Enzyme slurry composition

Abstract

The present invention provides an aqueous enzyme slurry comprising enzyme granules, a water soluble salt, and a natural polymer viscosity modifier.

Description

[0001] This application is a divisional application of the invention patent application filed on June 27, 2018, with application number 201880042716.4 and invention title "Enzyme Slurry Composition". Technical Field

[0002] This invention relates to an enzyme slurry composition that exhibits excellent stability and reduced production costs. Background Technology

[0003] Detergent compositions often contain enzymes along with other components such as surfactants. A variety of different enzymes can be used in such detergent compositions to help clean substrates, such as clothing or dishes. Enzymes may include proteases to break down protein materials, lipases to break down fatty materials, and amylases to break down carbohydrate-based materials.

[0004] During the preparation of detergent compositions, enzymes are typically added in liquid form as a solution or in solid form as granules. Granules can contain a large amount of active enzyme, while the concentration (enzyme activity) of liquid enzyme products is limited by enzyme solubility. Furthermore, liquid enzyme products require expensive formulation ingredients to maintain adequate enzyme stability. Summary of the Invention

[0005] In a first aspect, the present invention provides an enzyme slurry composition comprising:

[0006] (a) Enzyme particles;

[0007] (b) Water-soluble salts of sodium, potassium, magnesium, zinc or ammonium;

[0008] (c) Xanthan gum, guar gum, sophora bean gum, welan gum, gellan gum, alginate, carrageenan, or microcrystalline cellulose; and

[0009] (d) Water.

[0010] In one embodiment, the enzyme particles are spray-dried enzymes or enzyme crystals.

[0011] In another aspect, the present invention provides soap or detergent strips comprising enzyme particles, such as enzyme crystals.

[0012] Other aspects and embodiments of the invention will be apparent from the description and examples. Detailed Implementation

[0013] The aqueous enzyme slurry of the present invention is a product containing enzyme particles suspended in an aqueous stable solution. A viscosity modifier is added to maintain a uniform distribution of enzyme particles in the slurry (even after long-term storage).

[0014] The enzyme slurry dissolves rapidly after being added to the detergent manufacturing process, so it can be used without affecting the customer's detergent production procedures. The dissolution time of the enzyme slurry in detergent formulations can be further reduced by adding enzyme inhibitors.

[0015] Enzyme slurries offer superior enzyme stability compared to traditional liquid products. Furthermore, the relative amount of active enzyme can be higher than in traditional liquid products, resulting in lower production costs per unit of enzyme activity. Since the water content in enzyme slurries is typically around 60% w / w–80% w / w, the cost of formulation components is also lower.

[0016] Enzyme granules

[0017] The enzyme slurry of the present invention comprises enzyme particles. The enzyme particles may contain one or more enzymes, as described below. Different enzymes may be mixed or separated within the enzyme particles (e.g., arranged in layers).

[0018] Enzyme particles are any form of enzyme in the form of solid microparticles. They can be enzyme crystals, enzyme precipitates, spray-dried or lyophilized enzymes, or any form of granular enzyme. Typically, the particle size of enzyme particles, measured by equivalent spherical diameter (average particle size based on volume), is less than 2 mm, preferably less than 1 mm, less than 0.5 mm, less than 0.25 mm, or less than 0.1 mm; and greater than 0.05 μm, preferably greater than 0.1 μm, greater than 0.5 μm, greater than 1 μm, greater than 5 μm, or greater than 10 μm.

[0019] In a preferred embodiment, the particle size of the enzyme particles ranges from 0.5 μm to 100 μm.

[0020] The enzyme particles contain at least 1% w / w enzyme protein, preferably at least 5% w / w enzyme protein, at least 10% w / w enzyme protein, at least 20% w / w enzyme protein, at least 30% w / w enzyme protein, at least 40% w / w enzyme protein, at least 50% w / w enzyme protein, at least 60% w / w enzyme protein, at least 70% w / w enzyme protein, at least 80% w / w enzyme protein, or at least 90% w / w enzyme protein.

[0021] In a preferred embodiment, the enzyme particles are enzyme crystals, or the enzyme protein is in crystalline form.

[0022] Enzyme crystallization can be carried out in a variety of ways known in the art (e.g., as described in WO 91 / 09943 or WO 94 / 22903).

[0023] Enzymes can be formulated in enzyme particles as known in the art for use in solid enzyme formulations, such as formulations for reducing dust, improving stability, and / or altering enzyme release rates. Enzyme particles can also be formulated in a matrix or coated with a reagent that inhibits the dissolution of the enzyme particles in a PVOH / membrane solution used to prepare a water-soluble membrane.

[0024] The enzyme molecules on the surface of enzyme particles can also be cross-linked, such as CLEC (cross-linked enzyme crystals) or CLEA (cross-linked enzyme aggregates).

[0025] Water-soluble salts

[0026] One or more water-soluble salts are added to the (aqueous) enzyme slurry composition to prevent enzyme particles from dissolving in the aqueous slurry after storage. The water-soluble salts are salts of sodium, potassium, magnesium, zinc, or ammonium. Preferred salts are sulfates, nitrates, chlorides, acetates, citrates, and similar types.

[0027] In one embodiment, the water-soluble salt is selected from the group consisting of: sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, zinc nitrate, ammonium nitrate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, ammonium chloride, sodium acetate, potassium acetate, calcium acetate, magnesium acetate, zinc acetate, ammonium acetate, sodium citrate, potassium citrate, calcium citrate, magnesium citrate, zinc citrate, ammonium citrate, sodium formate, potassium formate, calcium formate, magnesium formate, zinc formate, and ammonium formate.

[0028] Add water-soluble salt to the enzyme slurry in an amount sufficient to prevent the enzyme particles from dissolving after storage. This is easy to measure because if the enzyme particles dissolve, the residual enzyme activity will decrease after storage.

[0029] The appropriate amount of water-soluble salt is at least 1% w / w. The upper limit is the solubility limit of the salt, for example, 10% w / w.

[0030] Viscosity modifiers and other ingredients

[0031] To prevent the sedimentation of enzyme particles, the enzyme slurry of the present invention contains a viscosity modifier, such as a natural polymer. Preferably, the viscosity modifier is selected from the group consisting of: alginate, carrageenan, xanthan gum, guar gum, sophora bean gum, vetiver gum, gelatan gum, and microcrystalline cellulose.

[0032] The viscosity modifier is added to the enzyme slurry in an amount sufficient to prevent enzyme particle sedimentation after slurry storage. The amount of viscosity modifier needed is adjusted according to the desired storage time (e.g., four weeks). As shown in Example 2, this can be determined by incubating samples with different amounts of viscosity modifier for the desired time and then measuring the sedimentation profiles.

[0033] The appropriate amount of viscosity modifier is at least 0.1% w / w, for example at least 0.25% w / w, depending on the type of viscosity modifier. This upper limit is determined by the tolerable viscosity level in the application, for example 2.5% w / w.

[0034] Enzyme slurries may also contain preservatives, such as phenoxyethanol, sorbate, or benzoate, to prevent microbial contamination / degradation after storage.

[0035] Water is used as the carrier liquid in the enzyme slurry. The exact amount of water depends on the amount of other components in the enzyme slurry, but the enzyme slurry contains at least 50% w / w water. In one embodiment, the amount of water is at least 60% w / w, preferably at least 70% w / w, more preferably at least 80% w / w, and most preferably at least 90%. In a preferred embodiment, the water content is 50% w / w-90% w / w, 60%-90%, or 70%-90%.

[0036] One or more enzymes

[0037] The enzyme particles used in the enzyme slurry of the present invention (see above) contain one or more enzymes, particularly suitable for enzymes included in laundry or dishwashing detergents (detergent enzymes), such as proteases (e.g., subtilisin or metalloproteinases), lipases, keratinases, amylases, glycosylases, cellulases, pectinases, mannanases, arabinosylases, galactanases, xanthan gums, xylanases, DNases, hydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxygenases, and / or halogenated peroxidases).

[0038] Preferred detergent enzymes are proteases (e.g., subtilisin or metalloproteinases), lipases, amylases, lyases, cellulases, pectins, mannanases, DNases, hydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxyses, and / or halogenated peroxidases); or combinations thereof.

[0039] More preferred detergent enzymes are proteases (e.g., subtilisin or metalloproteinase), lipases, amylases, cellulases, pectinases, and mannanases; or combinations thereof.

[0040] Enzyme particles can be contained in enzyme slurry by adding individual particles, each containing one enzyme, or by adding combination particles containing two or more of these enzymes.

[0041] protease

[0042] The protease used in this invention is a serine protease, such as subtilisin, metalloproteinase, and / or trypsin-like protease. Preferably, the protease is subtilisin or metalloproteinase; more preferably, the protease is subtilisin.

[0043] Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds and have an essential serine residue at their active site (White, Handler, and Smith, 1973, “Principles of Biochemistry,” 5th ed., McGraw-Hill Book Company, New York, pp. 271-272). Subtilis proteases comprise, preferably, subgroups I-S1 and I-S2, as defined by Siezen et al., Protein Engineering 4 (1991) 719-737; and Siezen et al., Protein Science 6 (1997) 501-523. Due to the highly conserved structure of the active site of serine proteases, the subtilis proteases according to the present invention can be functionally equivalent to the specified subgroup of subtilase proposed by Siezen et al. (ibid.).

[0044] Substantia protease can be of animal, plant, or microbial origin, including chemically or genetically modified mutants (protein-engineered variants), preferably alkaline microbial substantia protease. Examples of substantia protease are those derived from the genus Bacillus, such as substantia protease Novo, substantia protease Carlsberg, substantia protease BPN', substantia protease 309, substantia protease 147, and substantia protease 168 (described in WO89 / 06279) and protease PD138 (WO 93 / 18140). Examples are described in WO 98 / 020115, WO 01 / 44452, WO01 / 58275, WO 01 / 58276, WO 03 / 006602, and WO 04 / 099401.

[0045] Other examples of useful proteases are described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO07 / 006305, WO 11 / 036263, WO The variants in 11 / 036264, especially those with substituted variants in one or more of the following positions: 3, 4, 9, 15, 27, 36, 43, 57, 61, 62, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130 156, 158, 160, 161, 167, 170, 182, 185, 188, 191, 194, 195, 199, 204, 205, 206, 209, 212, 217, 218, 224, 232, 235, 236, 245, 248, 252, 261, 262, 274 and 275 (using BPN' numbering).

[0046] The protease may contain substitutions at one or more positions of 171, 173, 175, 179 or 180 of SEQ ID NO: 3 corresponding to WO 2004 / 067737.

[0047] More preferred protease variants may contain one or more of the following substitutions: S3T, V4I, S9R, S9E, A15T, K27R, *36D, N43R, G61E, G61D, N62D, N62E, V68A, N76D, N87S,R, *97E, A98S, S99G, S99D, S99A, S99AD, S101E, S101D, S101G, S101M, S101N, S101R, S101H, S103A, V104I, V104Y, V104N, S106A, G118V, G118R, H120D, H120N, N123S, S1 28L, P129Q, S130A, S156D, A158E, G160D, G160P, S161E, Y167A, R170S, Q182E, N185E, S188E, Q191N, A194P, G195E, V199M, N204D, V205I, Y209W, S212G, L217Q, L217D, N218D, N218S, A232V, K235L, Q236H, Q245R, N252K, N261W, N261D, N261E, L262E, L262D, T274A, R275H (using BPN' numbering).

[0048] Examples of commercially available proteases include those sold under the following trademarks: Alcalase™, Relase™, Relase™ Ultra, Savinase™, Savinase™ Ultra, Duralase™, Durazym™, Everlase™, Primase™, Polarzyme™, Kannase™, Liquanase™, Liquanase™ Ultra, Ovozyme™, Coronase™, Coronase™ Ultra, Blaze™, Blaze Evity™ 100T, Blaze Evity™ 125T, Blaze Evity™ 150T, Neutrase™, Esperase™, Carnival™, Progress Uno™, and ProgressExcel™ (Novozymes A / S); and those sold under the following trademarks: Maxatase™, Maxacal™, Puramax™, FN2™, FN3™, FN4™, Excellase™, Maxapem™, and Purafect. Ox™, PurafectOxP™, Effectenz™ P1050, Effectenz™ P1060, Excellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz™ P100, Purafect Prime™, Preferenz™ P110, Effectenz™ P1000, Purafect™, Effectenz™ P2000, Purafast™, Properase™, Opticlean™, and Optimase™ (Genencor / Danisco / DuPont); Axapem™ (Gist-Brocases NV); BLAP (the sequence shown in Figure 29 of US 5352604) and its variants (Henkel). AG); and KAP (Alkaliophilic Bacillus subtilis protease) from Kao Corporation.

[0049] Lysing enzyme

[0050] The lyase can be a pectin lyase derived from the genus Bacillus, particularly Bacillus licheniformis or Bacillus agaradhaerens, or a variant derived from any of these sources. For example, commercially available pectin lyases, as described in US6124127, WO 99 / 027083, WO 99 / 027084, WO 02 / 006442, WO 02 / 092741, and WO 03 / 095638, are XPect™, Pectawash™, and Pectaway™ (Novozymes).

[0051] Mannanase

[0052] Mannanases can be basic mannanases of family 5 or 26. They can be wild-type from the genera *Bacillus* or *Pythium*, particularly *Bacillus mucosa*, *Bacillus licheniformis*, *Bacillus alkalophilus*, *Bacillus clausti*, or specific *Pythium*. Suitable mannanases are described in WO 99 / 064619. Commercially available mannanases are Mannaway™ (Novozymes A / S) and Mannastar™ (DuPont).

[0053] Cellulase

[0054] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Cladosporium*, such as fungal cellulases produced by specific *Pyrophyllus*, *Thermophyllus*, and *Fusarium* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0055] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0056] Commercially available cellulases include Celluzyme™, Carezyme™, Carezyme Premium™, Whitezyme™, and Cellluclean™ (Novozymes); Clazinase™, Revitaranz™, and Puradax HA™ (DuPont); Biotouch™ DCL and FCL (AB Enzyme); and KAC-500(B)™ (Kao Corporation).

[0057] Lipase and keratinase

[0058] Suitable lipases and keratins include those derived from bacteria or fungi. This includes chemically modified mutants or protein-engineered mutants. Examples include lipases from the genus *Thermomyces*, such as *T. lanuginosus* (formerly named *Pyrophyllus lanuginosus*) as described in EP258 068 and EP 305 216; cutinases from the genus *Pyrophyllus*, such as *Pyrophyllus specificus* as described in WO 96 / 13580; and lipases from the genus *Pseudomonas*, such as those from *P. alcaligenes* or *P. pseudoalcaligenes* (EP 218 272), *P. cepacia* (EP 331 376), *P. stutzeri* (GB 1,372,034), *P. fluorescens*, and *Pseudomonas* strain SD 705 (WO 95 / 06720 and WO...). 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); Bacillus lipases, such as those from Bacillus subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta [Chinese Journal of Biochemistry and Biophysics], 1131: 253-360), thermophilic steatobacterium (JP64 / 744992) or Bacillus pumilus (WO 91 / 16422).

[0059] Other examples are, for instance, those lipase variants described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260 105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 060063, WO 2007 / 087508, and WO 2009 / 109500.

[0060] Preferred commercially available lipases include Lipolase™, Lipolase Ultra™, and Lipex™; Lecitase™, Lipolex™; Lipoclean™, and Lipoprime™ (Novozymes). Other commercially available lipases include Lumafast (DuPont); Lipomax (Gist Brocardis / DuPont); and Bacillus lipase from Solvay.

[0061] amylase

[0062] Suitable amylases (α and / or β) include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Amylases include, for example, α-amylases obtained from Bacillus species (e.g., specific strains of Bacillus licheniformis described in more detail in GB 1,296,839).

[0063] Examples of suitable amylases include the amylase having SEQ ID NO: 2 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO: 3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO: 4 in WO 99 / 019467, such variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0064] Suitable amylases include the amylase having SEQ ID NO: 6 in WO 02 / 010355 or a variant thereof having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those with deletions at positions 181 and 182 and substitutions at position 193. Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of the Bacillus licheniformis α-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those having substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase comprising residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 is those having the following substitutions:

[0065] M197T;

[0066] H156Y+A181T+N190F+A209V+Q264S; or

[0067] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0068] Another suitable amylase is the amylase having SEQ ID NO: 6 in WO 99 / 019467 or a variant thereof having 90% sequence identity with SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions, or insertions in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184.

[0069] Other amylases that can be used are those of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 7 with WO 96 / 023873, or variants thereof having 90% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants are those with deletions at positions 181 and 182 or positions 183 and 184. The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those with deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0070] Other amylases that may be used are amylases having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 of WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity with SEQ ID NO: 10 of WO 01 / 66712. Preferred variants of SEQ ID NO: 10 of WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0071] Another suitable amylase is the amylase of SEQ ID NO: 2 having WO 09 / 061380 or a variant thereof having 90% sequence identity with SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having C-terminal truncation and / or substitution, deletion, or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO: 2 are those having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those having deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those having the following substitutions:

[0072] N128C+K178L+T182G+Y305R+G475K;

[0073] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0074] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0075] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein the variant is C-terminally truncated and optionally further includes a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0076] Other suitable amylases are α-amylases having SEQ ID NO: 12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, as well as variants having substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with the most preferred being variants having substitutions at all of these positions.

[0077] Other examples are amylase variants, such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0078] Commercially available amylases include Stainzyme™; Stainzyme Plus™; Duramyl™, Termammyl™, Termammyl Ultra™; Natalase™, Fungammyl™, and BAN™ (Novozymes); Rapidase™ and Purastar™ / Effectenz™; Powerase™, Amplify™, Amplify Prime™, Preferenz™ S100, and Preferenz™ S110 (DuPont).

[0079] Deoxyribonuclease (DNAase)

[0080] Suitable deoxyribonucleases (DNases) are any enzymes that catalyze the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. According to the invention, DNases obtainable from bacteria are preferred; particularly, DNases obtainable from the genus *Bacillus* are preferred; especially, DNases obtainable from *Bacillus subtilis* or *Bacillus licheniformis* are preferred. Examples of such DNases are described in patent applications WO 2011 / 098579 or PCT / EP2013 / 075922.

[0081] hydrolytic enzymes

[0082] Suitable perhydrolases catalyze perhydrolysis reactions that result in the production of peracids from carboxylic acid ester (acyl) substrates in the presence of a peroxide source (e.g., hydrogen peroxide). While many enzymes carry out these reactions at low levels, perhydrolases exhibit high perhydrolysis:hydrolysis ratios (typically greater than 1). Suitable perhydrolases can be of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants.

[0083] Examples of useful perhydrolysins include naturally occurring mycobacterial perhydrolysins or variants thereof. One exemplary enzyme is derived from Mycobacterium smegmatis. The enzyme's properties, structure, and variants are described in WO 2005 / 056782, WO2008 / 063400, US 2008 / 145353, and US 2007167344.

[0084] Oxidase / Peroxidase

[0085] Suitable oxidases and peroxidases (or oxidoreductases) include various sugar oxidases, laccases, peroxidases, and halogenated peroxidases.

[0086] Suitable peroxidases include those peroxidases included in the enzyme classification EC 1.11.1.7 as stated by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment thereof that exhibits peroxidase activity.

[0087] Suitable peroxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *C. cinerea* (EP 179,486), and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0088] Peroxidases used in this invention also include halogenated peroxidases, such as chloride peroxidase, bromoperoxidase, and compounds exhibiting chloride or bromoperoxidase activity. Halogenated peroxidases are classified according to their specificity for halide ions. Chloride peroxidase (EC1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

[0089] In one embodiment, the halogenated peroxidase is a chloride peroxidase. Preferably, the halogenated peroxidase is a vanadium haloperoxidase, i.e., a vanadate-containing halogenated peroxidase. In a preferred method of the invention, the vanadate-containing halogenated peroxidase is combined with a chloride ion source.

[0090] Halogenated peroxidases have been isolated from many different fungi, particularly from the dematiaceous hyphomycete fungal group, such as Caldariomyces (e.g., C. fumago), Alternaria, Curvularia (e.g., C. verruculosa and C. inaequalis), Helicobacter, Fibrosporium, and Botrytis.

[0091] Halogenated peroxidases have also been isolated from bacteria such as Pseudomonas (e.g., P. pyrrocinia) and Streptomyces (e.g., Streptomyces aureofaciens).

[0092] In a preferred embodiment, the halogenated peroxidase may be derived from the genus Curvularia, particularly Curvularia verruculosa or Curvularia anisotropis, such as Curvularia anisotropis CBS 102.42 described in WO 95 / 27046; or Curvularia verruculosa CBS 147.63 or Curvularia verruculosa CBS 444.70 described in WO 97 / 04102; or derived from species of the genus Geniculosporium, such as Drechslerahartlebii described in WO 01 / 79459, Dendryphiellasalina described in WO 01 / 79458, Phaeochrichoconiscrotalarie described in WO 01 / 79461, or Geniculosporium described in WO 01 / 79460.

[0093] The oxidases according to the invention specifically include any laccase or fragment thereof exhibiting laccase activity or exhibiting similar activity as defined in enzyme classification EC 1.10.3.2, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0094] The preferred laccase is a microbial enzyme. This enzyme can be derived from plants, bacteria, or fungi (including filamentous fungi and yeast).

[0095] Suitable examples of fungi include laccases derived from the following strains: *Aspergillus*, *Neurospora* (e.g., *Neurospora crassa*), *Stenocystis*, *Botrytis*, *Collybia*, *Fomes*, *Lentinula*, *Pleurotus*, *Coralus* (e.g., *Coralus longifolius* and *Coralus discolor*), *Rhizoctonia* (e.g., *Rhizoctonia solani*), *Coprinus* (e.g., *Coprinus comatus*, *Coprinus friesii*, and *Coprinus plicatilis*), *Psathyrella* (e.g., *P. condelleana*), *P. papilionaceus* (e.g., *P. papilionaceus*), *Schytalidium* (e.g., *S. thermophilum*), and *Polyporus* (e.g., *P.*). Pinsitus), genus P. radiata (e.g., P. radiata) (WO 92 / 01046) or genus C. hirsutus (e.g., C. hirsutus) (JP 2238885).

[0096] Suitable examples from bacteria include laccases that may be derived from strains of the genus Bacillus.

[0097] The preferred laccases are those derived from the genera *Coprinus* or *Hypericum*; particularly those derived from *Coprinus gracilistylus*, as disclosed in WO 97 / 08325; or from *Hypericum thermophilum*, as disclosed in WO 95 / 33836.

[0098] Other examples of oxidases include, but are not limited to, amino acid oxidases, glucose oxidases, lactate oxidases, galactose oxidases, polyol oxidases (e.g., WO 2008 / 051491), and aldose oxidases. Oxidases and their corresponding substrates can be used as hydrogen peroxide generating enzyme systems, thereby serving as a source of hydrogen peroxide. Several enzymes, such as peroxidases, halogenated peroxidases, and hydrolases, require a source of hydrogen peroxide. Other examples of such combinations of oxidases and substrates will be readily identifiable by those skilled in the art through study of EC 1.1.3, EC 1.2.3, EC 1.4.3, and EC 1.5.3 or similar categories (under the International Society for Biochemistry).

[0099] As mentioned above, amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1–30 amino acids; small N-terminal or C-terminal extensions, such as methionine residues at the N-terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function (e.g., polyhistidine fragments, antigenic epitopes, or binding domains).

[0100] Examples of conserved substitutions are found in the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0101] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the enzyme activity of the resulting mutant molecule is tested to identify amino acid residues essential for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contract site. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0102] Using known mutagenesis, recombination, and / or tampering methods, followed by an associated screening procedure, one or more amino acid substitutions, deletions, and / or insertions can be made and tested. These associated screening procedures are disclosed, for example, those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7:127).

[0103] The association between two amino acid sequences is described by the parameter “sequence identity”. For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The “longest identity” output of the Niedelt tag (obtained using the non-simplified (-nobrief) option) is used as the identity percentage and calculated as follows: (identical residues x 100) / (alignment length - total number of vacancies in the alignment).

[0104] Enzyme stabilizers / inhibitors:

[0105] As described above, one or more enzymes can be stabilized using conventional stabilizers, such as polyols (e.g., glycerol), (mono, di, or tri) propylene glycol, sugar alcohols, polypropylene glycol, and / or polyethylene glycol, preferably polyethylene glycol or polypropylene glycol with a molecular weight in the range of 200-1000; or compounds that work by temporarily reducing protease activity (reversible inhibitors).

[0106] Therefore, the compositions of the present invention may further comprise a protease inhibitor / stabilizer, which is a reversible inhibitor of protease activity (e.g., serine protease activity). Preferably, the protease inhibitor is a (reversible) subtilisin inhibitor. In particular, the protease inhibitor may be a peptide aldehyde, orthoboric acid, or boronic acid; or a derivative thereof.

[0107] The inhibition constant Ki (mol / L) of the protease inhibitor against serine proteases can be from 1E-12 to 1E-03; more preferably from 1E-11 to 1E-04; even more preferably from 1E-10 to 1E-05; even more preferably from 1E-10 to 1E-06; and most preferably from 1E-09 to 1E-07.

[0108] boric acid

[0109] The protease inhibitor may be boric acid or a derivative thereof; preferably phenylboronic acid or a derivative thereof. In embodiments of the present invention, the phenylboronic acid derivative has the following formula:

[0110]

[0111] R is selected from the group consisting of: hydrogen, hydroxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, and substituted C1-C6 alkenyl. Preferably, R is hydrogen, CH3, CH3CH2, or CH3CH2CH2.

[0112] In a preferred embodiment, the protease inhibitor (phenylboronic acid derivative) is 4-formyl-phenyl-boronic acid (4-FPBA).

[0113] In another specific embodiment, the protease inhibitor is selected from the group consisting of: thiophene-2-boronic acid, thiophene-3-boronic acid, acetamide phenylboronic acid, benzofuran-2-boronic acid, naphthalene-1-boronic acid, naphthalene-2-boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thiathrapheneboronic acid, 4-dibenzofuranboronic acid, 5-methylthiophene-2-boronic acid, thionaphtheneboronic acid, furan-2-boronic acid, furan-3-boronic acid, 4,4-biphenyl-diboronic acid, 6-hydroxy-2-naphtalene, 4-(methylthio)phenylboronic acid, 4 (Trimethyl-silyl)phenylboronic acid, 3-bromothiopheneboronic acid, 4-methylthiopheneboronic acid, 2-naphthylboronic acid, 5-bromothiopheneboronic acid, 5-chlorothiopheneboronic acid, dimethylthiopheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thiathroneboronic acid, dibenzothiopheneboronic acid, 4-carboxyphenylboronic acid, 9-anthraylboronic acid, 3,5-dichlorophenylboronic acid, di... Phenylated boric anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, p-tolylboronic acid, o-tolylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-di-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, and 4-methoxyphenylboronic acid.

[0114] Other boric acid derivatives suitable as protease inhibitors in detergent compositions are described in US 4,963,655, US 5,159,060, WO 95 / 12655, WO 95 / 29223, WO 92 / 19707, WO 94 / 04653, WO 94 / 04654, US 5442100, US 5488157 and US 5472628.

[0115] Peptide aldehyde or ketone

[0116] Protease stabilizers may have the formula: P-(A)yL-(B)x-B0-R*, where:

[0117] R* is H (hydrogen), CH3, CX3, CHX2, or CH2X. Preferably, R* = H, thus making the stabilizer a peptide aldehyde having the formula P-(A)yL-(B)x-B0-H;

[0118] X is a halogen atom, especially F (fluorine);

[0119] B0 is a single amino acid residue having the L- or D-configuration of the formula -NH-CH(R)-C(=O)-;

[0120] x is 1, 2, or 3;

[0121] Bx is independently a single amino acid residue that is attached to the next B or to B0 via its C-terminus;

[0122] L is absent or independently a linking group having the formula -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=O)-;

[0123] If L is absent, then A is absent or is independently a single amino acid residue attached to L via the N-terminus of an amino acid;

[0124] P is selected from the group consisting of: hydrogen, or, if L is absent, an N-terminal protecting group;

[0125] y is 0, 1, or 2.

[0126] R is independently selected from the group consisting of: C that is optionally substituted with one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Aryl groups;

[0127] R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2; and

[0128] R'' is C 1-6 Alkyl groups.

[0129] x can be 1, 2, or 3, and therefore B can correspondingly be 1, 2, or 3 amino acid residues. Thus, B can represent B1, B2-B1, or B3-B2-B1, where B3, B2, and B1 each represent one amino acid residue. y can be 0, 1, or 2, so A can be absent, or it can have 1 or 2 amino acid residues of the formula A1 or A2-A1, where A2 and A1 each represent one amino acid residue.

[0130] B0 can be a single amino acid residue with an L- or D-configuration, attached to an H via the C-terminus of an amino acid. B0 has the formula –NH-CH(R)-C(=O)-, where R is C 1-6 Alkyl, C 6-10 Aryl or C 7-10 The aryl alkyl side chain is such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, or benzyl, and wherein R may optionally be substituted by one or more identical or different substituents R'. Specific examples of B0 are arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (Ile), leucine (Leu), methionine (Met), ortholeucine (Nle), orthovaline (Nva), phenylalanine (Phe), meta-tyrosine, para-tyrosine (Tyr), and valine (Val). A specific embodiment is when B0 is leucine, methionine, phenylalanine, para-tyrosine, and valine.

[0131] B1, which is attached to B0 via the C-terminus of an amino acid, can be an aliphatic, hydrophobic, and / or neutral amino acid. Examples of B1 are alanine (Ala), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), n-leucine (Nle), n-valine (Nva), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Specific examples of B1 are alanine, glycine, isoleucine, leucine, and valine. A specific example is when B1 is alanine, glycine, or valine.

[0132] If present, B2, attached to B1 via the C-terminus of an amino acid, can be an aliphatic, hydrophobic, neutral, and / or polar amino acid. Examples of B2 are alanine (Ala), arginine (Arg), cyprodinium (Cpd), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), n-leucine (Nle), n-valine (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val). Specific examples of B2 are alanine, arginine, cyprodinium, glycine, isoleucine, leucine, phenylalanine, and valine. A specific example is when B2 is arginine, glycine, leucine, phenylalanine, or valine.

[0133] B3 (if present), attached to B2 via the C-terminus of an amino acid, can be a large, aliphatic, aromatic, hydrophobic, and / or neutral amino acid. Examples of B3 are isoleucine (Ile), leucine (Leu), ortholeucine (Nle), orthovaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). Specific examples of B3 are leucine, phenylalanine, tyrosine, and tryptophan.

[0134] The linking group L may be absent, or may be selected from the group consisting of: -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)-, or -C(=S)-C(=O)-. A specific embodiment of the invention is when L is absent or L is a carbonyl group -C(=O)-.

[0135] A1, which is attached to L via the N-terminus of an amino acid (if present), can be an aliphatic, aromatic, hydrophobic, neutral, and / or polar amino acid. Examples of A1 are alanine (Ala), arginine (Arg), cyproheptadine (Cpd), glycine (Gly), isoleucine (Ile), leucine (Leu), ortholeucine (Nle), valine (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp), and valine (Val). Specific examples of A1 are alanine, arginine, glycine, leucine, phenylalanine, tyrosine, tryptophan, and valine. A specific example is when B2 is leucine, phenylalanine, tyrosine, or tryptophan.

[0136] The A2 residue (if present) attached to A1 via the N-terminus of an amino acid can be a large, aliphatic, aromatic, hydrophobic, and / or neutral amino acid. Examples of A2 are arginine (Arg), isoleucine (Ile), leucine (Leu), ortholeucine (Nle), orthovaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp), and valine (Val). Specific examples of A2 are phenylalanine and tyrosine.

[0137] The N-terminal protecting group P (if present) may be selected from formyl, acetyl (Ac), benzyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic ethyl carbamate protecting groups such as fluorenylmethoxycarbonyl (Fmoc), methoxycarbonyl (Moc), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and adamantyloxycarbonyl; p-methoxybenzylcarbonyl, benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylaminocarbonyl, methanesulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphatamido (MeOP(OH)(=O)), and benzylphosphatamido (PhCH2OP(OH)(=O)).

[0138] In the case of a tripeptide aldehyde with a protecting group (i.e., x=2, L is absent and A is absent), P is preferably acetyl, methoxycarbonyl, benzyloxycarbonyl, methylaminocarbonyl, methanesulfonyl, benzylsulfonyl, and benzylphosphatidyl. In the case of a tetrapeptide aldehyde with a protecting group (i.e., x=3, L is absent and A is absent), P is preferably acetyl, methoxycarbonyl, methanesulfonyl, ethylsulfonyl, and methylphosphatidyl.

[0139] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO98 / 13460, WO 98 / 13461, WO 98 / 13462, WO 07 / 141736, WO 07 / 145963, WO 09 / 118375, WO10 / 055052 and WO 11 / 036153. More specifically, the peptide aldehyde can be Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF3, Moc-Val-Ala-Leu-CF3, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala- Phe-CF3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr -H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-G ly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu -H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeSO2-Phe-Gly-Ala-Leu- H, MeSO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Val-Ala-Leu-H, EtSO2-Phe-Gly-Ala-Leu-H, PhCH2SO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Leu-Ala-Leu-H, PhCH2O-P(OH)(O)-Phe-Ala-Leu-H, or MeO-P(OH)(O)-Leu-Gly-Ala-Leu-H. A preferred stabilizer for use in the liquid compositions of the present invention is Cbz-Gly-Ala-Tyr-H, or its bisulfite adduct, wherein Cbz is a benzyloxycarbonyl group.

[0140] Other examples of such peptide aldehydes include α-MAPI, β-MAPI, Phe-C(=O)-Arg-Val-Tyr-H, Phe-C(=O)-Gly-Gly-Tyr-H, Phe-C(=O)-Gly-Ala-Phe-H, Phe-C(=O)-Gly-Ala-Tyr-H, Phe-C(=O)-Gly-Ala-LH, Phe-C(=O)-Gly-Ala-Nva-H, and Phe-C(=O)-Gl y-Ala-Nle-H, Tyr-C(=O)-Arg-Val-Tyr-H, Tyr-C(=O)-Gly-Ala-Tyr-H, Phe-C(=S)-Arg-Val-Phe-H, Phe-C(=S)-Arg-Val-Tyr-H, Phe-C(=S)-Gly-Ala-Tyr-H, Anti-analgesin, GE20372A, GE20372B, Chymostatin A, Chymostatin B, and Chymostatin C.

[0141] The protease stabilizer can be a bisulfite adduct of the peptide aldehyde described above, for example, as described in WO2013 / 004636. This adduct can have the formula P-(A)yL-(B)xN(H)-CHR-CH(OH)-SO3M, where P, A, y, L, B, x, and R are as defined above, and M is H or an alkali metal, preferably Na or K.

[0142] Aqueous solutions of bisulfite adducts can be prepared by reacting the corresponding peptide aldehyde with aqueous solutions of sodium bisulfite (NaHSO3) and potassium bisulfite (KHSO3) using known methods, for example as described in WO 98 / 47523; US 6,500,802; US 5,436,229; J. Am. Chem. Soc. (1978) 100,1228; Org. Synth., Coll. Vol. 7: 361.

[0143] Particularly preferred peptide aldehyde protease stabilizers have the formula P-B3-B2-B1-B0-H, or are bisulfite adducts having the formula P-B3-B2-B1-N(H)-CHR-CHOH-SO3M, wherein

[0144] i) H is hydrogen;

[0145] ii) B0 is a single amino acid residue having the L- or D-configuration of the formula -NH-CH(R)-C(=O)-;

[0146] iii) B1 and B2 are independently single amino acid residues;

[0147] iv) B3 is a single amino acid residue, or it is absent;

[0148] v) R is independently selected from the group consisting of: C that is optionally substituted with one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Aryl alkyl groups;

[0149] vi) R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2;

[0150] vii) R'' is C 1-6 alkyl groups;

[0151] viii) P is an N-terminal protecting group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz); and

[0152] ix) M is H or an alkali metal, preferably Na or K.

[0153] In one or even more preferred embodiment, the peptide aldehyde protease stabilizer has the formula P-B2-B1-B0-H or an adduct having the formula P-B2-B1-N(H)-CHR-CHOH-SO3M, wherein

[0154] i) H is hydrogen;

[0155] ii) B0 is a single amino acid residue having the L- or D-configuration of the formula -NH-CH(R)-C(=O)-;

[0156] iii) B1 and B2 are independently single amino acid residues;

[0157] iv) R is independently selected from the group consisting of: C that is optionally substituted with one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Aryl alkyl groups;

[0158] v) R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2;

[0159] vi) R'' is C 1-6 alkyl groups;

[0160] vii) P is an N-terminal protecting group, preferably methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz); and

[0161] viii) M is H or an alkali metal, preferably Na or K.

[0162] Preferred embodiments of B0, B1, B2, B3 and P are as described above.

[0163] The molar ratio of the aforementioned peptide aldehyde (or bisulfite adduct) to the protease can be at least 1:1 or 1.5:1, and it can be less than 1000:1, more preferably less than 500:1, even more preferably from 100:1 to 2:1 or from 20:1 to 2:1, or most preferably, from 10:1 to 2:1.

[0164] Formates (e.g., sodium formate) and formic acid have also shown good performance as inhibitors of protease activity. Formates can be used synergistically with the aforementioned protease inhibitors, as shown in WO 2013 / 004635. Formates are present in the slurry composition in an amount of at least 0.1% w / w or 0.5% w / w, for example at least 1.0%, at least 1.2%, or at least 1.5%. The amounts are typically less than 5% w / w, less than 4%, or less than 3%.

[0165] In the embodiments, the protease is a metalloproteinase and the inhibitor is a metalloproteinase inhibitor, such as a protein hydrolysate-based inhibitor (e.g., as described in WO 2008 / 134343).

[0166] Detergent composition

[0167] The enzyme slurry of the present invention can be added to any form of detergent composition (such as liquid detergents, as well as soaps and detergent bars (e.g., synthetic detergent bars)) and thus form part of them. Soap and detergent bar compositions are described in more detail below.

[0168] The enzyme slurry (as described above) can be added to the liquid detergent composition in an amount corresponding to 0.0001% to 5% (w / w) of active enzyme protein (AEP); preferably from 0.0005% to 5%, more preferably from 0.0005% to 2%, even more preferably from 0.0005% to 1%, and most preferably from 0.001% to 1%, and most preferably from 0.005% to 1% (w / w) of active enzyme protein.

[0169] The liquid detergent composition has a physical form; it is not a solid (or gas). It can be a pourable liquid, paste, pourable gel, or non-pourable gel. It can be isotropic or structural, preferably isotropic. It can be a formulation for washing in an automatic washing machine or for hand washing, or for (automatic) dishwashing. It can also be a personal care product, such as shampoo, toothpaste, or hand soap.

[0170] This liquid detergent composition typically contains at least 20% and up to 95% water by weight, such as up to 70%, 50%, 40%, or 30% water. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in the aqueous liquid detergent. The aqueous liquid detergent may contain from 0% to 30% organic solvents.

[0171] The selection of detergent components may include (for textile care) the type of textile to be cleaned, the type and / or extent of soiling, the temperature at which cleaning is performed, and considerations for the formulation of the detergent product. Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as components may contain additional functionality as would be understood by a person skilled in the art.

[0172] The selection of other components is within the capabilities of those skilled in the art and includes conventional components, including the exemplary non-limiting components described below.

[0173] surfactants

[0174] The detergent composition may contain one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In a specific embodiment, the detergent composition contains a mixture of one or more nonionic surfactants and one or more anionic surfactants. These one or more surfactants are typically present at levels from about 0.1% to 60% by weight (e.g., about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%). The one or more surfactants are selected based on the desired cleaning application, and the surfactants include any one or more conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be used.

[0175] When included therein, the detergent will typically contain anionic surfactants ranging from about 1% to about 40% by weight (e.g., from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25%). Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl sulfonate (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0176] When included therein, the detergent will typically contain from about 0.1% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0177] When included therein, the detergent will typically contain from about 0.2% to about 40% by weight (e.g. from about 0.5% to about 30%, particularly from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%) of a nonionic surfactant. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)), together with products available under the trade names SPAN and TWEEN, and combinations thereof.

[0178] When included therein, the detergent will typically contain from about 0.1% to about 20% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) (such as alkyl dimethyl amine oxides), N-(cocoylalkyl)-N,N-dimethyl amine oxides and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0179] When included therein, the detergent will typically contain from about 0.1% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

[0180] Water-soluble additives

[0181] Water-soluble additives are compounds that dissolve hydrophobic compounds in aqueous solutions (or conversely, polar substances in nonpolar environments). Typically, water-soluble additives exhibit both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants); however, the molecular structure of water-soluble additives is generally unfavorable for spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Water-soluble additives do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and the formation of micelles, thin layers, or other well-defined intermediate phases by lipids occur. Instead, many water-soluble additives exhibit a continuous type of aggregation process in which the size of the aggregates increases with increasing concentration. However, many water-soluble additives alter the phase behavior, stability, and colloidal properties of systems containing substances with both polar and nonpolar characteristics, including mixtures of water, oils, surfactants, and polymers. Water-soluble additives are routinely used in a variety of industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble additives in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0182] The detergent may contain 0-5% by weight, such as about 0.5% to about 5%, or about 3% to about 5%, of a water-soluble additive. Any water-soluble additive known in the art for use in detergents may be used. Non-limiting examples of water-soluble additives include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthylformate, sodium hydroxynaphthylsulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0183] Builders and co-builders

[0184] The detergent composition may contain about 0-65% (e.g., about 5% to about 50%) of a detergent builder or co-builder, or a mixture thereof, by weight. In dishwashing detergents, the level of the builder is typically 40%-65%, particularly 50%-65%. The builder and / or co-builder may specifically be chelating agents that form water-soluble complexes having Ca and Mg ions. Any builder and / or co-builder known in the art for use in laundry detergents may be used. Non-limiting examples of builder include citrates, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2”-nitrotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0185] The detergent composition may also contain 0-50% (e.g., about 5% to about 30%) of a detergent co-adjuvant or a mixture thereof by weight. The detergent composition may include the co-adjuvant alone or in combination with a builder (e.g., a citric acid builder). Non-limiting examples of co-adjuvants include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents (such as aminocarboxylates, aminopolycarboxylates, and phosphates), and alkylsuccinic acids or alkenylsuccinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-dibutanoic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta(methylenephosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N- (2-Sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta(methylene phosphate) (DTPMP), aminotri(methylene phosphate) (ATMP), and combinations thereof and salts thereof. Further exemplary builders and / or co-builders are described in, for example, WO 09 / 102854, US 5977053.

[0186] polymer

[0187] The detergent may contain 0-10% (e.g., 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1%) of a polymer by weight. Any polymer known in the art for use in detergents may be used. The polymer may function as a co-adjuvant as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or antifoaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicone, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575 and US 5,955,415. Salts of the polymers mentioned above are also considered.

[0188] Fabric colorant

[0189] The detergent compositions of the present invention may further include fabric colorants, such as dyes or pigments, which, when formulated in the detergent composition, can deposit on the fabric upon contact with a washing liquid containing the detergent composition, and thus alter the color of the fabric through the absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants alter the color of a surface when they absorb at least a portion of the visible spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling into the Colour Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, such as those described in WO 2005 / 03274, WO 2005 / 03275, WO2005 / 03276, and EP 1876226 (incorporated herein by reference). The detergent composition preferably contains a fabric toner from 0.0001% w / w to 0.2% w / w. Suitable toners are also disclosed, for example, in WO 2007 / 087257 and WO2007 / 087243.

[0190] auxiliary materials

[0191] Any detergent component known in the art for use in laundry detergents may also be used. Other optional detergent components include corrosion inhibitors, shrinkage inhibitors, anti-fouling redeposition agents, anti-wrinkle agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including the enzyme stabilizers / inhibitors mentioned above, CMCs and / or polyols such as propylene glycol), fabric conditioners (including clays), fillers / processing aids, optical brighteners / brighteners, foaming agents, foam (foam) regulators, fragrances, soil suspending agents, softeners, defoaming agents, dulling inhibitors, and wicking agents, used alone or in combination. Any ingredient known in the art for use in laundry detergents may be used. The selection of such ingredients is entirely within the skill of a person skilled in the art.

[0192] dispersant- The detergent compositions of the present invention may also contain dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactantscience series, Volume 71, Marcel Dekker, Inc.

[0193] Dye transfer inhibitors - The detergent compositions of the present invention may further include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole, or mixtures thereof. When present in the subject composition, the dye transfer inhibitor may be present at a level from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0194] Fluorescent whitening agent- The detergent compositions of the present invention will also preferably contain additional components that can color the cleaned item, such as optical brighteners or fluorescent whitening agents. When present, the brightening agent is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions may be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents include the following sodium salts: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2,4-diphenylamino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2'-disulfonate, and sodium 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl)-disulfonate. Also preferred is Parawhite KX, commercially available and supplied by Paramount Minerals and Chemicals in Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-aminoalkylcoumarin.

[0195] Suitable levels of fluorescent brighteners include lower levels from about 0.01, from 0.05, from about 0.1, or even higher levels from about 0.2% w / w to 0.5% or even 0.75% w / w.

[0196] Fouling release polymers- The detergent compositions of the present invention may also include one or more soil-releasing polymers that help remove soiling from fabrics, such as cotton or polyester-based fabrics, particularly hydrophobic soiling from polyester-based fabrics. Soil-releasing polymers may be, for example, nonionic or anionic terephthalic acid-based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, PowderedDetergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Decker. Another type of soil-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to that core structure. The core structure may comprise a polyalkylimide structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (incorporated herein by reference). Furthermore, random graft copolymers are suitable soil-releasing polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (both incorporated herein by reference). Other fouling-releasing polymers are substituted polysaccharide structures, particularly substituted cellulose structures, such as modified cellulose derivatives, as described in EP 1867808 or WO 2003 / 040279 (both incorporated herein by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0197] Anti-redeposition agent The detergent compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under the category of dirt-releasing polymers may also function as anti-redeposition agents.

[0198] rheology modifiersIt is a structural agent or thickener, distinct from a viscosity reducer. Rheology modifiers are selected from the group consisting of: non-polymeric crystals, hydroxyl functional materials, and polymeric rheology modifiers, which impart shear-thinning characteristics to the aqueous liquid matrix of the composition. The rheology and viscosity of detergents can be modified and adjusted by methods known in the art, for example, as shown in EP2169040.

[0199] Other suitable auxiliary materials Including but not limited to shrink-proof agents, wrinkle-resistant agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam modifiers, water-soluble additives, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastic agents used in liquid detergents.

[0200] bleaching system

[0201] Examples of liquid detergents combining bleach and enzymes include, for example, US 5,275,753 and WO 99 / 00478. The detergent may contain 0-50% bleaching system. Any bleaching system known in the art for use in laundry detergents can be utilized. Suitable bleaching system components include bleaching catalysts such as MnTACN, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, pre-formed peracids, and mixtures thereof.

[0202] Soap and detergent bars

[0203] Laundry soaps and detergent bars may contain surfactants (e.g., anionic synthetic surfactants), builders (e.g., complexing agents, such as EDTA and HEDP (1-hydroxyethane-1,1-diphosphonic acid)), polymeric detergents, detergent chelators, polyols (e.g., glycerol), pH-controlling compounds, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachants, bleaching activators, clay removers, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances, and / or other compounds known in the art.

[0204] The laundry soap bars of the present invention can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, mills, pressing machines, extruders, cutting machines, and packaging machines. The soaps and detergent bars of the present invention are not limited to being prepared by any single method.

[0205] Enzyme particles may be present in laundry soap bars in amounts from 0.00001% w / w to 2.0% w / w. Typical amounts, based on the total composition of the laundry soap bars, are in the range of 0.0001% to 0.4%, preferably in the range of 0.001% to 0.1%, and most preferably in the range of 0.004% to 0.08%.

[0206] When enzyme slurries are used in the manufacture of soap or detergent bars, it is advantageous that the enzyme particles (e.g., enzyme crystals) remain undissolved during the manufacturing process. Soap and detergent bars with such undissolved enzyme particles exhibit higher residual enzyme activity after storage compared to soap and detergent bars made using dissolved enzymes. Preferably, the soap and detergent bars contain enzyme crystals, as shown in Example 5.

[0207] polyols

[0208] Polyols, as useful components of detergent soap bars, are characterized by water solubility, a carbon backbone length between C2 and C6, and multiple hydroxyl groups, preferably containing between 2 and 6 hydroxyl groups per molecule.

[0209] Useful polyols herein include, but are not limited to, 1,2-butanediol, 3-chloro-1,2-propanediol, ethylene glycol, 1,2-hexanediol, glycerol, mannose, propylene glycol, sorbitol, sucrose, and mixtures thereof. The polyol level, by weight of the final composition, will generally be between 0.5% and 10%, and preferably between 1% and 5%, and more preferably between 1.5% and 3%.

[0210] glycerin

[0211] During soap making, glycerin may be removed for commercial reasons or it may remain in the soap. Therefore, soap can be described as either high-glycerin or low-glycerin soap. Low-glycerin soap contains from 0 to 2% glycerin, and if more glycerin is needed, it can be added during soap making while the soap remains liquid, thus ensuring proper absorption in or into the soap noodle. In one specific embodiment, the laundry soap noodle contains from 1% to 6% glycerin. In a more specific embodiment of the invention, the laundry soap noodle contains from 2% to 5% glycerin by weight of the total laundry soap noodle. In a most preferred embodiment, the laundry soap noodle contains about 4% glycerin.

[0212] pH control compounds

[0213] It has been found that it may be necessary to lower the pH to improve enzyme stability. A pH control agent can be any suitable compound capable of controlling the pH. In one specific embodiment, the pH control agent is an acid.

[0214] In one specific embodiment, the pH-controlling compound may be selected from the group consisting of: fatty acids, acetic acid, aconitic acid, adipic acid, arachidic acid, arachidonic acid, aspartic acid, benzyl acid, butyric acid, capric acid, hexanoic acid, ceric acid, citric acid, formic acid, trans-butenedioic acid, glutamic acid, glutaric acid, glyceric acid, glyceryl-3-phosphate, glyoxylic acid, isocitrate, α-ketoglutaric acid, lactic acid, lauric acid, linoleic acid, linolenic acid, maleic acid, malic acid, malonic acid, myristic acid, oleic acid, oxalic acid, oxaloacetic acid, palmitic acid, palmitoleic acid, phosphatidic acid, phosphoenolpyruvate, pimelic acid, propionic acid, pyruvate, stearic acid, succinic acid, tartaric acid, and valeric acid.

[0215] The pH control agent will generally be present at a level between 0.01% and 8%, preferably between 0.1% and 6%, and more preferably between 0.2% and 2% based on the weight of the final composition.

[0216] Moisture content

[0217] Moisture provides the laundry soap bar with an acceptable feel and other physical properties. Moisture can be added to the premix by including it with the ingredients and / or adding it as free water to the premix. The laundry soap bar contains less than 30% w / w of water (moisture content) of the final laundry soap bar, or from 10% to 30%, preferably from 20% to 30% of the final laundry soap bar.

[0218] soap

[0219] Soap contains water-soluble salts of higher fatty acids. The soap can be derived from animal and / or plant sources. Soap can be made by direct saponification of fats and oils or by neutralization of free fatty acids. Suitable soaps are sodium, potassium, ammonium, and alkylammonium salts containing higher fatty acids with from about 8 to about 24 carbon atoms, such as from 12 to about 18 carbon atoms. In one specific embodiment, the soap is selected from sodium and potassium salts of fatty acids derived from coconut oil and animal fats, such as sodium or potassium animal fats and palm oil. Additionally, nut oils, such as coconut or palm kernel oil, can be added in amounts of 5% to 30% by weight of the final product.

[0220] Synthetic surfactants

[0221] Synthetic anionic surfactants suitable for detergent soap bars include water-soluble salts of organosulfur reaction products, preferably alkali metals, ammonium, and alkylammonium salts, having in their molecular structure an alkyl group containing from about 10 to about 20 carbon atoms, and a sulfonic acid or sulfate ester group. Examples of the group of synthetic surfactants are sodium alkyl sulfates and potassium alkyl sulfates, especially those obtained by sulfation of higher alcohols (C8-18 carbon atoms), such as those produced by reducing glycerides of animal fats or coconut oils; and sodium alkylbenzene sulfonates and potassium alkylbenzene sulfonates, wherein the alkyl group contains from about 9 to about 15 carbon atoms in a straight-chain or branched configuration. Of particular value are linear straight-chain alkylbenzene sulfonates (LAS), wherein the average number of carbon atoms in the alkyl group is from about 11 to 13, abbreviated as C11-13 LAS. Alkali metal salts, particularly sodium salts, of these surfactants are preferred.

[0222] Other examples of anionic synthetic detergents applicable herein are sodium alkyl glycerol ether sulfonates (AES), especially those ethers derived from animal fats and coconut oils of higher alcohols; sodium coconut oil fatty acid monoglyceride sulfonate and sodium coconut oil fatty acid monoglyceride sulfate; and sodium or potassium salts of alkyl ethylene oxide ether sulfates containing about 1 to about 10 units of ethylene oxide per molecule and wherein the alkyl group contains from about 10 to about 20 carbon atoms.

[0223] In addition, suitable anionic synthetic detergents also include water-soluble salts of esters of α-sulfonated fatty acids containing from about 6 to about 20 carbon atoms in the fatty acid group and from about 1 to about 10 carbon atoms in the ester group; water-soluble salts of 2-acyloxyalkyl-1-sulfonic acids containing from about 2 to about 9 carbon atoms in the acyl group and from 9 to about 23 carbon atoms in the alkyl group; water-soluble salts of olefins and paraffin sulfonates containing from about 12 to about 20 carbon atoms; and water-soluble salts of β-alkoxyalkyl sulfonates containing from about 1 to about 3 carbon atoms in the alkyl group and from about 8 to about 20 carbon atoms in the alkyl group.

[0224] Preferred examples of anionic synthetic surfactants are C10-18 alkyl sulfates (AS), C10-18 linear alkylbenzene sulfonates (LAS), C10-14 alkyl glycerol ether sulfonates (AES), and mixtures thereof.

[0225] Examples of ingredients found in regular laundry soap bars (synthetic detergent bars) are:

[0226] Linear alkylbenzene sulfonates, coconut oil fatty alcohol sulfates, soda ash, calcium carbonate, coconut oil fatty alcohol, TiO2, cellulase, diethylenetriamine (methylenephosphonic acid), coconut oil monoethanolamide, fluorescent whitening agents, substituted methylcellulose, fragrances, and moisture.

[0227] Examples of detergent ingredients contained in regular laundry soap bars (combination bars) are:

[0228] LAS, soap, cellulase, protease, borax, sodium silicate, citric acid, sodium carbonate, glycerol, propylene glycol, sugar (sorbitol), MgSO4, soda ash, talc, moisture.

[0229] The present invention relates to the following embodiments:

[0230] 1. An enzyme slurry composition comprising:

[0231] (a) Enzyme particles;

[0232] (b) Water-soluble salts of sodium, potassium, magnesium, zinc, or ammonium; and

[0233] (c) Xanthan gum, guar gum, sophora bean gum, vegan gum, gelatan gum, alginate, carrageenan, or microcrystalline cellulose; and

[0234] (d) Water.

[0235] 2. The enzyme slurry as described in Scheme 1, wherein the enzyme slurry comprises xanthan gum, guar gum, sophora bean gum, vegan gum, gelacanth gum, alginate, carrageenan, or microcrystalline cellulose in an amount of 0.25% w / w to 2.5% w / w.

[0236] 3. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme slurry contains an amount of water-soluble salt of 1% w / w to 10% w / w.

[0237] 4. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme slurry contains at least 5% w / w of enzyme particles.

[0238] 5. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme slurry contains at least 1% w / w of active enzyme protein, preferably at least 5% w / w of active enzyme protein.

[0239] 6. The enzyme slurry according to any one of the foregoing embodiments, wherein the enzyme is selected from proteases (e.g., subtilisin or metalloproteinases), lipases, keratinases, amylases, glycosylases, cellulases, pectinases, mannanases, arabinosylases, galactanases, xanthan gumases, xylanases, DNases, hydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxyses and / or halogenated peroxidases); preferably proteases, lipases, amylases, cellulases, pectinases, mannanases and / or DNases.

[0240] 7. The enzyme slurry as described in any of the preceding embodiments, further comprising a polyol selected from the group consisting of glycerol, (mono, di, or tri) propylene glycol, sugar alcohols, polypropylene glycol, and / or polyethylene glycol, preferably polyethylene glycol or polypropylene glycol with a molecular weight in the range of 200 to 800.

[0241] 8. The enzyme slurry as described in any of the preceding embodiments, wherein the water-soluble salt is selected from the group consisting of: sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, zinc nitrate, ammonium nitrate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, ammonium chloride, sodium acetate, potassium acetate, calcium acetate, magnesium acetate, zinc acetate, ammonium acetate, sodium citrate, potassium citrate, calcium citrate, magnesium citrate, zinc citrate, ammonium citrate, sodium formate, potassium formate, calcium formate, magnesium formate, zinc formate, and ammonium formate.

[0242] 9. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme particles comprise enzyme crystals.

[0243] 10. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme particles are composed of enzyme crystals.

[0244] 11. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme particles comprise a spray-dried enzyme.

[0245] 12. The enzyme slurry as described in any of the preceding embodiments, wherein the enzyme particles are composed of a spray-dried enzyme composition.

[0246] 13. The enzyme slurry as described in any of the foregoing embodiments, wherein the enzyme slurry further comprises an enzyme inhibitor.

[0247] 14. The enzyme slurry as described in embodiment 13, wherein the enzyme is a protease and the inhibitor is a peptidaldehyde or a derivative thereof, or boric acid.

[0248] 15. The enzyme slurry as described in embodiment 13 or 14, wherein the protease inhibitor is Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF3, Moc-Val-Ala-Leu-CF3, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF3, Cbz-Gly- Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly -Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Phe-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met- H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H , MeSO2-Phe-Gly-Ala-Leu-H, MeSO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Val-Ala-Leu-H, EtSO2-Phe-Gly-Ala-Leu-H, PhCH2SO2-Val-A la-Leu-H, PhCH2O-P(OH)(O)-Leu-Ala-Leu-H, PhCH2O-P(OH)(O)-Phe-Ala-Leu-H, or MeO-P(OH)(O)-Leu-Gly-Ala-Leu-H, or a bisulfite adduct of any of these, wherein Cbz is a benzyloxycarbonyl and Moc is a methoxycarbonyl; preferably, the protease inhibitor is Cbz-Gly-Ala-Tyr-H or Moc-Val-Ala-Leu-H, or a bisulfite adduct thereof, wherein Cbz is a benzyloxycarbonyl and Moc is a methoxycarbonyl.

[0249] 16. A method for preparing an enzyme-containing detergent composition, the method comprising adding an enzyme slurry according to any one of embodiments 1-15 to the composition, the composition comprising one or more detergent ingredients, such as surfactants and / or builder agents.

[0250] 17. The method of embodiment 16, wherein the detergent is soap or detergent strip.

[0251] 18. A detergent composition, which can be obtained by the method described in embodiment 16 or 17.

[0252] 19. A soap or detergent bar containing enzyme particles, such as enzyme crystals.

[0253] 20. The soap or detergent bar as described in embodiment 19, wherein the enzyme particles are protease crystals.

[0254] Some embodiments of the present invention include:

[0255] Example 1. An enzyme slurry composition comprising:

[0256] (a) Enzyme particles;

[0257] (b) Water-soluble salts of sodium, potassium, magnesium, zinc, or ammonium; and

[0258] (c) Xanthan gum, guar gum, sophora bean gum, vegan gum, gelatan gum, alginate, carrageenan, or microcrystalline cellulose; and

[0259] (d) Water.

[0260] Example 2. The enzyme slurry according to Example 1, wherein the enzyme slurry contains xanthan gum, guar gum, sophora bean gum, vegan gum, gelacanth gum, alginate, carrageenan or microcrystalline cellulose in an amount of 0.1% w / w-2.5% w / w.

[0261] Example 3. The enzyme slurry according to Example 1, wherein the enzyme slurry contains xanthan gum, guar gum, sophora bean gum, vegan gum, gelacanth gum, alginate, carrageenan or microcrystalline cellulose in an amount of 0.25% w / w-2.5% w / w.

[0262] Example 4. An enzyme slurry according to any one of Examples 1-3, wherein the enzyme slurry contains at least 1% w / w to 10% w / w of water-soluble salt.

[0263] Example 5. An enzyme slurry according to any one of Examples 1-4, wherein the enzyme slurry contains at least 1% w / w amount of enzyme particles.

[0264] Example 6. An enzyme slurry according to any one of Examples 1-4, wherein the enzyme slurry contains at least 5% w / w amount of enzyme particles.

[0265] Example 7. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains at least 1% w / w of active enzyme protein.

[0266] Example 8. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains 1% w / w-50% w / w of active enzyme protein.

[0267] Example 9. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains 1% w / w-25% w / w of active enzyme protein.

[0268] Example 10. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains at least 5% w / w of active enzyme protein.

[0269] Example 11. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains at least 5% w / w to 50% w / w of active enzyme protein.

[0270] Example 12. An enzyme slurry according to any one of Examples 1-6, wherein the enzyme slurry contains at least 5% w / w-25% w / w of active enzyme protein.

[0271] Example 13. An enzyme slurry according to any one of Examples 1-12, wherein the enzyme is selected from proteases (e.g., subtilisin or metalloproteinases), lipases, keratinases, amylases, glycosylases, cellulases, pectinases, mannanases, arabinosylases, galactanases, xanthan gums, xylanases, DNases, hydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxyses and / or halogenated peroxidases).

[0272] Example 14. An enzyme slurry according to any one of Examples 1-12, wherein the enzyme is selected from protease, lipase, amylase, cellulase, pectinase, mannanase, and / or DNase.

[0273] Example 15. An enzyme slurry according to any one of Examples 1-12, wherein the enzyme is a protease, such as subtilisin.

[0274] Example 16. An enzyme slurry according to any one of Examples 1-15, wherein the enzyme particles comprise enzyme crystals.

[0275] Example 17. An enzyme slurry according to any one of Examples 1-15, wherein the enzyme particles are composed of enzyme crystals.

[0276] Example 18. An enzyme slurry according to any one of Examples 1-15, wherein the enzyme particles comprise a spray-dried enzyme.

[0277] Example 19. An enzyme slurry according to any one of Examples 1-15, wherein the enzyme particles are composed of a spray-dried enzyme composition.

[0278] Example 20. An enzyme slurry according to any one of Examples 1-19, wherein the enzyme slurry contains more than one enzyme.

[0279] Example 21. An enzyme slurry according to any one of Examples 1-20, the enzyme slurry further comprising a polyol selected from the group consisting of: glycerol, (mono, di, or tri) propylene glycol, sugar alcohol, polypropylene glycol and / or polyethylene glycol, preferably polyethylene glycol or polypropylene glycol with a molecular weight in the range of 200 to 800.

[0280] Example 22. An enzyme slurry according to any one of Examples 1-21, wherein the polyol is glycerol.

[0281] Example 23. An enzyme slurry according to any one of Examples 1-21, wherein the polyol is (mono, di, or tri) propylene glycol.

[0282] Example 24. An enzyme slurry according to any one of Examples 1-21, wherein the polyol is a sugar alcohol, such as sorbitol.

[0283] Example 25. An enzyme slurry according to any one of Examples 1-21, wherein the polyol is polyethylene glycol, preferably polyethylene glycol with a molecular weight in the range of 200 to 800.

[0284] Example 26. An enzyme slurry according to any one of Examples 1-21, wherein the polyol is polypropylene glycol, preferably polypropylene glycol with a molecular weight in the range of 200 to 800.

[0285] Example 27. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt of sodium is selected from the group consisting of: sodium sulfate, sodium nitrate, sodium chloride, sodium acetate, sodium citrate, and sodium formate.

[0286] Example 28. An enzyme slurry according to any one of Examples 1-27, wherein the water-soluble salt of potassium is selected from the group consisting of: potassium sulfate, potassium nitrate, potassium chloride, potassium acetate, potassium citrate, and potassium formate.

[0287] Example 29. An enzyme slurry according to any one of Examples 1-28, wherein the water-soluble salt of calcium is selected from the group consisting of: calcium nitrate, calcium chloride, calcium acetate, calcium citrate, and calcium formate.

[0288] Example 30. An enzyme slurry according to any one of Examples 1-29, wherein the water-soluble salt of magnesium is selected from the group consisting of: magnesium sulfate, magnesium nitrate, magnesium chloride, magnesium acetate, magnesium citrate, and magnesium formate.

[0289] Example 31. An enzyme slurry according to any one of Examples 1-30, wherein the water-soluble salt of zinc is selected from the group consisting of: zinc sulfate, zinc nitrate, zinc chloride, zinc acetate, zinc citrate, and zinc formate.

[0290] Example 32. An enzyme slurry according to any one of Examples 1-31, wherein the water-soluble salt of ammonium is selected from the group consisting of: ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium acetate, ammonium citrate, and ammonium formate.

[0291] Example 33. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, and ammonium sulfate.

[0292] Example 34. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, zinc nitrate, and ammonium nitrate.

[0293] Example 35. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, and ammonium chloride.

[0294] Example 36. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium acetate, potassium acetate, calcium acetate, magnesium acetate, zinc acetate, and ammonium acetate.

[0295] Example 37. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium citrate, potassium citrate, calcium citrate, magnesium citrate, zinc citrate, and ammonium citrate.

[0296] Example 38. An enzyme slurry according to any one of Examples 1-26, wherein the water-soluble salt is selected from the group consisting of: sodium formate, potassium formate, calcium formate, magnesium formate, zinc formate, and ammonium formate.

[0297] Example 39. An enzyme slurry according to any one of Examples 1-38, wherein the enzyme slurry further comprises an enzyme inhibitor.

[0298] Example 40. The enzyme slurry according to Example 39, wherein the enzyme is a protease and the enzyme inhibitor is a peptidoprotease inhibitor, such as Z-GAY-H.

[0299] Example 41. The enzyme slurry according to Example 39, wherein the enzyme is a protease and the enzyme inhibitor is a borate protease inhibitor, such as 4-FPBA.

[0300] Example 42. An enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor has the formula P-(A)yL-Bx-B0-R* or its bisulfite adduct, wherein:

[0301] a) R* is H (hydrogen), CH3, CX3, CHX2, or CH2X;

[0302] b) X is a halogen atom;

[0303] c) B0 is a single amino acid residue having the formula -NH-CH(R)-C(=O)-;

[0304] d) x is 1, 2, or 3;

[0305] e) Bx (B1, B2, B3) are each a single amino acid residue that is attached to the next B or to B0 via its C-terminus;

[0306] f) L is absent or independently a linking group having the formula -C(=O)-, -C(=O)-C(=O)-, -C(=S)-, -C(=S)-C(=S)- or -C(=S)-C(=O)-;

[0307] g) If L is absent, then A is absent or is independently a single amino acid residue attached to L via the N-terminus of an amino acid;

[0308] h) P is selected from the group consisting of: hydrogen, or, if L is absent, an N-terminal protecting group;

[0309] i) y is 0, 1, or 2;

[0310] j) R is independently selected from the group consisting of: C that is optionally substituted by one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Arylalkyl;

[0311] k) R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2; and

[0312] l) R'' is C 1-6 alkyl.

[0313] m) x can be 1, 2 or 3.

[0314] Example 43. The enzyme slurry according to Example 42, wherein x=2, L is absent, A is absent, and P is p-methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz).

[0315] Example 44. An enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is a peptide aldehyde having the formula P-B3-B2-B1-B0-H or a bisulfite adduct having the formula P-B3-B2-B1-N(H)-CHR-CHOH-SO3M, wherein

[0316] a) H is hydrogen;

[0317] b) B0 is a single amino acid residue having the formula -NH-CH(R)-C(=O)-;

[0318] c) B1 and B2 are each a single amino acid residue;

[0319] d) B3 is a single amino acid residue, or it is absent;

[0320] d) R is independently selected from the group consisting of: C that is optionally substituted with one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Arylalkyl;

[0321] e) R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2;

[0322] f) R'' is C 1-6 alkyl groups;

[0323] g) P is an N-terminal protecting group; and

[0324] h) M is H or an alkali metal, preferably Na or K.

[0325] Example 45. An enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is a peptide aldehyde having the formula P-B2-B1-B0-H or a bisulfite adduct having the formula P-B2-B1-N(H)-CHR-CHOH-SO3M, wherein

[0326] a) H is hydrogen;

[0327] b) B0 is a single amino acid residue having the formula -NH-CH(R)-C(=O)-;

[0328] c) B1 and B2 are each a single amino acid residue;

[0329] d) R is independently selected from the group consisting of: C that is optionally substituted with one or more identical or different substituents R'. 1-6 Alkyl, C 6-10 Aryl or C 7-10 Arylalkyl;

[0330] e) R' is independently selected from the following group, which consists of: halogen, -OH, -OR'', -SH, -SR'', -NH2, -NHR'', -NR''2, -CO2H, -CONH2, -CONHR'', -CONR''2, -NHC(=N)NH2;

[0331] f) R'' is C 1-6 alkyl groups;

[0332] g) P is an N-terminal protecting group; and

[0333] h) M is H or an alkali metal, preferably Na or K.

[0334] Example 46. An enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is a peptide aldehyde having the formula P-B2-B1-B0-H or a bisulfite adduct having the formula P-B2-B1-B0-SO3M, wherein

[0335] a) H is hydrogen;

[0336] b) B0 is Phe, Tyr, or Leu;

[0337] c) B1 and B2 are each a single amino acid residue;

[0338] d) P is an N-terminal protecting group; and

[0339] e) M is H or an alkali metal, preferably Na or K.

[0340] Example 47. An enzyme slurry according to any one of Examples 42-44, wherein B3 is leucine, phenylalanine, tyrosine, or tryptophan.

[0341] Example 48. The enzyme slurry according to Example 47, wherein B3 is leucine, phenylalanine or tyrosine.

[0342] Example 49. An enzyme slurry according to any one of Examples 42-48, wherein B0 is leucine, methionine, phenylalanine, p-tyrosine, or valine.

[0343] Example 50. The enzyme slurry according to Example 49, wherein B0 is leucine, phenylalanine or p-tyrosine.

[0344] Example 51. An enzyme slurry according to any one of Examples 42-50, wherein B1 is alanine, glycine or valine.

[0345] Example 52. An enzyme slurry according to any one of Examples 42-51, wherein B2 is arginine, glycine, leucine, phenylalanine or valine.

[0346] Example 53. The enzyme slurry according to Example 52, wherein B2 is arginine, glycine or valine.

[0347] Example 54. An enzyme slurry according to any one of Examples 44-53, wherein P is p-methoxycarbonyl (Moc) or benzyloxycarbonyl (Cbz).

[0348] Example 55. The enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF3, Moc-Val-Ala-Leu-CF3, Cbz-Gly-Ala-Phe-H, Cbz-Gl y-Ala-Phe-CF3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr- H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Leu-H, Ac-Phe-Gly-Ala-Ph e-H, Ac-Phe-Gly-Val-Tyr-H, Ac-Phe-Gly-Ala-Met-H, Ac-Trp-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala- Leu-H, MeO-CO-Phe-Gly-Ala-Leu-H, MeO-CO-Phe-Gly-Ala-Phe-H, MeSO2-Phe-Gly-Ala-Leu-H, MeSO2-Val-Ala-Leu-H, PhC H2O-P(OH)(O)-Val-Ala-Leu-H, EtSO2-Phe-Gly-Ala-Leu-H, PhCH2SO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Leu-Ala-Leu-H, PhCH2O-P(OH)(O)-Phe-Ala-Leu-H or MeO-P(OH)(O)-Leu-Gly-Ala-Leu-H, or any of these bisulfite adducts, wherein Cbz is a benzyloxycarbonyl group and Moc is a methoxycarbonyl group.

[0349] Example 56. The enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is Cbz-Arg-Ala-Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-CF3, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Val-Ala-Leu-CF3, Moc-Val-Ala-Leu-CF3, Cbz-Gly-Ala-Phe-H, Cbz-Gly-Ala-Phe-CF3, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly -Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, MeO-CO-Val-Ala-Leu-H, MeNCO-Val-Ala-Leu-H, MeSO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Val-Ala-Leu-H, PhCH2SO2-Val-Ala-Leu-H, PhCH2O-P(OH)(O)-Leu-Ala-Leu-H or PhCH2O-P(OH)(O)-Phe-Ala-Leu-H, or any of these bisulfite adducts, wherein Cbz is a benzyloxycarbonyl group and Moc is a methoxycarbonyl group.

[0350] Example 57. An enzyme slurry according to Example 39 or 40, wherein the enzyme inhibitor is Cbz-Gly-Ala-Tyr-H or Moc-Val-Ala-Leu-H, or a bisulfite adduct thereof, wherein Cbz is benzyloxycarbonyl and Moc is methoxycarbonyl.

[0351] Example 58. A method for preparing an enzyme-containing detergent composition, the method comprising adding an enzyme slurry to an aqueous composition comprising one or more detergent ingredients, such as surfactants and / or builder agents.

[0352] Example 59. The method according to Example 58, wherein the enzyme slurry is as defined in any one of Examples 1-57.

[0353] Example 60. The method according to Example 58 or 59, wherein the aqueous composition is agitated to aid in the dissolution of the enzyme slurry.

[0354] Example 61. A soap or detergent bar containing enzyme particles.

[0355] Example 62. The soap or detergent bar according to Example 61, wherein the enzyme particles are enzyme crystals.

[0356] Example 62. A soap or detergent bar according to Example 61 or 62, wherein the enzyme is a protease.

[0357] Example 63. A method for preparing an enzyme-containing soap or detergent bar, the method comprising adding an enzyme slurry according to any one of Examples 1-57 to a premixed composition of the soap or detergent bar, said premixed composition comprising one or more detergent ingredients, such as surfactants, soap and / or glycerin.

[0358] Example 64. The method according to Example 63, wherein the enzyme particles of the enzyme slurry do not dissolve during the manufacturing process.

[0359] Example 65. The method according to Example 63 or 64, wherein the enzyme particles of the enzyme slurry are enzyme crystals.

[0360] Example 66. The method according to any one of Examples 63-65, wherein the enzyme is a protease.

[0361] The invention is further described through the following examples, which should not be construed as limiting the scope of the invention.

[0362] Example

[0363] Protease inhibitors (also referred to as "inhibitors" in this example):

[0364] Cbz-Gly-Ala-NHCH(CH2C6H4pOH)CH(OH)(SO3)Na, where Cbz is a benzyloxycarbonyl group (the bisulfite / bisulfite form of the peptide aldehyde).

[0365] The protease 1 used in the examples below is Savinase™ having the amino acid sequence shown in SEQ ID NO: 1. Protease 2 is another protease having the amino acid sequence shown in SEQ ID NO: 2. 'Active enzyme protein' means an enzyme protein that exhibits catalytic activity.

[0366] The preparation process of enzyme slurry can be divided into three steps:

[0367] 1. Mix all ingredients except the viscosity modifier and enzyme granules in water to form a premix. Stir with an anchor mixer at room temperature.

[0368] 2. Gradually add the viscosity modifier over 10 minutes, while stirring at a gradually increasing speed until reaching 800 rpm. Keep stirring at room temperature for 1 hour using an anchor stirrer.

[0369] 3. Add the enzyme granule concentrate to the mixture. After stirring the final mixture at 800 rpm for 16 h at room temperature using an anchor stirrer, the product is ready.

[0370] Example 1

[0371] Effect of xanthan gum concentration on the sedimentation curve of crystal enzyme slurry

[0372] Crystalline protease slurry mixtures were prepared using xanthan gum of various concentrations. The formulations for each mixture are listed in Table 1.

[0373] Table 1. Formulations of mixtures with different xanthan gum concentrations

[0374]

[0375] After 8 weeks of storage at 25°C, sedimentation of the protease particles was observed and evaluated by calculating the percentage of clear liquid in the test vials (supernatant length to total slurry length) (Table 2). The results indicate that low sedimentation can be obtained using a xanthan gum concentration of approximately 0.125% w / w. When higher amounts of xanthan gum were used, almost no sedimentation occurred.

[0376] Table 2. Sedimentation curves of saveinase slurries containing xanthan gum crystals of various concentrations (25℃)

[0377]

[0378] Example 2

[0379] Rapid dissolution of enzyme slurry in liquid detergents with additives

[0380] To reduce processing time in detergent manufacturing, it is desirable to achieve rapid dissolution of enzyme slurries in commercial liquid detergents. Crystalline protease slurries with protease inhibitors and / or PEG400 as additives were investigated to evaluate the time required for complete dissolution in commercial detergents (Table 3).

[0381] Table 3. Formulations of crystal protease slurries with or without additives.

[0382]

[0383] The results showed that the addition of DSAA significantly reduced the dissolution time of crystalline saveinase slurry in three liquid detergents, two of which are commercially available in China (Table 4). Increasing the temperature to 50°C further reduced the dissolution time to within 1 hour in all given liquid detergents. It was also noted that the crystalline enzyme slurry dissolved completely in the given detergents without stirring after several days (data not shown here). The addition of PEG did not improve the dissolution rate.

[0384] Table 4. Dissolution time of crystal protease slurry in liquid detergent (with stirring)

[0385]

[0386] NA: Not obtained.

[0387] Detergent 1 is a standard liquid detergent base of China (CN20) based on a standard formulation.

[0388] Detergent 2 is LibyQuanxiao, a commercial liquid detergent purchased from a supermarket in January 2015.

[0389] Detergent 3 is Blue Moon Deep Clean, a commercial liquid detergent purchased from a supermarket in January 2015.

[0390] Example 3

[0391] Effect of salt on the sedimentation curve of crystal enzyme slurry

[0392] Enzyme slurries require water-soluble salts, such as potassium or sodium salts, to prevent enzyme particles from dissolving after storage in the slurry. Different types and concentrations of salts were investigated to elucidate their effects on the sedimentation curves of crystalline protease slurries (Table 5).

[0393] Table 5. Formulation of the mixture of crystalline Savinase slurry and salt.

[0394]

[0395] The results showed that 2.5%, 5%, and 10% potassium acetate and 5% sodium formate had no difference in their effects on the sedimentation curves. At the same concentration (based on visual observation), the effect of sodium formate on the sedimentation curve was the same as that of potassium acetate.

[0396] Example 4

[0397] Stability of the enzyme slurry itself

[0398] The stability of the crystal protease slurries in various formulations was evaluated. The results showed that they were all very stable and comparable to commercial liquid protease products (Table 6). SavinaseEvity16L contains a protease inhibitor, while Savinase16L does not.

[0399] Table 6. Stability of crystal protease slurry in various formulations.

[0400]

[0401] NA: Not obtained.

[0402] Example 5

[0403] Laundry soap bars

[0404] Soap bars are made by mixing dissolved protease 2 or crystalline protease 2 with soap materials using the following steps:

[0405] 1. Pre-press the soap granules into smaller granules using a Sunlab Sampler-U benchtop plodding machine with a batch size of 1-kg;

[0406] 2. Add 1 kg of soap granules to the Murenking SM-168S kitchen mixer, then add the following ingredients and mix in the mixer for 10 minutes:

[0407] (a) A solution of 30 g glycerol and 2 g proteinase 2, wherein the solution contains 0.17% w / w proteinase inhibitor and 5% w / w proteinase 2 (dissolved enzyme protein); or

[0408] (b) 2 g of proteinase 2 crystal slurry, the solution containing 5% w / w sodium formate, 0.17% w / w protease inhibitor and 5% w / w proteinase 2 (crystal enzyme protein).

[0409] 3. Turn on the Sunlab Sampler-U, add the mixture from step 2 into the hopper, and press it into fine soap granules; repeat twice;

[0410] 4. Replace the screen of the Sunlab Sampler-U with a high-shear screen (16 mesh, 1.2 mm spacing), then press the soap granules into thin soap strips; repeat twice.

[0411] 5. Replace the Sampler-U with a regular sieve (5 mm diameter holes) and press the soap strips into soap granules; repeat twice.

[0412] 6. Turn on the heater to heat and maintain the extrusion cylinder at 40°C; when ready, add the extrusion cylinder to the head of the Sampler-U to set it to extrusion mode;

[0413] 7. Turn on Sampler-U and add soap granules to the hopper; adjust the speed or drum temperature as needed to ensure smooth extrusion of the soap bar without cracking; and

[0414] 8. Cut the extruded soap bars into certain lengths so that each bar weighs about 50 g, and store them in plastic bags.

[0415] Table 7. Enzymatic stability of dissolved and crystalline proteases in soap bars.

[0416]

[0417] As shown in Table 7, the enzyme stability in soap bars is much better when using enzyme crystals compared to using dissolved enzymes.

Claims

1. An enzyme slurry composition comprising: (a) Enzyme particles; (b) Water-soluble salts of sodium, potassium, magnesium, zinc, or ammonium; and (c) Xanthan gum, guar gum, sophora bean gum, vegan gum, gellan gum, alginate, carrageenan, or microcrystalline cellulose; and (d) Water.

2. The enzyme slurry as described in claim 1, wherein the enzyme slurry comprises xanthan gum, guar gum, sophora bean gum, vegan gum, gelacanth gum, alginate, carrageenan, or microcrystalline cellulose in an amount of 0.25% w / w to 2.5% w / w.

3. The enzyme slurry as described in any of the preceding claims, wherein the enzyme slurry contains an amount of water-soluble salt of 1% w / w to 10% w / w.

4. The enzyme slurry as described in any of the preceding claims, wherein the enzyme slurry contains at least 5% w / w of enzyme particles.

5. The enzyme slurry as described in any of the preceding claims, wherein the enzyme slurry contains at least 1% w / w of active enzyme protein, preferably at least 5% w / w of active enzyme protein.

6. The enzyme slurry according to any one of the preceding claims, wherein the enzyme is selected from proteases (e.g., subtilisin or metalloproteinase), lipases, keratinases, amylases, glycosylases, cellulases, pectinases, mannanases, arabinoseases, galactanases, xanthan gumases, xylanases, DNases, hydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxygenases, and / or halogenated peroxidases); preferably proteases, lipases, amylases, cellulases, pectinases, mannanases, and / or DNases.

7. The enzyme slurry as described in any of the preceding claims, further comprising a polyol selected from the group consisting of glycerol, (mono, di, or tri) propylene glycol, sugar alcohols, polypropylene glycol, and / or polyethylene glycol, preferably polyethylene glycol or polypropylene glycol with a molecular weight in the range of 200 to 800.

8. The enzyme slurry as claimed in any of the preceding claims, wherein the water-soluble salt is selected from the group consisting of: sodium sulfate, potassium sulfate, magnesium sulfate, zinc sulfate, ammonium sulfate, sodium nitrate, potassium nitrate, calcium nitrate, magnesium nitrate, zinc nitrate, ammonium nitrate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, zinc chloride, ammonium chloride, sodium acetate, potassium acetate, calcium acetate, magnesium acetate, zinc acetate, ammonium acetate, sodium citrate, potassium citrate, calcium citrate, magnesium citrate, zinc citrate, ammonium citrate, sodium formate, potassium formate, calcium formate, magnesium formate, zinc formate, and ammonium formate.

9. The enzyme slurry as claimed in any of the preceding claims, wherein the enzyme particles comprise enzyme crystals.

10. The enzyme slurry as described in any of the preceding claims, wherein the enzyme particles are composed of enzyme crystals.

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