High alkaline textile detergent containing protease

Abstract

The present invention relates to a textile detergent, in particular a liquid textile detergent, comprising a) at least one protease having proteolytic activity and comprising an amino acid sequence having over its entire length at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 and having, in each case based on the numbering of SEQ ID NO: 1, (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, in particular the amino acid substitutions 3T, 4I, 99E and 199I, and (ii) at least one further amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, in particular selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R153D, R153E, R153F, R153S 43Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, 256D, L256E and L256Q, and b) at least one detergent ingredient, preferably in an amount of 0.01 to 99.9% by weight, wherein the fabric detergent has a pH value of about 9 to about 12, measured in a 1% by weight solution in deionized water at 20° C. The present invention also relates to a corresponding method for cleaning textiles, in which the agent according to the invention is used, and to the use of the agent according to the invention for removing protease-sensitive stains from textiles.

Description

[0001] The present invention relates to the field of detergents, in particular to the field of detergents comprising at least one enzyme. The present invention relates to a textile detergent, in particular a liquid textile detergent, comprising at least one protease and at least one detergent ingredient, wherein the amino acid sequence of the protease has been modified, in particular for application in a textile detergent having a pH value of about 9 to about 12. The present invention also includes corresponding washing methods, the use of the agents described herein and the use of the protease in a textile detergent having a pH value of about 9 to about 12, and the use of the protease in a textile detergent having a pH value of about 9 to about 12 for removing protease-sensitive stains on textiles.

[0002] For decades, the use of enzymes in detergents has been established in the prior art. They are used to expand the performance range of related agents according to their special activity. These include hydrolases such as proteases, amylases, lipases and cellulases in particular. The first three mentioned enzymes hydrolyze proteins, starches and fats, thereby directly contributing to the removal of dirt. Due to the action of cellulases on fabrics, cellulases are particularly used. Another group of detergent enzymes are oxidative enzymes, in particular oxidases, which are optionally preferably used together with other components for bleaching stains or generating bleaching agents in situ. In addition to these enzymes that undergo continuous optimization, other enzymes for use in detergents are also continuously provided, such as pectinases, beta-glucanases, mannanases or other hemicellulases (glycosidases), in particular in order to be able to optimally treat specific stains, in particular hydrolyze specific plant polymers.

[0003] Proteases are the oldest enzymes contained in almost all modern effective detergents. This makes them one of the most important enzymes in technology. Among these, the proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important, because they are serine proteases due to the catalytically active amino acids. They act as non-specific endopeptidases and hydrolyze any amide bonds located inside peptides or proteins. Their optimal pH values ​​are usually in the alkaline range of about pH 9. For example, R. Siezen's article "Subtilases: Subtilisin-like Proteases", "Subtilisin enzymes" pp. 75-95, R. Bott and C. Betzel, ed., New York, 1996, gives a review of this family. Subtilases are naturally formed by microorganisms. In particular, subtilisins formed and secreted by Bacillus species are the most important class of subtilases.

[0004] Examples of subtilisins which are preferably used in detergents are subtilisins BPN' and Carlsberg, protease PB92, subtilisins 147 and 309, the proteases from Bacillus lentus, in particular the protease from Bacillus lentus DSM 5483, subtilisin DY and thermitase, proteinase K and proteases TW3 and TW7, which can be classified as subtilases but are no longer subtilisins in the strict sense, and variants of said proteases whose amino acid sequence has been modified relative to the starting protease.

[0005] International patent applications WO 95 / 23221 A1, WO 92 / 21760 A1 and WO 2013 / 060621 A1 disclose variants of proteases from Bacillus lentus DSM 5483, which are suitable for use in detergents or cleaning agents. In addition, international patent applications WO 2011 / 032988 A1 and WO 2016 / 096714 A1 and European patent application EP 3044302 A1 disclose detergents and cleaning agents containing protease variants from Bacillus lentus DSM 5483. Among other positions, the protease variants disclosed in these documents can be modified at positions 3, 4, 99 and / or 199 in the numbering of the protease from Bacillus lentus DSM 5483, and for example have amino acids 3T, 4I, 99E or 199I at the positions. However, other modified combinations as described below have not been found in these documents. In particular, it was not discovered from these documents that certain Bacillus lentus protease variants are suitable for use in highly alkaline textile detergents.

[0006] Generally, in any case, only selected proteases are suitable for use in liquid formulations containing surfactants. Many proteases do not show sufficient catalytic performance in such formulations, or they are not sufficiently stable. Therefore, for the use of proteases in textile detergents, high catalytic activity and stability under wash cycle conditions are particularly desirable.

[0007] Solid textile detergents typically have a pH of about 8 to about 10, while liquid textile detergents may have a pH of about 7 to about 9. Alkaline liquid detergents are relatively common and are particularly popular in North America. However, alkaline pH makes it difficult to find suitable enzymes and formulate them in alkaline liquid detergents, because high pH values ​​not only adversely affect the performance of the enzymes, but also their storage stability. In addition, enzymes are generally less stable in liquid detergents when the detergent has a high water content.

[0008] Therefore, the liquid formulations of the prior art containing proteases and surfactants have the following disadvantages: the proteases contained do not have satisfactory proteolytic activity or sufficient storage stability under standard washing conditions (e.g., in the temperature range of 20 to 40° C.), especially at higher pH, and therefore the formulations do not show optimal cleaning performance on protease-sensitive stains.

[0009] Therefore, one goal in developing detergent formulations is to selectively or randomly modify the enzymes contained using methods known in the prior art to optimize them for use in detergents. These methods include, for example, point mutagenesis, deletion or insertion mutagenesis, or fusion with other proteins or protein fragments.

[0010] Although many proteases for detergent use are known in the art, there remains a need for proteases for use in highly alkaline textile detergents, particularly liquid textile detergents having a pH of about 9 to about 12.

[0011] It is therefore an object of the present invention to further improve the stability of proteases in highly alkaline textile detergents, thereby further improving the cleaning performance of textile detergents.

[0012] Surprisingly, it has now been found that the protease defined herein or a protease sufficiently similar thereto (in terms of sequence identity) is improved in storage stability compared to conventional proteases and / or compared to the wild-type form (SEQ ID NO: 1) and / or compared to the starting variant (SEQ ID NO: 2 of WO 2013 / 060621 A1) and is therefore particularly suitable for use in textile detergents at a pH of about 9 to about 12.

[0013] Thus, in a first aspect, the present invention relates to a textile detergent comprising: a) at least one protease having proteolytic activity and comprising an amino acid sequence which over its entire length has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 and has (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, in particular the amino acid substitutions 3T, 4I, 99E and 199I, and (ii) at least one further amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, in particular selected from the group consisting of 74D, 74E, 74Q, 136Q, 143L, 143W, 143Y, 154D, 154Q, 161T, 163G, 171L, 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212S, 212T, 256D, 256E and 256Q, and b) at least one detergent component, preferably in an amount of 0.01 to 99.9% by weight, the textile detergent having a pH value of about 9 to about 12, the pH value being measured in a 1% by weight solution in deionized water at 20° C. The detergent according to the invention is preferably a liquid textile detergent. More preferably, the textile detergent according to the invention has a pH of 9 to 12, in particular 9.5 to 11.5, more preferably 10 to 11, particularly preferably pH 10 in a 1% by weight solution in deionized water at 20° C.

[0014] In particular, when the textile detergent contains at least one other enzyme of the same or different type, such as amylase, cellulase, lipase, mannanase or pectinase (the enumeration of other enzymes is not complete), the textile detergent according to the present invention also shows performance advantages over other textile detergents. Therefore, it is preferred that the textile detergent according to the present invention contains at least one other enzyme of the same type (i.e. another protease) or other enzymes of different types. The detergent preferably comprises at least one amylase having amylolytic activity and selected from: a) an alpha-amylase comprising an amino acid sequence that is at least 80% identical over its entire length to the amino acid sequence given in SEQ ID NO: 2, and optionally having at least one amino acid substitution at one of positions 172, 202, 208, 255 and 261 according to the numbering of SEQ ID NO: 2, preferably selected from the group consisting of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, R172Q and combinations thereof; and / or b) an alpha-amylase comprising an amino acid sequence that is at least 60% identical over its entire length to the amino acid sequence given in SEQ ID NO: 3, and at least one amino acid substitution at one of positions 172, 202, 208, 255 and 261 according to the numbering of SEQ ID NO: 2. In the numbering of NO:3, optionally at position 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, at least one amino acid substitution at one of positions 441, 444, 445, 446, 447, 450, 458, 461, 471, 482 and 484, and / or a deletion at one of positions 183 and 184, preferably at least one amino acid substitution at one of positions 9, 26, 149, 182, 186, 202, 257, 295, 299, 323, 339 and 345, and / or particularly preferably at least one amino acid substitution or deletion selected from the group consisting of R118K, D183*, G184*, N195F, R320K, R458K and a combination thereof;and / or c) an alpha-amylase comprising an amino acid sequence that is at least 90% identical over its entire length to the amino acid sequence given in SEQ ID NO: 4, and optionally in the presence of a polypeptide according to SEQ ID NO: NO: 4 has at least one substitution and / or deletion at one of positions 93, 116, 118, 129, 133, 134, 140, 142, 146, 147, 149, 151, 152, 169, 174, 183, 184, 186, 189, 193, 195, 197, 198, 200, 203, 206, 210, 212, 213, 235, 243, 244, 260, 262, 284, 303, 304, 320, 338, 347, 359, 418, 431, 434, 439, 447, 458, 469, 476 and 477, preferably at positions 183 and 184. ;

[0015] The invention also relates to a method for preparing such a textile detergent and to a method for cleaning textiles, in which the detergent according to the invention is used.

[0016] The invention also relates to the use of such a textile detergent for cleaning textiles.

[0017] The present invention also relates to the use of a protease in a textile detergent, preferably a liquid textile detergent, for removing protease-sensitive stains from textiles, said protease having over its entire length at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 and having, in each case based on the numbering according to SEQ ID NO: 1: (i) at least two amino acid substitutions at at least two of the positions corresponding to position 3, 4, 99 or 199, in particular the amino acid substitutions 3T, 4I, 99E and 199I, and (ii) at least one additional amino acid substitution at at least one of the positions corresponding to position 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, in particular selected from 74 , 74E, 74Q, 136Q, 143L, 143W, 143Y, 154D, 154Q, 161T, 163G, 171L, 200A, 200L, 200S, 200T, 203K, 203V, 203W, 209W, 212S, 212T, 256D, 256E and 256Q, the textile detergent having a pH of about 9 to about 12 measured in a 1 wt % solution in deionized water at 20°C.

[0018] Particularly preferred proteases for use in the agents, methods and uses according to the invention have over their entire length at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 and in each case have, based on the numbering according to SEQ ID NO: 1: (i) the amino acid substitutions S3T, V4I, R99E and V199I at the positions corresponding to positions 3, 4, 99 or 199, and (ii) at least one further amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, which is selected from the group consisting of N74D , N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, 256D, L256E and L256Q

[0019] These and further aspects, features and advantages of the present invention will become clear to those skilled in the art by studying the following detailed description and claims. Any feature from one aspect of the present invention may be used in any other aspect of the present invention. In addition, it will be readily understood that the embodiments contained herein are intended to describe and illustrate the present invention, but not to limit the present invention, and in particular, the present invention is not limited to these embodiments.

[0020] Unless otherwise indicated, all percentages are by weight (wt %).

[0021] Numerical ranges given in the format "from x to y" include the values. If multiple preferred numerical ranges are given in this format, it is readily understood that all ranges generated by combinations of the different endpoints are also included.

[0022] As used herein, "at least one" means one or more, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

[0023] The term "detergent" as used herein is synonymous with the term "textile detergent" or "agent" and denotes a composition for cleaning textiles as explained in the specification.

[0024] As used herein, "approximately", "about" or "substantially" with respect to a numerical value refers to ±10%, preferably ±5%, of the corresponding numerical value.

[0025] As used herein, "liquid" includes liquids and gels, as well as pasty compositions. Preferably, liquid compositions are flowable and pourable at room temperature, but they may also have a flowability limit.

[0026] As used herein, "overalkaline" refers to a pH of about 9 to about 12 measured in a 1 wt % solution in deionized water at 20°C.

[0027] The present invention is based on the surprising finding of the inventors that amino acid substitutions at the positions described herein lead to improved storage stability of these modified proteases in highly alkaline detergents compared to conventional proteases and / or compared to the wild type and / or compared to the starting variants which have been improved compared to the wild type.

[0028] This is particularly surprising since no such protease or such amino acid substitution has hitherto been associated with an improved storage stability of the corresponding protease in overbased detergents, in particular in liquid overbased detergents.

[0029] In a preferred embodiment, the modification according to the invention of the protease used according to the invention, in each case based on the numbering according to SEQ ID NO: 1, (i) at least two of the positions corresponding to position 3, 4, 99 or 199, and (ii) at least one of the positions corresponding to position 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, results in improved storage stability of the modified protease in highly alkaline textile detergents (pH 9 to 12).

[0030] In a further preferred embodiment of the protease for use according to the invention, the protease has (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, which are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, in each case based on the numbering according to SEQ ID NO: 1. A generation selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0031] In a preferred embodiment, the protease used according to the invention has (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, which are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, which are selected from the group consisting of N74D, N74E, N74Q, A136Q, The group consisting of R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q, a combination of at least two amino acid substitutions selected from group (i) and at least one amino acid substitution selected from group (ii) resulting in improved storage stability of the modified protease in a high alkaline textile detergent (pH 9 to 12).

[0032] In a particularly preferred embodiment, the detergent according to the invention contains a protease having one of the following amino acid substitution variants:

[0033] (i)S3T+V4I+R99E+V199I+Q200L+Y203W;

[0034] (ii)S3T+V4I+R99E+V199I+N212S;

[0035] (iii)S3T+V4I+R99E+V199I+N74D;

[0036] (iv)S3T+V4I+R99E+V199I+S154D+L256E;

[0037] (v)S3T+V4I+R99E+V199I+Q200L+Y203W+S154D+L256E;

[0038] (vi)S3T+V4I+R99E+V199I+N74D+Q200L+Y203W;

[0039] (vii)S3T+V4I+R99E+V199I+N74D+S154D+Q200L+Y203W+L256E;

[0040] (viii)S3T+V4I+R99E+V199I+N74D+N212S;

[0041] (ix)S3T+V4I+R99E+V199I+N74D+S154D+Y203W+L256E;

[0042] (x)S3T+V4I+R99E+V199I+N74D+Y203W;

[0043] (xi)S3T+V4I+R99E+V199I+N74D+S154D+Q200L+L256E;

[0044] (xii)S3T+V4I+R99E+V199I+N74D+Q200L;

[0045] (xiii)S3T+V4I+R99E+V199I+S154D+Q200L+Y203W;

[0046] (xiv)S3T+V4I+R99E+V199I+Q200L+Y203W+L256E;

[0047] (xv)S3T+V4I+R99E+V199I+A136Q+R143W+Y161T+Q200L;

[0048] (xvi)S3T+V4I+R99E+V199I+N74D+R143Y+A209W+N212S+L256E;

[0049] (xvii)S3T+V4I+R99E+V199I+A136Q+S154D+V171L+Q200L,

[0050] The numbering is in each case based on the numbering according to SEQ ID NO: 1.

[0051] Some embodiments of the proteases used according to the invention have improved storage stability. Compared to conventional proteases and / or wild-type enzymes (SEQ ID NO: 1) and / or starting variants of proteases (SEQ ID NO: 2 of WO 2013 / 060621 A1), they have improved stability in highly alkaline textile detergents, particularly when stored for 3 days or more, 4 days or more, 7 days or more, 10 days or more, 12 days or more, 14 days or more, 21 days or more, or 28 days or more, the pH of the textile detergent measured in a 1 wt % solution in deionized water at 20° C. is from about 9 to about 12.

[0052] Certain embodiments of the protease according to the present invention may have increased catalytic activity in highly alkaline textile detergents, independent of or in addition to increased storage stability. In many embodiments, the protease used according to the present invention may have a proteolytic activity of at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109% or 110% based on the wild type (SEQ ID NO: 1) and / or the starting variant (SEQ ID NO: 2 of WO 2013 / 060621 A1) with improved performance. This improved performance of the protease allows improved washing results to be achieved for protease-sensitive stains in different temperature ranges, particularly in the temperature range of 20°C to 40°C.

[0053] In addition, the preferred embodiments of the protease used according to the present invention have special stability in detergents, for example relative to surfactants and / or bleaching agents and / or chelating agents, and / or relative to temperature influences, in particular relative to high temperatures, for example 50-65°C, in particular 60°C, and / or relative to pH changes and / or relative to denaturants or oxidants and / or relative to proteolytic degradation and / or relative to changes in the redox ratio. Therefore, particularly preferred embodiments of the present invention provide protease variants with improved performance and / or protease variants with improved temperature stability. Therefore, further particularly preferred embodiments of the present invention provide protease variants with improved performance and / or protease variants with improved temperature stability. Therefore, this advantageous embodiment of the protease according to the present invention allows improved washing results to be achieved for protease-sensitive stains over a wide temperature range.

[0054] The proteases used according to the invention show enzymatic activity, i.e. they are able to hydrolyze peptides and proteins, in particular in detergents. Therefore, the proteases used according to the invention are enzymes which catalyze the hydrolysis of amide / peptide bonds in protein / peptide substrates and are therefore able to cleave proteins or peptides. In addition, the proteases according to the invention are preferably mature proteases, i.e. catalytically active molecules without signal peptides and / or propeptides. Unless otherwise indicated, the sequences also relate to mature (processed) enzymes in each case.

[0055] In various embodiments of the present invention, the protease used according to the present invention is a free enzyme. This means that the protease can directly act on all components of the reagent, and if the reagent is a liquid reagent, the protease directly contacts the solvent (e.g., water) of the reagent. In other embodiments, the reagent may include a protease that forms an interaction complex with other molecules or contains a "coating". In this case, a single protease molecule or a plurality of protease molecules may be separated from other groups of the reagent by a surrounding structure. This separation structure may come from, but is not limited to, a vesicle, such as a micelle or a liposome. However, the surrounding structure may also be a viral particle, a bacterial cell, or a eukaryotic cell. In various embodiments, the reagent may include a cell of Bacillus pumilus or Bacillus gibsonii or Bacillus subtilis expressing a protease according to the present invention, or a cell culture supernatant of such a cell.

[0056] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% and 98.8% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least two amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein The at least one amino acid substitution is selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0057] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98% and 98.5% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least three amino acid substitutions at at least three of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least three amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein The at least one amino acid substitution is selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0058] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% and 98% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO: 1, having (i) amino acid substitutions S3T, V4I, R99E and V199I, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the at least one amino acid substitution is selected from N74D, N74E, N The group consisting of 74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0059] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98% and 98.5% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least two amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least two amino acid substitutions at at least two of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least two amino acid substitutions are selected from the group consisting of N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0060] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% and 98% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least three amino acid substitutions at at least three of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least three amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least three amino acid substitutions at at least two of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least two amino acid substitutions are selected from the group consisting of N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0061] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97% and 97.5% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO: 1, having (i) amino acid substitutions S3T, V4I, R99E and V199I, and (ii) at least two amino acid substitutions at at least two of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the at least two amino acid substitutions are selected from N74D, N74E, N The group consisting of 74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0062] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% and 98% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least two amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least three amino acid substitutions at at least three of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least three amino acid substitutions are selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0063] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97% and 97.5% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least three amino acid substitutions at at least three of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least three amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least three amino acid substitutions at at least three of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least three amino acid substitutions are selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0064] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% and 97% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO: 1, having (i) amino acid substitutions S3T, V4I, R99E and V199I, and (ii) at least three amino acid substitutions at at least three of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the at least three amino acid substitutions are selected from N74D, N74E, N The group consisting of 74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0065] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97% and 97.5% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least two amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least four amino acid substitutions at at least four of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least four amino acid substitutions are selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0066] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% and 97% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO:1, having (i) at least three amino acid substitutions at at least three of the positions corresponding to positions 3, 4, 99 or 199, wherein the at least three amino acid substitutions are selected from the group consisting of S3T, V4I, R99E and V199I, and (ii) at least four amino acid substitutions at at least four of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the The at least four amino acid substitutions are selected from the group consisting of N74D, N74E, N74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0067] In a preferred embodiment of the invention, the protease used according to the invention comprises an amino acid sequence which is at least 70%, and in increasing order of preference at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% and 97% identical over its entire length to the amino acid sequence given in SEQ ID NO: 1, and, in each case based on the amino acid sequence according to SEQ ID NO: 1, having (i) amino acid substitutions S3T, V4I, R99E and V199I, and (ii) at least four amino acid substitutions at at least four of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, wherein the at least four amino acid substitutions are selected from N74D, N74E, N The group consisting of 74Q, A136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, L256D, L256E and L256Q.

[0068] In a particularly preferred embodiment, the protease used according to the invention contains one of the following amino acid substitution variants:

[0069] (i)S3T+V4I+R99E+V199I+Q200L+Y203W;

[0070] (ii)S3T+V4I+R99E+V199I+N212S;

[0071] (iii)S3T+V4I+R99E+V199I+N74D;

[0072] (iv)S3T+V4I+R99E+V199I+S154D+L256E;

[0073] (v)S3T+V4I+R99E+V199I+Q200L+Y203W+S154D+L256E;

[0074] (vi)S3T+V4I+R99E+V199I+N74D+Q200L+Y203W;

[0075] (vii)S3T+V4I+R99E+V199I+N74D+S154D+Q200L+Y203W+L256E;

[0076] (viii)S3T+V4I+R99E+V199I+N74D+N212S;

[0077] (ix)S3T+V4I+R99E+V199I+N74D+S154D+Y203W+L256E;

[0078] (x)S3T+V4I+R99E+V199I+N74D+Y203W;

[0079] (xi)S3T+V4I+R99E+V199I+N74D+S154D+Q200L+L256E;

[0080] (xii)S3T+V4I+R99E+V199I+N74D+Q200L;

[0081] (xiii)S3T+V4I+R99E+V199I+S154D+Q200L+Y203W;

[0082] (xiv)S3T+V4I+R99E+V199I+Q200L+Y203W+L256E;

[0083] (xv)S3T+V4I+R99E+V199I+A136Q+R143W+Y161T+Q200L;

[0084] (xvi)S3T+V4I+R99E+V199I+N74D+R143Y+A209W+N212S+L256E;

[0085] (xvii)S3T+V4I+R99E+V199I+A136Q+S154D+V171L+Q200L,

[0086] The numbering is in each case based on the numbering according to SEQ ID NO: 1.

[0087] In the context of the present invention, a protease characterized as having at least one given amino acid substitution means that it contains one or more (given) amino acid substitutions at the relevant position, ie at least the given position has not been further mutated or deleted, for example by fragmentation of the protease.

[0088] The identity of nucleic acid or amino acid sequence is determined by sequence comparison. The sequence comparison is based on the BLAST algorithm established and commonly used in the prior art (see, for example, Altschul et al. (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410, and Altschul et al. (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, pp. 3389-3402), and in principle, by assigning the nucleotides or amino acid similar sequences in the nucleic acid or amino acid sequence to each other. The table assignment of the relevant position is called alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparison (alignment), particularly multiple sequence comparison, is performed using a computer program. For example, the Clustal series (see, e.g., Chenna et al. (2003) "Multiple sequence alignment with the Clustal series of programs", Nucleic Acid Res. 31: 3497-3500), T-Coffee (see, e.g., Notredame et al. (2000) "T-Coffee: A novel method for multiple sequence alignments", J. Mol. Biol. 302: 205-217) or programs based on these programs or algorithms are often used. The computer program Vector Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA), and its program for sequence comparison is the AlignX module based on ClustalW. Unless otherwise stated, sequence identities given herein are determined by the BLAST algorithm.

[0089] This comparison also allows conclusions to be drawn about the similarity of the compared sequences. It is usually given as a percentage of identity, i.e. the ratio of identical nucleotides or amino acid functional groups in the alignment of the sequence or corresponding position. A broader concept of homology takes into account conservative amino acid exchanges in the case of amino acid sequences, i.e. amino acids with similar chemical activity, because they usually show similar chemical activity in proteins. Therefore, the similarity of the compared sequences can also be expressed as a percentage of homology or similarity. Identity and / or homology information about the entire polypeptide or gene or only about individual regions can be provided. Therefore, the homology or identical regions of different nucleic acid or amino acid sequences are defined by the matching in the sequence. Such regions usually have the same function. They can be very small and contain only a few nucleotides or amino acids. Generally, such small regions play an important role in the overall activity of the protein. Therefore, it may be advantageous to associate sequence matching only with a separate (optionally small) region. However, unless otherwise stated, the identity or homology information in this application refers to the total length of the specified specific nucleic acid or amino acid sequence.

[0090] In the context of the present invention, the statement that an amino acid position corresponds to a numbered position in SEQ ID NO: 1 thus means that in the alignment as defined above, the corresponding position correlates with the numbered position in SEQ ID NO: 1.

[0091] To describe substitutions involving exactly one amino acid position (amino acid substitutions), the following convention is used here: first, the naturally occurring amino acid is represented in the form of the single-letter code used internationally, then the relevant sequence position, and finally the inserted amino acid. Multiple substitutions within the same polypeptide chain are separated by slashes. For insertions, the additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol such as an asterisk or a dash, or is indicated by Δ before the corresponding position. For example, A95G describes the replacement of the alanine at position 95 by glycine, A95AG describes the insertion of a glycine after the amino acid alanine at position 95, and A95* or ΔA59 describes the deletion of the alanine at position 95. This nomenclature is known to those skilled in the art in the field of enzyme technology.

[0092] The present invention also relates to a detergent comprising a protease, characterized in that the protease can be obtained from the protease according to the present invention as a starting molecule by one or more conservative amino acid substitutions. The term "conservative amino acid substitution" refers to the replacement (substitution) of one amino acid functional group with another amino acid functional group, which replacement does not lead to a change in the polarity or charge of the replaced amino acid position, such as the replacement of a non-polar amino acid functional group with another non-polar amino acid functional group. Conservative amino acid substitutions within the scope of the present invention include, for example: G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V=L=M=Y=F=W=P=S=T.

[0093] The present invention also relates to a detergent comprising a protease, characterized in that the protease can be obtained from the protease according to the present invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis, and comprises an amino acid sequence that matches the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 consecutive amino acids, and the protease (i) has at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256.

[0094] Thus, the lack of proteolytic activity is not lost or reduced. In addition, such fragmentation, deletion mutagenesis, insertion mutagenesis or substitution mutagenesis for example can also reduce the allergenicity of the relevant enzyme, thereby improving their overall suitability. Advantageously, the enzyme even keeps their proteolytic activity after mutagenesis, i.e., their proteolytic activity at least corresponds to the proteolytic activity of the starting enzyme, i.e., in a preferred embodiment, the proteolytic activity is at least 80% of the activity of the starting enzyme, preferably at least 90%. Other replacements can also show beneficial effects. Single and multiple continuous amino acids can be replaced by other amino acids.

[0095] In this case, the amino acid positions are defined by aligning the amino acid sequence of the protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as given in SEQ ID NO: 1. Furthermore, the assignment of the positions depends on the mature protein. In particular, this assignment will also be used if the amino acid sequence of the protease according to the invention contains a higher or lower number of amino acid functional groups than the protease from Bacillus lentus according to SEQ ID NO: 1. Starting from the mentioned positions in the amino acid sequence of the protease from Bacillus lentus, the modified positions in the protease according to the invention are those which are exactly assigned to these positions in the alignment.

[0096] The correct assignment of the amino acids to be modified, i.e. in particular their functional correspondence, can be further confirmed by comparative tests, according to which two positions assigned to one another based on the alignment are modified in the same way in two compared proteases and it is observed whether the enzyme activity is altered in the same way in both cases. If, for example, the amino acid substitution at a specific position of the protease according to SEQ ID NO: 1 is accompanied by a change in the enzyme parameters, e.g. M The values ​​of K and K were increased, and corresponding changes in enzyme parameters were observed in the protease variants according to the invention in which amino acid substitutions have been made by the same introduced amino acids, such as K M If the value of is also increased, this can be considered as confirmation of the correct allocation.

[0097] Protease according to the present invention can be stabilized in addition, in particular by one or more mutations, for example substitution, or by coupling with a polymer. The increase in stability during storage and / or during use (for example in a washing process) leads to longer enzyme activity, thus improving cleaning performance. In principle, all stabilization options described in the prior art and / or suitable for can be considered. Those stabilizations achieved by mutations of the enzyme itself are preferred, because this stabilization does not require any further working steps after the enzyme is recovered. Other possibilities of stabilization are, for example:

[0098] - altering the binding of metal ions, in particular the calcium binding site, for example by replacing one or more amino acids involved in calcium binding with one or more negatively charged amino acids and / or by introducing a sequence change in at least one of the sequences of the two amino acids arginine and glycine;

[0099] - protection against the effects of denaturing agents such as surfactants by mutations that cause changes in the amino acid sequence at or on the surface of the protein;

[0100] - Replace amino acids near the N-terminus with those that are likely to contact the rest of the molecule through non-covalent interactions, thus helping to maintain the globular structure.

[0101] Preferred embodiments are those in which the enzyme is stabilized in several ways, because multiple stabilizing mutations act additively or synergistically.

[0102] The present invention also relates to proteases as described above, characterized in that they have at least one chemical modification. Proteases with such modifications are referred to as derivatives, i.e., proteases are derivatized. In the context of the present application, derivatives are therefore understood to refer to those proteins in which pure amino acid chains have been chemically modified. This derivatization can be achieved, for example, in vivo by a host cell expressing a protein. In this respect, the coupling of low molecular weight compounds such as lipids or oligosaccharides is particularly noteworthy. However, derivatization can also be carried out in vitro, for example, by chemical conversion of amino acid side chains or by covalent binding to proteins by another compound. For example, amines can be coupled to the carboxyl group of the enzyme to change the isoelectric point. Another such compound can also be another protein, which is, for example, combined with a protein according to the present invention by a bifunctional compound. Derivatization can also be understood to be covalently bonded to a macromolecular carrier, or non-covalently included in a suitable macromolecular cage structure. For example, when the coupled substance is an inhibitor, derivatization can affect the specificity of the substrate or the binding strength to the substrate, or cause a temporary blocking of the enzyme activity. This may be advantageous, for example, for shelf life. This modification can further affect stability or enzyme activity. They can also be used to reduce the allergenicity and / or immunogenicity of proteins, thereby, for example, improving their skin compatibility. For example, coupling with macromolecular compounds such as polyethylene glycol can improve the stability and / or skin compatibility of proteins. The derivatives of proteins according to the present invention can also be understood in the broadest sense to refer to preparations of these proteins. Depending on recovery, processing or preparation, proteins can be combined with various other substances, such as cultures from production microorganisms. Proteins can also be intentionally added to other substances, for example, to increase their storage stability. Therefore, all preparations of proteins according to the present invention are also according to the present invention. This is also irrelevant to whether it actually shows this enzyme activity in a specific preparation. This is because it may be desired that it has no activity or only low activity during storage, and only shows its enzyme function when used. For example, this can be controlled by suitable accompanying substances. In particular, in this regard, a combined preparation of protease and a specific inhibitor is possible.

[0103] Of all the abovementioned proteases or protease variants and / or derivatives, particular preference is given in the context of the present invention to those which have an improved storage stability and / or cleaning performance compared to the starting variant, wherein the cleaning performance is determined as described below in a wash system.

[0104] Many proteases and in particular subtilisin are formed as so-called proproteins, i.e., formed together with propeptides and signal peptides, wherein the function of the signal peptide is generally to ensure that the protease is released from the cells that produce them into the periplasm or the medium around the cells, and the propeptide is generally necessary for the correct folding of the protease. The signal peptide and propeptide are generally the N-terminal parts of the proprotein. The signal peptide is cleaved from the rest of the protease by a signal peptidase under natural conditions. Subsequently, with the support of the propeptide, the correct final folding of the protease occurs. The protease then exists in its active form and cleaves the propeptide itself. After the cleavage of the propeptide, the mature protease (particularly subtilisin) then exerts its catalytic activity in the absence of the N-terminal amino acid that was initially present. For general industrial applications, in particular in the context of the present invention, mature proteases, i.e., enzymes processed after their production, are preferred relative to the proprotein. The protease can also be modified by the cell that produces the protease after the polypeptide chain is produced, for example, by connecting sugar molecules, formylation or amination, etc. This type of modification is a post-translational modification, and can but does not necessarily have an impact on the function of the protease.

[0105] "Variant" as used herein refers to a natural or artificially produced variant of a natural protease, which has an amino acid sequence modified from a reference form. In addition to the amino acid changes discussed above, the protease according to the present invention may have other amino acid changes, particularly amino acid substitutions, insertions or deletions. For example, such proteases are developed by targeted genetic modification, i.e., by mutagenesis, and are optimized for specific applications or for specific properties (e.g., for their catalytic activity or stability, etc.). In addition, nucleic acids according to the present invention can be introduced into recombinant methods, so that they can be used to produce completely new types of proteases or other polypeptides. The purpose is to introduce targeted mutations such as substitutions, insertions or deletions into known molecules, so as to, for example, improve the cleaning performance of the enzyme. For this reason, in particular, the surface charge and / or isoelectric point of the molecule can be changed, thereby changing their interaction with the substrate. For example, the net charge of the enzyme can be changed to affect substrate binding, particularly for detergents and cleaning agents. Alternatively or in addition, one or more corresponding mutations can increase the stability or catalytic activity of the enzyme, thereby improving its cleaning performance. The favorable properties of single mutations, such as single substitutions, can complement each other. Therefore, within the scope of the present invention, proteases that have been optimized in certain properties can be further developed, for example, in terms of their stability to surfactants and / or bleaching agents and / or other components.

[0106] The detergent according to the invention contains, in increasing order of preference, 1×10 -8 % to 5% by weight, 0.0001 to 1% by weight, 0.0005 to 0.5% by weight, 0.001 to 0.1% by weight, based in each case on active protein and on the total weight of the detergent.

[0107] In another embodiment of the present invention, the protease is characterized in that its cleaning performance (after storage, for example for more than 2 weeks) is not significantly reduced compared to a conventional protease and / or a wild-type enzyme (SEQ ID NO: 1) and / or a corresponding starting variant with improved performance (SEQ ID NO: 2 from WO 2013 / 060621 A1), i.e., it has at least 80%, preferably at least 100%, more preferably at least 110% or more of the reference washing performance.

[0108] In the context of the present invention, cleaning performance is understood to mean the ability of a reagent to partially or completely remove existing stains, particularly the mitigation performance of one or more stains on a textile. The example of such stains is blood on cotton or chocolate milk / cigarette ash on cotton, cocoa powder on cotton or porridge on cotton. In the context of the present invention, the detergent comprising the protease or the washing liquid formed by the reagent and the protease itself all have cleaning performance. Therefore, the cleaning performance of the protease contributes to the cleaning performance of the reagent or the washing liquid formed by the reagent. Cleaning performance is preferably measured as described below.

[0109] Washing liquor is understood to mean a use solution containing detergent, which is acted on textiles or fabrics and thereby comes into contact with stains present on the textiles or fabrics. Washing liquor is usually formed at the beginning of the washing process and when the detergent is diluted with water, for example in a washing machine or in another suitable container.

[0110] Cleaning performance can be measured in a washing system containing a detergent and protease with a dosage of 2.0 to 8.0 g / L of washing liquid. The protease to be compared is used at the same concentration (based on active protein). Using the corresponding protease with comparable activity ensures that even if there is a difference in the ratio of active substance to total protein (value of specific activity), the corresponding enzyme properties, such as the cleaning performance to certain stains, can be compared. Usually, low specific activity can be compensated by adding a larger amount of protein. In addition, if the enzyme to be detected has different affinities to the test substrate in the activity test, the enzyme to be detected can also be used with the same amount or weight. In this article, the term "same amount" refers to the molar dosage of the enzyme to be detected. The term "same weight" refers to the enzyme to be detected using the same weight.

[0111] The concentration of the protease in the detergent used in the washing system is 0.0001-0.1% by weight, preferably 0.001-0.1% by weight, more preferably 0.01-0.06% by weight, particularly preferably 0.001-0.02% by weight, based on active protein.

[0112] The preferred liquid detergent composition for this washing system is as follows (all data are in weight percentage): <1% defoamer, 1-4% citric acid, 0.5-3% glycerol, 0.3-2% NaOH, 4-8% 1,2-propylene glycol, 2-5% FAEOS (fatty alcohol ether sulfate), 5-9% nonionic surfactant (FAEO), 7-12% anionic surfactant (LAS), 0.5-1.5% protease stabilizer, 1-3% palm kernel oil fatty acid, 0.5-2% HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 2-6% monoethanolamine (MEA), 0.2-1% soil release polymer, and the balance is softened water. The dosage of liquid detergent is preferably 2.0-8.0 g / l of washing liquid, for example 2.5 g / l, 3.2 g / l, 3.5 g / l, 4.0 g / l, 4.7 g / l, 4.9 g / l, 5.5 g / l or 5.9 g / l of washing liquid or about 55 g / work. Washing is preferably carried out in a pH range of about 9 to about 12, preferably about 10 to 11, particularly preferably at a pH of 10.

[0113] The whiteness as a measure of the cleaning performance, i.e. the degree of lightening of stains, is determined by optical measuring methods, preferably photometry. A suitable device for this purpose is, for example, a Minolta CM508d spectrometer. Typically, the device used for the measurement is calibrated in advance with a white standard, preferably a provided white standard.

[0114] Methods for determining protease activity are well known to those skilled in the art of enzyme technology and are often used. For example, such methods are disclosed in Tenside ["Surfactants"], Vol. 7 (1970), pp. 125-132. Alternatively, protease activity can be determined by releasing the chromophore p-nitroaniline (pNA) from the substrate Suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (AAPF). The protease cuts the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410nm, and its time course is a measure of enzyme activity (see Del Mar et al., 1979). The measurement is carried out at a temperature of 25°C, a pH of 8.6, and a wavelength of 410nm. The measurement time is 5min, and the measurement interval is 20s to 60s. Protease activity is usually expressed in protease units (PE). For example, suitable protease activity totals 2.25, 5 or 10 PE / ml washings. However, protease activity is not equal to zero.

[0115] An alternative test for determining the proteolytic activity of the protease according to the present invention is an optical measurement method, preferably a photometric method. Suitable tests include the protease-dependent cleavage of the substrate protein casein. It is cleaved into many smaller partial products by the protease. Compared with uncracked casein, the overall of these partial products has an increased absorption at 290nm, and a photometer can be used to measure this increased absorption, thereby drawing conclusions about the enzymatic activity of the protease.

[0116] Protein concentration can be determined using known methods, such as the BCA method (bicinchoninic acid; 2,2'-bisquinolinyl-4,4'-dicarboxylic acid) or the biuret method (Gornall et al. (1948), J. Biol. Chem., 177: 751-766). In this regard, the active protein concentration can be determined by titrating the active center with a suitable irreversible inhibitor and determining the residual activity (Bender et al. (1966), J. Am. Chem. Soc. 88 (24): 5890-5913).

[0117] Preferred embodiments of the protease according to the invention achieve this advantageous cleaning performance even at low temperatures, in particular in the temperature range of 10-60°C, preferably 15-50°C, particularly preferably 20-40°C.

[0118] According to the present invention, detergent is to be understood as detergent of all possible types, including concentrates and undiluted agents, for commercial scale, for washing machines or for hand washing or cleaning. These include detergents for textiles, carpets or natural fibers, for which the term detergent is used. In the context of the present invention, detergent also includes auxiliary detergents added to the actual detergent to realize other effects when washing textiles manually or using a machine. In addition, detergent in the context of the present invention also includes textile pre-treatment agents and post-treatment agents, i.e. agents contacted with the washings before the actual washing cycle, for example agents for loosening stubborn stains, and agents for further desired characteristics of the washings in the step after the actual textile washing, such as comfort, wrinkle resistance or low static charge. The last-mentioned agent especially includes fabric softener.

[0119] In addition to the protease according to the invention, the detergents according to the invention, which can be present as powdered or granular solids, in the form of compressed or recompressed particles, as homogeneous solutions or suspensions, can contain all known ingredients customary in such detergents, wherein the detergents preferably contain such other ingredients in an amount of 0.01 to 99.9% by weight. The detergents according to the invention can contain in particular surfactants, builders, polymers, glass corrosion inhibitors, corrosion inhibitors, bleaches such as peroxy compounds, bleach activators or bleach catalysts. They can also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, chelating agents, electrolytes, pH regulators and / or other auxiliaries such as optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators, dyes and perfumes and combinations thereof.

[0120] In particular, anionic surfactants, nonionic surfactants and their mixtures are suitable. The agent according to the invention can contain one or more surfactants, of which anionic surfactants, nonionic surfactants and their mixtures are particularly suitable, but can also contain cationic surfactants, zwitterionic surfactants and / or amphoteric surfactants. The agent preferably contains 5-70% by weight of surfactant, preferably 5-55% by weight and more preferably 5-35% by weight of surfactant. The detergent according to the invention particularly preferably contains no more than 30% by weight of surfactant, very particularly preferably no more than 20% by weight of surfactant.

[0121] Suitable anionic surfactants are in particular soaps and those containing sulfate or sulfonate groups, preferably with alkali metal ions as cations. Soaps which can be used are preferably saturated or unsaturated C 12-18 Alkali metal salts of fatty acids. Such fatty acids may also be used in an incompletely neutralized form. Suitable sulfate surfactants include C 12-18 Salts of sulfuric acid half esters of fatty alcohols and sulfated products of the nonionic surfactants mentioned with a low degree of ethoxylation. Surfactants of the sulfonate type which can be used include, for example, C 9-14 Alkylbenzene sulfonates, from C 12-18 Alkylsulfonates obtained from alkanes (e.g. by sulfochlorosulfonation or sulfoxidation and subsequent hydrolysis or neutralization), C 12-18 Olefin sulfonates, mixtures of olefin and hydroxyalkane sulfonates, disulfonates such as those obtained from C 12-18 Monoolefins which are obtained by sulfonation with gaseous sulfur trioxide and subsequent alkaline or acidic hydrolysis of the sulfonation products, and α-sulfo fatty acid esters (ester sulfonates) formed by sulfonation of fatty acid methyl or ethyl esters, for example the α-sulfonated methyl esters of hydrogenated coconut oil fatty acids, hydrogenated palm kernel oil fatty acids or hydrogenated tallow fatty acids.

[0122] The agent preferably has 2-55% by weight, more preferably 3-35% by weight, of anionic surfactants; very preferably the agent contains 3-15% by weight of alkylbenzenesulfonates. In addition, the agent may preferably also contain other anionic surfactants, in particular alkyl ether sulfates, and nonionic surfactants, in particular fatty alcohol alkoxylates. These may therefore constitute the remainder of the surfactants.

[0123] Suitable alkylbenzene sulfonates are preferably selected from linear or branched alkylbenzene sulfonates of the formula

[0124]

[0125] wherein R' and R" are independently H or alkyl and together contain 6 to 19, preferably 7 to 15, in particular 9 to 13 C atoms. A very particularly preferred representative is sodium dodecylbenzenesulfonate.

[0126] As alkyl (alkenyl) sulfates, alkali metal salts, in particular sodium salts, of the sulfuric acid half esters of the following compounds are preferred: 12-18 Fatty alcohols (such as coconut oil fatty alcohol, tallow fatty alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol or stearyl alcohol) or C 10-20 Carbonyl alcohols and secondary alcohols with these chain lengths. Alkyl (alkenyl) sulfates with the chain lengths mentioned, which contain synthetic linear alkyl functions based on petrochemical preparations and have a degradation behavior similar to that of suitable compounds based on fat chemical raw materials, are also preferred. From a washing point of view, C 12-16 Alkyl sulfate and C 12-15 Alkyl sulfate and C 14-15 Alkyl sulfate.

[0127] Fatty alcohol ether sulfates, such as linear or branched C 7-21 Alcohols (such as 2-methyl-branched C 9-11 Alcohol or C with 1-4 EO 12-18 Sulfate monoesters of fatty alcohols are also suitable.

[0128] Examples of suitable alkyl ether sulfates are those of the formula R 1 -O-(AO) n -SO3 - X + In this formula, R 1 represents a linear or branched, substituted or unsubstituted alkyl functional group, preferably a linear, unsubstituted alkyl functional group, particularly preferably a fatty alcohol functional group. 1is selected from the group consisting of decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl functional groups and mixtures thereof, wherein representatives with an even number of C atoms are preferred. Particularly preferred functional groups R 1 Derived from C 12 -C 18 Fatty alcohols, for example, derived from coconut oil, tallow, lauryl, myristyl, cetyl or stearyl alcohol or derived from C 10 -C 20 Oxyl alcohol. AO represents an ethylene oxide (EO) group or a propylene oxide (PO) group, preferably an ethylene oxide group. The index n represents an integer from 1 to 50, preferably from 1 to 20, in particular from 2 to 10. Very particularly preferably, n represents the number 2, 3, 4, 5, 6, 7 or 8. X + represents a monovalent cation or the nth part of an n-valent cation, preferably an alkali metal ion, including Na + or K + In this case, Na + is the most preferred. Other cations X + Can be selected from NH4 + 、1 / 2Zn 2+ 、1 / 2Mg 2+ 、1 / 2Ca 2+ 、1 / 2Mn 2+ and mixtures thereof.

[0129] In various embodiments, the alkyl ether sulfate may be selected from fatty alcohol ether sulfates of the following formula:

[0130]

[0131] wherein k=11 to 19 and n=2, 3, 4, 5, 6, 7 or 8. Very particularly preferred representatives are C 12-14 Sodium fatty alcohol ether sulfate (k=11-13, n=2). The stated degree of ethoxylation is a statistical average value, which for a particular product can be an integer or a fraction. The stated degree of alkoxylation is a statistical average value, which for a particular product can be an integer or a fraction. Preferred alkoxylates / ethoxylates have a narrow homolog distribution (narrow range ethoxylates, NRE).

[0132] It has been shown that the cold wash performance is excellent if the detergents additionally contain soap(s) as anionic surfactants. Preferred detergents are therefore characterized in that they contain soap(s). Suitable are saturated fatty acid soaps, for example the salts of lauric acid, myristic acid, palmitic acid, stearic acid, hydrogenated erucic acid and behenic acid, and in particular soap mixtures derived from natural fatty acids, for example coconut oil fatty acid, palm kernel oil fatty acid or tallow fatty acid.

[0133] Suitable nonionic surfactants are in particular alkyl glucosides and ethoxylation and / or propoxylation products of alkyl glucosides or linear or branched alcohols, each having 8 to about 18 C atoms in the alkyl moiety and having 3 to 20, preferably 4 to 10, alkyl ether groups. Also suitable are the corresponding ethoxylation and / or propoxylation products of N-alkylamines, vicinal diols, fatty acid esters and fatty acid amides, which correspond with respect to the alkyl moiety to the long-chain alcohol derivatives mentioned, and alkylphenols having 5 to 12 C atoms in the alkyl function.

[0134] Nonionic surfactants preferably used are alkoxylated, advantageously ethoxylated, in particular primary alcohols, preferably having 8 to 18 C atoms and an average of 1 to 12 mol of ethylene oxide (EO) per mol of alcohol, wherein the alcohol function may be linear or preferably methyl-branched in the 2-position, or may contain linear and methyl-branched functional groups, such as are usually present in carbonyl alcohol functions, in the form of a mixture. However, alcohols of natural origin having 12 to 18 C atoms (e.g. coconut alcohol, palmityl alcohol, tallow alcohol or oleyl alcohol) and alcohol ethoxylates with linear functional groups having an average of 2 to 8 EO per mol of alcohol are particularly preferred. Preferred ethoxylated alcohols include, for example, C 3 or 4 EO, C 4 or 5 EO, C 5 or 6 EO, C 6 or 7 EO, C 7 or 8 EO, C 8 or 9 EO. 12-14 Alcohol, C with 7 EO 9-11 Alcohols, C with 3 EO, 5 EO, 7 EO or 8 EO 13-15 Alcohols, C with 3 EO, 5 EO or 7 EO 12-18 Alcohols, and mixtures thereof, such as C 12-14 Alcohol and C with 5 EO 12-18 The degree of ethoxylation is a statistical average which can be an integer or a fraction for a specific product. Preferred alcohol ethoxylates have a narrow homologue distribution (narrow range ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples of these are tallow fatty alcohols with 14 EO, 25 EO, 30 EO or 40 EO.

[0135] Another class of nonionic surfactants which are preferably used are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, which preferably have 1 to 4 carbon atoms in the alkyl chain, in particular fatty acid methyl esters, which are used either as the sole nonionic surfactant or in combination with other nonionic surfactants.

[0136] Another class of nonionic surfactants that can be advantageously used are alkyl polyglycosides (APGs). Alkyl polyglycosides that can be used have the general formula RO(G) z , in which R represents a linear or branched, in particular methyl-branched in the 2-position, saturated or unsaturated, aliphatic function having 8 to 22, preferably 12 to 18 C atoms, and G is a symbol for a monosaccharide unit having 5 or 6 C atoms, preferably glucose. The degree of glycosidation z is 1.0 to 4.0, preferably 1.0 to 2.0, in particular 1.1 to 1.4. Preference is given to using linear alkyl polyglycosides, i.e. alkyl polyglycosides in which the polyglycol functions are glucose functions and the alkyl functions are n-alkyl functions.

[0137] Nonionic surfactants of the amine oxide type (e.g. N-coconut alkyl-N,N-dimethylamine oxide and N-tallow alkyl-N,N-dihydroxyethylamine oxide), and nonionic surfactants of fatty acid alkanolamides, may also be suitable. The amount of these nonionic surfactants is preferably no more than the amount of ethoxylated fatty alcohols, in particular no more than half thereof.

[0138] The nonionic surfactants which can be preferably used in the detergents according to the invention have the formula R 1 –CH(OH)CH2O-(AO) w -(A'O) x -(A”O) y -(A”'O) z -R 2 , where R 1 represents a straight chain or branched, saturated, monounsaturated or polyunsaturated C 6-24 Alkyl or alkenyl functional group; R 2 represents a linear or branched hydrocarbon functional group having 2 to 26 carbon atoms; A, A', A"' and A"" are independently selected from the group consisting of -CH2CH2, -CH2CH2-CH2, -CH2-CH(CH3), -CH2-CH2-CH2-CH2, -CH2-CH(CH3)-CH2-, -CH2-CH(CH2-CH3), and w, x, y and z represent values ​​between 0.5 and 120, wherein x, y and / or z may also be 0. By adding the above formula R 1 –CH(OH)CH2O-(AO) W -(A'O)x-(A”O)y -(A”'O) z -R 2 The cleaning performance of the enzyme-containing preparations according to the invention can be significantly improved not only compared to systems without surfactants, but also compared to systems containing alternative nonionic surfactants (for example selected from the group of polyalkoxylated fatty alcohols).

[0139] By using these nonionic surfactants which have one or more free hydroxyl groups on one or both terminal alkyl groups, the stability of the enzymes comprised in the detergent formulations according to the invention can be significantly improved.

[0140] Particularly preferred are terminally capped poly(oxyalkylated) nonionic surfactants according to formula R 1 O[CH2CH2O] X CH2CH(OH)R 2 , except for the functional group R which represents a linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon functional group having 2 to 30 carbon atoms, preferably having 4 to 22 carbon atoms 1 In addition, it also contains a linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon functional group R having 1 to 30 carbon atoms 2 , wherein x represents a value between 1 and 90, preferably a value between 30 and 80, in particular a value between 30 and 60.

[0141] Formula R 1 O[CH2CH(CH3)O] x [CH2CH2O] y CH2CH(OH)R 2 Surfactants of the type R 1 represents a linear or branched aliphatic hydrocarbon functional group of 4 to 18 carbon atoms or a mixture thereof, R 2 represents a linear or branched hydrocarbon functional group having 2 to 26 carbon atoms or a mixture thereof, x represents a value between 0.5 and 1.5, and y represents a value of at least 15. These groups of nonionic surfactants include, for example, C 2-26 Fatty alcohol-(PO)1-(EO) 15-40 -2-Hydroxyalkyl ethers, especially including C 8-10 Fatty alcohol-(PO)1-(EO) 22 -2-Hydroxydecyl ether.

[0142] Particularly preferably, the formula R 1 O[CH2CH2O] x [CH2CH(R 3 )O] y CH2CH(OH)R2 The terminal-capped poly(oxyalkylated) nonionic surfactant, wherein R 1 and R 2 independently of one another represent a linear or branched, saturated or monounsaturated or polyunsaturated hydrocarbon functional group having 2 to 26 carbon atoms, R 3 Independently selected from -CH3, -CH2CH3-, -CH2CH2-CH3, -CH(CH3)2, but preferably represents -CH3, x and y independently represent values ​​between 1-32, among which very particularly preferred is a non-ionic surfactant in which R3 = -CH3 and x is a value of 15-32 and y is a value of 0.5-15.

[0143] Other nonionic surfactants that can be preferably used are those of formula R 1 O[CH2CH(R 3 )O] x [CH2] k CH(OH)[CH2] j OR 2 The terminal-capped poly(oxyalkylated) nonionic surfactant, wherein R 1 and R 2 represents a linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon functional group having 1 to 30 carbon atoms, R 3 represents H or a methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl or 2-methyl-2-butyl functional group, x represents a value between 1 and 30, k and j represent a value between 1 and 12, preferably a value between 1 and 5. If the value of x is ≥ 2, then the above formula R 1 O[CH2CH(R 3 )O] x [CH2] k CH(OH)[CH2] j OR 2 Each R in 3 Can be different. 1 and R 2 Preference is given to linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon functional groups having 6 to 22 carbon atoms, with functional groups having 8 to 18 carbon atoms being particularly preferred. 3 , particularly preferably H, -CH3 or -CH2CH3. Particularly preferred values ​​for x are in the range of 1 to 20, in particular 6 to 15. As mentioned above, if x ≥ 2, then each R in the above formula 3 can be different. In this way, the oxyalkylene units in the square brackets can be varied. For example, if x represents 3, the functional group R 3 To form ethylene oxide (R 3 =H) or propylene oxide (R3 =CH3) units, which can be linked together in any order, for example (EO)(PO)(EO), (EO)(EO)(PO), (EO)(EO)(EO), (PO)(EO)(PO), (PO)(PO)(EO) and (PO)(PO)(PO). Here, for example, a value of 3 for x is selected, and it is entirely possible to be larger, wherein the range of variation increases with increasing values ​​of x and includes combinations of a large number of (EO) groups with a small number of (PO) groups, or vice versa. Particularly preferred terminally capped poly(oxyalkylated) alcohols of the above formula have values ​​of k=1 and j=1, so that the above formula is simplified to R 1 O[CH2CH(R 3 )O] x CH2CH(OH)CH2OR 2 In the last mentioned formula, R 1 , R 2 and R 3 As defined above, x represents a number from 1 to 30, preferably from 1 to 20, in particular from 6 to 18. 1 and R 2 With 9 to 14 C atoms, R 3 Surfactants representing H, and x taking values ​​of 6 to 15 are particularly preferred.

[0144] Finally, the general formula R 1 –CH(OH)CH2O-(AO) w -R 2 Nonionic surfactants have proven to be particularly effective, among which R 1 represents a straight chain or branched, saturated, monounsaturated or polyunsaturated C 6-24 Alkyl or alkenyl functional group; R 2 represents a linear or branched hydrocarbon functional group having 2 to 26 carbon atoms; A represents a functional group selected from -CH2CH2, -CH2CH2-CH2, -CH2-CH(CH3), and w represents a value of 1 to 120, preferably 10 to 80, in particular 20 to 40. These groups of nonionic surfactants include, for example, C 4-22 Fatty alcohol-(EO) 10 -80-2-Hydroxyalkyl ethers, especially C 8-12 Fatty alcohol-(EO) 22 -2-Hydroxydecyl ether and C 4-22 Fatty alcohol-(EO) 40-80 -2-Hydroxyalkyl ether.

[0145] Preferred detergents are characterized in that they contain at least one nonionic surfactant, preferably a nonionic surfactant selected from the group of hydroxy mixed ethers, in a weight ratio of 0.2 to 20% by weight, preferably 1 to 18% by weight, more preferably 2 to 15% by weight and particularly preferably 5 to 10% by weight, relative to the total weight of the detergent.

[0146] Suitable amphoteric surfactants are, for example, those of the formula (R iii )(R iv )(R v )N + CH2COO - Betaines, of which R iii represents an alkyl group having 8 to 25, preferably 10 to 21, carbon atoms, which is optionally interrupted by heteroatoms or heteroatom groups, and R iv and R v represents identical or different alkyl groups having 1 to 3 carbon atoms, in particular C 10-18 Alkyl dimethyl carboxymethyl betaine and C 11-17 Alkylamidopropyl dimethyl carboxymethyl betaine.

[0147] Suitable cationic surfactants are especially those of the formula (R vi )(R vi ')(R viii )(R ix )N + X - A quaternary ammonium compound, wherein R vi To R ix are four identical or different, in particular two long-chain and two short-chain alkyl groups, and X - The cationic surfactants are anions, in particular halogen ions, such as didecyldimethylammonium chloride, alkylbenzyldidecylammonium chloride and mixtures thereof. Other suitable cationic surfactants are tetravalent surface-active compounds, in particular tetravalent surface-active compounds with sulfonium, phosphonium, iodonium or arsonium groups, which are also known as antimicrobial active ingredients. By using tetravalent surface-active compounds with antimicrobial action, an antimicrobial action can be provided to the agent, or an antimicrobial action that may already be present due to other ingredients can be improved.

[0148] Complexing agents are another preferred component of detergents according to the present invention. Particularly preferred complexing agents are phosphonates, provided that their use is permitted by law. In addition to 1-hydroxyethane-1,1-diphosphonic acid, complexed phosphonates include many different compounds such as diethylenetriamine penta (methylenephosphonic acid) (DTPMP). Hydroxyalkane or aminoalkane phosphonates are particularly preferred in this application. Among hydroxyalkane phosphonates, 1-hydroxyethane-1,1-diphosphonate (HEDP) is particularly important as a co-builder. It is preferably used in the form of a sodium salt, wherein the disodium salt reacts in a neutral manner, and the tetrasodium salt reacts in an alkaline (pH 9) manner. Possibly preferred aminoalkane phosphonates include ethylenediaminetetramethylenephosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and their higher homologues. They are preferably used in the form of neutrally reacted sodium salts, such as the hexasodium salt of EDTMP or the heptasodium and octasodium salts of DTPMP. Among the phosphonates, HEDP is preferably used as a builder. In addition, aminoalkanephosphonates have a significant heavy metal binding capacity. Therefore, in particular if the agent also contains a bleaching agent, it is preferred to use aminoalkanephosphonates, in particular DTPMP, or a mixture of the phosphonates mentioned. Preferred detergents in the context of the present application contain one or more phosphonates selected from aminotrimethylenephosphonic acid (ATMP) and / or its salts; ethylenediaminetetra(methylenephosphonic acid) (EDTMP) and / or its salts; diethylenetriaminepenta(methylenephosphonic acid) (DTPMP) and / or its salts; 1-hydroxyethane-1,1-diphosphonic acid (HEDP) and / or its salts; 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC) and / or its salts; hexamethylenediaminetetra(methylenephosphonic acid) (HDTMP) and / or its salts; nitrilotri(methylenephosphonic acid) (NTMP) and / or its salts. Detergents containing 1-hydroxyethane-1,1-diphosphonic acid (HEDP) or diethylenetriamine penta(methylenephosphonic acid) (DTPMP) as phosphonic acid esters are particularly preferred. Of course, the detergents according to the invention can contain two or more different phosphonic acid esters. Preferred detergents according to the invention are characterized in that the detergent contains at least one complexing agent selected from the group of phosphonic acid esters, preferably 1-hydroxyethane-1,1-diphosphonic acid ester, the weight ratio of the phosphonic acid ester relative to the total weight of the detergent is preferably 0.1-8.0% by weight, more preferably 0.2-5.0% by weight, even more preferably 0.3-3.0% by weight, particularly preferably 0.5-2.0% by weight.

[0149] The detergents according to the invention also preferably contain a builder, preferably at least one water-soluble and / or water-insoluble organic and / or inorganic builder. Such builders include in particular silicates, carbonates and organic co-builders.

[0150] Polycarboxylates / polycarboxylic acids, polymeric polycarboxylates, aspartic acid, polyacetals, dextrins, other organic co-builders and phosphonates are particularly noteworthy as organic co-builders. These classes of substances are described below. If desired, such organic co-builder substances may be contained in an amount of up to 40 wt %, in particular up to 25 wt %, preferably 1 to 8 wt %.

[0151] Suitable organic builder substances are, for example, polycarboxylic acids which can be used in the form of the free acids and / or their sodium salts, where polycarboxylic acids are understood to be carboxylic acids with more than one acid function. These are, for example, citric acid, adipic acid, succinic acid, glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid, sugar acids, carboxylmethylinulin, monomeric and polymeric aminopolycarboxylic acids, in particular glycinediacetic acid. The organic builder substances may be present in an amount of up to 50% by weight, in particular up to 25% by weight, preferably from 10 to 20% by weight and particularly preferably from 1 to 5% by weight.

[0152] In addition to their builder action, free acids also have the characteristics of acidifying components usually and are therefore also used to provide detergents with lower and milder pH values. Citric acid, succinic acid, glutaric acid, adipic acid, gluconic acid and any mixture thereof are particularly mentioned here. Citric acid or citric acid salts are particularly preferably used as builder substances. Other particularly preferred builder substances are selected from methylglycine diacetic acid (MGDA), glutamic acid diacetate (GLDA), aspartic acid diacetate (ASDA), hydroxyethyliminodiacetate (HEIDA), iminodisuccinate (IDS), ethylenediamine disuccinate (EDDS), carboxymethyl inulin and polyaspartate.

[0153] In a preferred embodiment, citric acid and / or citrate are used as water-soluble organic builders. Particularly preferably, 0.5-25% by weight, preferably 0.75-12.5% ​​by weight, more preferably 1-4% by weight of citric acid and / or 0.5-25% by weight, preferably 0.75-12.5% ​​by weight, more preferably 1-4% by weight of citrate, preferably alkali metal citrate, more preferably sodium citrate is used. Citric acid / citrate can each be used in the form of its hydrate, for example citric acid can be used in the form of a monohydrate and citrate can be used in the form of trisodium citrate dihydrate.

[0154] Polymeric polycarboxylates are also suitable as builders. These are, for example, alkali metal salts of polyacrylic acid or polymethacrylic acid, for example those having a relative molecular weight of 500 to 70,000 g / mol. For the purposes of this application, the molar masses indicated for polymeric polycarboxylates are the weight-average molar masses M of the specific acid form. w , which in principle uses the gel permeation chromatography (GPC) determination using a UV detector. The measurement is carried out relative to an external polyacrylic acid standard, which produces a true molecular weight value due to its structural relationship with the test polymer. These specifications are very different from the molecular weight specifications using polystyrene sulfonic acid as a standard. The molar mass measured relative to polystyrene sulfonic acid is usually significantly higher than the molar mass provided in the present application. Suitable polymers are particularly preferably polyacrylates with a molecular weight of 2,000 to 20,000 g / mol. In this group, due to its excellent solubility, short-chain polyacrylates with a molar mass of 2,000 to 10,000 g / mol, particularly preferably 3,000 to 5,000 g / mol can be preferred again. In addition, copolymerized polycarboxylates are suitable, particularly those of acrylic acid and methacrylic acid and those of acrylic acid or methacrylic acid and maleic acid. The copolymer of acrylic acid and maleic acid containing 50% by weight to 90% by weight of acrylic acid and 50% by weight to 10% by weight of maleic acid has been found to be particularly suitable. Their relative molecular weight, based on the free acid, is generally from 2,000 to 70,000 g / mol, preferably from 20,000 to 50,000 g / mol, and in particular from 30,000 to 40,000 g / mol.

[0155] Solid reagent according to the present invention preferably contains at least one water-soluble and / or water-insoluble organic and / or inorganic builder. Water-soluble organic builder materials include the above-mentioned organic builder materials. In addition to the above-mentioned water-soluble organic builder, reagent of the present invention can also further contain inorganic water-soluble builders. In particular, alkali metal silicates, alkali metal carbonates, alkali metal bicarbonates, alkali metal phosphates and / or sesquicarbonates, which can exist in the form of their alkaline, neutral or acidic sodium or potassium salts, can be used as water-soluble inorganic builder materials. A small amount of calcium carbonate can also be optionally included in the solid textile detergent. For example, water-soluble crystalline and / or amorphous alkali metal silicates are suitable. Alkali metal silicates suitable as builders in reagents according to the present invention preferably have an alkali metal oxide and SiO2 molar ratio less than 0.95, particularly 1:1.1 to 1:12, and can exist in amorphous or crystalline form. The preferred alkali metal silicate is sodium silicate, especially amorphous sodium silicate, with a Na2O:SiO2 molar ratio of 1:2 to 1:2.8. x O 2x+1 Crystalline phyllosilicates of ·y H2O, in which x, referred to as the modulus, is a number from 1.9 to 22, in particular from 1.9 to 4, and y is a number from 0 to 33, with preferred values ​​for x being 2, 3 or 4, are preferably used as crystalline silicates, which may be present alone or in a mixture with amorphous silicates. Preferred crystalline phyllosilicates are those in which x in the general formula is 2 or 3. β- and δ-sodium disilicate (Na2Si2O5·y H2O) are particularly preferred. Virtually water-free crystalline alkali metal silicates of the above general formula, in which x is a number from 1.9 to 2.1 and are prepared from amorphous alkali metal silicates, can also be used in the agents according to the invention. In another embodiment according to the invention, crystalline phyllosilicates having a modulus of 2 to 3 are used, which can be prepared from sand and soda. In another embodiment of the agent according to the invention, crystalline sodium silicates having a modulus in the range of 1.9 to 3.5 are used. In the reagent containing amorphous and crystalline alkali metal silicates, the weight ratio of amorphous alkali metal silicate to crystalline alkali metal silicate is preferably 1:2 to 2:1, in particular 1:1 to 2:1. The crystalline layered silicate of the above formula (I) is sold by Clariant GmbH under the trade name Na-SKS, for example Na-SKS-1 (Na2Si 22 O 45 ·x H2O, Kenyait), Na-SKS-2 (Na2Si 14 O 29 ·x H2O, magadiite), Na-SKS-3 (Na2Si8O 17·x H2O) or Na-SKS-4 (Na2Si4O9·x H2O, macatite). Of these, Na-SKS-5 (α-Na2Si2O5), Na-SKS-7 (δ-Na2Si2O5, natrosilite), Na-SKS-9 (NaHSi2O5·3H2O), Na-SKS-10 (NaHSi2O5·3H2O, kanemite), Na-SKS-11 (t-Na2Si2O5) and Na-SKS-13 (NaHSi2O5), but especially Na-SKS-6 (5-Na2Si2O5), are particularly suitable. In one embodiment of the reagent according to the invention, a granular compound made of a crystalline layered silicate and a citrate, a crystalline layered silicate and the above-mentioned (co)polymerized polycarboxylic acid or an alkali metal silicate and an alkali metal carbonate is used, for example, which can be named 15 commercially available. The agent according to the invention preferably contains such water-soluble inorganic builder materials in an amount of 1 to 20% by weight, in particular 5 to 15% by weight. Equally important as water-soluble inorganic builder substances are carbonates (and bicarbonates), in particular sodium carbonate, and phosphonic acid / phosphonates. Phosphonic acid is also understood to mean optionally substituted alkylphosphonic acids, which may also have a plurality of phosphonic acid groups (so-called polyphosphonic acids). They are preferably selected from hydroxyl and / or aminoalkylphosphonic acids and / or their alkali metal salts, for example dimethylaminomethanediphosphonic acid, 3-aminopropane-1-hydroxy-1,1-diphosphonic acid, 1-amino-1-phenylmethanediphosphonic acid, 1-hydroxyethane-1,1-diphosphonic acid (HEDP), aminotri(methylenephosphonic acid), N,N,N',N'-ethylenediaminetetra(methylenephosphonic acid), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP) and acylated derivatives of phosphorous acid, which may also be used in any mixture.

[0156] In various embodiments, the builder system preferably comprises the following components, in each case based on the total mass of the agents:

[0157] a) 0 to 10 wt. %, preferably 1 to 4 wt. % citric acid; and / or

[0158] b) 0 to 10% by weight, preferably 1 to 4% by weight, of a citrate, preferably an alkali metal citrate; and / or

[0159] c) 0 to 40% by weight, preferably 0 to 15% by weight, particularly preferably 1 to 3% by weight, of alkali metal carbonate, which may also be at least partly replaced by alkali metal hydrogencarbonate, in particular sodium carbonate; and / or

[0160] d) 0 to 20% by weight, preferably 3 to 10% by weight, of alkali metal silicate;

[0161] e) 0 to 10% by weight, preferably 0.02 to 2% by weight, particularly preferably 0.5 to 2% by weight, of phosphonic acid and / or alkali metal phosphonates, in particular HEDP and / or DTPMP; and / or

[0162] f) 0 to 10% by weight, preferably 0.5 to 3% by weight, of polymeric polycarboxylates, in particular polyacrylates.

[0163] For component f), for example, alkali metal salts of polyacrylic acid or polymethacrylic acid are suitable, for example those with a relative molecular weight of 500 to 70,000 g / mol. This substance class has been described in detail above. The (co)polymeric polycarboxylic acid esters can be used as powders or as aqueous solutions.

[0164] The agents according to the invention are preferably free of phosphate builders, ie they contain less than 1% by weight, preferably no intentionally added phosphate builders.

[0165] The agent may also contain water-insoluble builder substances. As water-insoluble inorganic builder materials, in particular crystalline or amorphous water-dispersible alkali metal aluminosilicates are used, in an amount of up to 50% by weight, preferably not more than 40% by weight, in particular from 3 to 20% by weight, particularly preferably from 1 to 15% by weight. Of these, crystalline sodium aluminosilicates of detergent quality, in particular zeolite A, zeolite P, zeolite MAP and optionally zeolite X, alone or in the form of a mixture, for example in the form of cocrystals of zeolites A and X ( Aluminosilicates are preferably selected from the group consisting of Aluminosilicates of the invention. Aluminosilicates of the invention ...

[0166] In addition to the above-described builders, the detergent may also contain polymers with a cleaning effect. The weight ratio of the polymer is preferably 0.1 to 20% by weight, more preferably 1.0 to 15% by weight, even more preferably 2.0 to 12% by weight, relative to the total weight of the detergent according to the invention. Polymers containing sulfonic acid groups, in particular from the group of copolymerized polysulfonates, are preferably used as polymers with a cleaning effect. In addition to the monomers containing sulfonic acid groups, these copolymerized polysulfonates also contain at least one monomer selected from the group of unsaturated carboxylic acids. As unsaturated carboxylic acids, it is particularly preferred to use polymers of the formula R 1 (R 2 )C=C(R 3 )COOH unsaturated carboxylic acid, wherein R 1 To R3 independently of one another represent -H, -CH3, a linear or branched saturated alkyl functional group having 2 to 12 carbon atoms, a linear or branched, monounsaturated or polyunsaturated alkenyl functional group having 2 to 12 carbon atoms, an alkyl or alkenyl functional group as defined above substituted by -NH2-, -OH-, or -COOH-, or represent -COOH or -COOR 4 , where R 4 is a saturated or unsaturated, straight-chain or branched hydrocarbon functional group having 1 to 12 carbon atoms. Particularly preferred unsaturated carboxylic acids are acrylic acid, methacrylic acid, ethacrylic acid, α-chloroacrylic acid, α-cyanoacrylic acid, crotonic acid, α-phenylacrylic acid, maleic acid, maleic anhydride, fumaric acid, itaconic acid, citraconic acid, methylenemalonic acid, sorbic acid, cinnamic acid or mixtures thereof. Of course, unsaturated dicarboxylic acids can also be used. For monomers containing sulfonic acid groups, preferred are those of formula R 5 (R 6 )C=C(R 7 )-X-SO3H, where R 5 To R 7 independently of one another represent -H, -CH3, a linear or branched saturated alkyl functional group having 2 to 12 carbon atoms, a linear or branched, monounsaturated or polyunsaturated alkenyl functional group having 2 to 12 carbon atoms, an alkyl or alkenyl functional group substituted with -NH2-, OH-, or -COOH-, or represent -COOH or -COOR 4 , where R 4 is a saturated or unsaturated, linear or branched hydrocarbon functional group having 1 to 12 carbon atoms, and X represents an optional spacer group selected from -(CH2) n -, wherein n = 0 to 4, -COO-(CH2) k -, wherein k = 1 to 6, -C(O)-NH-C(CH3)2-, -C(O)-NH-C(CH3)2-CH2- and -C(O)-NH-CH(CH2CH3)-CH2-. Among these monomers, preferred are the formulas H2C=CH-X-SO3H, H2C=C(CH3)-X-SO3H and HO3S-X-(R 6 )C=C(R 7 )-X-SO3H, where R 6 and R 7 are independently selected from -H, -CH3, -CH2CH3, -CH2CH2CH3 and -CH(CH3)2, and X represents an optional spacer group selected from -(CH2) n -, wherein n = 0 to 4, -COO-(CH2) k-, wherein k = 1 to 6, -C(O)-NH-C(CH3)2-, -C(O)-NH-C(CH3)2-CH2- and -C(O)-NH-CH(CH2CH3)-. Particularly preferred monomers containing sulfonic acid groups are 1-acrylamido-1-propanesulfonic acid, 2-acrylamido-2-propanesulfonic acid, 2-acrylamido-2-methyl-1-propanesulfonic acid, 2-methacrylamido-2-methyl-1-propanesulfonic acid, 3-methacrylamido-2-hydroxy-propanesulfonic acid, allylsulfonic acid, methallylsulfonic acid, allyloxybenzenesulfonic acid, methallyloxybenzenesulfonic acid, 2-hydroxy-3-(2-propenyloxy)propanesulfonic acid, 2-methyl-2-propene-1-sulfonic acid, styrenesulfonic acid, vinylsulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, sulfomethylacrylamide, sulfomethylmethacrylamide, and mixtures of the above acids or their water-soluble salts. In the polymer, the sulfonic acid groups may be present in a fully or partially neutralized form. According to the present invention, preferably, a copolymer containing sulfonic acid groups with partially or completely neutralized sulfonic acid groups is used. The molar masses of the sulfonic copolymers preferably used according to the invention can be varied in order to adapt the properties of the polymers to the desired intended use. 1 (R 2 )C=C(R 3 )-XR 4 The monomers, R 1 To R 3 independently represent -H, -CH3 or -C2H5, X represents an optional spacer group selected from -CH2-, -C(O)O- and -C(O)-NH-, and R 4 denotes a linear or branched saturated alkyl functional group having 2 to 22 carbon atoms or an unsaturated, preferably aromatic functional group having 6 to 22 carbon atoms. Particularly preferred nonionic monomers are butene, isobutene, pentene, 3-methylbutene, 2-methylbutene, cyclopentene, hexene, 1-hexene, 2-methylpentene-1, 3-methylpentene-1, cyclohexene, methylcyclopentene, cycloheptene, methylcyclohexene, 2,4,4-trimethylpentene-1, 2,4,4-trimethylpentene-2, 2,3-dimethylhexene-1, 2,4-dimethylhexene-1, 2,5-dimethylhexene-1, 3,5-dimethylhexene-1, 4,4-dimethylhexane-1, ethylcyclohexene, 1-octene, α-olefins having more than 10 carbon atoms such as 1-decene, 1-dodecene, 1-hexadecene, 1-octadecene and C 22α-olefin, 2-styrene, α-methylstyrene, 3-methylstyrene, 4-propylstyrene, 4-cyclohexylstyrene, 4-dodecylstyrene, 2-ethyl-4-benzylstyrene, 1-vinylnaphthalene, 2-vinylnaphthalene, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, pentyl acrylate, hexyl acrylate, methyl methacrylate, N-(meth)acrylamide, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, N-(2-ethylhexyl)acrylamide, octyl acrylate, octyl methacrylate, N-(octyl)acrylamide, lauric acrylate, lauryl methacrylate, N-(lauryl)acrylamide, stearyl acrylate, stearyl methacrylate, N-(stearyl)acrylamide, behenyl acrylate, behenyl methacrylate and N-(behenyl)acrylamide, or a mixture thereof. The weight ratio of the copolymer containing sulfonic acid groups is preferably 0.1 to 15 wt %, more preferably 1.0 to 12 wt %, even more preferably 2.0 to 10 wt %, relative to the total weight of the detergent according to the present invention.

[0167] In one embodiment, the detergent comprises:

[0168] - 5 to 70% by weight, in particular 5 to 30% by weight, of surfactants, and / or

[0169] - 0 to 10% by weight, especially 1 to 3% by weight, of water-soluble or water-dispersible inorganic builder materials, and / or

[0170] - 0 to 10% by weight, in particular 1 to 4% by weight, of water-soluble organic builder substances, and / or

[0171] - 0.01-5% by weight of a complexing agent, and / or

[0172] - 0.01 to 5% by weight of graying inhibitors, and / or

[0173] - 0.01 to 5% by weight of foam inhibitors, and / or

[0174] - 0.01-5% by weight of optical brighteners.

[0175] In a preferred embodiment, the liquid detergent comprises:

[0176] - 5 to 20% by weight, in particular 9 to 17% by weight, of anionic surfactants, and / or

[0177] - 1 to 20% by weight, in particular 5 to 9% by weight, of a nonionic surfactant, and / or

[0178] - 0.5 to 10% by weight, in particular 1 to 4% by weight, of water-soluble organic builder substances, in particular citric acid or citrates, and / or

[0179] 0.01 to 5% by weight, in particular 0.5 to 2% by weight, of a complexing agent, in particular HEDP, and / or

[0180] 0.01 to 5% by weight, in particular 0.2 to 1% by weight, of graying inhibitors, and / or

[0181] - 0.01 to 5% by weight of foam inhibitors.

[0182] In a further preferred embodiment, the liquid detergent comprises:

[0183] - 0 to 10% by weight, in particular 1 to 3% by weight, of anionic surfactants, and / or

[0184] - 5 to 20% by weight, in particular 10 to 18% by weight, of a nonionic surfactant, and / or

[0185] - 1 to 10% by weight, especially 1.5 to 3% by weight, of water-soluble or water-dispersible inorganic builder materials, and / or

[0186] 0 to 1% by weight, in particular 0.01 to 0.5% by weight, of fragrance and / or 0.01 to 5% by weight, in particular 0.05 to 0.5% by weight, of optical brightener.

[0187] Suitable graying inhibitors or detergent active ingredients (detergent polymers) are cellulose ethers such as carboxymethylcellulose, methylcellulose, hydroxyalkylcellulose and mixed cellulose ethers, such as methylhydroxyethylcellulose, methylhydroxypropylcellulose and methylcarboxymethylcellulose. Preferably, sodium carboxymethylcellulose, hydroxypropylmethylcellulose and mixtures thereof, and optionally mixtures thereof with methylcellulose are used. Commonly used detergent active ingredients include copolyesters containing dicarboxylic acid units, alkylene glycol units and polyalkylene glycol units. The proportion of graying inhibitors and / or detergent active ingredients in the agent according to the invention is usually not more than 2% by weight, preferably 0.5-1.5% by weight, particularly preferably 0.5-2% by weight.

[0188] For example, derivatives of diaminostilbene disulfonic acid or alkali metal salts thereof may be contained as optical brighteners, particularly for textiles made of cellulose fibers (e.g., cotton). For example, salts of 4,4'-bis(2-anilino-4-morpholino-1,3,5-triazine-6-ylamino)stilbene-2,2'-disulfonic acid, or compounds of similar structures with diethanolamino, methylamino, or 2-methoxyethylamino groups instead of morpholino groups are suitable. In addition, substituted 4,4'-distyryl biphenyl type brighteners may be present, such as 4,4'-bis(4-chloro-3-sulfostyryl) biphenyl. A mixture of brighteners may also be used. 1,3-diaryl-2-pyrazoline type brighteners, such as 1-(p-sulfonylphenyl)-3-(p-chlorophenyl)-2-pyrazoline, and compounds with similar structures are particularly suitable for polyamide fibers. The content of the optical brightener or whitening agent mixture in the agent is usually not more than 1% by weight, preferably 0.05-0.5% by weight. In a preferred embodiment of the present invention, the agent does not contain such active ingredients.

[0189] Conventional foam regulators which can be used in the agents according to the invention include, for example, silicone-silicic acid mixtures, the finely divided silicic acid contained in which is preferably silanized or otherwise hydrophobized. The silicones can consist of linear compounds and crosslinked silicone resins and mixtures thereof. Other antifoaming agents are paraffin hydrocarbons, in particular microparaffins and paraffins having a melting point above 40° C., in particular saturated fatty acids or soaps having 20 to 22 carbon atoms, for example sodium behenate, and alkali metal salts of mono- and / or dialkyl phosphates, in which the alkyl chains each have 12 to 22 carbon atoms. Of these, preference is given to using alkali metal salts of mono- and / or dialkyl phosphates having a carbon chain of 12 to 22 carbon atoms. 16-18 Alkyl monoalkyl sodium phosphate and / or dialkyl sodium phosphate. The proportion of foam regulators can preferably be 0.2 to 2% by weight, particularly preferably not more than 1% by weight.

[0190] In order to adjust the desired pH value, the reagent according to the invention can contain an acid compatible with the system and the environment, in particular citric acid, acetic acid, tartaric acid, malic acid, lactic acid, glycolic acid, succinic acid, glutaric acid and / or adipic acid, and an inorganic acid, in particular sulfuric acid or alkali metal hydrogensulfate, or a base, in particular ammonium or alkali metal hydroxide, preferably sodium hydroxide. The reagent according to the invention contains a total of preferably not more than 10% by weight, in particular 0.5-6% by weight, particularly preferably 0.3-2% by weight of these types of pH regulators.

[0191] Peroxy compounds that can be suitable for use in the agents according to the invention include, in particular, organic peracids or persalts of organic acids, such as salts of phthalimide perhexanoic acid, perbenzoic acid or diperdodecane dioic acid, hydrogen peroxide and inorganic salts that release hydrogen peroxide under washing conditions, the salts including perborates, percarbonates, persilicates and / or persulfates such as caroate, and hydrogen peroxide inclusion compounds, such as H2O2-urea adducts. Hydrogen peroxide can also be produced by enzyme systems, i.e., oxidases and their substrates. If solid peroxy compounds are intended to be used, they can be used in the form of powders or granules, and they can also be coated in a manner known in principle. The peroxy compound can be added to the washing liquor itself or in the form of an agent containing the peroxy compound, which in principle can contain all conventional washing ingredients, cleaning ingredients or disinfecting components. It is particularly preferred to use alkali metal percarbonates or alkali metal perborate monohydrates. If the agent according to the invention contains peroxy compounds, their presence is preferably at most 50% by weight, in particular 5-30% by weight, more preferably 0.1-20% by weight.

[0192] Compounds which under perhydrolysis conditions generate aliphatic peroxycarboxylic acids and / or optionally substituted perbenzoic acids preferably having 1 to 10 carbon atoms, in particular 2 to 4 carbon atoms, can be used as bleach activators in the agents described. Substances with O-acyl and / or N-acyl and / or optionally substituted benzoyl groups with the stated number of carbon atoms are suitable. Preference is given to polyacylated alkylenediamines, in particular tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycolurils, in particular tetraacetyl glycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates or carboxylates or their sulfonic or carboxylic acids, in particular nonanoyloxybenzenesulfonate or isononanoyloxybenzenesulfonate or lauroyloxybenzenesulfonate (NOBS or isononoyloxybenzenesulfonate or LOBS), 4-(2-decanoyloxyethoxycarbonyloxy)-benzenesulfonate (DECOBS) or decanoyloxy The acyl acetals and acyl lactams are preferably acetylated bleach activators. Such bleach activators, especially in the presence of the above-mentioned hydrogen peroxide-generating bleaches, may be present in the customary amount range, preferably in an amount of 0.5 to 10% by weight, especially 1 to 8% by weight, based on the total agent, but are preferably completely absent when percarboxylic acids are used as the sole bleach.

[0193] In addition to or instead of the customary bleach activators, the solid agents may also contain sulfonimides and / or bleach-boosting transition metal salts or transition metal complexes as so-called bleach catalysts.

[0194] Washing composition according to the present invention can contain organic solvent as other components.The adding of organic solvent has a favorable influence on the cleaning performance of enzyme stability and these reagents.Preferred organic solvent is derived from the group of monohydric alcohol or polyhydric alcohol, alkanolamine or glycol ether.Described solvent is preferably selected from ethanol, n-propyl alcohol or isopropyl alcohol, butanol, ethylene glycol, propylene glycol, butylene glycol, glycerine, diethylene glycol, propylene glycol, butylene glycol, hexylene glycol, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether, diethylene glycol methyl ether, diethylene glycol ethyl ether, propylene glycol methyl ether, propylene glycol ethyl ether, propylene glycol propyl ether, dipropylene glycol methyl ether, dipropylene glycol ethyl ether, methoxy triglycol, ethoxy triglycol, butoxy triglycol, 1-butoxy ethoxy-2-propanol, 3-methyl-3-methoxybutanol, propylene glycol tert-butyl ether and the mixture of these solvents. Relative to the total weight of the detergent according to the present invention, the weight ratio of these organic solvents is preferably 0.1 to 10% by weight, more preferably 0.2 to 8.0% by weight, even more preferably 0.5 to 5.0% by weight. Particularly preferred organic solvents that are particularly effective in stabilizing detergents are glycerol and 1,2-propylene glycol. The liquid detergent preferably contains at least one polyol, preferably selected from the group of glycerol and 1,2-propylene glycol, and the amount of the polyol is preferably 0.1 to 10% by weight, preferably 0.2 to 8.0% by weight, more preferably 0.5 to 5.0% by weight, based on the total weight of the detergent according to the present invention. Other preferred organic solvents are organic amines and alkanolamines. In all cases, based on the total weight of the detergent according to the present invention, the detergent according to the present invention preferably contains a total of 0.1 to 10% by weight, more preferably 0.2 to 8.0% by weight, even more preferably 0.5 to 5.0% by weight of these amines. Ethanolamine is a particularly preferred alkanolamine.

[0195] Another preferred component of the detergent according to the invention is sugar alcohol (aldol). Aldols include those of the formula HOCH2[CH(OH)] n The non-cyclic polyol of CH2OH. The alditol includes, for example, mannitol, isomalt, lactitol, sorbitol and xylitol, threitol, erythritol and arabitol. Sorbitol has been considered to be particularly advantageous for enzyme stability. Relative to the gross weight of the detergent, the weight ratio of sugar alcohol is preferably 0.1 to 10 weight %, more preferably 2.0 to 8.0 weight %, even more preferably 3.0 to 6.0 weight %.

[0196] The detergents according to the invention may contain only the proteases defined herein. Alternatively, they may also contain other enzymes in concentrations that are advantageous for the effectiveness of the agent. Therefore, another embodiment of the invention is an agent that further comprises one or more other enzymes. The other enzymes preferably used are all enzymes that are capable of showing catalytic activity in the agent according to the invention, in particular lipases, amylases, cellulases, hemicellulases, mannanases, tannases, xylanases, xanthanases, xyloglucanases, β-glucosidases, pectinases, carrageenases, perhydrolases, oxidases, oxidoreductases or other proteases different from the protease according to the invention, and mixtures thereof. Based on the active protein, the other enzymes are advantageously present in an amount of 1×10 -8 In order of increasing preference, each additional enzyme is present in an amount of 1×10 -7 % by weight, 0.00001 to 1 % by weight, 0.00005 to 0.5 % by weight, 0.0001 to 0.1 % by weight and particularly preferably 0.0001 to 0.05 % by weight are present in the agent according to the present invention. Particularly preferably, the enzyme shows a synergistic cleaning performance to certain stains or spots, i.e. the enzymes contained in the agent composition support each other in terms of its cleaning performance. Very particularly preferably, this synergistic effect exists between the protease contained according to the present invention and other enzymes of the agent according to the present invention, including in particular between the protease and amylase and / or lipase and / or mannanase and / or cellulase and / or pectinase. Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the present invention. According to the present invention, the preferred textile detergent has at least one protease and at least one amylase. In another preferred embodiment of the present invention, the textile detergent has at least one protease and at least one cellulase. In a further preferred embodiment, the textile detergent has at least one protease and at least one lipase. Textile detergents having 3 to 10 different enzymes are particularly preferred and may be particularly preferred in terms of cleaning performance on a very broad range of stains.

[0197] Examples of proteases are subtilisin BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, protease PB92, subtilisin 147 and 309, protease from Bacillus lentus, subtilisin DY and the enzymes thermoprotease, proteinase K and proteases TW3 and TW7, which belong to the subtilase class but no longer to the subtilisin class in the narrow sense. Subtilisin Carlsberg is available under the trade name Subtilisin 147 and 309 are available from Novozymes under the trade names and The protease variants are derived from the protease from Bacillus lentus DSM 5483. Suitable other proteases are, for example, available under the trade name Progress Uno and The enzymes obtained from Novozymes are available under the trade name OxP, Prime, and Prefer and Preferenz The enzyme is available from Danisco / DuPont under the trade name Lavergy pro 104 The enzyme was obtained from BASF and is available under the trade name The enzyme was obtained from Advanced Biochemicals Ltd. under the trade name The enzyme was obtained from Wuxi Snyder Bioproducts Ltd. and is available under the trade name and Protease Enzymes available from Amano Pharmaceuticals Ltd., and enzymes available from Kao Corp under the name Proteinase K-16. Particular preference is given to using the proteases from Bacillus gibberellus and Bacillus pumilus disclosed in international patent applications WO 2008 / 086916 A1 and WO 2007 / 131656 A1. Other proteases that can advantageously be used are disclosed in patent applications WO 91 / 02792 A1, WO 2008 / 007319 A2, WO 93 / 18140 A1, WO 01 / 44452 A1, GB 1243784 A, WO 96 / 34946 A1, WO 02 / 029024 A1 and WO 03 / 057246 A1. Other proteases that can be used are those which occur naturally in the microorganism Stenotrophomonas maltophilia, in particular Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus.

[0198] Examples of amylases are alpha-amylases from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus, and in particular developments thereof which have been adapted for use in detergents or cleaners. The enzyme from Bacillus licheniformis is available from Novozymes under the name and from Danisco / DuPont under the name ST. These α-amylase development products are available under the trade names and ultra (both from Novozymes), OxAm (Danisco / DuPont) and (Daiwa Seiko Inc.). α-Amylase from Bacillus amyloliquefaciens was obtained from Novozymes under the trade name and derivative variants of the alpha-amylase from Bacillus stearothermophilus are also marketed by Novozymes under the trade name and Sold. In addition, for this purpose, alpha-amylases from Bacillus sp. A7-7 (DSM 12368) and cyclodextrin glucanotransferases (CGTases) from Bacillus agaradherens (DSM9948) should be emphasized. In addition, the decomposing amylases disclosed in international patent applications WO 95 / 26397A1, WO 96 / 23873A1, WO 99 / 23211A1, WO 00 / 60060A2, WO 03 / 002711A2, WO 03 / 054177A2, WO 2006 / 002643A2, WO 2007 / 079938A2, WO 2011 / 100410A2 and WO 2013 / 003659A1 can be used. Fusion products of all mentioned molecules can also be used. In addition, it is available from Novozymes under the trade name Developments of alpha-amylases obtained from Aspergillus niger and Aspergillus oryzae are suitable. Other commercially available products that can be advantageously used are, for example, and or ultra or plus and Amplify TM 12L or Amplify Prime TM 100L, the latter also from Novozymes, and PREFERENZ from Danisco / DuPont series, including for example PREFERENCE PREFERENCE or PREFERENCE According to the invention, variants of these enzymes which can be obtained by point mutations can also be used.

[0199] Preferred amylases include a) an alpha-amylase comprising an amino acid sequence which is at least 80%, and in increasing order of preference at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over its entire length to the amino acid sequence given in SEQ ID NO: 2, and having at least one amino acid substitution at one of positions 172, 202, 208, 255 and 261 in the numbering according to SEQ ID NO: 2, preferably selected from the group consisting of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, R172Q and combinations thereof. More preferably the amylase comprises the amino acid substitution M202L or M202T.

[0200] Further preferred amylases include b) alpha-amylases comprising an amino acid sequence which is at least 60%, and in increasing order of preference at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over its entire length to the amino acid sequence given in SEQ ID NO: 3, and in increasing order of preference at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over its entire length to the amino acid sequence given in SEQ ID NO: 3 The position in the number of NO:3 is 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305 5, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482 and 484, and / or a deletion at one of positions 183 and 184. Particularly preferred amylases have at least one amino acid substitution at one of positions 9, 26, 149, 182, 186, 202, 257, 295, 299, 323, 339 and 345, and / or particularly preferably at least one amino acid substitution or deletion selected from the group consisting of R118K, D183*, G184*, N195F, R320K, R458K and combinations thereof. Very particularly preferred amylases comprise an amino acid sequence which is at least 60%, in increasing order of preference at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical over its entire length to the amino acid sequence given in SEQ ID NO: 3 and has one of the following amino acid substitution or deletion combinations:

[0201] (i)M9L+M323T;

[0202] (ii) M9L+M202L / T / V / I+M323T;

[0203] (iii)M9L+N195F+M202L / T / V / I+M323T;

[0204] (iv)M9L+R118K+D183*+G184*+R320K+M323T+R458K;

[0205] (v)M9L+R118K+D183*+G184*+M202L / T / V / I+R320K+M323T+R458K;

[0206] (vi)M9L+G149A+G182T+G186A+M202L+T257I+Y295F+N299Y+M323T+A339S+E345R;

[0207] (vii)M9L+G149A+G182T+G186A+M202I+T257I+Y295F+N299Y+M323T+A339S+E345R;

[0208] (viii)M9L+R118K+G149A+G182T+D183*+G184*+G186A+M202L+T257I+Y295F+N299Y+R320K+M323T+A339S+E345R+R458K;

[0209] (ix)M9L+R118K+G149A+G182T+D183*+G184*+G186A+M202I+T257I+Y295F+N299Y+R320K+M323T+A339S+E345R+R458K;

[0210] (x)M9L+R118K+D183*+D184*+N195F+M202L+R320K+M323T+R458K;

[0211] (xi)M9L+R118K+D183*+D184*+N195F+M202T+R320K+M323T+R458K;

[0212] (xii)M9L+R118K+D183*+D184*+N195F+M202I+R320K+M323T+R458K;

[0213] (xiii)M9L+R118K+D183*+D184*+N195F+M202V+R320K+M323T+R458K;

[0214] (xiv)M9L+R118K+N150H+D183*+D184*+N195F+M202L+V214T+R320K+M323T+R458K;

[0215] (xv)M9L+R118K+D183*+D184*+N195F+M202L+V214T+R320K+M323T+E345N+R458K.

[0216] Further preferred amylases include c) alpha-amylases comprising an amino acid sequence which is at least 90% identical over its entire length to the amino acid sequence given in SEQ ID NO: 4, and optionally in the presence of a residue according to SEQ ID NO: 47, 458, 469, 476, and 477. Preferred amino acid substitutions include E260A / D / C / Q / L / M / F / P / S / W / V / G / H / I / K / N / R / T / Y, G304R / K / E / Q, W140Y / F, W189E / G / T, D134E, F262G / P, W284D / H / F / Y / R, W347H / F / Y, W439R / G, G476E / Q / R / K, G477E / Q / K / M / R, N195F / Y, N197F / L, Y 198N, Y200F, Y203F, I206H / L / N / F / Y, H210Y, E212V / G, V213A, M116T, Q129L, G133E, E134Y, K142R, P146 S, G147E, G149R, N151R, Y152H, Q169E, N174R, A186R, Y243F, S244Q, G303V, R320N, R359I, N418D and A447V.

[0217] Examples of cellulases (endoglucanases, EG) include fungal cellulase preparations enriched with endoglucanases (EG) and their development types, which are marketed by Novozymes under the trade name Available. Also available from Novozymes and are based on 50 kD-EG and 43 kD-EG from Humicola insolens DSM 1800, respectively. Other commercial products from this company that can be used are and Cellulases may also be used, for example, available from AB Enzymes under the trade name and obtained from AB Enzymes, which is based at least in part on 20 kD-EG from Melanocarpus. Other cellulases from AB Enzymes are and Other suitable cellulases are derived from Bacillus sp. CBS 670.93 and CBS 669.93, wherein the cellulase derived from Bacillus sp. CBS 670.93 is available from Danisco / DuPont under the trade name Other commercial products from Danisco / DuPont that may be used are "Genencor detergent cellulase L" and Neutra.

[0218] Other enzymes that can be used are, for example, lipases or cutinases, in particular due to their triglyceride cleavage activity, and for the in situ production of peroxyacids from suitable precursors. These include, for example, lipases that are initially available from Humicola lanuginosa (Thermomyces lanuginosus) or have been developed therefrom, in particular starting from said lipase, those with one or more of the following amino acid exchanges at positions D96L, T213R and / or N233R, particularly preferably at T213R and N233R. Lipase is available, for example, from the company Novozymes under the trade name Ultra, and Another lipase that can be advantageously used is available from Novozymes under the trade name In addition, for example, the cutinases originally isolated from Fusarium solani pisi and Humicola insolen may also be used. Likewise suitable lipases are available from Amano under the name Lipase Lipase Lipase or Lipase Lipase Bacillus Lipase Lipase and Lipase For example, lipases or cutinases from the company Danisco / DuPont can be used, the starting enzymes originally being isolated from Pseudomonas mendocina and Fusarium solanii. As other important commercial products, mention should be made of the preparation M1 sold by Danisco / Genencor. and By Meito Sangyo KK in the name Lipase and Lipase Enzymes for sale, and products from Danisco / DuPont

[0219] To improve the bleaching effect, according to the invention, oxidoreductases such as oxidases, oxygenases, catalases, peroxidases such as halogenated, chloroated, bromoated, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases) can be used. Advantageously, organic compounds that interact with the enzymes, particularly preferably aromatic compounds, are additionally added to enhance the activity of the relevant oxidoreductases (enhancers) or, in the case of significantly different redox potentials, to ensure electron flow between the oxidase and the stain (mediator).

[0220] The enzyme used within the scope of the present invention may, for example, be originally derived from a microorganism, such as Bacillus, Streptomyces, Humicola or Pseudomonas, and / or may be produced by suitable microorganisms using biotechnological methods known per se, such as by transgenic expression hosts, such as Escherichia or Bacillus, or by filamentous fungi. It should be emphasized that in particular industrial enzyme preparations of the relevant enzymes may also be involved, i.e. accompanying substances may be present. Therefore, the enzyme may be packaged and used together with accompanying substances, such as substances from fermentation or with other stabilizers.

[0221] The agent according to the invention advantageously contains the protease in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg and most particularly preferably 50 μg to 10 mg per gram of the agent. In various embodiments, the concentration of the protease described herein (active enzyme) in the agent is >0 to 1% by weight, preferably 0.001 to 0.1% by weight, based on the total weight of the agent.

[0222] In addition, the protease contained in the reagent and / or other components of the reagent can be coated with a substance that is impermeable to the enzyme at room temperature or in the absence of water, which substance becomes permeable to the enzyme under the conditions of use of the reagent. Therefore, this embodiment of the present invention is characterized in that the protease is coated with a substance that is impermeable to the protease at room temperature or in the absence of water. In addition, the detergent itself can be packaged in a container, preferably a breathable container, from which the detergent is released shortly before use or during the washing process.

[0223] The same or different types of proteases contained in the detergent and / or the optional presence of further enzymes can be adsorbed on a carrier material and / or embedded in a coating material to prevent their premature inactivation. The enzyme is then released into the washing liquid, i.e. under the conditions of use, and can exert its catalytic action. Preferred packaging forms have 0.05 to 15% by weight, in particular up to 10% by weight, of active protein of the protease.

[0224] The detergent according to the invention contains, in order of increasing preference, 1×10 -8 % to 5% by weight, 0.0001 to 1% by weight, 0.0005 to 0.5% by weight, 0.001 to 0.1% by weight, based in each case on active protein.

[0225] In the reagent described herein, the enzyme to be used can also be formulated with accompanying substances such as from fermentation. In liquid formulations, enzymes are preferably used as enzyme liquid formulations. Enzymes are usually not provided in the form of pure protein, but in the form of stable, storable and transportable preparations. These pre-packaged preparations include solid preparations such as obtained by granulation, extrusion or freeze-drying, or particularly in the case of liquid or gel reagents, enzyme solutions, which are advantageously concentrated as much as possible, have low water content and / or are supplemented with stabilizers or other auxiliary agents. Alternatively, for solid and liquid application forms, enzymes can also be encapsulated, such as by spray drying or extruding the enzyme solution with preferably natural polymers, or encapsulated in the form of capsules, such as those wherein the enzyme is encapsulated in a solidified gel, or those wherein the core-shell type of the enzyme-containing core is coated with a protective layer impermeable to water, air and / or compounds. In the covering layer, other active ingredients, such as stabilizers, emulsifiers, pigments, bleaching agents or dyes can be applied in addition. This capsule is applied by methods known per se, such as by shaking or roller granulation or in a fluidized bed process. Advantageously, such granules are low in dust, for example due to the application of a polymeric film former, and are storage stable due to the coating. In addition, two or more enzymes may be formulated together, such that a single granule exhibits multiple enzyme activities.

[0226] The enzyme can also be incorporated into a water-soluble film, such as those used in detergents formulated in unit dosage forms. Such films allow the enzyme to be released upon contact with water. As used herein, "water-soluble" refers to a film structure that is preferably completely water-soluble. Preferably, such a film is composed of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).

[0227] Embodiments of the present invention include all solid, powder, granule, tablet form, liquid, gel or paste application forms of reagent according to the present invention, which optionally can also be composed of multiphase, and can exist in compressed or non-compressed form. The reagent can exist as a free-flowing powder, particularly with a bulk density of 300 to 1,200g / L, particularly 500 to 900g / L or 600 to 850g / L. The solid dosage form of the reagent also includes extrudates, granules, tablets or sachets containing solid reagents, which can exist in the form of large packages and portioned packages. Alternatively, the reagent can also be liquid, gel-like or pasty, such as in the form of non-aqueous liquid detergents or non-aqueous pastes or in the form of aqueous liquid detergents or aqueous pastes. In addition, the reagent can exist as a single-component system. This reagent consists of a single phase. Alternatively, the reagent can also consist of multiple phases (multi-component system). This reagent is therefore divided into several components, such as two liquids, two solids or a liquid and a solid phase. Liquid presentation forms based on water and / or organic solvents can be present in gel form, in thickened form.

[0228] According to the definition of the present invention, a substance, such as a composition or an agent, is a solid if it is in a solid aggregate state at 25° C. and 1013 mbar.

[0229] According to the definition of the present invention, a substance, such as a composition or an agent, is a liquid if it is in a liquid aggregate state at 25° C. and 1013 mbar. Liquids also include gel forms.

[0230] If the detergents according to the invention are in liquid form, they preferably contain more than 40% by weight, preferably from 50 to 90% by weight and particularly preferably from 60 to 80% by weight, of water, based on their total weight.

[0231] The invention also relates to a method for cleaning textiles, in which a textile detergent according to the invention, in particular a liquid textile detergent, is used in at least one method step. This includes manual and machine methods, preferably machine methods, because they can be controlled more precisely, for example in terms of the amount used and the contact time. The method for cleaning textiles is generally characterized by the fact that in one or more method steps, different cleaning active substances are applied to the material to be cleaned and washed off after an exposure time, or the material to be cleaned is treated in another way with a detergent or a solution or dilution of the agent.

[0232] In various embodiments, the method is characterized in that the protease is used at a temperature of 0 to 100° C., preferably 10 to 70° C., more preferably 30 to 50° C. and most preferably 20 to 40° C. In particular, in the method carried out at a temperature of 10 to 60° C., preferably 15 to 50° C., more preferably 20 to 40° C., the consumer can see the advantages of using the textile detergent according to the present invention compared to textile detergents comprising commonly used proteases.

[0233] All elements, themes and embodiments described for the textile detergent according to the invention are also applicable to the subject of the invention. Therefore, when it is pointed out that this disclosure is also applicable to the above-mentioned method according to the invention, reference is explicitly made to the disclosure of the corresponding position at this point.

[0234] Alternative embodiments of the subject matter of the present invention also include methods for treating textile raw materials or for textile care, in which the protease according to the invention becomes active in at least one method step. Among these, preference is given to methods for textile raw materials, fibers or textiles comprising natural components, very particularly preferably for such materials, fibers or textiles comprising wool or silk.

[0235] The proteases used in the detergents according to the invention can advantageously be used in textile detergents and methods for washing textiles according to the above statements. Thus, they can advantageously be used to provide proteolytic activity in corresponding agents and methods.

[0236] Finally, the present invention also comprises the use of the proteases described herein in detergents, e.g. as described above, for the (improved) removal of protease-sensitive stains, e.g. from textiles. In a preferred embodiment of this use, the protease is stored in the detergent for 3 days or more, 4 days or more, 7 days or more, 10 days or more, 12 days or more, 14 days or more, 21 days or more or 28 days or more before washing.

[0237] Therefore, the present invention further relates to

[0238] - use of an agent according to the invention for cleaning textiles, and / or

[0239] - Use of a protease comprising an amino acid sequence having at least 70% sequence identity over its entire length to the amino acid sequence given in SEQ ID NO: 1 and having, in each case based on the numbering according to SEQ ID NO: 1: (i) at least two amino acid substitutions at at least two of the positions corresponding to positions 3, 4, 99 or 199, in particular the amino acid substitutions 3T, 4I, 99E and 199I, and (ii) at least one further amino acid substitution at at least one of the positions corresponding to positions 74, 136, 143, 154, 161, 163, 171, 200, 203, 209, 212 or 256, in particular selected from N74D, N74E, N74Q, A 136Q, R143L, R143W, R143Y, S154D, S154Q, Y161T, A163G, V171L, Q200A, Q200L, Q200S, Q200T, Y203K, Y203V, Y203W, A209W, N212S, N212T, 256D, L256E and L256Q, the textile detergent having a pH of from about 9 to about 12, the pH being measured in a 1 wt % solution in deionized water at 20°C,

[0240] In particular, the protease is used in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg and most particularly preferably 50 μg to 10 mg, in each case per gram of the reagent.

[0241] Particularly preferred embodiments are, for example, hand washing, manual removal of stains from textiles or in combination with mechanical methods.

[0242] All elements, themes and embodiments described for the protease according to the invention and the reagents containing them are also applicable to the subject of the invention. Therefore, when it is pointed out that this disclosure is also applicable to the above-mentioned use according to the invention, explicit reference is made to the disclosure of the corresponding position. Example

[0243] Determination of storage stability in liquid detergents

[0244] Detergent base used

[0245] Commercially available liquid detergents as shown in Table 1 were used.

[0246] Table 1a: Formulation A

[0247] Element Formula A (by weight %) Citric Acid 1-4％ Defoaming agent <1％ FAEOS(anionic surfactant) 2-5％ FAEO (nonionic surfactant) 5-9％ LAS (anionic surfactant) 7-12％ Palm kernel oil fatty acids 1-3％ Monoethanolamine 2-6％ NaOH 50% 0.3-2％ glycerin 0.5-3％ 1,2-Propanediol 4-8％ HEDP 0.5-2％ Soil Release Polymers 0.2-1％ Boric acid 0.5-1.5％ Softened water Add to 100%

[0248] No fragrance, enzymes and optical brighteners; dosage 3.17g / L

[0249] The pH of Formulation A measured at 1 wt% solution in deionized water at 20°C was pH 10.

[0250] Table 1b: Formulation B

[0251]

[0252] The pH of Formulation B measured at 1 wt % solution in deionized water at 20°C was pH 11.

[0253] The following proteases were used:

[0254] C1: Protease according to SEQ ID NO: 2 from WO 2013 / 060621 A1

[0255] C2: Subtilisin 309 from Bacillus lentus

[0256] E1: SEQ ID NO:1+S3T+V4I+R99E+V199I+Q200L+Y203W;

[0257] E2: SEQ ID NQ:1+S3T+V4l+R99E+V199l+Q200L+Y203W+S154D+L256E;

[0258] store

[0259] The protease is present in the Bacillus licheniformis in the supernatant produced in the bioreactor. The protease is diluted to the same activity level. 90% detergent matrix (Formula A or Formulation B) is mixed with 10% appropriately diluted Bacillus licheniformis supernatant and mixed thoroughly. The enzyme activity (starting value) in each batch is determined before storage in a sealed container at 30°C or 40°C for two weeks, four weeks or ten weeks. The sample amount removed is dissolved in 0.1M Tris / HCl (pH 8.6) by stirring at room temperature for 20min. Then, the AAPF test is performed as described below.

[0260] Protease activity test (AAPF test)

[0261] The activity of the protease is determined by the release of the p-nitroaniline chromophore from the substrate succinylalanine-alanine-proline-phenylalanine-p-nitroaniline (AAPFpNA; Bachem L-1400). The release of pNA causes an increase in absorbance at 410 nm, which changes over time as a measure of enzyme activity.

[0262] The measurement was carried out at a temperature of 25° C., a pH of 8.6 and a wavelength of 410 nm. The measurement time was 5 minutes with a measurement interval of 20-60 seconds.

[0263] Measurement method:

[0264] 10μL AAPF solution (70mg / mL)

[0265] 1000 μL Tris / HCl (0.1 M, pH 8.6, with 0.1% Brij 35)

[0266] 10 μL diluted protease solution

[0267] Kinetics generated in 5 min at 25 °C (410 nm)

[0268] result

[0269] The remaining activity in % after storage in the above-mentioned detergent matrix is ​​shown below. The remaining activity in % is based on the relevant enzyme activity before storage (starting value).

[0270] Table 2: Storage at 30°C or 40°C for two weeks in Formulation A (pH 10)

[0271]

[0272] Table 3: Storage for two weeks at 30°C or 40°C in Formulation B (pH 10; adjusted by addition of citric acid)

[0273]

[0274] Table 4: Storage at 30°C for 2, 4 or 10 weeks in Formulation B (pH 11)

[0275]

[0276] Proteases E1 and E2 according to the invention show significantly higher storage stability in various high alkaline detergent formulations when stored at 30°C and 40°C for 2 to 10 weeks compared to comparative proteases C1 and C2.

Claims

1. A textile detergent comprising a) at least one protease, wherein the protease has proteolytic activity and, based on the numbering according to SEQ ID NO: 1, is one of the following amino acid substitution variants: S3T+V4I+R99E+V199I+Q200L+Y203W, and S3T+V4I+R99E+V199I+Q200L+Y203W+S154D+L256E; and b) at least one detergent ingredient, The textile detergent has a pH of 9 to 12 measured in a 1 wt% solution in deionized water at 20°C.

2. The detergent according to claim 1, wherein the textile detergent is a liquid textile detergent.

3. The detergent according to claim 1, wherein the at least one detergent ingredient is present in an amount of 0.01-99.9 wt%.

4. The detergent according to claim 1, wherein the detergent contains 1×10 -8 to 5 wt % of the protease.

5. The detergent according to claim 1, wherein the detergent contains the protease in an amount of 0.0001-1 wt% based on the total weight of the detergent.

6. The detergent according to claim 1, wherein the detergent contains the protease in an amount of 0.0005-0.5 wt% based on the total weight of the detergent.

7. The detergent according to claim 1, wherein the detergent contains the protease in an amount of 0.001-0.1 wt% based on the total weight of the detergent.

8. The detergent according to claim 1, wherein the detergent comprises at least one other enzyme selected from the group consisting of amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase, oxidoreductase, lipase and combinations thereof.

9. The detergent according to claim 8, wherein the at least one other enzyme is an amylase and wherein the amylase is selected from: a) an alpha-amylase comprising an amino acid sequence which is at least 80% identical over its entire length to the amino acid sequence given in SEQ ID NO: 2, and optionally having at least one amino acid substitution at one of positions 172, 202, 208, 255 and 261 according to the numbering of SEQ ID NO: 2; and / or b) an alpha-amylase comprising an amino acid sequence which over its entire length is at least 60% identical to the amino acid sequence given in SEQ ID NO: 3 and, in the numbering according to SEQ ID NO: 3, optionally having at positions 9, 26, 30, 33, 82, 37, 106, 118, 128, 133, 149, 150, 160, 178, 182, 186, 193, 195, 202, 203, 214, 231, 256, 257, 258, 269, 270, 272, 283, 295, 296, 298, 299, 303, 304, 305 4, 305, 311, 314, 315, 318, 319, 320, 323, 339, 345, 361, 378, 383, 419, 421, 437, 441, 444, 445, 446, 447, 450, 458, 461, 471, 482, and 484, and / or a deletion at one of positions 183 and 184; and / or c) an alpha-amylase comprising an amino acid sequence that is at least 90% identical over its entire length to the amino acid sequence given in SEQ ID NO: 4, and optionally in the presence of a residue according to SEQ ID NO: 47, 484, 492, 503, 514, 526, 537, 538, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580 10. The detergent according to claim 9, wherein at least one amino acid substitution in SEQ ID NO:2 is selected from the group consisting of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N, R172Q, and combinations thereof; At least one amino acid substitution or deletion in SEQ ID NO: 3 is selected from R118K, D183*, G184*, N195F, R320K, R458K and combinations thereof; and / or At least one amino acid in SEQ ID NO:4 is deleted at positions 183 and 184.

11. The detergent according to claim 1, wherein the detergent ingredients are selected from the group consisting of surfactants, builders, complexing agents, polymers, corrosion inhibitors, bleaching agents, bleach activators or bleach catalysts, water-miscible organic solvents, enzyme stabilizers, chelating agents, electrolytes, pH adjusters, optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators, dyes and perfumes, and combinations thereof.

12. The detergent according to claim 1, comprising a) 5-20 wt% of anionic surfactant, and / or b) 1-20% by weight of a nonionic surfactant, and / or c) 0.5 to 10% by weight of water-soluble organic builder substances, and / or d) 0.01-5% by weight of a complexing agent, and / or e) 0.01 to 5% by weight of a graying inhibitor, and / or f) 0.01 to 5% by weight of foam inhibitors.

13. The detergent according to claim 12, comprising a) 9-17 wt. % of anionic surfactants, and / or b) 5-9% by weight of a nonionic surfactant, and / or c) 1 to 4% by weight of water-soluble organic builder substances, and / or d) 0.5-2% by weight of a complexing agent, and / or e) 0.2 to 1% by weight of graying inhibitors.

14. A detergent according to claim 12 or 13, wherein the water-soluble organic builder substance is citric acid and / or a citrate, and / or the complexing agent is HEDP.

15. The detergent according to claim 1, comprising a) 0-10 wt% of anionic surfactant, and / or b) 5-20% by weight of a nonionic surfactant, and / or c) 1 to 10% by weight of water-soluble or water-dispersible inorganic builder materials, and / or d) 0-1% by weight of fragrance, and / or e) 0.01 to 5% by weight of an optical brightener.

16. The detergent according to claim 15, comprising a) 1-3 wt% of anionic surfactant, and / or b) 10-18 wt% of a nonionic surfactant, and / or c) 1.5 to 3% by weight of water-soluble or water-dispersible inorganic builder materials, and / or d) 0.01-0.5 wt. % of fragrance, and / or e) 0.05-0.5 wt. % of an optical brightener.

17. The detergent according to claim 11, wherein the corrosion inhibitor comprises a glass corrosion inhibitor.

18. The detergent according to any one of claims 1 to 13 and 15-16, wherein the pH thereof in a 1 wt% solution in deionized water at 20°C is from 9.5 to 11.

5.

19. The detergent according to any one of claims 1 to 13 and 15-16, wherein the pH thereof in a 1 wt% solution in deionized water at 20°C is 10 to 11.

20. The detergent according to any one of claims 1 to 13 and 15-16, wherein the pH thereof in a 1 wt% solution in deionized water at 20°C is pH 10.

21. The detergent of claim 11, wherein the bleaching agent comprises a peroxygen compound.

22. A method for cleaning textiles, characterized in that In at least one process step, a washing agent according to any one of claims 1 to 21 is used.

23. Use of a detergent according to any one of claims 1 to 21 for removing protease-sensitive stains from textiles.

24. Use of a protease in a textile detergent for removing protease-sensitive stains on textiles, wherein the protease is one of the following amino acid substitution variants, in each case based on the numbering according to SEQ ID NO: 1: S3T+V4I+R99E+V199I+Q200L+Y203W, and S3T+V4I+R99E+V199I+Q200L+Y203W+S154D+L256; The textile detergent has a pH value of 9 to 12, measured in a 1% by weight solution in deionized water at 20° C.

25. Use according to claim 24, wherein the textile detergent is a liquid textile detergent.

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