Protease variants ix with enhanced performance

Abstract

The present invention relates to a protease having proteolytic activity comprising an amino acid sequence having at least 70% and increasingly preferably 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% identity with the amino acid sequence set forth in SEQ ID NO: 1 over its entire length, wherein the protease is based in each case on a number according to SEQ ID NO: 1, (i) has amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) has at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one position corresponding to position 6, 61, 62, 101, 170 and 188 and increasingly preferably two, three, four, five or six positions. The invention also relates to the production and use of said protease. The protease of this type shows very good cleaning performance.

Description

[0001] The present invention belongs to the field of enzyme technology. The present invention relates to proteases whose amino acid sequences have been altered, in particular for use in detergents and cleaning agents, in particular liquid detergents and cleaning agents, to improve their cleaning performance, and to nucleic acids encoding them and their production. The present invention also relates to the use of the proteases and methods of using them, as well as agents containing the proteases, in particular detergents and cleaning agents, in particular liquid detergents and cleaning agents.

[0002] Protease is one of the most important enzymes in technology. For detergents and cleaning agents, they are the oldest enzymes, and almost all modern high-performance detergents and cleaning agents contain proteases. They can degrade protein stains on items to be cleaned. Then, the proteases of subtilisin type (subtilases, subtilisin, EC 3.4.21.62) are particularly important, and these proteases belong to serine proteases due to the amino acids with catalytic activity. They act as non-specific endopeptidases and hydrolyze any amide bonds in peptides or proteins. Their optimum pH value is usually in a significant alkaline range. For example, R.Siezen's article "Subtilase: Subtilisin-like Proteases" ("Subtilases: Subtilisin-like Proteases"), pp. 75-95 (R.Bott and C.Betzel published, New York, 1996) outlines the family. Subtilase is naturally formed by microorganisms. In particular, the subtilisins formed and secreted by Bacillus species are the most important class of subtilases.

[0003] Examples of subtilisin-type proteases preferably used in detergents and cleansers are subtilisin BPN' and Carlsberg, protease PB92, subtilisin 147 and 309, alkaline proteases from Bacillus lentus (particularly Bacillus lentus DSM 5483), subtilisin DY and thermophilic proteases, proteinase K and proteases TW3 and TW7, which should be classified as subtilisins, but are no longer subtilisins in the narrow sense, and variants of the proteases, whose amino acid sequences are altered compared to the starting proteases. Proteases are altered in a targeted or random manner by methods known in the prior art, and are therefore optimized for use in, for example, detergents and cleansers. This includes point mutagenesis, deletion mutagenesis or insertion mutagenesis, or fusion with other proteins or protein parts. Therefore, for most proteases known in the prior art, appropriately optimized variants are known. For example, EP 2016175 and WO 2021 / 175697 disclose a protease from Bacillus pumilus or a variant thereof for use in detergents and cleaning agents.

[0004] Generally speaking, 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 stable enough. Therefore, for the use of proteases in detergents, it is particularly desirable that they have high catalytic activity and stability under the conditions of the wash cycle.

[0005] Therefore, the disadvantages of the liquid formulations containing proteases and surfactants in the prior art are that the proteases contained do not have satisfactory proteolytic activity and / or insufficient storage stability under standard washing conditions (e.g., in the temperature range of about 20°C to about 40°C), and thus the formulations do not show optimal cleaning performance on protease-sensitive stains. There is still a need to improve the cleaning performance of enzyme-containing detergents and cleaners, especially the cleaning performance of protease-containing detergents and cleaners, especially with respect to protease-sensitive stains, especially to improve the cleaning performance of enzyme-containing detergents and cleaners in the temperature range of about 20°C to about 40°C.

[0006] Surprisingly, it has now been found that a protease from Bacillus pumilus or a protease sufficiently similar thereto (based on sequence identity) is present according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, which are identical to the wild-type form (SEQ ID NO: 1) or the initial variant (reference protease) has improved washing performance and is therefore particularly suitable for use in detergents or cleaning agents, in particular liquid detergents and cleaning agents.

[0007] The present invention therefore provides a protease having proteolytic activity comprising an amino acid sequence which is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the amino acid sequence according to SEQ ID NO: NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one position and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188.

[0008] A preferred subject matter of the present invention is a protease with proteolytic activity comprising an amino acid sequence which is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the amino acid sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one position and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, wherein the amino acid substitutions are selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0009] The present invention also relates to a method for producing a protease as defined above, comprising introducing into the starting molecule: (i) based on the numbering according to SEQ ID NO: 1, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) based on the numbering according to SEQ ID NO: NO:1, at least one and increasingly preferably two, three, four, five or six amino acid substitutions are introduced at at least one position and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, and the amino acid substitutions are preferably selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, and the starting molecule has an amino acid sequence that has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO:1 over its entire length.

[0010] Therefore, proteases in the sense of this patent application include both proteases per se and proteases prepared using the method of the present invention. Therefore, all statements about proteases relate to proteases per se and proteases prepared by the corresponding methods.

[0011] Other aspects of the invention relate to nucleic acids encoding these proteases, non-human host cells containing the proteases or nucleic acids of the invention, and reagents, in particular detergents and cleaning agents, in particular liquid detergents and cleaning agents, comprising the proteases of the invention, to washing and cleaning methods, and to the use of the proteases according to the invention in detergents or cleaning agents for removing protease-sensitive stains.

[0012] These and other aspects, features and advantages of the present invention will be apparent to those skilled in the art by studying the detailed description and claims below. Any feature of one aspect of the present invention may be used in any other aspect of the present invention. In addition, it should be readily understood that the embodiments contained herein are intended to describe and illustrate, but not to limit the present invention, and in particular, the present invention is not limited to these embodiments.

[0013] Unless otherwise indicated, all percentages are expressed as weight percent (wt. %).

[0014] Numerical ranges expressed in the format "from x to y" also include the values. If several preferred numerical ranges are specified in this format, it is readily understood that any range resulting from combinations of the various endpoints is also included.

[0015] 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.

[0016] The terms "detergents and cleaners" or "detergents or cleaners" as used herein are synonymous with the term "agent" and denote compositions for cleaning textiles and / or hard surfaces, in particular dishes, as explained in the specification.

[0017] As used herein, "approximately", "about" or "approximately" when referring to a numerical value refers to ±10%, preferably ±5% of the corresponding numerical value.

[0018] “Substantially free” means that the composition or agent contains less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight, and particularly preferably less than 0.1% by weight of the corresponding substance relative to the total weight of the composition / agent.

[0019] As used herein, "liquid" includes liquids and gels as well as pasty compositions. Liquid compositions are preferably flowable and pourable at room temperature, but they may also have limits of fluidity. According to the definition of the present invention, a substance (e.g., a composition or an agent) is solid if it is in a solid state of aggregation at 25°C and 1,013 mbar. According to the definition of the present invention, a substance (e.g., a composition or an agent) is liquid if it is in a liquid state of aggregation at 25°C and 1,013 mbar. Liquids also include gel forms.

[0020] As used herein, "variant" refers to a naturally occurring or artificially produced variant of a native protease having an amino acid sequence modified from a reference form.

[0021] The present invention is based on the surprising discovery of the inventors that amino acid substitutions at the positions described herein bring about improved cleaning performance of such altered proteases in detergents and cleaners.

[0022] In a preferred embodiment, the protease according to the invention (i) based on the numbering according to SEQ ID NO: 1 at the position corresponding to position 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271 changes and (ii) based on the numbering according to SEQ ID NO: 1 at at least one position corresponding to position 6, 61, 62, 101, 170 and 188 changes, resulting in improved cleaning performance of this improved protease in detergents and cleaning agents, in particular liquid detergents and cleaning agents, on at least one and increasingly preferably two, three, four or five protease-sensitive stains, preferably selected from stains containing blood, egg (egg yolk), milk and other proteins. Thus, the protease of the present invention allows better removal of at least one, preferably multiple, protease-sensitive stains on textiles and / or hard surfaces (e.g., tableware).

[0023] In a preferred embodiment, the protease according to the invention (i) has the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) has at least one and more preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188. Increasingly preferred are two, three, four, five or six amino acid substitutions selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein the combination of an amino acid substitution from group (i) and at least one amino acid substitution from group (ii) results in improved cleaning performance of this improved protease in detergents and cleaners on at least one and increasingly preferably two, three, four or five protease-sensitive stains, preferably selected from stains containing blood, egg (egg yolk), milk and other proteins.

[0024] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0025] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least two amino acid substitutions at at least two positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0026] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least three amino acid substitutions at at least three positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0027] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least four amino acid substitutions at at least four positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0028] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least five amino acid substitutions at at least five positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0029] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having amino acid substitutions at positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0030] In a further preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence which has at least 70%, and increasingly preferably 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% identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the amino acid sequence according to SEQ ID NO: 1, having one of the following amino acid substitution combinations: (a) P9T-S89A-D101S-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (b) P9T-S89A-D101E-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (c) P9T-S89A-D101A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (d) Y6W-P9T-S89A-N130D-T133A-N144K -S189T-Y217M-S224A-N252T-Q271E; (e)Y6W-P9T-F61G-Q62N-S89A-N130D- T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (f) P9T-F61G-Q62-N-S89 A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E; and (g)Y6W -P9T-S89A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E.

[0031] In a particularly preferred embodiment, the protease according to the present invention comprises one of the following amino acid substitution combinations: (a) P9T-S89A-D101S-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (b) P9T-S89A-D101E-N130D-T133A-N144K 4K-S189T-Y217M-S224A-N252T-Q271E; (c)P9T-S89A-D101A-N130D-T133A-N144 K-S189T-Y217M-S224A-N252T-Q271E; (d) Y6W-P9T-S89A-N130D-T133A-N144K-S1 89T-Y217M-S224A-N252T-Q271E; (e)Y6W-P9T-F61G-Q62N-S89A-N130D-T133A-N 144K-S189T-Y217M-S224A-N252T-Q271E; (f)P9T-F61G-Q62-N-S89A-N130D-T133 A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E; and (g) Y6W-P9T-S89A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E, wherein the numbering is in each case based on the numbering according to SEQ ID NO: 1, and wherein the protease contains no further changes besides the mentioned amino acid substitutions.

[0032] In a preferred embodiment, the combination of amino acid substitutions from groups (i) and (ii) described herein in the present invention results in such altered proteases having improved cleaning performance in detergents and cleaners, in particular liquid detergents and cleaners, on at least one and increasingly preferably two, three, four, five or six protease-sensitive stains, preferably selected from stains containing blood, egg (yolk), milk and other proteins. Thus, the proteases of the present invention allow for improved removal of at least one, preferably multiple, protease-sensitive stains on textiles and / or hard surfaces (e.g., tableware). Typical protease-sensitive stains include egg (yolk), blood, milk and other protein-containing stains. When a protease shows improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, there is an improvement in cleaning performance, in particular proteolytic cleaning performance, according to the present invention, said protease-sensitive stains preferably selected from stains containing blood, egg (yolk), milk and other proteins, as described herein.

[0033] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six at positions corresponding to positions 6, 61, 62, 101, 170 and 188 and more preferably Increasingly preferably two, three, four, five or six amino acid substitutions, preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein the protease has improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, preferably selected from the group consisting of stains containing blood, egg (egg yolk), milk and other proteins, the cleaning performance being determined as described in Example 2.

[0034] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least two amino acids at at least two positions corresponding to positions 6, 61, 62, 101, 170 and 188 Substitution, said amino acid substitution is preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein said protease has improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, said protease-sensitive stains preferably being selected from the group consisting of stains containing blood, egg (egg yolk), milk and other proteins, said cleaning performance being determined as described in Example 2.

[0035] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least three amino acids at at least three positions corresponding to positions 6, 61, 62, 101, 170 and 188 A protease comprising: a ...

[0036] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least four amino acids at at least four positions corresponding to positions 6, 61, 62, 101, 170 and 188 Substitution, said amino acid substitution is preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein said protease has improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, said protease-sensitive stains preferably being selected from the group consisting of stains containing blood, egg (egg yolk), milk and other proteins, said cleaning performance being determined as described in Example 2.

[0037] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least five amino acids at at least five positions corresponding to positions 6, 61, 62, 101, 170 and 188 Substitution, said amino acid substitution is preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein said protease has improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, said protease-sensitive stains preferably being selected from the group consisting of stains containing blood, egg (egg yolk), milk and other proteins, said cleaning performance being determined as described in Example 2.

[0038] In a preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence that is at least 70%, and increasingly preferably 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 to the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having amino acid substitutions at positions corresponding to positions 6, 61, 62, 101, 170 and 188, The amino acid substitutions are preferably selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, wherein the protease has improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, the protease-sensitive stains preferably being selected from stains containing blood, egg (egg yolk), milk and other proteins, the cleaning performance being determined as described in Example 2.

[0039] In a further preferred embodiment, the protease according to the invention is a protease with proteolytic activity and comprises an amino acid sequence which has at least 70%, and increasingly preferably 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% identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length, and in each case based on the amino acid sequence according to SEQ ID No.: 1, having one of the following amino acid substitution combinations: (a) P9T-S89A-D101S-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (b) P9T-S89A-D101E-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (c)P9T-S89A-D101A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (d) Y6W-P9T- S89A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (e) Y6W-P9T-F61G-Q62N-S89 A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (f)P9T-F61G-Q62-N-S89A-N130 D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E; and (g) Y6W-P9T-S89A-N130D-T133 A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E, wherein the protease has improved cleaning performance on at least one and increasingly preferably two, three, four or five protease-sensitive stains relative to a reference protease, the protease-sensitive stains preferably being selected from stains containing blood, egg (egg yolk), milk and other proteins, the cleaning performance being determined as described in Example 2.

[0040] In a particularly preferred embodiment, the protease according to the present invention comprises one of the following amino acid substitution combinations: (a) P9T-S89A-D101S-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (b) P9T-S89A-D101E-N130D-T133A-N144K 4K-S189T-Y217M-S224A-N252T-Q271E; (c)P9T-S89A-D101A-N130D-T133A-N144 K-S189T-Y217M-S224A-N252T-Q271E; (d) Y6W-P9T-S89A-N130D-T133A-N144K-S1 89T-Y217M-S224A-N252T-Q271E; (e)Y6W-P9T-F61G-Q62N-S89A-N130D-T133A-N 144K-S189T-Y217M-S224A-N252T-Q271E; (f)P9T-F61G-Q62-N-S89A-N130D-T133 A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E; and (g) Y6W-P9T-S89A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E, wherein the numbering is in each case based on the numbering according to SEQ ID NO: 1, wherein the protease does not contain any other changes besides the mentioned amino acid substitutions, and wherein the protease has improved cleaning performance on at least one and increasingly preferably two, three, four or five protease-sensitive stains, the protease-sensitive stains are preferably selected from stains containing blood, egg (egg yolk), milk and other proteins, and the cleaning performance is determined as described in Example 2.

[0041] The protease according to the present invention has enhanced catalytic activity in detergents or cleaning agents. In various embodiments, the protease of the present invention may have at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185 %, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150% or more proteolytic activity. Such enhanced performance proteases provide improved cleaning of stains sensitive to proteolysis over a range of temperatures, particularly over a temperature range of about 20°C to about 40°C.

[0042] In addition, the preferred embodiments of the protease according to the present invention have special stability in detergents or cleaning agents, for example, compared with surfactants and / or bleaches and / or chelating agents, and / or in terms of temperature effects, especially at high temperatures, for example between about 50°C and about 65°C, especially at about 60°C, and / or in acidic or alkaline conditions and / or in pH changes and / or under denaturants or oxidants and / or proteolytic degradation and / or changes in redox ratios. Therefore, a particularly preferred embodiment of the present invention provides a protease variant with enhanced performance and / or a protease variant with increased temperature stability. A further very particularly preferred embodiment of the present invention provides a protease variant with enhanced performance and / or a protease variant with increased temperature stability. Therefore, this advantageous embodiment of the protease according to the present invention allows to improve the washing effect of stains sensitive to protease in a wide temperature range.

[0043] Cleaning performance within the scope of the present invention is understood to refer to the lightening performance of one or more stains (especially stains on clothing or dishes). Within the scope of the present invention, the detergent or cleaning agent containing protease or the washing liquid or cleaning liquid formed by the detergent or cleaning agent and the protease itself all have respective cleaning performance. Therefore, the cleaning performance of the enzyme contributes to the cleaning performance of the agent or the washing liquid or cleaning liquid formed by the agent. The cleaning performance is preferably determined as described below.

[0044] Washing liquor refers to a ready-to-use solution containing a detergent or cleaning agent, which is applied to textiles or fabrics or hard surfaces, thereby coming into contact with stains present on the textiles or fabrics or hard surfaces. Washing liquor is usually produced at the beginning of the washing or cleaning process, and the detergent or cleaning agent is diluted with water, for example in a dishwasher, washing machine or other suitable container.

[0045] The proteases of the present invention are enzymatically active, i.e. they are capable of hydrolyzing peptides and proteins, in particular in detergents or cleaning agents. Thus, the proteases of the present invention are enzymes which catalyze the hydrolysis of amide / peptide bonds in protein / peptide substrates and are thus capable of cleaving proteins or peptides. In addition, the proteases of the present invention are preferably mature proteases, i.e. catalytically active molecules without signal peptides and / or propeptides. Unless otherwise indicated, the sequences shown in each case also refer to mature (processed) enzymes.

[0046] In various embodiments of the present invention, the protease is a free enzyme. This means that the protease can directly act on all the ingredients 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 comprise a protease that forms an interaction complex with other molecules or a protease that comprises a "coating". In this case, one or more protease molecules can be separated from other components of the reagent by the structure surrounding them. This separation structure can be due to, but not limited to, vesicles such as micelles or liposomes. However, the surrounding structure can also be a viral particle, a bacterial cell, or a eukaryotic cell. In various embodiments, the reagent may comprise a cell of Bacillus pumilus or Bacillus subtilis expressing a protease according to the present invention, or a cell culture supernatant of such a cell.

[0047] For the purposes of the present invention, a protease characterized by a given substitution means that it comprises one or more (given) substitutions at the relevant position, i.e., no other mutations or deletions occur at least at the given position - e.g., cleavage of the protease. In various embodiments, the proteases described herein, except for the explicitly mentioned substitutions, have the sequence of SEQ ID NO: 1, i.e., except for the substituted positions, they have 100% identity with the sequence according to SEQ ID NO: 1.

[0048] 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: 3389-3402), and in principle occurs by assigning similar nucleotides or amino acid sequences to each other in nucleic acid or amino acid sequences. The tabular assignment of related positions is called alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparison (alignment), particularly multiple sequence comparison, is generated 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, e.g., these programs or algorithms are often used. The computer program Vector Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) using specified standard parameters for sequence comparison (alignment), wherein the AlignX module for sequence comparison is based on ClustalW, or Clone Manager 10 (sequence alignment at the amino acid level using the scoring matrix BLOSUM 62). Unless otherwise stated, sequence identities indicated herein are determined using the BLAST algorithm.

[0049] Such comparisons also allow conclusions to be drawn about the similarity of the compared sequences to each other. It is usually given as a percentage identity, i.e., the ratio of identical nucleotides or amino acid residues at the same position or positions corresponding to each other in the comparison. In the case of amino acid sequences, the broader concept of homology takes into account the exchange of conservative amino acids, i.e., the exchange of amino acids with similar chemical activities, because they usually perform similar chemical activities in proteins. Therefore, the similarity of the compared sequences can also be expressed as a percentage homology or percentage similarity. Identity and / or homology information about the entire polypeptide or gene can be provided, or only identity and / or homology information about a single region can be provided. Therefore, the homology or same region of different nucleic acids or amino acid sequences is 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 single (optionally small) region. However, unless otherwise stated, the identity or homology information in this application relates to the entire length of the indicated specific nucleic acid or amino acid sequence.

[0050] In the context of the present invention, an indication that an amino acid position corresponds to a numerically designated position in SEQ ID NO: 1 thus means that the corresponding position is associated with the numerically designated position in SEQ ID NO: 1 in the alignment as defined above. Furthermore, the assignment of the positions is based on the mature protein. This assignment also applies in particular when the amino acid sequence of the protease according to the invention comprises a greater number of amino acid groups than the protease according to SEQ ID NO: 1. The positions that are altered in the protease according to the invention, based on said positions in the amino acid sequence of the protease according to SEQ ID NO: 1, are the positions assigned to said positions in the alignment.

[0051] In addition to the amino acid changes explained above, the protease of the present invention can also have other amino acid changes, particularly amino acid substitutions, insertions or deletions. For example, by targeted genetic changes, such proteases are developed by mutagenesis methods and optimized for specific uses or specific properties (for example, with respect to its catalytic activity, stability, etc.). In addition, the nucleic acid of the present invention can be introduced into a recombinant method to produce brand-new proteases or other polypeptides. The purpose is to introduce targeted mutations (for example, substitutions, insertions or deletions) into known molecules, such as to improve the cleaning performance of the enzyme of the present invention. For this reason, the surface charge and / or isoelectric point of the molecule can be particularly changed to change 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 additionally, the stability or catalytic activity of the protease can be increased by one or more corresponding mutations, so that its cleaning performance can be improved. The favorable properties of a single mutation (for example, a single substitution) can complement each other. Therefore, a protease optimized for certain properties (for example, with respect to its stability during storage) can be additionally developed in the context of the present invention.

[0052] For substitutions involving only one amino acid position (amino acid exchange), the following convention is used herein: first, the naturally occurring amino acid is designated in the form of the internationally accepted single-letter code, followed by the relevant sequence position, and finally the inserted amino acid. Multiple or alternative exchanges within the same polypeptide chain are separated by slashes. Thus, "130D / V" means that position 130 has been mutated to D or V. In the case of an insertion, the additional amino acid is named according to the sequence position. In the case of a deletion, the deleted amino acid is replaced by a symbol (e.g., an asterisk or a dash), or Δ is marked before the corresponding position. For example, P9T describes the replacement of proline at position 9 by threonine, P9TH describes the insertion of histidine after the amino acid threonine at position 9, and P9* or ΔP9 describes the deletion of proline at position 9. This nomenclature is known to those skilled in the art of enzyme technology.

[0053] Therefore, the present invention also relates to a protease, characterized in that the protease is obtained by using the protease described herein as a starting molecule through one or more conservative amino acid substitutions, and the protease has at least one of the above amino acid substitutions according to the numbering of SEQ ID NO: 1. The term "conservative amino acid substitution" refers to exchanging (substituting) one amino acid functional group with another amino acid functional group, and this exchange does not cause a change in the polarity or charge at the position of the amino acid exchange, such as exchanging a non-polar amino acid functional group with another non-polar amino acid functional group. Within the scope of the present invention, conservative amino acid substitutions 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.

[0054] Alternatively or additionally, the protease is characterized in that it is obtained from a protease according to the invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence corresponding to a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273 or 274 or 275 consecutive amino acids of the starting molecule, wherein the protease is in each case based on the sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0055] Thus, for example, a single amino acid in the end or ring of the enzyme can be deleted without causing a loss or reduction of proteolytic activity. In addition, such fracture or deletion, insertion or substitution mutagenesis can also be used, for example, to reduce the allergenicity of the relevant enzyme, thereby improving its availability as a whole. Advantageously, even after mutagenesis, the enzyme still retains its proteolytic activity, that is, its proteolytic activity is at least corresponding to the proteolytic activity of the starting enzyme, that is, in a preferred embodiment, the proteolytic activity is at least 100% of the activity of the starting enzyme, preferably at least 120%. Further substitution can also show a favorable effect. Single and multiple continuous amino acids can be replaced with other amino acids.

[0056] Thus, advantageous positions for sequence changes (particularly substitutions) of the protease according to SEQ ID NO: 1 are positions corresponding to the positions described in the alignment herein (i.e. according to the numbering of SEQ ID NO: 1), which preferably have significance when transferred to the homologous positions of the protease according to the invention and confer advantageous functional properties to the protease. The following amino acid residues are located at the mentioned positions in the wild-type molecule of the protease: 6Y, 9P, 61F, 62Q, 89A, 101D, 130N, 133T, 144K, 170K, 188N, 189S, 217Y, 224S, 252N, 271Q.

[0057] The correct assignment of the amino acids to be varied, i.e. in particular their functional correspondence, can be further confirmed by comparative tests, based on which two positions which were assigned to one another on the basis of an alignment of two proteases to be compared are altered in the same manner and observations are made to determine whether the enzymatic activity in both proteases is altered in the same manner. For example, if an amino acid substitution at a certain position of the protease from Bacillus pumilus according to SEQ ID NO: 1 is accompanied by a change in enzyme parameters, e.g. K M The values ​​of K and K were increased, and corresponding changes in enzyme parameters were observed in the protease variants of the present invention, such as K M If an increase in the value of , wherein an amino acid exchange of a protease variant according to the invention is achieved by introducing an identical amino acid, this can be regarded as confirmation of the correct assignment.

[0058] All these aspects also apply to the method for producing protease according to the present invention. Therefore, the method according to the present invention also comprises one or more of the following method steps:

[0059] (a) introducing single or multiple conservative amino acid substitutions, wherein the protease, in each case based on the numbering according to SEQ ID NO: 1, (i) has the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) has at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions being preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G;

[0060] (b) modifying the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence corresponding to a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 consecutive amino acids of the starting molecule, wherein the protease is in each case based on the amino acid sequence of SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0061] All embodiments also apply to the method according to the invention.

[0062] In another embodiment of the present invention, the protease described previously is stabilized, particularly by one or more mutations, such as substitutions, or by a method coupled to a polymer. The increase in stability during storage and / or use (e.g., in a washing process) causes the enzyme activity to last longer, thereby improving cleaning performance. In principle, all stabilization options described and / or suitable in the prior art are conceivable. Preferably, those stabilizations achieved by the mutation of the enzyme itself are preferred, because this stabilization does not require any further working steps after the enzyme is recovered. The example of a sequence change suitable for this purpose is stated above. Further suitable sequence changes are known from the prior art.

[0063] Other possibilities for stabilization are, for example:

[0064] - 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 / glycine;

[0065] - protection against the effects of denaturing agents such as surfactants by mutations that result in changes in the amino acid sequence on or on the surface of the protein;

[0066] - Replace amino acids close to the N-terminus with amino acids that are likely to contact the rest of the molecule through non-covalent interactions, thereby helping to maintain the globular structure.

[0067] Preferred embodiments are those in which the enzyme is stabilized in multiple ways, in that the multiple stabilizing mutations act in an additive or synergistic manner.

[0068] Alternatively or additionally, the protease of the present invention may be combined with at least one reversible inhibitor compound selected from the group consisting of: polyols, in particular glycerol and 1,2-propylene glycol, benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters or derivatives, in particular phenylboronic acid derivatives or 4-formylphenylboronic acid (4-FPBA), compounds of formula (I) or (II)

[0069]

[0070] wherein R is in each case selected from -COOH, C 1-6 Alkyl substituted or unsubstituted C 2-6 Dicarboxylic acid, C 1-6 Alkyl substituted or unsubstituted C 2-6 Carboxylic acid and -OOC-NR 2 2, where R 2 The same or different and selected from C 1-6 Alkyl or H, and salts, esters or derivatives thereof, preferably benzoic acid, phenylmalonic acid, benzylmalonic acid, phenylsuccinic acid, benzylsuccinic acid, methyl 3-benzoylpropionate, (S)-3-phenylbutyric acid and benzyl carbamate, and combinations thereof, to further improve the stability of the protease in detergents and cleaners.

[0071] In the context of the present invention, "phenylboronic acid derivatives" are understood to be compounds of formula (III). The compound of formula (III) has the following structural formula:

[0072]

[0073] Where R is hydrogen, hydroxyl, C 1-6 Alkyl, substituted C 1-6 Alkyl, C 1-6 Alkenyl or substituted C 1-6 Preferably, the functional group R in the phenylboronic acid derivative is C 1-6 Alkyl, more preferably -CH3, -CH3CH2 or -CH3CH2CH2. More preferably, the functional group R in the phenylboronic acid derivative is hydrogen. Particularly preferably, the phenylboronic acid derivative is 4-formylphenylboronic acid (4-FPBA).

[0074] The reversible inhibitor compound used may be a boronic acid.

[0075] The reversible inhibitor compound used may be a compound of formula (I) or (II)

[0076]

[0077] wherein R is in each case selected from -COOH, C 1-6 Alkyl substituted or unsubstituted C 2-6 Dicarboxylic acid, C 1-6 Alkyl substituted or unsubstituted C 2-6 Carboxylic acid and -OOC-NR 2 2, where R 2 The same or different and selected from C 1-6 Alkyl or H, and salts, esters or derivatives thereof, and combinations thereof, are preferably selected from benzoic acid, phenylmalonic acid, benzylmalonic acid, phenylsuccinic acid, benzylsuccinic acid, methyl 3-benzoylpropionate, (S)-3-phenylbutyric acid and benzyl carbamate.

[0078] In a particularly preferred embodiment, the protease according to the invention is used in an agent or composition which is essentially free of boron-containing compounds. In this context, "essentially free of boron-containing compounds" means that the respective agent or composition contains less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight, particularly preferably less than 0.1% by weight of boron-containing compounds, based on the total weight of the agent / composition. In a very particularly preferred embodiment, these agents / compositions contain no boron-containing compounds, i.e. in particular they contain no boric acid and / or phenylboronic acid derivatives.

[0079] In another embodiment of the present invention, the protease is characterized in that its cleaning performance is not significantly reduced compared to the wild-type enzyme (SEQ ID NO: 1) or the starting variant (reference protease), i.e., it has at least 80% of the reference washing performance, preferably at least 100%, more preferably at least 110%, and particularly preferably at least 120% or more.

[0080] Cleaning performance can be measured in a washing system containing detergent (dosage is 2.0 to 8.0 grams per liter of washing liquid) and enzyme. The enzyme to be compared is used at the same concentration (based on active protein). The use of the activity equivalent of the related enzymes ensures that even if the ratio of active substance to total protein (specific activity value) deviates, the respective enzyme characteristics, such as the cleaning performance to certain stains, can be compared. Generally, low specific activity can be compensated by adding a larger amount of protein. In addition, if the enzyme to be detected has different affinities for the test substrate in the activity test, the enzyme to be detected can also be used with the same amount or weight. In this context, the expression "same amount" refers to the use of the enzyme to be detected by molar amount. The expression "same weight" refers to the use of the same weight of the enzyme to be detected.

[0081] The protease concentration in the detergent used for such washing systems is from 0.001 to 0.1% by weight, preferably from 0.01 to 0.06% by weight, relative to the active protein.

[0082] Washing or cleaning performance refers to the ability of a detergent or cleaning agent to partially or completely remove existing stains, particularly the lightening performance of one or more stains on a textile (particularly a cotton textile, a polyester textile and / or a mixed cotton-polyester textile). Examples of such stains are bloodstains on cotton or chocolate milk / cigarette ash on cotton, cocoa on cotton, egg yolk on cotton, milk / cigarette ash on cotton or porridge on cotton etc. Further examples also include cotton-polyester blended textiles or the above-mentioned stains on polyester textiles or other blended textiles. Within the scope of the present invention, a detergent or cleaning agent comprising protease or a washing liquid or cleaning solution formed by the reagent and protease itself all have cleaning performance. Therefore, the cleaning performance of protease contributes to the cleaning performance of the reagent or the washing liquid or cleaning solution formed by the reagent.

[0083] Washing liquor or cleaning liquor refers to a solution containing a detergent or cleaning agent, which is applied to textiles or hard surfaces to come into contact with stains on the textiles or hard surfaces. Washing liquor or cleaning liquor is usually produced at the beginning of the washing or cleaning process, the detergent or cleaning agent being diluted with water, for example in a washing machine, dishwasher or other suitable container.

[0084] A liquid reference detergent for such a washing system may, for example, be composed as follows (all figures are weight percentages (wt%)): 4.4% alkylbenzenesulfonic acid, 5.6% other anionic surfactants, 2.4% C 12-18Fatty acid sodium salt (soap), 4.4% nonionic surfactant, 0.2% phosphonate, 1.4% citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerol, 0.08% preservative, 1% ethanol, the remainder is deionized water. The dosage of the liquid detergent is preferably 4.5 to 6.0 grams per liter of washing liquid, for example 4.7, 4.9 or 5.9 grams per liter of washing liquid. The washing process is preferably carried out in a pH range between pH 7 and pH 10.5, preferably between pH 8 and pH 9.

[0085] The cleaning performance for stains on cotton is determined by measuring the degree of cleanliness of washed textiles. For example, the washing process can be carried out for 60 minutes at a temperature of about 20°C or about 40°C, and the hardness of the water can be between 15.5°dH and 16.5°dH (German hardness). In the context of the present invention, the cleaning performance is determined, for example, at 20°C or 40°C using a liquid detergent (such as the above-mentioned liquid detergent), wherein the washing process is preferably carried out for 60 minutes at 600rpm.

[0086] Optical measurement methods, preferably photometry, are used to determine whiteness (i.e. the degree of lightening of stains) as a measure of cleaning performance. An apparatus suitable for this purpose is, for example, a Minolta CM508d spectrometer. Typically, the apparatus used for the measurement is calibrated in advance using a white standard, preferably a provided white standard.

[0087] Preferred embodiments of the protease according to the invention achieve this advantageous cleaning performance even at low temperatures, in particular in a temperature range of about 10°C to about 60°C, preferably in a temperature range of about 15°C to about 50°C, particularly preferably in a temperature range of about 20°C to about 40°C.

[0088] Methods for determining protease activity are familiar to those skilled in the art of enzyme technology and are routinely used. For example, such methods are disclosed in Tenside, volume 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 cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410nm, and its time curve is a measure of enzyme activity (see Del Mar et al., 1979). The measurement is carried out at a temperature of 25°C, pH 8.6 and a wavelength of 410nm. The measurement time is 5 minutes and the measurement interval is 20 seconds to 60 seconds. Protease activity is usually expressed in protease units (PU). Suitable protease activity reaches, for example, 2.25, 5 or 10 PE per milliliter of washing solution. However, protease activity is not equal to zero.

[0089] According to the present invention, an alternative test for determining the proteolytic activity of a protease is an optical measurement method, preferably a photometric method. A test suitable for this purpose comprises the protease-dependent cleavage of the substrate protein casein. Casein is cleaved into a plurality of smaller partial products by the protease. Relative to uncut casein, the entirety of these partial products has an increased absorbance at 290 nm, wherein the absorbance of this increase can be measured using a photometer, thereby a conclusion about the enzymatic activity of the protease can be drawn.

[0090] The protein concentration can be determined by known methods, such as the BCA method (bicinchoninic acid; 2,2'-bicinchoniyl-4,4'-dicarboxylic acid) or the biuret method (Gornall et al., J. Biol. Chem. 177 (1948): 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 (see Bender et al., J. Am. Chem. Soc. 88, 24 (1966): 5890-5913).

[0091] The present invention also relates to proteases as described above, characterized in that it has at least one chemical modification. Proteases with such modifications are called derivatives, i.e., proteases are derivatized. In the context of the present application, derivatives are therefore understood to be proteins whose pure amino acid chains have been chemically modified. This derivatization can be achieved, for example, in vivo by a host cell expressing the protein. In this regard, coupling with 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 covalently binding another compound to the protein. 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, bound to the protein of the present invention by a bifunctional compound. Derivatization is also understood to refer to covalent binding or non-covalent inclusion in a suitable macromolecular cage structure with a macromolecular carrier. Derivatization can affect, for example, substrate specificity or binding strength with a substrate, or if the coupled substance is an inhibitor, a temporary blocking of the enzyme activity is caused. For example, this may be advantageous for the shelf life. This modification may further affect stability or enzyme activity. They can also be used to reduce the allergenicity and / or immunogenicity of proteins, thereby increasing, for example, 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 the proteins of the present invention can also be understood as preparations of these proteins in the broadest sense. Proteins can be combined with various other substances, such as cultures from producing microorganisms, according to their recovery, processing or preparation. Proteins can also be deliberately mixed with other substances, for example to increase their storage stability. Therefore, all preparations of the proteins of the present invention are also in line with the present invention. This is also independent of 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 a small amount of activity during storage, and only shows its enzyme function when used. This can be controlled by, for example, corresponding accompanying substances. In this regard, proteases can be prepared in combination with specific inhibitors, in particular. Among all the above-mentioned proteases or protease variants and / or derivatives, those proteases or protease variants and / or derivatives having improved storage stability and / or cleaning performance compared to the starting variants are particularly preferred in the context of the present invention, wherein the cleaning performance in the washing system is determined as described herein.

[0092] The present invention also relates to nucleic acids encoding proteases of the present invention, and vectors containing such nucleic acids, in particular cloning vectors or expression vectors. These vectors can be DNA or RNA molecules. They can exist in the form of single strands, single strands complementary to the single strands, or double strands. In particular, in the case of DNA molecules, the sequences of the two complementary strands must be considered in all three possible reading frames. In addition, it must be taken into account that different codons (i.e., base triplets) can encode the same amino acid, so that a certain amino acid sequence can be encoded by multiple different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences that can encode any of the above-mentioned proteases are included in the subject matter of the present invention. Those skilled in the art can undoubtedly determine these nucleic acid sequences, because despite the degeneracy of the genetic code, a determined amino acid can be assigned to a single codon. Therefore, based on the amino acid sequence, those skilled in the art can easily determine the nucleic acid encoding the amino acid sequence. In addition, in the case of the nucleic acids of the present invention, one or more codons can be replaced by synonymous codons. This aspect particularly relates to the heterologous expression of the enzyme of the present invention. Therefore, each organism, such as the host cell of the production strain, has a certain codon selection. "Codon selection" refers to the translation of the genetic code into amino acids by the relevant organism. If a codon on an organism's nucleic acid is faced with a relatively small number of loaded tRNA molecules, a bottleneck may occur in protein biosynthesis. This causes the codon to be translated less efficiently in the organism than a synonymous codon encoding the same amino acid, despite encoding the same amino acid. Since a larger number of tRNA molecules exist for the synonymous codon, this can be translated more efficiently in the organism.

[0093] Those skilled in the art can utilize currently generally known methods, such as chemical synthesis or polymerase chain reaction (PCR), in combination with standard methods of molecular biology and / or protein chemistry, to produce corresponding nucleic acids or even complete genes based on known DNA and / or amino acid sequences. Such methods can be obtained, for example, from Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd edition, Cold Spring Laboratory Press.

[0094] For the purposes of the present invention, "vector" refers to an element consisting of nucleic acid, which comprises the nucleic acid of the present invention as a characteristic nucleic acid region. They can establish these nucleic acids as stable genetic elements in species or cell lines in several generations or cell divisions. Vectors, particularly when used in bacteria, are special plasmids, i.e. circular genetic elements. In the context of the present invention, nucleic acids of the present invention are cloned into vectors. Vectors include, for example, vectors derived from bacterial plasmids, viruses or bacteriophages, or are mainly synthetic vectors or plasmids with elements from various sources. In each case, by other genetic elements present, vectors can establish themselves as stable units in specific host cells in several generations. They can be present as separate units outside the chromosome, or can be integrated into chromosomes or chromosomal DNA. Expression vectors include nucleic acid sequences that allow them to replicate in host cells containing them, preferably microorganisms, particularly preferably bacteria, and express the nucleic acids contained therein. The expression is particularly affected by one or more promoters that regulate transcription. In general, expression can be carried out by a natural promoter that is initially located in front of the nucleic acid to be expressed, by a promoter of the host cell provided on the expression vector, or by a modified or completely different promoter of another organism or another host cell. In this article, at least one promoter is provided for the expression of the nucleic acid according to the invention and is used for its expression. The expression vector can also be regulatable, for example by changing the culture conditions or when the host cells containing them reach a certain cell density or by adding certain substances, in particular gene expression activators. An example of such a substance is the galactose derivative isopropyl β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to the expression vector, the nucleic acid contained is not expressed in the cloning vector.

[0095] The present invention also relates to a non-human host cell, which contains a nucleic acid of the present invention or a vector of the present invention, or contains a protease of the present invention, in particular a non-human host cell that secretes a protease into a culture medium surrounding the host cell. Preferably, the nucleic acid of the present invention or the vector of the present invention is transformed into a microorganism, which then represents the host cell of the present invention. Alternatively, a single component, i.e. a part of a nucleic acid of the present invention or a nucleic acid fragment, can also be introduced into a host cell in such a way that the resulting host cell contains a nucleic acid of the present invention or a vector of the present invention. This procedure is particularly suitable when the host cell already contains one or more components of a nucleic acid of the present invention or a vector of the present invention and then supplements other components accordingly. Methods for transforming cells have been established in the prior art and are well known to those skilled in the art. In principle, all cells, i.e. prokaryotic cells or eukaryotic cells, are suitable as host cells. Preferably, a host cell that can be managed in a genetically advantageous manner, such as a host cell that is advantageous for transformation with a nucleic acid or a vector and its stable establishment, such as a unicellular fungus or a bacterium. In addition, a preferred host cell feature is good microbiological and biotechnological manageability. This relates to, for example, easy cultivation, high growth rate, low requirements for fermentation medium, and good production and secretion rate of foreign proteins. According to the present invention, preferred host cells secrete the (transgenic) expressed protein into the culture medium surrounding the host cells. In addition, proteases can be modified by the cells producing them after their production, for example by attachment of sugar molecules, formylation, amination, etc. Such post-translational modifications can have a functional impact on proteases.

[0096] A further preferred embodiment is a host cell whose activity can be regulated by genetic control elements, which are provided, for example, on a vector, but which may also be present in the cells from the outset. For example, expression in the cells can be induced by controlled addition of a chemical compound used as an activator, by changing the culture conditions or when a specific cell density is reached. This allows the economical production of proteins according to the invention. An example of such a compound is IPTG as described above.

[0097] Prokaryotic or bacterial cells are preferred host cells. Bacteria are characterized by short generation time and low requirements for culture conditions. This makes it possible to establish an economical and effective culture method or production method. In addition, those skilled in the art have extensive experience in the fermentation technology of bacteria. Gram-negative or Gram-positive bacteria can be suitable for specific production due to many different reasons, which need to be determined by experiments according to various specific circumstances, such as nutrient sources, product formation rate, required time, etc. For Gram-negative bacteria, such as Escherichia coli, a large amount of protein is secreted into the periplasmic space, that is, secreted into the compartment between the two membranes surrounding the cell. This can be advantageous for specific applications. In addition, Gram-negative bacteria can also be designed so that they not only release the expressed protein into the periplasmic space, but also into the culture medium around the bacteria. On the contrary, Gram-positive bacteria, such as bacillus or actinomycetes or other representative bacteria of Actinomycetales do not have outer membranes, so secreted proteins will be immediately released into the culture medium around the bacteria, usually nutrient medium, from which the expressed protein can be purified. They can be directly separated from the culture medium or further processed. In addition, Gram-positive bacteria are related or identical to most of the origin organisms in terms of technologically important enzymes, and usually they themselves form comparable enzymes, so that they have similar codon selections, and their protein synthesis apparatus is naturally arranged accordingly. The host cell according to the present invention can be changed in terms of its requirements for culture conditions, with different or additional selection markers, or also express different or additional proteins. In particular, this can also relate to those host cells expressing multiple proteins or enzymes. The present invention can be applied in principle to all microorganisms, in particular all fermentable microorganisms, particularly preferably microorganisms of the genus Bacillus, and allows the use of such microorganisms to produce proteins according to the present invention. Then, for the purposes of the present invention, such microorganisms represent host cells.In another embodiment of the present invention, the host cell is characterized in that it is a bacterium, preferably a bacterium selected from the following genera: Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably a bacterium selected from the following genera: Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alkaliphilic alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

[0098] However, the host cell can also be a eukaryotic cell, characterized in that it has a nucleus. Therefore, the present invention also relates to a host cell, characterized in that it has a nucleus. In contrast to prokaryotes, eukaryotic cells are able to modify the formed protein after translation. Examples thereof are fungi, such as actinomycetes or yeasts, such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the protein undergoes a specific modification during its synthesis, the modification makes this system possible. The modifications carried out by the eukaryotic system, particularly modifications related to protein synthesis, include, for example, binding to low molecular weight compounds, such as membrane anchors or oligosaccharides. This oligosaccharide modification can be desired, for example, to reduce the allergenicity of the expressed protein. Coexpression with enzymes (such as cellulases) naturally formed by this cell can also be advantageous. In addition, for example, thermophilic fungi expression systems are particularly suitable for expressing thermostable proteins or variants.

[0099] The host cells of the present invention are cultured and fermented in the usual manner, for example in a batch or continuous system. In the first case, the host cells are inoculated into a suitable nutrient medium and the product is harvested from the medium after a period of time to be determined experimentally. Continuous fermentation is characterized by achieving a flow equilibrium, in which the cells partially die after a relatively long period of time, but also re-grow, and at the same time, the formed protein can be removed from the medium.

[0100] The host cell of the present invention is preferably used to produce the protease of the present invention. Therefore, the present invention also relates to a method for producing the protease, comprising

[0101] a) culturing the host cell of the present invention, and

[0102] b) Isolating the protease from the culture medium or the host cells.

[0103] The subject of the present invention preferably comprises fermentation process. Fermentation process itself is known from the prior art, and represents actual large-scale production step, and is usually followed by suitable purification method for the product (for example according to the protease of the present invention) produced. All fermentation processes based on the corresponding method for producing the protease according to the present invention represent the embodiment of the subject of the present invention. It is particularly contemplated that fermentation process characterized by fermentation by feeding strategy. In this case, the medium components consumed by continuous culture are added. As a result, a significant increase in cell density and cell mass or dry mass, and / or a significant increase in the activity of the target protease in particular can be achieved. In addition, fermentation can also be designed to filter out undesirable metabolites or neutralize by adding buffer or suitable counter ions. The prepared protease can be harvested from the fermentation medium. This fermentation process is superior to isolating protease from host cells (that is, preparing products from cell mass (dry mass)), but it is necessary to provide suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems so that the host cells secrete the protease into the fermentation medium. If there is no secretion, it is also possible to alternatively isolate protease from host cells, that is, purify it from the cell mass, for example by precipitation with ammonium sulfate or ethanol or by chromatographic purification.

[0104] All of the above aspects can be combined to form a method for producing the protease of the present invention.

[0105] The present invention also provides an agent, characterized in that it contains the protease according to the present invention as described above. The agent is preferably a detergent or a cleaning agent. Particularly preferably, the detergent and cleaning agent are substantially free of boron-containing compounds. In this context, "substantially free of boron-containing compounds" means that, based on the total weight of the agent, the agent according to the present invention contains less than 2% by weight, preferably less than 1% by weight, more preferably less than 0.5% by weight, and particularly preferably less than 0.1% by weight of boron-containing compounds. In a very particularly preferred embodiment, the detergents and cleaning agents according to the present invention do not contain boron-containing compounds, that is, in particular, they do not contain boric acid and / or phenylboronic acid derivatives.

[0106] According to the present invention, all conceivable detergent or cleaning agent types are understood as detergent or cleaning agent, including concentrates and undiluted agents, for commercial scale, washing machines or hand washing or cleaning. These include, for example, detergents for textiles, carpets or natural fibers, for which the term detergent is used. These include, for example, dishwashing detergents for dishwashers (dishwashing detergents) or manual dishwashing detergents or cleaning agents for hard surfaces (such as metal, glass, porcelain, ceramics, tiles, stones, painted surfaces, plastics, wood or leather), for which the term cleaning agent is used, i.e., in addition to manual and mechanical dishwashing detergents, there are also, for example, scrubbers, glass cleaners, toilet fragrances, etc. The detergents and cleaning agents of the present invention also include auxiliary detergents, which are added to the actual detergents in manual or automatic textile washing processes to achieve further effects. Furthermore, in the context of the present invention, detergents and cleaning agents also include textile pre- and post-treatment agents, i.e. those agents which come into contact with the laundry before the actual washing, for example to dissolve stubborn stains, and which impart further desired properties to the laundry in a downstream step of the actual textile washing, such as a pleasant feel, wrinkle resistance or low static charge. Furthermore, softeners are also included in the latter agents.

[0107] The detergent or cleaning agent of the present invention may be a powder or granular solid, compacted or further compacted particle form, homogeneous solution or suspension, and it may also contain all known conventional ingredients in such detergents or cleaning agents in addition to the protease of the present invention, preferably at least one other ingredient is present in the detergent or cleaning agent. The detergent or cleaning agent of the present invention may especially contain surfactants, builders, polymers, glass corrosion inhibitors, corrosion inhibitors, bleaching agents (e.g. peroxides), bleach activators or bleach catalysts. They may also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, chelating agents, electrolytes, pH adjusting agents and / or other auxiliaries, such as optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators, dyes and spices, and combinations thereof.

[0108] Advantageous ingredients of the agents of the invention are disclosed in the international patent application WO 2009 / 121725, starting from the second to last paragraph on page 5 and ending after the second paragraph on page 13. Reference is expressly made to this disclosure and the disclosure content therein is incorporated into the present patent application.

[0109] The agent of the present invention advantageously contains 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg, very particularly preferably 50 μg to 10 mg of the protease of the present invention per gram of the agent. In various embodiments, the concentration of the protease (active enzyme) described herein in the preparation is > 0 to 1% by weight, preferably 0.0001 or 0.001 to 0.1% by weight, based on the total weight of the agent or composition.

[0110] The reagent of the present invention contains protease, and its amount is increasingly preferably 1x 10 -8 % by weight, 0.0001 to 1 % by weight, 0.0005 to 0.5 % by weight, 0.001 to 0.1 % by weight, in each case relative to the active protein and relative to the total weight of the detergent.

[0111] Embodiments of the present invention include all solid, powder, liquid, gel or pasty application forms of reagent of the present invention, which also can be optionally composed of multiple phases, and can exist in compressed or uncompressed form.Described reagent can exist in free-flowing powder form, particularly with 300g / l to 1200g / l, particularly 500g / l to 900g / l or 600g / l to 850g / l of bulk density.The solid application form of described reagent also comprises extrudate, granule, tablet or sachet.Or, this reagent can also be liquid, gel or paste form, for example non-aqueous liquid detergent or non-aqueous paste form, or aqueous liquid detergent or aqueous paste form.Liquid preparation is usually preferred.In addition, this reagent can exist as a single component system.This reagent is composed of one phase.Or, this reagent can also be composed of multiple phases.Therefore, this reagent is divided into multiple components.

[0112] The proteases of the present invention are preferably used in liquid detergents for cleaning textiles, and are particularly preferably used in liquid detergents having a pH of about 8 to about 9.

[0113] The detergent or cleaning agent of the present invention may contain only one protease of the present invention. Alternatively, they may also contain other hydrolases or other enzymes, the concentration of which is conducive to the effectiveness of the reagent. Therefore, another embodiment of the present invention is represented by a reagent that also contains one or more other enzymes. Other enzymes that can preferably be used are all enzymes that can show catalytic activity in the reagent of the present invention, in particular lipases, amylases, cellulases, hemicellulases, mannanases, tannases, xylanases, xylanases, xanthanases, xyloglucanases, β-glucosidases, pectinases, carrageenases, perhydrolases, oxidases, oxidoreductases or other proteases different from the protease of the present invention, and mixtures thereof. The amount of other enzymes contained in the reagent is advantageously 1 x 10 based on active protein. -8 More preferably, the amount of each additional enzyme contained in the agent according to the invention is 1 x 10 -7The enzyme preferably has a synergistic cleaning performance for a particular dirt or stain, i.e., the enzymes contained in the reagent assist each other in their cleaning performance. Such synergy is particularly preferably present between the protease contained in the present invention and other enzymes of the reagent of the present invention, particularly including between the protease and amylase and / or lipase and / or mannanase and / or cellulase and / or pectinase. Synergy can occur not only between different enzymes, but also between one or more enzymes and other components of the reagent of the present invention.

[0114] According to the invention preferred textile detergents have at least one protease and at least one amylase. In another preferred embodiment of the 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. In a further preferred embodiment, the textile detergent has at least one protease, at least one amylase and at least one lipase. In a further preferred embodiment, the textile detergent has at least one protease, at least one amylase and at least one cellulase. In a further preferred embodiment, the textile detergent has at least one protease, at least one amylase and at least one cellulase. In a further preferred embodiment, the textile detergent has at least one protease, at least one amylase, at least one cellulase and at least one lipase. Textile detergents with 3 to 10 different enzymes are particularly preferred, and textile detergents with 3 to 10 different types of enzymes may be particularly preferred in terms of very broad stain cleaning performance.

[0115] Examples of proteases include subtilisin BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, protease PB92, subtilisin 147 and 309, a protease from Bacillus lentus, subtilisin DY, as well as thermophilic proteases, proteinase K and proteases TW3 and TW7, which are more narrowly related to subtilases but no longer to subtilisins. Subtilisin Carlsberg is marketed under the trade name Subtilisin 147 and 309 are supplied by Novozymes under the trade names and Protease variants described, for example, in WO 95 / 23221, WO 92 / 21760, WO 2013 / 060621 and EP3660151 are derived from the protease of Bacillus lentus DSM 5483. Other suitable proteases are available, for example, from Novozymes under the trade name Progress Uno and The enzyme was obtained from Danisco / DuPont under the trade name OxP, Prime, and Prefer and Preferenz The enzyme was obtained from BASF under the trade name Lavergy pro104 The enzyme was obtained from Advanced Biochemicals Ltd. under the trade name The enzyme was obtained from WuxiSnyder Bioproducts Ltd. under the trade name The enzyme was obtained from Amano Pharmaceuticals Ltd. under the trade name and Protease The enzyme obtained from , and the enzyme obtained from Kao Corp under the name Proteinase K-16. Particularly preferably, the proteases from Bacillus gibberellus and Bacillus pumilus are used, which are disclosed in WO 2008 / 086916, WO 2007 / 131656, WO 2017 / 215925, WO 2021 / 175696 and WO 2021 / 175697. Other proteases that can be used are those naturally present in the microorganism Stenotrophomonasmaltophilia, in particular Stenotrophomonasmaltophilia K279a, Bacillusintermedius and Bacillus sphaericus.

[0116] Examples of amylases include α-amylases from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus, and developments thereof which are particularly improved for use in detergents or cleaners. The enzyme from Bacillus licheniformis is available from Novozymes as The name was obtained from Danisco / DuPont The α-amylase developed by this company is available under the trade name and ultra (both from Novozymes), OxAm (Danisco / DuPont) and (Daiwa Seiko Inc.). α-Amylase from Bacillus amyloliquefaciens was obtained from Novozymes as and a derivative variant of the α-amylase from Bacillus stearothermophilus is also sold by Novozymes under the name and In addition, for this purpose, the alpha-amylases from Bacillus A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948) should be emphasized. Fusion products of all the molecules mentioned can also be used. In addition, the developed alpha-amylases from Aspergillus niger and Aspergillus oryzae are also suitable, which are sold under the trade names Supplied by Novozymes. Other commercial 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.

[0117] Suitable cellulases include cellulases of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases are cellulases from Bacillus, Pseudomonas, Humicola, Fusarium, Thielvia, Acremonium, such as fungal cellulases from Humicola insolens, Mycelophthorathermophila and Fusarium oxysporum, which are disclosed in US4435307, US5648263, US5691178, US5776757 and WO89 / 09259. Particularly suitable cellulases are alkaline or neutral cellulases with color protection properties. Examples of such cellulases are cellulases described in EP 0495257, EP 0531372, WO96 / 11262, WO 96 / 29397 and WO 98 / 08940. Examples of cellulases having endo-1,4-glucanase activity (EC 3.2.1.4) are described in WO 2002 / 099091, such as a cellulase having a sequence having at least 97% identity to the amino acid sequence of positions 1 to 773 of SEQ ID NO: 2 of WO 2002 / 099091. Another example may include a GH44 xyloglucanase, such as a xyloglucanase having a sequence having at least 60% identity to positions 40 to 559 of SEQ ID NO: 2 of WO 2001 / 062903. Commercially available cellulases include Celluzyme TM 、Carezyme TM 、Carezyme Premium TM 、Celluclean TM (e.g. Celluclean TM 5000L and Celluclean TM 4000T), Celluclean Classic TM 、Cellusoft TM , and Whitezyme TM (NovozymesA / S), Clazinase TM and Puradax HA TM (Genencor International Inc.), KAC-500(B) TM (KaoCorporation), RevitalenzTM 1000、Revitalenz TM 2000 and Revitalenz TM 3000 (DuPont), and and (AB Enzymes).

[0118] Other enzymes that can be used are, for example, lipases or cutinases, particularly for their triglyceride cleaving activity, but also for the in situ generation of peroxyacids from suitable precursors. Suitable lipases and cutinases are those of bacterial or fungal origin. Chemically modified mutant enzymes produced by protein engineering are included. Examples include lipases from Thermomyces, e.g., lipases from T. Lanuginosus (formerly Humicola lanuginosa), as described in EP 0258068 and EP 0305216, cutinases from Humicola, e.g., H. insolens (WO 96 / 13580), lipases from strains of Pseudomonas (some of which are now renamed Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes, P. cepacia, Pseudomonas strains SD705, P. wisconsinensis, GDSL-type Streptomyces, lipases from Magnaporthe oryzae (Magnaporthe oryzae) and Pseudomonas sp. grisea), cutinase from Pseudomonas mendocina, lipase from Thermobida fusca, lipase from Geobacillus stearothermophilus, lipase from Bacillus subtilis and lipase from Streptomyces griseus and S. pristinaespiralis. Lipases originally obtainable from Humicola lanuginosus (Thermomyces lanuginosus) or developed therefrom, in particular lipases starting from the mentioned lipases with one or more amino acid exchanges at the following positions: D96L, T213R and / or N233R, particularly preferably T213R and N233R, are among the preferred lipases. Preferred commercial lipase products include Lipolase TM 、Lipex TM 、Lipolex TM and Lipoclean TM(Novozymes A / S), Lumafast (Genencor / DuPont) and Lipomax (Gist-Brocades).

[0119] To improve the bleaching effect, according to the invention, oxidoreductases can be used, for example oxidases, oxygenases, catalases, peroxidases, for example halogen, chlorine, bromine, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases). It is advantageous to additionally add organic compounds that interact with the enzymes, particularly preferably aromatic compounds, in order to enhance the activity of the relevant oxidoreductases (enhancers) or, in the case of very different redox potentials, to ensure the flow of electrons between the oxidase and the stain (mediators).

[0120] In the cleaning agents described herein, the enzymes used may be further formulated with accompanying substances, such as substances from fermentation. In liquid preparations, the enzymes are preferably used in the form of liquid enzyme preparations.

[0121] Enzymes are usually not provided in the form of pure proteins, but in the form of stable, storable and transportable preparations. These ready-to-use preparations include, for example, solid preparations obtained by granulation, extrusion or lyophilization, or, especially in the case of liquids or gels, solutions of the enzymes are provided, which are advantageously as concentrated as possible, have a low water content, and / or are mixed with stabilizers or other auxiliaries.

[0122] Alternatively, enzymes may also be encapsulated for solid and liquid application forms, for example, enzyme solutions with preferred natural polymers are spray dried or extruded or encapsulated in capsule form, for example, enzymes are encapsulated in solidified gels, or in core-shell capsules, wherein the enzyme-containing core is covered with a protective layer that is impermeable to water, air and / or chemicals. Other active ingredients, such as stabilizers, emulsifiers, pigments, bleaching agents or dyes, may be applied in the covering layer in addition. Such capsules are made using methods known per se, such as by vibrating granulation or rolling granulation or by fluidized bed processes. Advantageously, such particles, for example, cause less dust due to the application of polymer film formers, and are stably stored due to coating.

[0123] Furthermore, two or more enzymes can be formulated together so that a single particle exhibits multiple enzyme activities.

[0124] Enzymes can also be introduced into water-soluble films, such as films used to prepare unit dosage detergents and cleansers. Such films allow the enzyme to be released after contact with water. As used herein, "water-soluble" refers to a film structure that is preferably completely water-soluble. Preferably, such films are composed of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).

[0125] The present invention also relates to a method for cleaning textiles and / or hard surfaces, in particular dishes, characterized in that an agent according to the invention is used in at least one method step. In various embodiments, the method is characterized in that the protease is used at a temperature of about 0°C to about 100°C, preferably about 20°C to about 60°C and more preferably about 20°C to about 40°C.

[0126] This includes manual and machine methods, of which machine methods are preferred because they can be controlled more precisely, for example with regard to the amount used and the contact time. Textile cleaning methods are often characterized by the fact that in a number of method steps, various 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 with a detergent or a solution or dilution of such a detergent.

[0127] Since the proteases of the invention are intrinsically already hydrolytically active and exhibit said activity also in a medium which is otherwise devoid of cleaning power (e.g. in a simple buffer), a separate and / or single step of the method may consist of contacting the proteases of the invention with the stain as the sole cleaning ingredient, preferably in a buffer solution or in water. This represents a further embodiment of the subject matter of the invention.

[0128] An alternative embodiment of the subject matter of the invention also comprises a method for treating a textile raw material or for textile care, wherein the protease according to the invention is active in at least one method step. Among them, methods for textile raw materials, fibers or textiles containing natural ingredients are preferred, and methods for textiles containing wool or silk are particularly preferred.

[0129] Finally, the present invention also comprises the use of the proteases described herein in detergents or cleaning agents, e.g. as described above, for the (improved) removal of peptide- or protein-containing stains, e.g. from textiles and / or hard surfaces.

[0130] In another preferred embodiment, the present invention relates to the use of the protease of the present invention as described herein in a detergent or cleaner for improving the cleaning performance on stains sensitive to at least one and increasingly preferably two, three, four or five proteases, preferably selected from stains containing blood, egg (egg yolk), milk and other proteins, wherein the improvement in cleaning performance relative to a reference protease is determined as described in Example 2, in particular in the temperature range of about 20°C to about 40°C.

[0131] In another preferred embodiment, the present invention relates to the use of a protease in a detergent or cleaning agent, in particular a liquid detergent or cleaning agent, for improving the cleaning performance on stains sensitive to at least one and increasingly preferably two, three, four or five proteases, preferably selected from stains containing blood, egg (egg yolk), milk and other proteins, wherein the improvement in cleaning performance relative to a reference protease is determined as described in Example 2, in particular in the temperature range of about 20°C to about 40°C, wherein the protease is a protease having proteolytic activity and comprises the residue corresponding to SEQ ID %, 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 shown in NO: 1, wherein the protease is in each case based on the amino acid sequence according to SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six other amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the other amino acid substitutions being selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

[0132] All aspects, themes and embodiments described for the proteases of the invention and the reagents containing them also apply to the subject matter of the invention. Therefore, explicit reference is made here to the disclosures of the corresponding places, where it is pointed out that the disclosures also apply to the methods and uses of the invention. Example

[0133] Table 1: Detergent matrices used

[0134]

[0135]

[0136] Table 2: Proteases used:

[0137]

[0138] Example 1: Determination of protease activity

[0139] Protease activity was determined in a discontinuous assay using casein as substrate. The final concentration of the substrate solution was 12 mg / ml casein (prepared according to Hammarsten; Merck, Darmstadt, #2242) and 30 mM Tris in synthetic tap water. The synthetic tap water was a solution of 0.029% (w / v) CaCl2·2H2O, 0.014% (w / v) MgCl2·6H2O and 0.021% (w / v) NaHCO3 with a hardness of 15° dH (German hardness). The substrate solution was heated to 70°C and the pH was adjusted to 8.5 at 50°C using 0.1 N NaOH. A protease solution was prepared by adding 2% (w / v) anhydrous pentasodium tripolyphosphate to synthetic tap water and adjusting the pH to 8.5 with hydrochloric acid. 200 μl of the enzyme solution was added to 600 μl of the casein solution. The mixture was incubated at 50°C for 15 minutes. The reaction was terminated by adding 3% (w / v) solution of 600 μl 0.44M trichloroacetic acid (TCA), 0.22M sodium acetate. After cooling on ice for 15 minutes, TCA insoluble proteins were removed by centrifugation. 900 μl of remaining solution was mixed with 300 μl 2NNaOH, and the absorbance of the mixture containing TCA soluble proteins was measured at 290nm. By adding 600 μl TCA solution to 600 μl casein solution, 200 μl enzyme solution was then added to generate control values. Under these conditions, the protease solution causing the absorbance at 290nm to change to 0.500OD had an activity of 10HPE per ml (according to current designation).

[0140] Example 2: Determining Cleaning Performance

[0141] Microwash test

[0142] The cleaning performance was determined in a microwash test using Bacillus subtilis culture supernatants containing the expressed protease variants. The supernatants were used at the same activity as the benchmark control (1.15 μg / ml active enzyme protein in the wash).

[0143] Conditions: 20°C and 40°C, 16°dH water, 1 hour

[0144] Stains:

[0145] 1.CS38 (egg yolk / pigment (dry))

[0146] 2.C05 (blood / milk / ink)

[0147] 3. E111 (blood)

[0148] 4.PC10 (milk / oil)

[0149] 5.H-MR-B (milk / ash)

[0150] The punched fabric pieces (diameter = 10 mm) were placed in a microtiter plate, the detergent was preheated to 40°C, the final concentration was 3.17 g / L, the detergent and enzyme were added to the stain and incubated at 20°C and 40°C at 600 rpm for 1 hour, then the stain was rinsed with clean water several times and dried, and the brightness was measured using a colorimeter. The brighter the fabric, the better the cleaning performance. What is measured here is the Y value = brightness, the higher the Y value, the higher the brightness. All measured Y values ​​are corrected by the performance of the detergent only (without protease) and the performance of the detergent corrected with a virtual vector (Y 变体 –Y 空白 =ΔY 变体 ). For each stain, determine the ΔY for each protease 变体 , and all ΔY of all stains for each variant 变体 The values ​​are added to determine ΣΔY 变体 In order to compare the cleaning performance of the variants according to the invention with the reference control (=protease P1), ΔY P1 (for each stain) and ΣΔY P1 (Total cleaning performance) are all normalized to 100% and the relative cleaning performance of the variants according to the invention is calculated. An increase in the total performance of ≥ 15% is considered a significant improvement in performance.

[0151] The results are summarized in Tables 3 and 4 below.

[0152] Table 3: Washing performance at 20°C (%):

[0153] P1 P2 P3 P4 P5 P6 P7 P8 CS38 100％ 107％ 139％ 88％ 126％ 96％ 167％ 152％ C05 100％ 110％ 105％ 103％ 101％ 112％ 130％ 106％ E111 100％ 162％ 161％ 173％ 152％ 126％ 141％ 147％ PC10 100％ 205％ 172％ 148％ 198％ 158％ 235％ 129％ H-MR-B 100％ 144％ 171％ 162％ 172％ 170％ 212％ 181％ % Total Performance 100％ 147％ 151％ 140％ 149％ 129％ 169％ 144％

[0154] Table 4: Washing performance at 40°C (%):

[0155] P1 P2 P3 P4 P5 P6 P7 P8 CS38 100％ 117％ 118％ 89％ 105％ 113％ 121％ 91％ C05 100％ 133％ 122％ 128％ 137％ 169％ 156％ 101％ PC10 100％ 160％ 160％ 169％ 172％ 115％ 150％ 133％ H-MR-B 100％ 123％ 163％ 125％ 177％ 117％ 182％ 150％ % Total Performance 100％ 158％ 158％ 151％ 168％ 142％ 162％ 136％

[0156] Proteases P2 to P8 of the invention show significantly improved wash performance compared to the benchmark protease P1 at both 20°C and 40°C.

Claims

1. Protease, It has proteolytic activity and comprises the amino acid sequence, which has, over its entire length, at least 70%, and increasingly preferably 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% identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the protease is in each case numbered according to SEQ ID NO:

1. (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one, and increasingly preferably two, three, four, five or six amino acid substitutions at at least one, and increasingly preferably two, three, four, five or six, positions corresponding to positions 6, 61, 62, 101, 170 and 188.

2. The protease according to claim 1, It has proteolytic activity and comprises the amino acid sequence, which has, over its entire length, at least 70%, and increasingly preferably 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% identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the protease is in each case numbered according to SEQ ID NO:

1. (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) at least one, and increasingly preferably two, three, four, five or six, amino acid substitutions selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188 at at least one, and increasingly preferably two, three, four, five or six, positions corresponding to positions 6, 61, 62, 101, 170 and 188.

3. The protease according to claim 1 or 2, wherein the protease has one of the following amino acid substitution combinations, in each case based on the numbering according to SEQ ID NO: 1: (a)P9T-S89A-D101S-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (b)P9T-S89A-D101E-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (c)P9T-S89A-D101A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (d) Y6W-P9T-S89A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (e) Y6W-P9T-F61G-Q62N-S89A-N130D-T133A-N144K-S189T-Y217M-S224A-N252T-Q271E; (f) P9T-F61G-Q62-N-S89A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E; and (g) Y6W-P9T-S89A-N130D-T133A-N144K-N188G-S189T-Y217M-S224A-N252T-Q271E.

4. Protease, characterized in that (a) which is obtained by single or multiple conservative amino acid substitutions from a protease according to any one of claims 1 to 3 as a starting molecule, wherein the protease is in each case based on the amino acid sequence of SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G at at least one and increasingly preferably two, three, four, five or six amino acid substitutions at positions corresponding to positions 6, 61, 62, 101, 170 and 188; (b) which is obtained by fragmentation, deletion, insertion or substitution mutagenesis using a protease according to any one of claims 1 to 3 as a starting molecule and comprises an amino acid sequence corresponding to a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273 or 274 or 275 consecutive amino acids of the starting molecule, wherein the protease is in each case based on the sequence of SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

5. A protease according to any one of the preceding claims, wherein the protease shows improved cleaning performance relative to a reference protease on at least one and increasingly preferably two, three, four or five protease-sensitive stains, preferably selected from stains containing blood, egg (yolk), milk and other proteins, wherein the cleaning performance is determined as described in Example 2.

6. A method for producing a protease, comprising adding (i) introducing amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, based on the numbering according to SEQ ID NO: 1, and (ii) introducing at least one, and increasingly preferably two, three, four, five or six amino acid substitutions at at least one, and increasingly preferably two, three, four, five or six, positions corresponding to positions 6, 61, 62, 101, 170 and 188, based on the numbering according to SEQ ID NO: 1, the amino acid substitutions being preferably selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G, The starting molecule has an amino acid sequence that has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length.

7. The method according to claim 6, further comprising one or more of the following method steps: (a) introducing single or multiple conservative amino acid substitutions, wherein the protease, in each case based on the numbering according to SEQ ID NO: 1, (i) has the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) has at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G; (b) modifying the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence corresponding to at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 consecutive amino acids in length of the starting molecule, wherein the protease is in each case based on the amino acid sequence of SEQ ID NO: 1, (i) having amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) having at least one and increasingly preferably two, three, four, five or six amino acid substitutions at at least one and increasingly preferably two, three, four, five or six positions corresponding to positions 6, 61, 62, 101, 170 and 188, the amino acid substitutions preferably being selected from the group consisting of Y6W, F61G, Q62N, D101S, D101E, D101A, K170G and N188G.

8. Nucleic acid encoding a protease according to any one of claims 1 to 5 or encoding a protease obtainable according to the method of claims 6 to 7.

9. A vector comprising a nucleic acid according to claim 8, in particular a cloning vector or an expression vector.

10. A non-human host cell comprising a nucleic acid according to claim 8 or a vector according to claim 9, or comprising a protease according to any one of claims 1 to 5, or comprising a protease obtained according to the method of claims 6 to 7, in particular a non-human host cell which secretes the protease into the culture medium surrounding the host cell.

11. A method for producing a protease, comprising a) cultivating the host cell according to claim 10, and b) Isolating the protease from the culture medium or the host cells.

12. An agent, in particular a detergent or cleaning agent, in particular a liquid detergent or cleaning agent, characterized in that It contains at least one protease according to any one of claims 1 to 5 or a protease obtained by the method according to claims 6 to 7, the reagent is substantially free of boron-containing compounds and / or the pH value of the reagent is about 8 to about 9.

13. A method for cleaning textiles or hard surfaces, characterized in that The reagent according to claim 12 is used in at least one method step.

14. Use of a protease according to any one of claims 1 to 5 or a protease obtained according to the process of claims 6 to 7 in a detergent or cleaning agent, in particular a liquid detergent or cleaning agent, for removing peptide- or protein-containing stains.

15. Use of a protease according to any one of claims 1 to 5 or a protease obtained according to the process of claims 6 to 7 in a detergent or cleaning agent, in particular a liquid detergent or cleaning agent, for improving the cleaning performance on stains sensitive to at least one and increasingly preferably two, three, four or five proteases, preferably selected from stains containing blood, egg (egg yolk), milk and other proteins, the improvement in cleaning performance relative to a reference protease being determined as described in Example 2, in particular in the temperature range of about 20°C to about 40°C.

Images