH)-CH ₂ -COOH, , R ² and R ³ are -CH ₃ , R ⁴ isH, n=1 and m=0.

In one embodiment, the alpha-amylase and/or subtilisin-type protease is stabilized during stor age preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA by addition of a compound characterized by R ¹ and R ² in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH,

-CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (E)-CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5- trihydroxyphenyl, preferably-CH ₂ -C(OH)(COOH)-CH ₂ -COOH, R ² is -CH ₃ , R ³ is propyl, R ⁴ is H, n=1 and m=0.

In one embodiment, the alpha-amylase and/or subtilisin-type protease is stabilized during stor age preferably in the presence of a complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA by addition of a compound characterized by R ¹ in the compound according to formula (I) is selected from -CH(OH)-CH(OH)-COOH, -CH ₂ -C(OH)(COOH)-CH ₂ -COOH, (£)- CH=CHCOOH, and (Z)-CH=CHCOOH, and 3,4,5-trihydroxyphenyl, preferably

-CH ₂ -C(OH)(COOH)-CH ₂ -COOH, and R ² is two times methyl and one time butyl, R ³ and R ⁴ are H, n=1 , and m=0.

Use of enzyme preparation for formulation processes

The invention in one aspect relates to the use of the liquid enzyme preparation of the invention to be formulated into enzyme containing products such as detergent formulations e.g. I&l and homecare formulations for laundry and hard surface cleaning, wherein at least components (a) and (b) are mixed in no specified order in one or more steps with one or more detergent com ponents. In one embodiment, at least components (a), (b) and (c) as disclosed above are mixed in no specified order in one or more steps with one or more detergent components. In one aspect of the invention relates to a detergent formulation comprising the liquid enzyme preparation of the invention and one or more detergent components.

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.

Individual detergent components and usage in detergent formulations 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), 6 ^th edition (2015). Another reference book for those skilled in the art may be“Detergent Formulations Encyclopedia”, Solverchem Publications,

2016.

It is understood that the detergent components are in addition to the components comprised in the enzyme preparation of the invention. If a component comprised in the enzyme preparation of the invention is also a detergent component, it might be the concentrations that need to be adjusted that the component is effective for the purpose desired in the detergent formulation.

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/or effective cleaning conditions (e.g. pH, quantity of foaming), amounts of certain components to effectively provide optical benefits (e.g. optical brightening, dye transfer inhibition), and/or amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. viscosity modifiers, hy drotropes, desiccants). In one embodiment, a detergent formulation is a formulation of more than two detergent com ponents, 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.

In one embodiment of the present invention, detergent formulations, preferably liquid detergent formulations, comprise component (a) in amounts in the range of 0.1 % to 30% by weight, rela tive to the total weight of the detergent formulation. The enzyme preparation may comprise component (a) in amounts in the range of 0.1 % to 15% by weight, 0.25% to 10% by weight, 0.5% to 10% by weight, 0.5% to 6% by weight, or 1 % to 3% by weight, all relative to the total weight of the detergent formulation.

In one embodiment of the present invention, detergent formulations, preferably liquid detergent formulations, comprise 0.5 to 20% by weight, particularly 1 -10% by weight component (b) and 0.01 % to 10% of component (a), more particularly 0.05 to 5% by weight and most particularly 0.1 % to 2% by weight of component (a), all relative to the total weight of the detergent formula tion.

In one embodiment, the detergent formulation of the invention is liquid at 20°C and 101 .3 kPa. The liquid detergent formulation may comprise water or may be essentially free of water, mean ing that no significant amounts of water are present. Non-significant amounts of water herein means that the liquid detergent formulation comprises less than 15%, less than 10%, less than 7%, less than 5%, less than 4%, less than 3%, less than 2% by weight water, all relative to the total weight of the liquid detergent formulation, or no water. In one embodiment, enzyme con centrate free of water free of water means that the liquid detergent formulation does not com prise significant amounts of water but does comprise organic solvents in amounts of 30-80% by weight, relative to the total weight of the enzyme concentrate.

Water-comprising liquid detergent formulations may comprise water as sole solvent. In embod iments, mixtures of water with one or more water-miscible solvents are used as aqueous medi um. The term water-miscible solvent refers to organic solvents that are miscible with water at ambient temperature without phase-separation. Examples are ethylene glycol, 1 ,2-propylene glycol, isopropanol, and diethylene glycol. Preferably, at least 50% by volume of the respective aqueous medium is water, referring to the solvent.

Detergent formulations of the invention comprise at least one compound selected from surfac tants, builders, polymers, fragrances and dyestuffs.

The detergent formulation of the invention comprises at least one surfactant selected from non ionic surfactants, amphoteric surfactants, anionic surfactants, and cationic surfactants - com- pounds and effective amounts known to those skilled in the art. In one embodiment, the deter gent formulation of the invention comprises 5 to 30 % by weight of anionic surfactant and at least one non-ionic surfactant, for example in the range of from 3 to 20% by weight, all relative to the total weight of the detergent formulation, wherein the detergent formulation may be liquid.

At least one non-ionic surfactant may be selected from alkoxylated alcohols, di- and multiblock copolymers of ethylene oxide and propylene oxide and reaction products of sorbitan with eth ylene oxide or propylene oxide, alkyl polyglycosides (APG), hydroxyalkyl mixed ethers and amine oxides.

Further suitable non-ionic surfactants are selected from di- and multiblock copolymers, com posed of ethylene oxide and propylene oxide. Further suitable nonionic surfactants are selected from ethoxylated or propoxylated sorbitan esters. Amine oxides or alkyl polyglycosides, espe cially linear C4-Ci8-alkyl polyglucosides and branched Cs-Cis-alkyl polyglycosides are likewise suitable. Examples for amine oxides include but are not limited to lauryl dimethyl aminoxide, sometimes also called lauramine oxide, and more specifically cocamidylpropyl dimethylaminox- ide, sometimes also called cocamidopropylamine oxide.

The detergent formulation may comprise 0.1 to 60% by weight relative to the total weight of the detergent formulation of surfactant. The detergent formulation may comprise at least one com pound selected from anionic surfactants, non-ionic surfactants, amphoteric surfactants, and amine oxide surfactants as well as combinations of at least two of the foregoing. In one embod iment, the detergent formulation of the invention comprises 5 to 30% by weight of anionic sur factant and at least one non-ionic surfactant, for example in the range of from 3 to 20% by weight, all relative to the total weight of the detergent formulation, wherein the detergent formu lation may be liquid.

Non-ionic surfactant means a surfactant that contains neither positively nor negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, non-ionic surfac tants do not ionize in solution. At least one non-ionic surfactant may be selected from alkoxylat ed alcohols, di- and multiblock copolymers of ethylene oxide and propylene oxide and reaction products of sorbitan with ethylene oxide or propylene oxide, alkyl polyglycosides (APG), hy droxyalkyl mixed ethers and amine oxides.

Non-ionic surfactants may be compounds of the general formulae (Sla) and (Sib): R ¹ is selected from C1-C23 alkyl and C2-C23 alkenyl, wherein alkyl and/or alkenyl are linear (straight-chain; n-) or branched; examples are n-C ₇ Hi ₅ , n-CgHig, n-CnH ₂ 3, n-Ci3H ₂₇ , n-

C15H31 , n-Ci ₇ H ₃₅ , 1-C9H19, 1-C12H25·

R ² is selected from H, C1-C20 alkyl and C2-C20 alkenyl, wherein alkyl and/or alkenyl are linear (straight-chain; n-) or branched.

R ³ and R ⁴ , each independently selected from C1-C16 alkyl, wherein alkyl is linear (straight-chain; n-) 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 (straight-chain; n-) or branched. The integers of the general formulae (Sla) and (Sib) are defined as follows:

m is in the range of zero to 200, preferably 1 -80, more preferably 3-20; n and 0, each in dependently in the range of zero to 100; n preferably is in the range of 1 to 10, more pref erably 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.

Compounds according to formula (Sla) may be called alkyl polyethyleneglycol ether (AEO) herein. Compounds according to formula (Sib) may be called alkylphenol polyethyleneglycol ether (APEO) herein.

The detergent formulation may comprise at least one non-ionic surfactant selected from com pounds of general formula (Sla), wherein said non-ionic surfactant is characterized in R ¹ being n-Ci3H ₂ 7, R ² and R ⁵ being H, m being 3-20, n and 0 = 0. The detergent formulation may comprise at least one non-ionic surfactant selected from com pounds of general formula (Sla), wherein said non-ionic surfactant is characterized in R1 being linear or branched Cio alkyl, R ² and R ⁵ being H, m being 3-14, n and o = 0.

The detergent formulation may comprise at least two non-ionic surfactants, selected from com pounds of general formula (Sla), wherein one of said non-ionic surfactants is characterized in R ¹ being n-Ci ₅ H _3i , R ² and R ⁵ being H, m being 1 1 -80, n and o = 0, and the other surfactant is characterized in R ¹ being n-Ci ₇ H35, R ² and R ⁵ being H, m being 1 1 -80, n and 0 = 0.

In one embodiment, the detergent formulation comprises at least one non-ionic surfactant se lected from general formula (Sla), wherein m is in the range of 3 to 1 1 , preferably not more than 10, more preferably not more than 7; n and 0 is 0, R ¹ is linear C9-C17 alkyl, R ² and R ⁵ is H.

The detergent formulation may comprise at least two non-ionic surfactants, selected from com pounds of general formula (Sla), wherein one of said non-ionic surfactants is characterized in R ¹ being n-Ci2H ₂ 5, R ² and R ⁵ being H, m being 3-30, preferably 7, n and 0 = 0, and the other sur factant is characterized in R ¹ being n-Ci ₄ H ₂₉ , R ² and R ⁵ being H, m being 3-30, preferably 7, n and o = 0.

The detergent formulation may comprise at least two non-ionic surfactants, selected from com pounds of general formula (Sla), wherein one of said non-ionic surfactants is characterized in R ¹ being n-Cn H ₂₃ , R ² and R ⁵ being H, m being 4-10, n and 0 = 0, and the other surfactant is char acterized in R ¹ selected from n-Cn H ₂₃ and n-Ci ₇ H ₃₅ , R ² and R ⁵ being H, m being 4-10, n and o = 0.

The detergent formulation may comprise at least two non-ionic surfactants, selected from com pounds of general formula (Sla), wherein one of said non-ionic surfactants is characterized in R ¹ being n-CgHig, R ² and R ⁵ being H, m being 5-7, n and 0 = 0, and the other surfactant is charac terized in R ¹ being n-Ci ₇ H ₃₅ , R ² and R ⁵ being H, m being 5-7, n and 0 = 0. In one embodiment, the detergent formulation comprises at least two non-ionic surfactants, selected from com pounds of general formula (Sla), wherein one of said non-ionic surfactants is characterized in R ¹ being n-Cn H ₂₃ , R ⁵ being H, m is 7, n and 0 = 0, and the other surfactant is characterized in R ¹ being CI ₃ H ₂₇ , R ⁵ being H, m being 7, n and 0 = 0.

The non-ionic surfactants of the general formulae (Sla) and (Sib) may be of any structure, is it block or random structure, and is not limited to the displayed sequence of formulae (Sla) and (Sib).

In one embodiment, the detergent formulation according to the invention comprises at least one compound according to formula (Sla) or (Sib) in the range of about 0.3% to 30% by weight, in the range of about 0.4% to 20% by weight, or in the range of about 0.5% to 10%, all relative to the total weight of a detergent formulation. At least one non-ionic surfactant may be selected from a surfactant according to general formula (Sla), and wherein m is 7; n and 0 is 0, R ¹ is Ci ₂ - CM, R ² and R ⁵ is H. In an embodiment, the detergent formulation comprises two non-ionic sur factants, selected from compounds of general formula (Sla), wherein one of said non-ionic sur factants is characterized in R ¹ being C12, R ² and R ⁵ being H, m is 7, n and 0 = 0, and the other surfactant is characterized in R ¹ being C14, R ² and R ⁵ being H, m being 7, n and 0 = 0, wherein preferably the total amount of the non-ionic surfactants is in the range of about 0.3% to 30% by weight, in the range of about 0.4% to 20% by weight, or in the range of about 0.5% to 10%, all relative to the total weight of a detergent formulation.

Non-ionic surfactants may further be compounds of the general formula (SI I), which might be called alkyl-polyglycosides (APG):

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

R ¹ is selected from C ₁ -C ₁₇ alkyl and C ₂ -C ₁₇ alkenyl, wherein alkyl and/or alkenyl are linear (straight-chain; n-) or branched; examples are n-C Hi ₅ , n-CgHig, n-Cn H ₂₃ , n-Ci ₃ H ₂₇ , n- C1 ₅ H ₃₁ , n-Ci ₇ H ₃₅ , i-CgH ig, i-Ci ₂ H ₂₅ .

R ² is selected from H, C1 -C17 alkyl and C ₂ -Ci alkenyl, wherein alkyl and/or alkenyl are linear (straight-chain; n-) or branched.

G ¹ is selected from monosaccharides with 4 to 6 carbon atoms, such as glucose and xylose. The integer w of the general formula (Sll) is in the range of from 1 .1 to 4, w being an average number.

Non-ionic surfactants may further be compounds of general formula (Sill):

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

AO being identical or different alkylene oxides, selected from CH ₂ -CH ₂ -0, (CH ₂ ) ₃ -0,

(CH ₂ ) ₄ -0, CH ₂ CH(CH ₃ )-0, CH(CH ₃ )-CH ₂ -0- and CH ₂ CH(n-C ₃ H ₇ )-0.

R ¹ is selected from linear (straight-chain; n-) or branched C ₄ -C3o-alkyl, and from straight- chain or branched C ₄ -C3o-alkylene with at least one C-C double bond. R ¹ may be straight- chain or branched C ₄ -C3o-alkyl, n-C ₄ -C3o-alkyl, n-C ₇ -Ci ₅ alkyl, or n-Cio-Ci ₂ -alkyl.

R ² is selected from linear (straight-chain; n-) or branched Ci-C3o-alkyl, and from straight- chain or branched C ₂ -C3o-alkylene with at least one C-C double bond. R ² may be straight- chain or branched C6-C2o-alkyl, preferably straight-chain or branched C8-Ci2-alkyl, more preferably straight-chain or branched Cio-Ci2-alkyl.

The integer x of the general formula (Sill) may be a number in the range of 5 to 70, 10 to 60, 15 to 50, or 20 to 40.

In one embodiment of the present invention, (AO) _x is selected from (CH ₂ CH ₂ 0) _xi , x1 being se lected from one to 50.

In one embodiment of the present invention, (AO) _x is selected from

-(CH2CH ₂ 0) _X2 -(CH2CH(CH ₃ )-0) _x3 and -(CH ₂ CH20) _X2 -(CH(CH ₃ )CH2-0) _X3 , x2 and x3 being identi cal or different and selected from 1 to 30.

In one embodiment of the present invention, (AO) _x is selected from -(ChbChbO)^, x4 = being in the range of from 10 to 50, AO being EO, and R ¹ and R ² each being independently selected from C ₃ -Ci4-alkyl.

In the context of the present invention, x or x1 or x2 and x3 or x4 are to be understood as aver age values, the number average being preferred. Therefore, each x or x1 or x2 or x3 or x4 - if applicable - can refer to a fraction although a specific molecule can only carry a whole number of alkylene oxide units.

In one embodiment, the detergent formulation of the invention comprises at least one non-ionic surfactant according to formula (Sill), wherein R ¹ is n-C ₃ -Ci alkyl, R ² is linear or branched C ₃ - Ci4 alkyl. Preferably AO is selected from -(CH2CH20) _X2 -(CH ₂ CH(CH ₃ )-0) _x3 , -(CH ₂ CH ₂ 0) _X2 - (CH(CH ₃ )CH ₂ -0) _x3 , and -(CH ₂ CH ₂ 0) _X4 , wherein x2 and x4 is a number in the range of 15-50 and x3 is a number in the range of 1 to 15. At least one non-ionic surfactant may be a com pound according to formula (Sill), wherein R ¹ is n-C ₃ alkyl, R ² is branched Cn alkyl, AO is CH ₂ - CH2-O, and x is 22. At least one non-ionic surfactant may be a compound according to formula (Sill), wherein R ¹ is n-C ₃ alkyl, R ² is n-C ₃ -Cio alkyl, AO is CH2-CH2-O, and x is 40. At least one non-ionic surfactant may be a compound according to formula (Sill), wherein R ¹ is n-C ₃ alkyl, R ² is n-Cio alkyl, AO is selected from -(CH2CH20) _X2 -(CH ₂ CH(CH ₃ )-0) _x3 , -(CH ₂ CH ₂ 0) _X2 - (CH(CH ₃ )CH ₂ -0) _x3 , wherein x2 = 22 and x3 = 1 .

In one embodiment, the detergent formulation, preferably a liquid detergent formulation accord ing to the invention at least one compound according to formula (Sill) in the range of about 0.3% to 10% by weight, in the range of about 0.5% to 5% by weight, or in the range of about 1 % to 3%, all relative to the total weight of a detergent formulation. At least one non-ionic surfactant may be a compound according to formula (Sill), wherein R ¹ is n-C ₃ alkyl, R ² is branched Cn alkyl, AO is CH ₂ -CH ₂ -0, and x is 22.

Non-ionic surfactants may further be selected from sorbitan esters and/or ethoxylated or propoxylated sorbitan esters. Non-limiting examples are products sold under the trade names SPAN and TWEEN. Non-ionic surfactants may further be selected from alkoxylated mono- or di-alkylamines, fatty acid monoethanolamides (FAMA), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), and combinations thereof.

At least one amphoteric surfactant may be selected from surfactants that bear a positive and a negative charge in the same molecule under use conditions. Preferred examples of amphoteric surfactants are so-called betaine-surfactants. Many examples of betaine-surfactants bear one quaternized nitrogen atom and one carboxylic acid group per molecule. A particularly preferred example of amphoteric surfactants is cocamidopropyl betaine (lauramidopropyl betaine).

At least one anionic surfactant may be selected from alkali metal and ammonium salts of Cs- Cis-alkyl sulfates, of Cs-Cis-fatty alcohol polyether sulfates, of sulfuric acid half-esters of ethox ylated C4-Ci2-alkylphenols (ethoxylation: 1 to 50 mol of ethylene oxide/mol), C12-C18 sulfo fatty acid alkyl esters, for example of C12-C18 sulfo fatty acid methyl esters, furthermore of C12-C18- alkylsulfonic acids and of Cio-Cis-alkylarylsulfonic acids. Preference is given to the alkali metal salts of the aforementioned compounds, particularly preferably the sodium salts.

Further examples for suitable anionic surfactants are soaps, for example the sodium or potassi um salts of stearic acid, oleic acid, palmitic acid, ether carboxylates, and alkylether phosphates.

Detergent formulations of the invention may comprise one or more compounds selected from complexing agents (chelating agents, sequestrating agents), precipitating agents, and ion ex change compounds, which may form water-soluble complexes with calcium and magnesium. 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.

Non-phosphate based builders according to the invention include sodium gluconate, citrate(s), silicate(s), carbonate(s), phosphonate(s), amino carboxylate(s), polycarboxylate(s), polysul- fonate(s), and polyphosphonate(s). Compounds and effective amounts are known to those skilled in the art.

E.g. the detergent formulation of the invention may comprise citric acid in amounts in the range of 0.1 % to 10.0% by weight, in the range of 0.5% to 8.0% by weight, in the range of 1 .0% to 5.0% by weight, or in the range of 2.0 to 4.0% by weight, all relative to the total weight of the detergent formulation. The citric acid may be provided as a mixture with formiate, e.g. Na- citrate:Na-formiate=9:1 .

The detergent formulation of the invention may comprise at least one phosphonate, preferably selected from derivatives polyphosphonic acids such as of diphosphonic acid such as sodium salt of HEDP, derivatives of aminopolyphosphonic acid such as aminoalkylene phosphonic ac ids such as DTPMP in amounts in the range of 0.1 % to 5.0% by weight, in the range of 0.5% to 3.0% by weight, or in the range of 1 .0% to 2.0% by weight, all relative to the total weight of the detergent formulation.

Detergent formulations of the invention may comprise one or more aminocarboxylates. Com pounds and effective amounts are known to those skilled in the art. E.g. the detergent formula tion of the invention comprises at least one aminocarboxylate selected from ethylenediaminetet- raacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetate (MGDA), and glutamic acid diacetate (GLDA), which all may be (partially) neutralized with alkali, in amounts in the range of 0.1 % to 25.0% by weight, in the range of 1 .0% to 15.0% by weight, in the range of 2.0% to 12.0% by weight, or in the range of 2.5% to 10.0% by weight, all relative to the total weight of the detergent formulation. In one embodiment, the detergent formulation comprises at least one aminocarboxylate selected from ethylenediaminetetraacetic acid

(EDTA), diethylenetriaminepentaacetic acid (DTPA), methylglycine diacetate (MGDA), and glu tamic acid diacetate (GLDA), which all may be (partially) neutralized with alkali, in amounts in the range of 10.0% to 25.0% by weight, or in the range of 15.0% to 25.0% by weight, all relative to the total weight of the detergent formulation.

The term alkali refers to alkali metal cations, same or different, for example cations of lithium, sodium, potassium, rubidium, cesium, and combinations of at least two of the foregoing. Pre ferred examples of alkali metal cations are sodium and potassium and combinations of sodium and potassium.

In an embodiment, the detergent formulation of the invention comprises at least:

• one alkali metal salt of methyl glycine diacetic acid (MGDA), with an average of more than two and less than three of the carboxyl groups being neutralized with alkali, and/or

• one alkali metal salt of L- and D-enantiomers of glutamic acid diacetic acid (GLDA) or of enantiomerically pure L-GLDA, with an average of more than three of the carboxyl groups being neutralized with alkali, preferably an average of more than three and less than four of the carboxyl groups are neutralized with alkali.

In one embodiment of the present invention, alkali metal salts of MGDA are selected from com pounds of the general formula (Cl):

[CH ₃ -CH(COO)-N(CH ₂ -COO) ₂ ]M ₃ -xi- _yi (NH ₄ )zi Hxi (Cl)

The variables of formula (Cl) are defined as follows: M is selected from alkali metal cations, same or different, for example cations of lithium, so dium, potassium, rubidium, cesium, and combinations of at least two of the foregoing. Pre ferred examples of alkali metal cations are sodium and potassium and combinations of sodium and potassium.

x1 is selected from 0.0 to 1 .0, preferably 0.1 to 0.5, more preferably up to 0.1 to 0.3;

z1 is selected from 0.0 to 1 .0, preferably 0.0005 to 0.5;

however, the sum of x1 +z1 in formula (Cl) is greater than zero, for example 0.05 to 0.6.

Examples of M _3-xi-zi (NH ₄ ) _zi H _xi are Na _3-xi H _xi , [Nao _.7 (NH ₄ )o _.3 ] _3-xi H _xi , [(NH ₄ )o _.7 Nao _.3 ] _3-xi H _xi ,.

In one embodiment of the present invention, MGDA is selected from at least one alkali metal salt of racemic MGDA and from alkali metal salts of mixtures of L- and D-enantiomers according to formula (Cl), said mixture containing predominantly the respective L-isomer with an enantio meric excess (ee) in the range of from 5 to 99%, preferably 5 to 95 %, more preferably from 10 to 75% and even more preferably from 10 to 66%.

In one embodiment of the present invention, the total degree of alkali neutralization of MGDA is in the range of from 0.80 to 0.98 mol-%, preferred are 0.90 to 0.97%. The total degree of alkali neutralization does not take into account any neutralization with ammonium.

In one embodiment of the present invention, alkali metal salts of GLDA are selected from com pounds of the general formula (CM)

[OOC-(CH ₂ )2-CH(COO)-N(CH2-COO)2]M _4-X2-Z2 (NH ₄ ) _Z2 H _X2 (CM)

The variables of formula (CM) are defined as follows:

M is selected from alkali metal cations, same or different, as defined above for compounds of general formula (CM)

x2 is selected from 0.0 to 2.0, preferably from 0.02 to 0.5, more preferably from 0.1 to 0.3; z2 is selected from 0.0 to 1 .0, preferably 0.0005 to 0.5;

however, the sum of x2+z2 in formula (I) is greater than zero, for example 0.05 to 0.6.

Examples of M _3-X 2- _Z 2(NH ₄ ) _Z 2H _X I are Na _3-X 2H _X 2, [Nao _.7 (NH ₄ )o _.3 ] _3-x 2H _x 2, [(NH ₄ )o _.7 Nao _.3 ] _3-x 2H _X 2,.

In one embodiment of the present invention, alkali metal salts of GLDA may be selected from alkali metal salts of the L- and D- enantiomers according to formula (CM), said mixture contain ing the racemic mixture or preferably predominantly the respective L-isomer, for example with an enantiomeric excess (ee) in the range of from 5 to 99%, preferably 5 to 95%. The enantiomeric excess can be determined, e.g., by measuring the polarization (polarimetry) or preferably by chromatography, for example by HPLC with a chiral column, for example with one or more cyclodextrins as immobilized phase or with a ligand exchange (Pirkle-brush) con cept chiral stationary phase. Preferred is determination of the enantiomeric excess by HPLC with an immobilized optically active ammonium salt such as D-penicillamine.

Generally, in the context of the present invention, small amounts of MGDA and/or GLDA may also bear a cation other than alkali metal. It is thus possible that small amounts of builder, such as 0.01 % to 5 mol-% of total builder may bear alkali earth metal cations such as, e.g., Mg ²⁺ or Ca ²⁺ , or a transition metal cation such as, e.g., a Fe ²⁺ or Fe ³⁺ cation.“Small amounts” of MGDA and/or GLDA herein refer to a total of 0.1 % to 1 w/w%, relative to the respective builder.

In one embodiment of the present invention, MGDA and/or GLDA comprised in detergent for mulations may contain in the range of 0.1 % to 10% by weight relative to the respective builder of one or more optically inactive impurities, at least one of the impurities being at least one of the impurities being selected from iminodiacetic acid, formic acid, glycolic acid, propionic acid, acetic acid and their respective alkali metal or mono-, di- or triammonium salts.

Further examples of detergent builders are polymers with complexing groups like, for example, polyethylenimine in which 20 to 90 mole-% of the N-atoms bear at least one CH ₂ COO ^· group, and the respective alkali metal salts of the above sequestrants, especially their sodium salts.

Further examples of suitable detergent builders are polyalkylenimines, for example polyethyl- enimines and polypropylene imines. Polyalkylenimines may be used as such or as polyalkox- ylated derivatives, for examples ethoxylated or propoxylated. Polyalkylenimines comprise at least three alkylenimine units per molecule. Said alkylenimine unit is a C2-Cio-alkylendiamine unit, for example a 1 ,2-propylendiamine, preferably an a,w-C2-Cio-alkylendiamine, for example 1 ,2-ethylendiamine, 1 ,3-propylendiamine, 1 ,4-butylendiamine, 1 ,5-pentylendiaminne, 1 ,6-hex- andiamine (also being referred to as 1 ,6-hexylendiamine), 1 ,8-diamine or 1 ,10-decandiamine, even more preferred are 1 ,2-ethylendiamine, 1 ,3-propylendiamine, 1 ,4-butylendiamine, and 1 ,6- hexandiamine.

In another embodiment of the present invention, said polyalkylenimine is selected from poly- alkylenimine unit, preferably a polyethylenimine or polypropylenimine unit.

The term“polyethylenimine” in the context of the present invention does not only refer to poly ethylenimine homopolymers but also to polyalkylenimines comprising NH-CH2-CH2-NH structur al elements together with other alkylene diamine structural elements, for example NH-CH2-CH2- CH2-NH structural elements, NH-CH ₂ -CH(CH ₃ )-NH structural elements, NH-(CH ₂ )4-NH structural elements, NH-(CH ₂ ) ₆ -NH structural elements or (NH-(CH ₂ ) ₈ -NH structural elements but the NH- CH ₂ -CH ₂ - NH structural elements being in the majority with respect to the molar share.

The term“polypropylenimine” in the context of the present invention does not only refer to poly- propylenimine homopolymers but also to polyalkylenimines comprising NH-CH ₂ -CH(CH3)-NH structural elements together with other alkylene diamine structural elements, for example NH- CH ₂ -CH ₂ -CH ₂ -NH structural elements, NH-CH ₂ -CH ₂ -NH structural elements, NH-(CH ₂ )4-NH structural elements, NH-(CH ₂ ) ₆ -NH structural elements or (NH-(CH ₂ ) ₈ -NH structural elements but the NH-CH ₂ -CH(CH ₃ )-NH structural elements being in the majority with respect to the molar share.

Branches may be alkylenamino groups such as, but not limited to -CH ₂ -CH ₂ -NH ₂ groups or (CH ₂ ) ₃ -NH ₂ -groups. Longer branches may be, for examples, -(CH ₂ ) ₃ -N(CH ₂ CH ₂ CH ₂ NH ₂ ) ₂ or -(CH ₂ ) ₂ -N(CH ₂ CH ₂ NH ₂ ) ₂ groups. The degree of branching can be determined for example by ¹³ C-NMR or ¹⁵ N-NMR spectroscopy, preferably in D ₂ 0, and is defined as follows:

DB = D+T/D+T+L

with D (dendritic) corresponding to the fraction of tertiary amino groups, L (linear) corresponding to the fraction of secondary amino groups and T (terminal) corresponding to the fraction of pri mary amino groups.

Preferred branched polyethylenimine units are polyethylenimine units with DB in the range from 0.25 to 0.95, particularly preferably in the range from 0.30 to 0.90% and very particularly prefer ably at least 0.5, and those that exhibit little or no branching, thus predominantly linear or linear polyethylenimine units. In the context of the present invention, CH ₃ -groups are not being con sidered as branches.

In one embodiment of the present invention alkoxylated polyalkylenimine is selected from poly- ethoxylated polyethylenimine, ethoxylated polypropylenimine, ethoxylated a,w-hexandiamines, ethoxylated and propoxylated polyethylenimine, ethoxylated and propoxylated polypropyl enimine, and ethoxylated and poly-propoxylated a,w-hexandiamines.

In one embodiment of the present invention the average molecular weight M _n (number average) of alkoxylated polyethylenimine is in the range of from 2,500 to 1 ,500,000 g/mol, determined by GPC, preferably up to 500,000 g/mol.

In one embodiment of the present invention, the average alkoxylated polyalkylenimine are se lected from ethoxylated a,w-hexanediamines and ethoxylated and poly-propoxylated a,w- hexanediamines, each with an average molecular weight M _n (number average) in the range of from 800 to 500,000 g/mol, preferably 1 ,000 to 30,000 g/mol. Detergent formulations of the invention may comprise one or more complexing agent other than EDTA, DTPA, MGDA and GLDA, e.g. citrate, phosphonic acid derivatives, for example the disodium salt of hydroxyethane-1 ,1 -diphosphonic acid (“HEDP”), for example trisodium citrate, and phosphates such as STPP (sodium tripolyphosphate).

In one embodiment, a detergent formulation of the invention comprises a builder system com prising

• ethylenediaminetetraacetic acid (EDTA) and/or diethylenetriaminepentaacetic acid

(DTPA) and/or methylglycine diacetate (MGDA) and/or glutamic acid diacetate (GLDA), as disclosed above in amounts in the range of 0.1% to 25.0% by weight, in the range of 1.0% to 15.0% by weight, or in the range of 3.0% to 10.0% by weight, all relative to the total weight of the detergent formulation;

• optionally citric acid in amounts in the range of 0.1 % to 10.0% by weight, in the range of 0.5% to 8.0% by weight, in the range of 1 .0% to 5.0% by weight, or in the range of 2.0% to 4% by weight, all relative to the total weight of the detergent formulation; the citric acid may be provided as a mixture with formiate, e.g. Na-citrate:Na-formiate=9:1 ;

• optionally at least one phosphonate, preferably selected from derivatives polyphosphonic acids such as of diphosphonic acid such as sodium salt of HEDP, and derivatives of aminopolyphosphonic acid such as aminoalkylene phosphonic acids such as DTPMP in amounts in the range of 0.1% to 5.0% by weight, in the range of 0.5% to 3.0% by weight, or in the range of 1.0% to 2.0% by weight, all relative to the total weight of the detergent formulation;

• optionally at least one polycarboxylate selected from homopolymers with the repeating monomer being the same unsaturated carboxylic acid, such as polyacrylic acid (PAA) and copolymers with the repeating monomers being at least two different unsaturated carbox ylic acids, such as copolymers of acrylic acid with methacrylic acid, copolymers of acrylic acid or methacrylic acid and maleic acid and/or fumaric acid, in amounts in the range of 0.1 % to 10% by weight, 0.25% to 5% by weight, or 0.3% to 2.5% by weight, all relative to the total weight of the detergent formulation;

In one embodiment, a detergent formulation of the invention comprises a builder system com prising

• ethylenediaminetetraacetic acid (EDTA) and/or diethylenetriaminepentaacetic acid

(DTPA) and/or methylglycine diacetate (MGDA) and/or glutamic acid diacetate (GLDA), as disclosed above in amounts in the range of 10.0% to 25.0% by weight, or in the range of 15.0% to 25.0% by weight, all relative to the total weight of the detergent formulation; • citric acid in amounts in the range of 0.5% to 8.0% by weight, or in the range of 1 .0% to 5.0% by weight, all relative to the total weight of the detergent formulation; the citric acid may be provided as a mixture with formiate, e.g. Na-citrate:Na-formiate=9:1 ;

• at least one phosphonate selected from derivatives polyphosphonic acids such as of di- phosphonic acid such as sodium salt of HEDP, and derivatives of aminopolyphosphonic acid such as aminoalkylene phosphonic acids such as DTPMP in amounts in the range of 0.5% to 3.0% by weight, or in the range of 1 .0% to 2.0% by weight, all relative to the total weight of the detergent formulation;

• at least one polycarboxylate selected from homopolymers with the repeating monomer be ing the same unsaturated carboxylic acid, such as polyacrylic acid (PAA) and copolymers with the repeating monomers being at least two different unsaturated carboxylic acids, such as copolymers of acrylic acid with methacrylic acid, copolymers of acrylic acid or methacrylic acid and maleic acid and/or fumaric acid, in amounts in the range of 2.5% to 5.0% by weight, all relative to the total weight of the detergent formulation;

In one embodiment, the detergent formulation of the invention comprises a builder system as disclosed above and at least one non-ionic surfactant of general formula (Sill):

In one embodiment, R ¹ is n-Cs alkyl, R ² is branched Cn alkyl, AO is CH2-CH2-O, and x is 22.

In one embodiment, R ¹ is n-Cs alkyl, R ² is n-Cs-Cio alkyl, AO is CH2-CH2-O, and x is 40.

In one embodiment, R ¹ is n-Cs alkyl, R ² is n-Cio alkyl, AO is selected from -(CH ₂ CH ₂ 0) _X2 - (CH ₂ CH(CH ₃ )-0) _X3 , -(CH2CH ₂ 0) _X2 -(CH(CH ₃ )CH2-0) _X3 , wherein x2 = 22 and x3 = 1 .

In one embodiment, the detergent formulation, preferably a liquid detergent formulation com prises at least one compound according to formula (Sill) in amounts in the range of about 0.3% to 10% by weight, in the range of about 0.5% to 5% by weight, or in the range of about 1 % to 3%, all relative to the total weight of a detergent formulation.

In one embodiment of the present invention, the formulation according to the invention is free from phosphates and polyphosphates, with hydrogenphosphates being subsumed, for example free from trisodiumphosphate, pentasodiumtripolyphosphate and hexasodiummetaphosphate.

In connection with phosphates and polyphosphates, in the context of the present invention,“free from” is to be understood as meaning that the content of phosphate and polyphosphate is in total in the range from 10 ppm to 0.2% by weight, determined by gravimetry and relative to the total weight of the detergent formulation.

Liquid detergent formulations of the invention may comprise one or more corrosion inhibitors. Non-limiting examples of suitable corrosion inhibitors include sodium silicate, triazoles such as benzotriazoles, bisbenzotriazoles, aminotriazoles, alkylaminotriazoles, phenol derivatives such as hydroquinone, pyrocatechol, hydroxyhydroquinone, gallic acid, phloroglucinol and pyrogallol, further polyethylenimine and salts of bismuth or zinc. Corrosion inhibitors may be formulated into liquid detergent formulations of the invention in amounts of 0.1 to 1.5 % w/w relative to the overall weight of the liquid detergent formulation.

Liquid detergent formulations of the invention may comprise at least one graft copolymer com posed of

(a) at least one graft base selected from nonionic monosaccharides, disaccharides, oligosac charides and polysaccharides,

and side chains obtained by grafting on of

(b) at least one ethylenically unsaturated mono- or dicarboxylic acid and

(c) at least one compound of the general formula (PI),

wherein the variables in formula (PI) are defined as follows:

R ¹ is selected from methyl and hydrogen,

A ¹ is selected from C2-C4-alkylene,

R ² are identical or different and selected from Ci-C4-alkyl,

X is selected from halide, mono-Ci-C4-alkyl sulfate and sulfate.

Liquid detergent formulations of the invention may comprise one or more buffers such as mo- noethanolamine and N,N,N-triethanolamine. Liquid detergent formulations of the invention may be adapted in sudsing characteristics for sat isfying various purposes. Hand dishwashing detergents usually request stable suds. Automatic dishwasher detergents are usually requested to be low sudsing. Laundry detergents may range from high sudsing through a moderate or intermediate range to low. Low sudsing laundry deter gents are usually recommended for front-loading, tumbler-type washers and washer-dryer com binations. Those skilled in the art are familiar with using suds stabilizers or suds suppressors as detergent components in detergent formulations which are suitable for specific applications. Examples of suds stabilizers include but are not limited to alkanolamides and alkylamine oxides. Examples of suds suppressors include but are not limited to alkyl phosphates, silicones and soaps.

Liquid detergent formulations may comprise at least one compound selected from organic sol vents, preservatives, viscosity modifiers, and hydrotropes. Compounds and effective amounts known to those skilled in the art.

E.g. liquid detergent formulations comprise amounts of organic solvents are 0.5 to 25% by weight, relative to the total weight of the liquid detergent formulation. Especially when inventive liquid detergent formulations are provided in pouches or the like, 8 to 25% by weight of organic solvent(s) relative to the total weight of the liquid detergent formulation may be comprised. Or ganic solvents are those disclosed above.

Liquid detergent formulations according to the invention may be provided in compartmented pouches or the like, the compartment comprising the liquid enzyme preparation of the invention is provided separated from the compartment comprising bleaches, such as inorganic peroxide compounds or chlorine bleaches such as sodium hypochlorite. In one embodiment, the com partment comprising the liquid enzyme preparation also comprises at least one complexing agent such as EDTA and/or DTPA and/or MGDA and/or GLDA, wherein MGDA and GLDA are as disclosed above.

Liquid detergent formulation may be called aqueous herein when the solvent comprised in the detergent formulation is essentially water. In one embodiment, water is the sole solvent. In other embodiments, mixtures of water with one or more water-miscible solvents are used. The term water-miscible solvent refers to organic solvents that are miscible with water at ambient temper ature without phase-separation. Examples are ethylene glycol, 1 ,2-propylene glycol, isopropa nol, and diethylene glycol. Preferably, at least 50% by volume referring to the whole solvent comprised in the aqueous detergent formulation is water.

“Detergent formulation” or“cleaning formulation” herein means formulations designated for cleaning soiled material. Cleaning may mean laundering or hard surface cleaning. Soiled mate rial according to the invention includes textiles and/or hard surfaces. 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, sheeps 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.

The term“hard surface cleaning” is defined herein as cleaning of hard surfaces wherein hard surfaces may include any hard surfaces in the household, such as floors, furnishing, walls, sani tary ceramics, glass, metallic surfaces including cutlery or dishes. The term“hard surface clean ing” may therefore may mean“dish washing” which refers to all forms of washing dishes, e.g. by hand or automatic dish wash (ADW). Dish washing includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, chi na, glass and acrylics.

In one aspect, the invention relates to the providing a liquid detergent formulation comprising at least the enzyme preparation of the invention and at least one detergent component.

In one embodiment, the invention provides a liquid detergent formulation comprising at least components (a) and (b) as disclosed above and at least one detergent component, wherein component (b) comprises at least one alpha-amylases (EC 3.2.1 .1 ), preferably selected from

• amylase from Bacillus sp.707 or variants thereof having amylolytic activity, preferably se lected from amylases having SEQ ID NO:6 as disclosed in WO 99/19467 and variants thereof having amylolytic activity; • amylase selected from those comprising amino acids 1 to 485 of SEQ ID NO:2 as de scribed in WO 00/60060 those having SEQ ID NO: 12 as described in WO 2006/002643, and variants thereof having amylolytic activity;

• amylase from Bacillus halmapalus or variants thereof having amylolytic activity, prefera bly selected from amylases having SEQ ID NO: 1 and 2 as disclosed in WO

2013/001078; having SEQ ID NO:6 as described in WO 201 1/098531 ; and variants thereof having amylolytic activity;

• amylase from Bacillus amyloliquefaciens or variants thereof having amylolytic activity, preferably selected from amylases according to SEQ ID NO: 3 of WO 2016/092009;

• hybrid amylases according to WO 2014/183920 with A and B domains having at least 90% similarity or identity to SEQ ID NO:2 of WO 2014/183920 and a C domain having at least 90% similarity or identity to SEQ ID NO:6 of WO 2014/183920, wherein the hybrid amylase has amylolytic activity; preferably the hybrid alpha-amylase is at least 95% simi lar or identical to SEQ ID NO: 23 of WO 2014/183920 and having amylolytic activity;

• hybrid amylase according to WO 2014/183921 with A and B domains having at least 75% similarity or identity to SEQ ID NO: 2, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 39 as disclosed in WO 2014/183921 and a C domain having at least 90% similarity or identity to SEQ ID NO: 6 of WO 2014/183921 , wherein the hybrid amylase has amylolytic activity; prefera bly, the hybrid alpha-amylase is at least 95% similar or identical to SEQ ID NO: 30 as disclosed in WO 2014/183921 and having amylolytic activity;

and optionally one or more further enzymes, selected from proteases, lipases, cellulases and mannanases - all as disclosed above.

In embodiments of the above embodiments, the liquid detergent formulation has increased stor age stability when compared to a liquid detergent formulation lacking component (a). Increased storage stability in this context may mean that there is no significant loss in enzymatic activity and therefore wash performance towards at least one enzyme-sensitive stain type, preferably towards at least amylase-sensitive stains, after storage of the detergent at 37°C formulation for 3, 7, 14, 21 or 28 days. Wash performance towards specified enzyme sensitive-stain type means that the respective enzyme is acting on the enzyme-sensitive parts of a specific stain. Different enzymes are able to breakdown different types of stains. For example, proteases are acting on proteinaceous material and thereby degrade proteins into smaller peptides. Amylase- sensitive stains are usually starch-based stains wherein the carbohydrates may be degraded into oligo- or monosaccharides by amylases. Lipase sensitive stains are usually comprising fatty compounds. Mannanase sensitive stains usually comprise mannan. Cellulases may clean indi rectly by hydrolyzing certain glycosidic bonds in cotton fibers. In this way, particulate soils at tached to microfibrils are removed.

In one embodiment, said liquid detergent formulation comprising at least components (a) and (b) and at least one detergent component preferably EDTA and/or DTPA in amounts up to 3% by weight, preferably up to 2.5% and/or MGDA and/or GLDA in amounts in the range of 10% to 30% by weight, preferably in the range of 15% to 25%, all relative to the total weight of the liquid detergent formulation, as disclosed above

has increased storage stability when compared to a liquid detergent formulation lacking compo nent (a).

In one embodiment, the liquid detergent formulation comprising at least components (a) and (b) and at least one detergent component has increased storage stability when compared to a liq uid detergent formulation lacking component (a), wherein component (b) comprises in addition to at least one alpha amylase as disclosed above, at least one protease as disclosed above, preferably selected from the group of serine endopeptidases (EC 3.4.21 ), more preferably se lected from the group of subtilisin type proteases (EC 3.4.21 .62). The protease may be selected from protease according to SEQ ID NO:22 as described in EP 1921 147 or variants thereof hav ing proteolytic activity as disclosed above and from subtilisin 309 as disclosed in Table I a) of WO 89/06279 or variants thereof having proteolytic activity as disclosed above.

In one aspect, the invention relates to a method to increase storage stability of a liquid deter gent formulation comprising at least one amylase and optionally:

EDTA and/or DTPA in amounts up to 3% by weight, preferably up to 2.5% and/or

MGDA and/or GLDA in amounts in the range of 10% to 30% by weight, preferably in the range of 15% to 25%, all relative to the total weight of the liquid detergent formulation, as disclosed above,

by adding at least one compound according to formulae (I) or (II) - component (a) as disclosed above - to the detergent formulation.

Storage stability of an inventive liquid detergent formulation is increased when stored at 37°C for 14, 21 , and/or 28 days when compared to a liquid detergent formulation lacking the com pound according to formula (I) stored under the same conditions. Increased storage stability within this invention may mean that the increase in amylolytic stability in the presence of com ponent (a) is at least 20%, or at least 25%, at least 30%, at least 40%, at least 50%, least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, when compared to the amylolytic activity in the absence of component (a).

Further use

The invention relates to a method for removing enzyme-sensitive stains comprising the steps of contacting a stain with a detergent formulation of the invention comprising components (a) and (b) and one or more detergent components - all as disclosed above. In one embodiment, the method for removing stains includes steps performed by an automatic device such as a laundry machine or an automatic dishwasher.

In one embodiment, the detergent formulation comprises the enzyme preparation of the inven tion.

In one aspect, the method relates to the removal of stains comprising starch. In one embodi ment, removing of stains comprising starch may be done at cleaning temperatures < 40°C, at cleaning temperatures < 30°C, at cleaning temperatures < 25°C, or at cleaning temperatures < 20°C.

In one embodiment, the invention relates to a method for removing stains comprising starch at a cleaning temperature of temperature < 30°C, wherein the method comprises the steps of con tacting the stain with a detergent formulation of the invention comprising components (a) and (b) and one or more detergent components. Components (a) and (b) are those as disclosed above.

Examples

The invention will be further illustrated by working examples.

General remarks: percentages are weight percent unless specifically noted otherwise.

Acetylcholine (A.12) was purchased from Sigma Aldrich. The counterion was chloride.

The precursor of (A.14) can be produced directly instead of use of HCI in the ethoxylation of trimethylamine or via reaction of choline hydrogencarbonate with methanesulfonic, see Con- stantinescu et al in Chem. Eng. Data, 2007, 52 1280-1285.

I. Synthesis of Salts (component (a))

Based upon the amounts of water distilled off and by IR spectroscopy it could be shown that the esterification reactions were complete.

90% methanesulfonic acid refers to a mixture from 10% water and 90% methanesulfonic acid. 1.1 Synthesis of inventive salt (A.1 ):

An amount of 150 g tartaric acid (1 .0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 mole). Water was removed within 45 minutes in a rotary evapo rator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 15 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously re duced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish sub stance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yel lowish liquid were obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.7 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.1 ) was obtained.

1.2 Synthesis of inventive salt (A.2):

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 374 g of a 75% by weight aqueous solution of choline chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation, the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 200 g diethylene glycol. An amount of 607 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 10.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.2) was obtained.

1.3 Synthesis of inventive salt (A.3):

An amount of 210 g citric acid monohydrate (1 .0 mol) was dissolved in 561 g of a 75% by weight aqueous solution of choline chloride (3.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 270 g diethylene glycol. An amount of 868 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 9.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.3) was obtained. 1.4 Synthesis of inventive salt (A.4):

An amount of 210 g citric acid monohydrate (1.0 mol) were dissolved in 485 g of a 75% by weight aqueous solution of choline methanesulfonate (2.0 mol). Water was removed within 45 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pres sure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 200g diethylene glycol. An amount of 663 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.5 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.4) was obtained.

1.5 Synthesis of inventive salt (A.5):

An amount of 98,1 g maleic anhydride (1.0 mol) were mixed with 363 g of choline methanesul fonate (2.0 moles) as dry substance. The mixture was heated in a rotary evaporator to 135°C. After one hour of mixing an amount of 12 g of methanesulfonic acid (pure) was added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of mixing the pres sure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 200g diethylene glycol. An amount of 653 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 8.9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.5) was obtained.

1.6 Synthesis of inventive salt (A.6):

An amount of 210 g citric acid monohydrate (1.0 mole) was dissolved in 437 g of a 70% by weight aqueous solution of beta-methyl choline chloride (HO-CH(CH ₃ )-CH2-N(CH ₃ )3 Cl, 2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask) - oil bath tem perature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% by weight methanesulfonic acid were added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 200g diethylene glycol. An amount of 676 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 12.3 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.6) was obtained.

1.7 Synthesis of inventive salt (A.7): An amount of 210 g citric acid monohydrate (1 .0 mole) was dissolved in 520 g of a 70% by weight aqueous solution of beta-n-propyl choline chloride (2.0 moles). Water was removed with in 45 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 250 g propylene glycol. An amount of 774 g of a yellowish liquid was obtained. An aliquot of 200 g of the liquid so obtained was neutralized with 1 1 .9 g ethanolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.7) was obtained.

1.8 Synthesis of inventive salt (A.8):

An amount of 210 g citric acid monohydrate (1 .0 mole) was dissolved in 520 g of a 70% aque ous solution of dimethylmonobutylcholine chloride (2.0 moles). Water was removed within 45 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 18 g of 90% methanesulfonic acid was added and the temperature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was contin uously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellow ish substance was obtained that was diluted with 200 g propylene glycol. An amount of 788 g of a yellowish liquid. An aliquot of 200 g of the liquid so obtained was neutralized with 1 1 .4 g etha nolamine to a pH value of 6 to 6.5 (10% in water). Inventive salt (A.8) was obtained.

1.9 Synthesis of inventive salt (A.9):

An amount of 85 g gallic acid (3,4,5-trihydroxybenzoic-acid, 0.5 moles) was dispersed in 121 g of a 75% by weight aqueous solution of choline methanesulfonate (0.5 moles). Water was re moved within 90 minutes in a rotary evaporator (2-l-flask) - oil bath temperature of 100 to 120°C, 50 to 80 mbar. An amount of 8 g of 90% methanesulfonic acid was added and the tem perature was raised to 145°C at a pressure of 800 mbar. After one hour of rotary evaporation the pressure was continuously reduced to 10 mbar while water was removed for another 4.5 h at 145°C. A light yellowish substance was obtained that was diluted with 100 g diethylene gly col. An amount of 271 g of a yellowish liquid was obtained. An aliquot of 100 g of the liquid so obtained was neutralized with 4.6 g ethanolamine to a pH value of 6 to 6.5 (10% in water). In ventive salt (A.9) was obtained.

Comparative salts:

C-(A.15): Choline chloride, 75% by weight aqueous solution, commercially available from BASF SE C-(A.10):

An amount of 75 g (0.5 mol) tartaric acid was portion-wise dissolved (15 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1 .0 mol). The solution was stirred until the CO2 evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-I- flask) - oil bath temperature of 120°C, 10 mbar. A clear substance was obtained that was dilut ed with 150 g diethylene glycol. 390 g of a clear solution were obtained, C-(A.10). No ester for mation could be detected.

C-(A.1 1 ): An amount of 105 g (0.5 mol) citric acid monohydrate was portion-wise dissolved (20 g units) in 206 g of an 80% by weight aqueous solution of choline bicarbonate (1.0 mol). The solution was stirred until the CO2 evolution ceased. Water was removed within 90 minutes by rotary evaporation (2-l-flask) - oil bath temperature of 120°C, 10 mbar. A clear substance was obtained that was diluted with 150 g diethylene glycol. 412 g of a clear solution were obtained, C-(A.1 1 ). No ester formation could be detected.

II. Application tests

11.1 Amylase and protease stability

The storage stability of amylase was assessed at 37°C. Base test formulations were manufac tured by making base formulations I to V by mixing the components according to Table 1 .

Amylases used: Amyl = Stainzyme, Amy2 = Amplify, Amy3 = Stainzyme Plus L (12L)

Protease used: (S) Savinase Ultra 16.0L (CAS-No. 9014-01 -1 , EC-No. 232-752-2) was pur chased from Sigma-Aldrich

The respective component (a) or comparative compound was added, if applicable, to the re spective base formulation in amounts as indicated in Table 1.

Amylase (component (b)) was added to the respective base formulation in amounts as indicated in Table 1 . The amount of amylase as provided in Table 1 refers to active protein.

Protease (component (b)) was added to the respective base formulation in amounts as indicat ed in Table 1 . The amount of protease as provided in Table 1 refers to active protein.

Water was added to accomplish the balance to 100. Table 1 : liquid formulation

(Comp.1 ): MGDA-Na3 (40 wt% in water) (Comp.2): Citric acid

(Comp.3): GLDA 50% solution

(Comp.4): PAA, Polyacrylic acid M _w 5.000 g/mol (Comp.5): Glycerol (G) or propanediol (P) (Comp.6): Dehypon WET

(Comp.7): Na ₄ HEDP

(Comp.8) Thickener (Rheocare XGN) ^** for comparative tests without inventive compounds those were replaced by the same amount of glycerol

Amylase activity at certain points in time as indicated in Table 2 was measured quantitatively by the release of the chromophore para- nitrophenol (pNP) from the substrate (Ethyliden-blocked- pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and a-glucosidase (Roche Applied Science 1 1626329103), which is present in excess compared to the a-amylase, process these smaller products until pNP is released; the release of pNP, measured via an increase of absorption at 405 nm, is directly proportional to the a-amylase activity of the sample. Amylase standard: Termamyl 120 L (Sigma 3403).

Table 2 displays amylase activity measured in liquid formulations after storage for 1 to 30 days at 37°C. The amylolytic activity values provided were calculated referring to the value deter mined in the reference formulation at the time 0.

The nomenclature of formulations is as follows: the Roman number before the full stop charac terizes the base formulation, the Arabian number the type of compound (A.# compound accord- ing to invention (component (a)); C-A.# comparative compound)). Zero (“0”): no salt, but diethy lene glycol.

Table 2: amylase activity in the course of time of storage at 37°C

Protease activity at certain points in time as indicated in Table 3 was be determined by employ ing Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which was determined by measuring OD405· Measurements were done at 20°C.

Table 3 displays protease activity measured in liquid formulations after storage for 1 to 30 days at 37°C. The proteolytic activity values provided in Table 3 were calculated referring to the value determined in the reference formulation at the time 0.

The nomenclature of formulations is as follows: the Roman number before the full stop charac- terizes the base formulation, the Arabian number the type of salt (A.# inventive salt (component (a)); C-A.# comparative compound). Zero (“0”): no salt, but diethylene glycol. Table 3: protease activity in the course of time of storage at 37°C

III. Stability in laundry formulation

The storage stability of amylase was assessed at 37°C.

Base test formulations were manufactured by making base formulations VI to IX by mixing the components according to Table 4.

Table 4: liquid laundry formulations

(Comp.1 ): n-Ci8-alkyl-(OCH ₂ CH2)25-OH

(Comp.2): Tallow oil soap C14-C18 Carbonic acid, sodium salt

(Comp.3): Sodium Cio-Ci2-alkyl benzenesulfonate

(Comp.4): Sodium laurethsulfate - n-Ci ₂ H ₂₅ -0-(CH ₂ CH ₂ 0) ₃ -SC> ₃ Na

(Comp.5): Complexing agent EDTA or DTPA

(Comp.6) mixture Na-citrate:Na-formiate 9:1

^** for comparative tests without inventive compounds those were replaced by the same amount of water. Amylase activity at certain points in time as indicated in Table 5 was measured quantitatively by the release of the chromophore para-nitrophenol (pNP) from the substrate (Ethyliden-blocked- pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and a-glucosidase (Roche Applied Science 1 1626329103), which is present in excess compared to the a-amylase, process these smaller products until pNP is released; the release of pNP, measured via an increase of absorption at 405 nm, is directly proportional to the a-amylase activity of the sample. Amylase standard: Termamyl 120 L (Sigma 3403).

Table 5 displays amylase activity measured in liquid formulations after storage for 1 to 28 days at 37°C. The amylolytic activity values provided were calculated referring to the value deter mined in the reference formulation at the time 0.

The nomenclature of formulations is as follows: the Roman number before the full stop charac terizes the base formulation, the Arabian number the type of compound (A.# compound accord ing to invention (component (a)); C-A.# comparative compound).

Table 5: amylase activity in the course of time of storage at 37°C

The detergent performance of formulations according to Table 4 in cleaning amylase-sensitive stains can be carried out on applicable types of test fabrics. Pre-soiled test fabrics may be pur chased from wfk test fabrics GmbH, Krefeld; EMPA = Swiss Federal Institute of Materials Test- ing; or CFT = Center for Test Material B.V.

The test can be performed as follows: a multi stain monitor comprising e.g. 8 standardized soiled fabric patches, each of 2.5 x 2.5 cm size and stitched on two sides to a polyester carrier is washed together in a launder-O-meter with 2.5 g of cotton fabric and 5g/L of the liquid test laundry detergent, Table 4.

The conditions may be chosen as follows: Device: Launder-O-Meter from SDL Atlas, Rock Hill, USA. Washing liquor: 250 ml, washing time: 60 minutes, washing temperature: 30°C. Water hardness: 2.5 mmol/L; Ca:Mg:HCC>3 4:1 :8; fabric to liquor ratio 1 :12; after the wash cycle, the multi stain monitors are rinsed in water, followed by drying at ambient temperature over a time period of 14 hours.

The total level of cleaning can be evaluated using color measurements: Reflectance values of the stains on the monitors are measured using a sphere reflectance spectrometer (SF 500 type from Datacolor, USA, wavelength range 360-700nm, optical geometry d/8°) with a UV cutoff filter at 460 nm. In this case, with the aid of the CIE-Lab color space classification, the bright- ness L ^* , the value a ^* on the red - green color axis and the b ^* value on the yellow - blue color axis, are measured before and after washing and averaged for the 8 stains of the monitor. The change of the color value (D E) value, can be defined and calculated automatically by the eval uation color tools on the following equation:

D E is a measure of the achieved cleaning effect. All measurements may be repeated six times to yield an average number. Note that higher D E values show better cleaning. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units easily.

The launder-O-meter tests can be executed with freshly prepared formulations according to Table 4 and/or with the same formulations after storage at 37°C for a defined time such as 3 days, about 7 days, about 14 days, about 21 days, about 28 days, about. As an approximation one week at 37°C is equivalent to 3½ weeks at 20°C.

Pre-condition for improved wash-performance is the availability of amylolytic and/or proteolytic activity after storage of the detergent formulation.