PERFORMANCE-ENHANCED PROTEASE VARIANTS X

Abstract

The invention relates to proteases which have proteolytic activity and comprise 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 indicated in SEQ ID NO:1 over its entire length, wherein the proteases, in each case based on the numbering according to SEQ ID NO: 1, (i) has 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 or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R at least at one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170. The invention also relates to the production and use of said proteases. Proteases of this type exhibit a very good cleaning performance.

Description

[0001] PERFORMANCE-ENHANCED PROTEASE VARIANTS X

[0002] The invention lies in the field of enzyme technology. The invention relates to proteases whose amino acid sequence has been modified, particularly with regard to use in detergents and cleaning agents, in particular liquid detergents and cleaning agents, in order to improve their cleaning performance, and to the nucleic acids encoding them and their preparation. The invention further relates to the uses of these proteases and methods in which they are used, as well as to agents containing them, in particular detergents and cleaning agents, in particular liquid detergents and cleaning agents.

[0003] Proteases are among the most technically important enzymes of all. They are the longest-established enzymes in detergents and cleaning agents and are contained in virtually all modern, high-performance detergents and cleaning agents. They degrade protein-containing soils on the items being cleaned. Among these, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important. These are serine proteases due to their catalytically active amino acids. They act as nonspecific endopeptidases and hydrolyze any acid amide bonds found within peptides or proteins. Their pH optimum is usually in the significantly alkaline range. An overview of this family is provided, for example, in the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996. Subtilases are naturally produced by microorganisms.Among these, the subtilisins produced and secreted by Bacillus species are particularly worth mentioning as the most important group within the subtilases.

[0004] Examples of the subtilisin-type proteases preferably used in detergents and cleaning agents are the subtilisins BPN' and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY, and the enzymes thermitase, proteinase K, and the proteases TW3 and TW7, which are classified as subtilases but no longer as subtilisins in the narrower sense, as well as variants of the aforementioned proteases that have an amino acid sequence that is altered compared to the parent protease. Proteases are modified in a targeted or random manner using methods known from the state of the art and thus optimized, for example, for use in detergents and cleaning agents. These include point, deletion, or insertion mutagenesis, or fusion with other proteins or protein fragments. For most of the proteases known from the state of the art, optimized variants are known.For example, EP 2016175 and WO 2021 / 175697 disclose a protease from Bacillus pumilus or variants thereof intended for washing and cleaning agents.

[0005] In general, only selected proteases are suitable for use in liquid surfactant-containing preparations. Many proteases either do not exhibit sufficient catalytic performance in such preparations or are not sufficiently stable. Therefore, high catalytic activity and stability under conditions such as those encountered during a washing process are particularly desirable for the use of proteases in detergents.

[0006] Consequently, prior art liquid formulations containing proteases and surfactants have the disadvantage that the proteases they contain do not exhibit satisfactory proteolytic activity under standard washing conditions (e.g., in a temperature range of approximately 20°C to approximately 40°C) and / or are not sufficiently stable in storage, and the formulations therefore do not exhibit optimal cleaning performance on protease-sensitive soils. There is still a need to improve the cleaning performance of enzyme-containing, especially protease-containing, detergents and cleaning agents, particularly with regard to cleaning performance on protease-sensitive soils, especially in a temperature range of approximately 20°C to approximately 40°C.

[0007] Surprisingly, it has now been found that a protease from Bacillus pumilus or a sufficiently similar protease (based on sequence identity) which, based on the numbering according to SEQ ID NO:1, contains (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitutions which selected from the group consisting of S63Q, N99H, S156H, G166A and K170R, with respect to their washing performance compared to the wild-type form (SEQ ID NO:1) oran initial variant (reference protease) and is therefore particularly suitable for use in detergents or cleaning agents, in particular liquid detergents and cleaning agents.

[0008] The invention therefore relates to a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitutions,selected from the group consisting of S63Q, N99H, S156H, G166A and K170R.,

[0009] The invention further relates to a process for the preparation of a protease as defined above, comprising introducing (i) the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E at the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) at least one and increasingly preferably two, three, four or five amino acid substitutions at at least one and increasingly preferably two, three, four or five of the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 63, 99, 156, 166 and 170, selected from the group consisting of S63Q, N99H, S156H, G166A and K170R, into a starting molecule having an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length.

[0010] A protease within the meaning of the present patent application therefore encompasses both the protease itself and a protease produced by a process according to the invention. All statements regarding the protease therefore refer to both the protease itself and the proteases produced by corresponding processes.

[0011] Further aspects of the invention relate to the nucleic acids encoding these proteases, proteases according to the invention or nucleic acids-containing non-human host cells, and agents comprising proteases according to the invention, in particular washing and cleaning agents, in particular liquid washing and cleaning agents, washing and cleaning processes, and uses of the proteases according to the invention in washing or cleaning agents for removing protease-sensitive soils.

[0012] These and other aspects, features, and advantages of the invention will become apparent to those skilled in the art from a study of the following detailed description and claims. Any feature of one aspect of the invention may be employed in any other aspect of the invention. Furthermore, it is to be understood that the examples contained herein are intended to describe and illustrate the invention, but not to limit it, and in particular, the invention is not limited to these examples.

[0013] Unless otherwise stated, all percentages are by weight (wt%).

[0014] Numerical ranges specified in the format "from x to y" include the specified values. If multiple preferred numerical ranges are specified in this format, it goes without saying that all ranges resulting from the combination of the different endpoints will also be included.

[0015] "At least one" as used herein means one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

[0016] The term "washing and cleaning agent" or "washing or cleaning agent" as used herein is synonymous with the term "agent" and refers to a composition for cleaning textiles and / or hard surfaces, in particular dishes, as explained in the description.

[0017] "About," "approximately," or "approximately," as used herein with reference to a numerical value, refers to the corresponding numerical value ±10%, preferably ±5%. "Substantially free of" 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 most preferably less than 0.1% by weight, of the corresponding substance, based on the total weight of the composition / agent.

[0018] "Liquid," as used herein, includes liquids and gels, as well as pasty compositions. It is preferred that the liquid compositions be flowable and pourable at room temperature, but it is also possible for them to have a yield point. A substance, e.g., a composition or an agent, is solid according to the definition of the invention if it is in the solid state at 25°C and 1013 mbar. A substance, e.g., a composition or an agent, is liquid according to the definition of the invention if it is in the liquid state at 25°C and 1013 mbar. Liquid also includes gel.

[0019] "Variant", as used herein, refers to natural or artificially produced variations of a native protease that have a modified amino acid sequence compared to the reference form.

[0020] The present invention is based on the surprising finding of the inventors that amino acid substitutions at the positions described herein result in improved cleaning performance of this modified protease in detergents and cleaning agents.

[0021] In preferred embodiments, the modifications according to the invention (i) at the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, and (ii) at least one of the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 63, 99, 156, 166 and 170, lead to an improved cleaning performance of this modified protease in washing and cleaning agents, in particular liquid washing and cleaning agents, on at least one and increasingly preferably two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils. Proteases according to the invention therefore enable improved removal of at least one, preferably several, protease-sensitive soil(s) on textiles and / or hard surfaces, for example dishes.

[0022] In preferred embodiments, the protease according to the invention has (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156H, G166A and K170R, wherein the Combination of the amino acid substitutions from group (i) and the at least one amino acid substitution from group (ii) to an improved cleaning performance of this modified protease in washing and cleaning agents on at least one and increasingly preferably two, three, four or five protease-sensitive soil(s), which preferably consist of blood,egg (yolk), milk and other protein-containing soils.

[0023] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three,four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156H, G166A and K170R.

[0024] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least two of the positions corresponding to positions 63, 99, 156, 166 and 170, at least two amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R,has.,

[0025] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least three of the positions corresponding to positions 63, 99, 156, 166 and 170, at least three amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R,has. In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least four of the positions corresponding to positions 63, 99, 156, 166 and 170, at least four amino acid substitutions selected from the group consisting of S63Q, N99H, S156H,G166A and K170R existing group.,

[0026] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at the positions corresponding to positions 63, 99, 156, 166 and 170, the amino acid substitutions S63Q, N99H, S156H, G166A and K170R.

[0027] In further preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case, based on the numbering according to SEQ ID NO:1, has one of the following amino acid substitution combinations: (a) P9T-S63Q-S89A-N130D-T133A-N144K-G166A-S189T-Y217M-S224A-N252T-Q271 E, (b) P9T-S89A-N99H-N130D-T133A-N144K-K170R-S189T-Y217M-S224A-N252T-Q271 E and (c) P9T- S89A-N130D-T133A-N144K-S156R-S189T-Y217M-S224A-N252T-Q271E.

[0028] In particularly preferred embodiments, the protease according to the invention comprises one of the following amino acid substitution combinations: (a) P9T-S63Q-S89A-N130D-T133A-N144K-G166A-S189T-Y217M-S224A-N252T-Q271 E, (b) P9T-S89A-N99H-N130D-T133A-N144K-K170R-S189T-Y217M-S224A-N252T-Q271 E and (c) P9T-S89A-N130D-T133A-N144K-S156R-S189T-Y217M-S224A-N252T-Q271 E, wherein the numbering is in each case based on the numbering according to SEQ ID NO:1 and wherein the protease does not comprise any further changes besides the amino acid substitutions mentioned.In preferred embodiments, the combinations of the amino acid substitutions according to the invention from group (i) and (ii) described herein lead to improved cleaning performance of this modified protease in washing and cleaning agents, in particular liquid washing and cleaning agents, on at least one and increasingly preferably on two, three, four, five or six protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils. Proteases according to the invention therefore enable improved removal of at least one, preferably of several, protease-sensitive soil(s) on textiles and / or hard surfaces, for example dishes. Typical protease-sensitive soils include, for example, egg (yolk), blood, milk and other protein-containing soils.An improvement in cleaning performance according to the invention, in particular in proteolytic cleaning performance, is present when the protease shows an improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, as described herein.

[0029] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three,four or five amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R, wherein the protease exhibits improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, the cleaning performance being determined as described in Example 2.

[0030] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least two of the positions corresponding to positions 63, 99, 156, 166 and 170, at least two amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R,wherein the protease exhibits improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, the cleaning performance being determined as described in Example 2.

[0031] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least three of the positions corresponding to positions 63, 99, 156, 166 and 170, at least three amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R,wherein the protease exhibits improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, the cleaning performance being determined as described in Example 2.

[0032] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least four of the positions corresponding to positions 63, 99, 156, 166 and 170, at least four amino acid substitutions selected from the group consisting of S63Q, N99H, S156H, G166A and K170R,wherein the protease exhibits improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, the cleaning performance being determined as described in Example 2.

[0033] In preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at the positions corresponding to positions 63, 99, 156, 166 and 170, the amino acid substitutions S63Q, N99H, S156H, G166A and K170R, wherein the protease has at least one and increasingly preferably two,three, four or five protease-sensitive soil(s), preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, exhibit improved cleaning performance compared to a reference protease, the cleaning performance being determined as described in Example 2.

[0034] In further preferred embodiments, the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, and in each case, based on the numbering according to SEQ ID NO:1, has one of the following amino acid substitution combinations: (a) P9T-S63Q-S89A-N130D-T133A-N144K-G166A-S189T-Y217M-S224A-N252T-Q271 E, (b) P9T-S89A-N99H-N130D-T133A-N144K-K170R-S189T-Y217M-S224A-N252T-Q271 E and (c) P9T- S89A-N130D-T133A-N144K-S156R-S189T-Y217M-S224A-N252T-Q271 E, wherein the protease is present on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s),which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, shows improved cleaning performance compared to a reference protease, the cleaning performance being determined as described in Example 2.

[0035] In particularly preferred embodiments, the protease according to the invention comprises one of the following amino acid substitution combinations: (a) P9T-S63Q-S89A-N130D-T133A-N144K-G166A-S189T-Y217M-S224A-N252T-Q271 E, (b) P9T-S89A-N99H-N130D-T133A-N144K-K170R-S189T-Y217M-S224A-N252T-Q271 E and (c) P9T-S89A-N130D-T133A-N144K-S156R-S189T-Y217M-S224A-N252T-Q271 E, wherein the numbering is in each case based on the numbering according to SEQ ID NO:1, wherein the protease comprises no further changes besides the amino acid substitutions mentioned and wherein the protease shows an improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the cleaning performance is determined as described in Example 2.

[0036] Proteases according to the invention have increased catalytic activity in detergents or cleaning agents. In various embodiments, the proteases according to the invention can have a proteolytic activity that, based on the wild type (SEQ ID NO:1) and / or an already performance-improved starting variant of the protease, is at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%,

[0037] 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%,

[0038] 134%, 135%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150% or more. Such performance-enhanced proteases enable improved washing results on proteolytically sensitive soils in various temperature ranges, in particular a temperature range from approximately 20°C to approximately 40°C.

[0039] Furthermore, preferred embodiments of proteases according to the invention have particular stability in washing or cleaning agents, for example towards surfactants and / or bleaching agents and / or chelators, and / or towards temperature influences, in particular towards high temperatures, for example between about 50°C and about 65°C, in particular about 60°C, and / or towards acidic or alkaline conditions and / or towards pH changes and / or towards denaturing or oxidizing agents and / or towards proteolytic degradation and / or towards a change in redox conditions. Particularly preferred embodiments of the invention therefore provide protease variants with improved performance and / or more temperature stability. Further very particularly preferred embodiments of the invention provide protease variants with improved performance and more temperature stability.Such advantageous embodiments of proteases according to the invention consequently enable improved washing results on protease-sensitive soils in a wide temperature range.

[0040] In the context of the invention, cleaning performance is understood to mean the brightening performance on one or more soils, in particular on laundry or dishes. Within the context of the invention, both the washing or cleaning agent comprising the protease or the washing or cleaning liquor formed by this agent, as well as the protease itself, exhibit a respective cleaning performance. The cleaning performance of the enzyme thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent. The cleaning performance is preferably determined as specified below.

[0041] Wash liquor is understood to mean the use solution containing the washing or cleaning agent which acts on textiles or fabrics or hard surfaces and thus comes into contact with the soiling present on textiles or fabrics or hard surfaces. The wash liquor is usually formed when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example in a dishwasher, a washing machine or in another suitable container. The proteases according to the invention have enzymatic activity, i.e. they are capable of hydrolyzing peptides and proteins, in particular in a washing or cleaning agent. A protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide / peptide bonds in protein / peptide substrates and is thus capable of cleaving proteins or peptides.Furthermore, a protease according to the invention is preferably a mature protease, i.e., the catalytically active molecule without signal and / or propeptide(s). Unless otherwise stated, the sequences indicated also refer to mature (processed) enzymes.

[0042] In various embodiments of the invention, the protease is a free enzyme. This means that the protease can interact directly with all components of a composition and, if the composition is a liquid composition, that the protease is in direct contact with the composition's solvent (e.g., water). In other embodiments, a composition may contain proteases that form an interaction complex with other molecules or that contain a "coat." In this case, a single or multiple protease molecules may be separated from the other components of the composition by a surrounding structure. Such a separating structure may be formed by, but is not limited to, vesicles, such as a micelle or a liposome. However, the surrounding structure may also be a virus particle, a bacterial cell, or a eukaryotic cell.In various embodiments, an agent may contain cells of Bacillus pumilus or Bacillus subtilis expressing the proteases of the invention, or cell culture supernatants of such cells.

[0043] In the context of the present invention, the feature that a protease has the specified substitutions means that it contains one (of the specified) substitution(s) at the respective position, i.e., at least the specified positions are not otherwise mutated or deleted, for example, by fragmentation of the protease. In various preferred embodiments, the proteases described herein, with the exception of the explicitly mentioned substitutions, have the sequence of SEQ ID NO:1, i.e., apart from the substituted positions, they are 100% identical to the sequence according to SEQ ID NO:1.

[0044] The identity of nucleic acid or amino acid sequences is determined by sequence comparison. This sequence comparison is based on the BLAST algorithm, which is established and commonly used in the state of the 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 is essentially achieved by matching similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another. A tabular assignment of the relevant positions is referred to as an alignment. Another algorithm available in the state of the art is the FASTA algorithm. Sequence comparisons (alignments), especially multiple sequence comparisons, are created using computer programs. Frequently used, for example, are 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 (cf. e.g. Notredame et al. (2000) T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol., 302:205-217) or programs based on these programs or algorithms. Furthermore, sequence comparisons (alignments) are possible with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the given standard parameters, whose AlignX module for sequence comparisons is based on ClustalW, or Clone Manager 10 (use of the BLOSUM 62 scoring matrix for sequence alignment at the amino acid level). Unless otherwise stated, sequence identity reported herein is determined using the BLAST algorithm.

[0045] Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. This is usually expressed as percent identity, i.e. the proportion of identical nucleotides or amino acid residues at the same positions or corresponding positions in an alignment. The broader term homology in amino acid sequences includes conserved amino acid substitutions, i.e. amino acids with similar chemical activity, since these usually exert similar chemical activities within the protein. Therefore, the similarity of the compared sequences can also be expressed as percent homology or percent similarity. Identity and / or homology statements can be made for entire polypeptides or genes, or just for individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences.Such regions often exhibit identical functions. They can be small and comprise only a few nucleotides or amino acids. Such small regions often perform essential functions for the overall activity of the protein. It may therefore be useful to refer sequence matches only to individual, possibly small regions. Unless otherwise stated, however, statements of identity or homology in this application refer to the entire length of the respective nucleic acid or amino acid sequence.

[0046] In the context of the present invention, the statement that an amino acid position corresponds to a numerically designated position in SEQ ID NO:1 therefore means that the corresponding position is assigned to the numerically designated position in SEQ ID NO:1 in an alignment as defined above. Furthermore, the assignment of the positions is based on the mature protein. This assignment is also to be applied in particular if the amino acid sequence of a protease according to the invention comprises a higher number of amino acid residues than the protease according to SEQ ID NO:1. Starting from the stated positions in the amino acid sequence of the protease according to SEQ ID NO:1, the change positions in a protease according to the invention are those that are assigned to precisely these positions in an alignment.

[0047] In addition to the amino acid modifications explained above, proteases according to the invention can exhibit further amino acid modifications, in particular amino acid substitutions, insertions, or deletions. Such proteases are further developed, for example, by targeted genetic modification, i.e., by mutagenesis methods, and optimized for specific applications or with regard to special properties (for example, with regard to their catalytic activity, stability, etc.). Furthermore, nucleic acids according to the invention can be incorporated into recombination approaches and thus used to generate completely novel proteases or other polypeptides. The aim is to introduce targeted mutations such as substitutions, insertions, or deletions into the known molecules, for example, to improve the purification performance of enzymes according to the invention.For this purpose, in particular the surface charges and / or the isoelectric point of the molecules and thus their interactions with the substrate can be changed. For example, the net charge of the enzymes can be changed in order to influence substrate binding, particularly for use in detergents and cleaning agents. Alternatively or additionally, one or more corresponding mutations can increase the stability or catalytic activity of the protease and thus improve its cleaning performance. Advantageous properties of individual mutations, e.g. individual substitutions, can complement each other. A protease that has already been optimized with regard to certain properties, for example with regard to its stability during storage, can therefore be further developed within the scope of the invention.

[0048] The following convention is used to describe substitutions that affect exactly one amino acid position (amino acid exchanges): first, the naturally occurring amino acid is named using the internationally accepted one-letter code, followed by the corresponding sequence position, and finally, the inserted amino acid. Multiple or alternative exchanges within the same polypeptide chain are separated by slashes. "130D / V" thus means that position 130 is mutated to D or V. In the case of insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, such as an asterisk or a dash, or an A is indicated in front of the corresponding position.For example, P9T describes the substitution of proline at position 9 by threonine, P9TH the insertion of histidine after the amino acid threonine at position 9, and P9* or AP9 the deletion of proline at position 9. This nomenclature is known to those skilled in the art of enzyme technology.

[0049] The invention therefore further provides a protease which is characterized in that it is obtainable from a protease as described herein as the starting molecule by single or multiple conservative amino acid substitution, wherein the protease has at least one of the amino acid substitutions described above in the numbering according to SEQ ID NO:1. The term "conservative amino acid substitution" means the exchange (substitution) of one amino acid residue for another amino acid residue, wherein this exchange does not lead to a change in the polarity or charge at the position of the exchanged amino acid, e.g., the exchange of one non-polar amino acid residue for another non-polar amino acid residue. Conservative amino acid substitutions within the scope of the invention include, for example: G=A=S, I=V=L=M, D=E, N=Q, K=R, Y=F, S=T, G=A=I=V=L=M=Y=F=W=P=S=T.

[0050] Alternatively or additionally, the protease is characterized in that it is obtainable from a protease according to the invention as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which corresponds to the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably at two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H,S156H, G166A and K170R, existing group is / are selected.,

[0051] For example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without losing or reducing the proteolytic activity. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also reduce the allergenicity of the enzyme in question and thus improve their overall usability. Advantageously, the enzymes retain their proteolytic activity even after mutagenesis, i.e. their proteolytic activity corresponds at least to that of the starting enzyme, i.e. in a preferred embodiment the proteolytic activity is at least 100%, preferably at least 120% of the activity of the starting enzyme. Other substitutions can also have advantageous effects. Both individual amino acids and multiple contiguous amino acids can be exchanged for other amino acids.

[0052] Advantageous positions for sequence changes, in particular substitutions, of the protease according to SEQ ID NO:1, which, when transferred to homologous positions of the proteases according to the invention, are preferably of importance and impart advantageous functional properties to the protease, are accordingly the positions which, in an alignment, correspond to the positions described herein, i.e., in the numbering according to SEQ ID NO:1. The following amino acid residues are located at the stated positions in the wild-type molecule of the protease: 9P, 63S, 89A, 99N, 130N, 133T, 144K, 156S, 166G, 170K, 189S, 217Y, 224S, 252N, 271Q.

[0053] Further confirmation of the correct assignment of the amino acids to be modified, i.e. in particular their functional correspondence, can be provided by comparative experiments, according to which the two positions assigned to one another on the basis of an alignment are modified in the same way in both compared proteases and it is observed whether the enzymatic activity is modified in the same way in both. If, for example, an amino acid exchange in a certain position of the protease from Bacillus pumilus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example with an increase in the KM value, and a corresponding change in the enzymatic parameter, for example also an increase in the KM value, is observed in a protease variant according to the invention whose amino acid exchange was achieved by the same introduced amino acid, this can be seen as confirmation of the correct assignment.

[0054] All of the above-mentioned facts are also applicable to the processes according to the invention for producing a protease. Accordingly, a process according to the invention further comprises one or more of the following process steps:

[0055] (a) introducing a single or multiple conservative amino acid substitution, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) which are selected from the group consisting of S63Q, N99H, S156H, G166A and K170R;

[0056] (b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence which corresponds to the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four, five or six of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156H, G166A and K170R.

[0057] All embodiments also apply to the methods according to the invention.

[0058] In a further embodiment of the invention, a previously described protease is stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer. Increasing stability during storage and / or use, for example during the washing process, results in longer-lasting enzymatic activity and thus improved cleaning performance. In principle, all stabilization options described and / or expedient in the prior art are contemplated. Preference is given to stabilizations achieved via mutations of the enzyme itself, since such stabilizations do not require any further steps following the isolation of the enzyme. Examples of suitable sequence modifications are mentioned above. Other suitable sequence modifications are known from the prior art.

[0059] Other options for stabilization include:

[0060] Altering the binding of metal ions, in particular the calcium binding sites, for example by exchanging one or more of the amino acid(s) involved in calcium binding for one or more negatively charged amino acids and / or by introducing sequence changes in at least one of the sequences of the two amino acids arginine / glycine;

[0061] Protection against the influence of denaturing agents such as surfactants through mutations that cause a change in the amino acid sequence on or at the surface of the protein;

[0062] Replacement of amino acids located near the N-terminus with those that presumably interact with the rest of the molecule via non-covalent interactions and thus contribute to maintaining the globular structure. Preferred embodiments are those in which the enzyme is stabilized in multiple ways, since multiple stabilizing mutations act additively or synergistically.

[0063] Alternatively or additionally, the protease according to the invention can be reacted 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 the formulas (I) or (II) (II), wherein R is each selected from -COOH, Ci-e-alkyl-substituted or unsubstituted C2-6-dicarboxylic acids, Ci-e-alkyl-substituted or unsubstituted C2-e-carboxylic acids and -OOC-NR 2 2, where R 2 are the same or different and selected from Ci-e-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 benzylcarbamate, and combinations thereof, in order to further increase the stability of the protease in detergents and cleaning agents.

[0064] For the purposes of the present invention, "phenylboronic acid derivative" refers to a compound of formula (III). The compound of formula (III) has the following structural formula: where R is hydrogen, a hydroxyl, a C1-6 alkyl, a substituted C1-6 alkyl, a C1-6 alkenyl, or a substituted C1-6 alkenyl group. Preferably, the R radical in the phenylboronic acid derivative is a C1-6 alkyl group, and among these, it is more preferably -CH3, -CH3CH2, or -CH3CH2CH2. More preferably, the R radical in the phenylboronic acid derivative is hydrogen. The phenylboronic acid derivative is particularly preferred as 4-formylphenylboronic acid (4-FPBA).

[0065] The reversible inhibitor compound used can be boric acid.

[0066] The reversible inhibitor compound used can be a compound of formulas (I) or (II) where R is each selected from -COOH, Ci-e-alkyl-substituted or unsubstituted C2-6-dicarboxylic acids, Ci-e-alkyl-substituted or unsubstituted C2-e-carboxylic acids and -OOC-NR 2 2, where R 2are the same or different and are selected from Ci-e-alkyl or H, as well as salts, esters or derivatives thereof, and combinations thereof, preferably selected from the group consisting of benzoic acid, phenylmalonic acid, benzylmalonic acid, phenylsuccinic acid, benzylsuccinic acid, methyl 3-benzoylpropionate, (S)-3-phenylbutyric acid and benzylcarbamate.

[0067] In particularly preferred embodiments, the protease according to the invention is used in agents or compositions that are essentially free of boron-containing compounds. "Essentially free of boron-containing compounds" in this context means that the corresponding agents or compositions contain less than 2 wt.%, preferably less than 1 wt.%, more preferably less than 0.5 wt.%, and particularly preferably less than 0.1 wt.%, of boron-containing compounds, based on the total weight of the agent / composition. In very particularly preferred embodiments, these agents / compositions are free of boron-containing compounds, i.e., they contain, in particular, no boric acid and / or phenylboronic acid derivatives.

[0068] In a further embodiment of the 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 a starting variant (reference protease), ie it has at least 80% of the reference washing performance, preferably at least 100%, more preferably at least 110%, particularly preferably at least 120% or more.

[0069] The cleaning performance can be determined in a washing system that contains a detergent at a dosage of between 2.0 and 8.0 grams per liter of wash liquor and the enzyme. The enzymes to be compared are used at the same concentration (based on active protein). By using the same activity of the respective enzymes, it is ensured that even if there is a possible discrepancy in the ratio of active substance to total protein (the specific activity values), the respective enzymatic properties, for example the cleaning performance on specific soils, can be compared. In general, a low specific activity can be compensated for by adding a larger amount of protein. Furthermore, the enzymes to be tested can also be used in the same amount of substance or weight if the enzymes to be tested have a different affinity for the test substrate in an activity test.In this context, the term "equal amount of substance" refers to the same mole of the enzymes being tested. The term "equal amount by weight" refers to the same weight of the enzymes being tested.

[0070] The concentration of protease in the detergent intended for such a washing system is 0.001 to 0.1 wt.%, preferably 0.01 to 0.06 wt.%, based on active protein.

[0071] Washing or cleaning performance is understood to mean the ability of a washing or cleaning agent to partially or completely remove an existing soiling, in particular the lightening performance on one or more soils on textiles, in particular cotton textiles, polyester textiles and / or cotton-polyester blended textiles. Examples of such soils are blood on cotton or chocolate-milk / soot on cotton, cocoa on cotton, egg yolk on cotton, milk / soot on cotton or porridge on cotton, etc. Further examples include the aforementioned soils on cotton-polyester blended textiles or polyester-containing textiles or other blended textiles. Within the scope of the invention, both the washing or cleaning agent comprising the protease, or the washing or cleaning liquor formed by this agent, and the protease itself have a respective cleaning performance.The cleaning performance of the protease thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent.

[0072] The term "washing or cleaning liquor" refers to the working solution containing the washing or cleaning agent that acts on the textiles or hard surfaces and thus comes into contact with the soiling present on the textiles or hard surfaces. The washing or cleaning liquor is typically created when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example, in a washing machine, dishwasher, or other suitable container.

[0073] A liquid reference detergent for such a washing system can, for example, be composed as follows (all data in weight percent (wt%)): 4.4% alkylbenzenesulfonic acid, 5.6% other anionic surfactants, 2.4% C12-18 Na salts of fatty acids (soaps), 4.4% non-ionic surfactants, 0.2% phosphonates, 1.4% citric acid, 0.95% NaOH, 0.01% defoamers, 2% glycerin, 0.08% preservatives, 1% ethanol, and the remainder demineralized water. The preferred dosage of the liquid detergent is between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9, or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 7 and pH 10.5, preferably between pH 8 and pH 9.

[0074] The cleaning performance is determined for soiling on cotton by measuring the degree of cleaning of the washed textiles. For example, the washing process can be carried out for 60 minutes at a temperature of approximately 20°C or approximately 40°C, and the water can have a water hardness between 15.5°dH and 16.5°dH (German hardness). Within the scope of the invention, the cleaning performance is determined, for example, at 20°C or 40°C using a liquid detergent such as the one specified above, with the washing process preferably taking place for 60 minutes at 600 rpm.

[0075] The degree of whiteness, i.e., the lightening of soils, as a measure of cleaning performance, is determined using optical measurement methods, preferably photometrically. A suitable device for this purpose is the Minolta CM508d spectrometer. The devices used for the measurement are usually calibrated beforehand with a white standard, preferably one supplied with the product.

[0076] Preferred embodiments of proteases according to the invention achieve such advantageous cleaning performance even at low temperatures, in particular in the temperature ranges between about 10°C and about 60°C, preferably between about 15°C and about 50°C, and particularly preferably between about 20°C and about 40°C. Methods for determining protease activity are familiar to those skilled in the art in the field of enzyme technology and are routinely used by them. For example, such methods are disclosed in Tenside, Volume 7 (1970), pp. 125-132. Alternatively, protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979).The measurement is performed at a temperature of 25°C, pH 8.6, and a wavelength of 410 nm. The measurement time is 5 minutes, and the measurement interval is 20 to 60 seconds. Protease activity is usually expressed in protease units (PU). Suitable protease activities are, for example, 2.25, 5, or 10 PU per ml of wash liquor. However, protease activity is not zero.

[0077] An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measurement method, preferably a photometric method. The test suitable for this purpose involves the protease-dependent cleavage of the substrate protein casein. This is cleaved by the protease into a multitude of smaller partial products. The totality of these partial products exhibits an increased absorption at 290 nm compared to non-cleaved casein. This increased absorption can be determined using a photometer, thus allowing conclusions to be drawn about the enzymatic activity of the protease.

[0078] The protein concentration can be determined using known methods, for example, the BCA method (bicinchoninic acid; 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (Gornall et al., J. Biol. Chem. 177 (1948): 751-766). The determination of the active protein concentration can be carried out by titrating the active sites using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J. Am. Chem. Soc. 88, 24 (1966): 5890-5913).

[0079] The invention further relates to a protease as described above, which is characterized in that it has at least one chemical modification. A protease with such a change is referred to as a derivative, i.e. the protease is derivatized. For the purposes of the present application, derivatives are understood to be proteins whose pure amino acid chain has been chemically modified. Such derivatizations can, for example, take place in vivo by the host cell that expresses the protein. Coupling of low-molecular-weight compounds such as lipids or oligosaccharides is particularly noteworthy in this regard. Derivatizations can, however, also be carried out in vitro, for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein.For example, the coupling of amines to carboxyl groups of an enzyme is possible to change the isoelectric point. Such another compound can also be another protein, which is bound to a protein according to the invention, for example, via bifunctional chemical compounds. Derivatization also refers to covalent binding to a macromolecular carrier or noncovalent inclusion in suitable macromolecular cage structures. Derivatizations can, for example, influence substrate specificity or the binding strength to the substrate, or temporarily block enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the storage period. Such modifications can also influence stability or enzymatic activity.They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, increase its skin compatibility. For example, coupling with macromolecular compounds, such as polyethylene glycol, can improve the protein in terms of stability and / or skin compatibility. Derivatives of a protein according to the invention can also be understood, in the broadest sense, to include preparations of these proteins. Depending on the method of extraction, processing, or preparation, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein can also be deliberately mixed with other substances, for example to increase its storage stability. Therefore, all preparations of a protein according to the invention are also according to the invention.This is also independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. It may be desired that it has no or only low activity during storage, and only exhibits its enzymatic function at the time of use. This can be controlled, for example, via appropriate accompanying substances. In particular, the co-preparation of proteases with specific inhibitors is possible in this regard. Of all the proteases or protease variants and / or derivatives described above, those are particularly preferred within the scope of the present invention whose storage stability and / or cleaning performance is improved compared to the starting variant, wherein the cleaning performance is determined in a washing system as described herein.

[0080] The invention further relates to a nucleic acid encoding a protease according to the invention, as well as to a vector containing such a nucleic acid, in particular a cloning vector or an expression vector. These can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand, or as a double strand. Particularly in the case of DNA molecules, the sequences of both complementary strands must be considered in all three possible reading frames. Furthermore, it must be taken into account that different codons, i.e., base triplets, can code for the same amino acids, so that a specific amino acid sequence can be encoded by several different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences that can encode one of the proteases described above are included in this subject matter of the invention.The skilled person is able to determine these nucleic acid sequences without doubt, since, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, starting from an amino acid sequence, the skilled person can easily determine nucleic acids coding for this amino acid sequence. Furthermore, in nucleic acids according to the invention, one or more codons can be replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Thus, every organism, for example a host cell of a production strain, has a specific codon usage. Codon usage is understood to mean the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can arise if the codons located on the nucleic acid in the organism are opposed to a comparatively small number of loaded tRNA molecules.Although they code for the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon coding for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be translated more efficiently in the organism.

[0081] Using methods commonly used today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or protein chemical methods, a person skilled in the art can produce the corresponding nucleic acids, including complete genes, based on known DNA and / or amino acid sequences. Such methods are known, for example, from Sambrook, J., Fritsch, EF, and Maniatis, T. (2001). Molecular cloning: a laboratory manual, 3rd Edition, Cold Spring Laboratory Press.

[0082] For the purposes of the present invention, vectors are understood to be elements consisting of nucleic acids which contain a nucleic acid according to the invention as a characterizing nucleic acid region. They are capable of establishing this nucleic acid as a stable genetic element in a species or cell line over several generations or cell divisions. Vectors are, particularly when used in bacteria, special plasmids, i.e., circular genetic elements. Within the scope of the present invention, a nucleic acid according to the invention is cloned into a vector. Vectors include, for example, those whose origin is bacterial plasmids, viruses, or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the additional genetic elements present in each case, vectors are capable of establishing themselves as stable units in the respective host cells over several generations.They can be present extrachromosomally as separate units or integrated into a chromosome or chromosomal DNA. Expression vectors comprise nucleic acid sequences that enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a contained nucleic acid there. Expression is influenced in particular by the promoter(s) that regulate transcription. In principle, expression can occur through the natural promoter originally located upstream of the nucleic acid to be expressed, but also through a promoter of the host cell provided on the expression vector or through a modified or completely different promoter of another organism or another host cell. In the present case, at least one promoter for the expression of a nucleic acid according to the invention is provided and used for its expression.Expression vectors can also be regulated, for example, by changing the cultivation conditions or upon reaching a certain cell density of the host cells they contain, or by adding certain substances, particularly activators of gene expression. An example of such a substance is the galactose derivative isopropyl-ß-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained in cloning vectors is not expressed.

[0083] The invention further relates to a non-human host cell which comprises a nucleic acid according to the invention or a vector according to the invention, or which comprises a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. Preferably, a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, individual components, i.e., nucleic acid parts or fragments of a nucleic acid according to the invention, can be introduced into a host cell in such a way that the resulting host cell contains a nucleic acid according to the invention or a vector according to the invention.This procedure is particularly suitable when the host cell already contains one or more components of a nucleic acid according to the invention or of a vector according to the invention, and the additional components are then added accordingly. Methods for transforming cells are established in the prior art and are well known to the person skilled in the art. In principle, all cells are suitable as host cells, i.e., prokaryotic or eukaryotic cells. Preferred host cells are those that can be handled genetically advantageously, for example, with regard to transformation with the nucleic acid or the vector and its stable establishment, such as unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological manageability.This includes, for example, easy cultivability, high growth rates, low requirements for fermentation media, and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenically) expressed protein into the medium surrounding the host cells. Furthermore, the proteases can be modified by the cells producing them after their production, for example, by attaching sugar molecules, formylations, aminations, etc. Such post-translational modifications can influence the protease's function.

[0084] Further preferred embodiments are host cells whose activity can be regulated by genetic regulatory elements, which are provided, for example, on the vector, but can also be present in these cells from the outset. These cells can be stimulated to express, for example, through the controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions, or upon reaching a certain cell density. This enables economical production of the proteins of the invention. An example of such a compound is IPTG as described above.

[0085] Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on cultivation conditions. This allows for the establishment of cost-effective cultivation or production processes. Furthermore, experts in bacterial fermentation technology have extensive experience. For a specific production process, gram-negative or gram-positive bacteria may be suitable for a variety of reasons, which can be determined experimentally in each individual case, such as nutrient sources, product formation rate, time requirements, etc. In gram-negative bacteria, such as Escherichia coli, a multitude of proteins are secreted into the periplasmic space, the compartment between the two membranes surrounding the cells. This can be advantageous for specific applications.Furthermore, Gram-negative bacteria can also be engineered to secrete expressed proteins not only into the periplasmic space but also into the medium surrounding the bacteria. Gram-positive bacteria, such as Bacilli or Actinomycetes, or other members of the Actinomycetales, in contrast, lack an outer membrane, so secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium, from which the expressed proteins can be purified. They can be directly isolated from the medium or further processed. Furthermore, Gram-positive bacteria are related to or identical to most of the source organisms for technically important enzymes and usually produce comparable enzymes themselves, so they have a similar codon usage and their protein synthesis apparatus is naturally oriented accordingly.Host cells according to the invention can be modified with regard to their culture requirements, have different or additional selection markers, or express different or additional proteins. In particular, they can also be host cells that transgenically express multiple proteins or enzymes. The present invention is applicable in principle to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and results in the production of proteins according to the invention through the use of such microorganisms. Such microorganisms then represent host cells within the meaning of the invention.In a further embodiment of the invention, the host cell is characterized in that it is a bacterium, preferably one selected from the group of the genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus camosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

[0086] The host cell can also be a eukaryotic cell, which is characterized by having a cell nucleus. The invention therefore further relates to a host cell characterized by having a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are capable of post-translationally modifying the proteins they produce. Examples of this are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis that enable such systems. The modifications that eukaryotic systems carry out, particularly in connection with protein synthesis, include, for example, the binding of low-molecular-weight compounds such as membrane anchors or oligosaccharides.Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with enzymes naturally produced by such cells, such as cellulases, may also be advantageous. Furthermore, thermophilic fungal expression systems, for example, may be particularly suitable for the expression of temperature-stable proteins or variants.

[0087] The host cells according to the invention are cultivated and fermented in a conventional manner, for example, in discontinuous or continuous systems. In the former case, a suitable nutrient medium is inoculated with the host cells, and the product is harvested from the medium after a period of time to be determined experimentally. Continuous fermentations are characterized by the achievement of a steady state in which cells partially die but also regrow over a comparatively long period of time, and the protein formed can be simultaneously removed from the medium.

[0088] Host cells according to the invention are preferably used to produce proteases according to the invention. The invention therefore further provides a process for producing a protease comprising a) culturing a host cell according to the invention, and b) isolating the protease from the culture medium or from the host cell.

[0089] This subject matter of the invention preferably comprises fermentation processes.

[0090] Fermentation processes are known per se from the prior art and represent the actual large-scale production step, usually followed by a suitable purification method for the manufactured product, for example, the proteases according to the invention. All fermentation processes based on a corresponding process for producing a protease according to the invention represent embodiments of this subject matter of the invention. Fermentation processes characterized in that the fermentation is carried out via a feed strategy are particularly suitable. In this case, the media components consumed by the ongoing cultivation are fed in. This can achieve considerable increases in both cell density and cell mass or dry mass and / or, in particular, in the activity of the protease of interest.Furthermore, the fermentation can also be designed in such a way that undesired metabolic products are filtered out or neutralized by adding buffer or appropriate counterions. The produced protease can be harvested from the fermentation medium. Such a fermentation process is preferable to isolating the protease from the host cell, i.e., preparing the product from the cell mass (dry mass), but requires the provision of 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. Without secretion, the protease can alternatively be isolated from the host cell, i.e., purified from the cell mass, for example, by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

[0091] All of the above-mentioned facts can be combined into processes for producing the protease according to the invention. The invention further provides an agent characterized in that it contains the protease according to the invention as described above. The agent is preferably a washing or cleaning agent. The washing and cleaning agent is particularly preferably substantially free of boron-containing compounds. "Substantially free of boron-containing compounds" in this context means that the agents according to the invention contain 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, based on the total weight of the agent. In very particularly preferred embodiments, the washing and cleaning agents according to the invention are free of boron-containing compounds, i.e.In particular, they do not contain boric acid and / or phenylboronic acid derivatives.

[0092] According to the invention, a washing or cleaning agent is understood to mean all conceivable types of washing or cleaning agents, both concentrated and undiluted, for use on a commercial scale, in washing machines or for hand washing or cleaning. These include, for example, washing agents for textiles, carpets, or natural fibers, for which the term "washing agent" is used. This also includes, for example, dishwashing detergents for dishwashers (machine dishwashing detergents) or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood, or leather, for which the term "cleaning agent" is used. Thus, in addition to manual and machine dishwashing detergents, this also includes, for example, scouring agents, glass cleaners, toilet air fresheners, etc.The washing and cleaning agents within the scope of the invention also include washing aids that are added to the actual washing agent during manual or machine washing to achieve an additional effect. Furthermore, the washing and cleaning agents within the scope of the invention also include textile pre- and post-treatment agents, i.e. agents with which the laundry is brought into contact before the actual washing, for example, to dissolve stubborn soils, and also agents that impart further desirable properties to the laundry, such as a pleasant feel, resistance to creasing, or low static charge, in a step following the actual textile washing. Fabric softeners, among others, are counted among the latter agents.

[0093] The detergents or cleaning agents according to the invention, which may be in the form of powdered or granular solids, in compacted or recompacted particle form, as homogeneous solutions or suspensions, may contain, in addition to a protease according to the invention, all known ingredients customary in such agents, with at least one further ingredient preferably being present in the agent. The agents according to the invention may contain, in particular, surfactants, builders, polymers, glass corrosion inhibitors, corrosion inhibitors, bleaching agents such as peroxygen compounds, bleach activators, or bleach catalysts. They may also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, sequestering agents, electrolytes, pH regulators, and / or other auxiliaries such as optical brighteners, graying inhibitors, dye transfer inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.Advantageous ingredients of the compositions according to the invention are disclosed in the international patent application WO 2009 / 121725, beginning on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. This disclosure is expressly incorporated by reference, and the disclosure content therein is incorporated into the present patent application.

[0094] An agent according to the invention advantageously contains the protease according to the invention in an amount of 2 pg to 20 mg, preferably 5 pg to 17.5 mg, particularly preferably 20 pg to 15 mg, and most particularly preferably 50 pg to 10 mg per g of the agent. In various embodiments, the concentration of the protease described herein (active enzyme) in the agent is >0 to 1 wt.%, preferably 0.0001 or 0.001 to 0.1 wt.%, based on the total weight of the agent or composition.

[0095] An agent according to the invention contains the protease increasingly preferably in an amount of 1 x 10 -8 up to 5 wt.%, from 0.0001 to 1 wt.%, from 0.0005 to 0.5 wt.%, from 0.001 to 0.1 wt.%, each based on active protein and based on the total weight of the detergent.

[0096] The embodiments of the present invention encompass all solid, powdered, liquid, gel-like, or pasty dosage forms of agents according to the invention, which may optionally also consist of multiple phases and may be present in compressed or uncompressed form. The agent may be in the form of a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. Solid dosage forms of the agent also include extrudates, granules, tablets, or pouches. Alternatively, the agent may also be liquid, gel-like, or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste, or in the form of an aqueous liquid detergent or a water-containing paste. Liquid agents are generally preferred. Furthermore, the agent may be in the form of a one-component system. Such agents consist of one phase.Alternatively, a remedy can also consist of multiple phases. Such a remedy is therefore divided into several components.

[0097] The proteases according to the invention are preferably used in liquid detergents for cleaning textiles, particularly preferably in liquid detergents with a pH of about 8 to about 9.

[0098] Detergents or cleaning agents according to the invention can contain exclusively one protease according to the invention. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention thus represents agents which further comprise one or more further enzymes. Preferably used further enzymes are all enzymes which can exhibit catalytic activity in the agent according to the invention, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ß-glucosidase, pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase or other proteases distinguishable from the proteases according to the invention, as well as mixtures thereof. Further enzymes are advantageously present in the agent in an amount of 1 x 10 -8to 5 wt.% based on active protein. Each additional enzyme is increasingly preferably present in an amount of 1 x 10 7to 3 wt.%, from 0.00001 to 1 wt.%, from 0.00005 to 0.5 wt.%, from 0.0001 to 0.1 wt.% and particularly preferably from 0.0001 to 0.05 wt.% in agents according to the invention, based on active protein. The enzymes particularly preferably display synergistic cleaning performance against certain soilings or stains, i.e. the enzymes contained in the agent support one another in their cleaning performance. Such a synergism very particularly preferably exists between the protease contained according to the invention and another enzyme in an agent according to the invention, including in particular between the said protease and an amylase and / or a lipase and / or a mannanase and / or a cellulase and / or a pectinase. Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the invention.

[0099] Textile detergents preferred according to the invention comprise at least one protease and at least one amylase. In a further preferred embodiment of the invention, textile detergents comprise at least one protease and at least one cellulase. In a further preferred embodiment, textile detergents comprise at least one protease and at least one lipase. In a further preferred embodiment, textile detergents comprise at least one protease, at least one amylase, and at least one lipase. In a further preferred embodiment, textile detergents comprise at least one protease, at least one amylase, and at least one cellulase. In a further preferred embodiment, textile detergents comprise at least one protease, at least one amylase, at least one cellulase, and at least one lipase.Particularly preferred are textile detergents which contain 3 to 10 different enzymes, whereby textile detergents which contain 3 to 10 different enzyme types may be particularly preferred with regard to cleaning performance against a very wide range of stains.

[0100] Examples of proteases are subtilisins BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus lentus formis, protease PB92, subtilisins 147 and 309, the protease from Bacillus lentus, subtilisin DY, and the enzymes thermitase, proteinase K, and proteases TW3 and TW7, which are classified as subtilases but no longer as subtilisins in the narrower sense. Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes. Subtilisins 147 and 309 are marketed by Novozymes under the trade names Esperase® and Savinase®, respectively. Protease variants are derived from the protease from Bacillus lentus DSM 5483, described in e.g. WO 95 / 23221, WO 92 / 21760, WO 2013 / 060621 and EP 3660151. Other useful proteases are e.g.the enzymes available under the trade names Durazym®, Relase®, Everlase®, Nafizym®, Natalase®, Kannase®, Progress Uno 101 L® and Ovozyme® from Novozymes, the enzymes available under the trade names Purafect®, Purafect® OxP, Purafect® Prime, Excellase®, Properase®, Preferenz P100® and Preferenz P300® from Danisco / DuPont, the enzyme available under the trade name Lavergy pro 104 LS® from BASF, the enzyme available under the trade name Protosol® from Advanced Biochemicals Ltd., the enzyme available under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., the enzyme available under the trade names Proleather® and Protease P® from Amano Pharmaceuticals Ltd. and the enzyme available under the name Proteinase K-16 from Kao Corp. The proteases from Bacillus gibsonii and Bacillus pumilus, which are disclosed in WO 2008 / 086916, WO 2007 / 131656, WO 2017 / 215925, WO 2021 / 175696 and WO 2021 / 175697, are also particularly preferably used.Other proteases that can be used are those that are naturally present in the microorganisms Stenotrophomonas maltophilia, especially Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus.

[0101] Examples of amylases are the α-amylases from Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus stearothermophilus, as well as their further developments that have been improved for use in detergents and cleaning agents. The enzyme from Bacillus licheniformis is available from Novozymes under the name Termamyl® and from Danisco / DuPont under the name Purastar® ST. Further developments of this α-amylase are available under the trade names Duramyl® and Termamyl® ultra (both from Novozymes), Purastar® OxAm (Danisco / DuPont), and Keistase® (Daiwa Seiko Inc.). The α-amylase from Bacillus amyloliquefaciens is marketed by Novozymes under the name BAN®, and derivatives of the α-amylase from Bacillus stearothermophilus are marketed under the names BSG® and Novamyl®, also from Novozymes. Furthermore, the a-amylase from Bacillus sp. is used for this purpose.A 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948) are particularly suitable. Fusion products of all of these molecules can also be used. Furthermore, the further developments of α-amylase from Aspergillus niger and A. oryzae, available under the trade name Fungamyl® from Novozymes, are also suitable. Other commercial products that can be used advantageously include Amylase-LT® and Stainzyme® or Stainzyme® ultra or Stainzyme® plus, as well as Amplify™ 12L or Amplify Prime™ 100L, the latter also from Novozymes, and the PREFERENZ S® series from Danisco / DuPont, including PREFERENZ S100®, PREFERENZ S1000®, and PREFERENZ S210®. Variants of these enzymes obtainable by point mutations can also be used according to the invention.

[0102] Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are also included. Suitable cellulases are cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, e.g., the fungal cellulases from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259. Particularly suitable cellulases are alkaline or neutral cellulases with color-care properties. Examples of such cellulases are cellulases described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Examples of cellulases with endo-1,4-glucanase activity (EC 3.2.1.4) are described in WO 2002 / 099091, e.g., those with a sequence of 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 comprise a GH44 xyloglucanase, e.g. a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40 to 559 of SEQ ID NO:2 of WO 2001 / 062903. Commercially available cellulases include Celluzyme™, Carezyme™, Carezyme Premium™, Celluclean™ (e.g. Celluclean™ 5000L and Celluclean™ 4000T), Celluclean Classic™, Cellusoft™, Endolase®, Renozyme® and Whitezyme™ (Novozymes A / S), Clazinase™ and Puradax HA™ (Genencor International Inc.), KAC-500(B)™ (Kao Corporation), Revitalenz™ 1000, Revitalenz™ 2000 and Revitalenz™ 3000 (DuPont), as well as Ecostone® and Biotouch® (AB Enzymes).

[0103] Other enzymes that can be used include lipases or cutinases, particularly because of their triglyceride-cleaving activities, but also to generate peracids from suitable precursors in situ. Suitable lipases and cutinases are those of bacterial or fungal origin. Chemically modified or mutated enzymes produced by protein engineering are included. Examples are lipase from Thermomyces, e.g. from T. lanuginosus (formerly called Humicola lanuginosa), as described in EP 0258068 and EP 0305216, cutinase from Humicola, e.g. H. insolens (WO 96 / 13580), lipase from strains of Pseudomonas (some of which have now been renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes, P. cepacia, P. sp. strain SD705, P.wisconsinensis, Streptomyces lipases of the GDSL type, cutinase from Magnaporthe grisea, cutinase from Pseudomonas mendocina, lipase from Thermobifida fusca, lipase from Geobacillus stearothermophilus, lipase from Bacillus subtilis, and lipase from Streptomyces griseus and S. pristinaespiralis. Preferred lipases include, for example, those originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or developed therefrom, in particular those with one or more of the following amino acid substitutions starting from the aforementioned lipase at positions D96L, T213R, and / or N233R, particularly preferably T213R and N233R. Preferred commercial lipase products include Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (Genencor / DuPont) and Lipomax (Gist-Brocades).

[0104] To increase the bleaching effect, oxidoreductases, e.g., oxidases, oxygenases, catalases, peroxidases such as halo-, chloro-, bromo-, lignin-, glucose-, or manganese peroxidases, dioxygenases, or laccases (phenol oxidases, polyphenol oxidases) can be used according to the invention. Advantageously, organic compounds, particularly aromatic ones, that interact with the enzymes are also added to enhance the activity of the respective oxidoreductases (enhancers) or to ensure electron flow in the event of significantly different redox potentials between the oxidizing enzymes and the soils (mediators).

[0105] In the cleaning agents described herein, the enzymes to be used can also be formulated together with accompanying substances, such as those from fermentation. In liquid formulations, the enzymes are preferably used as liquid enzyme formulation(s).

[0106] The enzymes are generally not provided in the form of pure protein, but rather in the form of stabilized, storable, and transportable preparations. These prefabricated preparations include, for example, solid preparations obtained by granulation, extrusion, or lyophilization or, particularly in the case of liquid or gel-like products, solutions of the enzymes, preferably as concentrated as possible, with little water content, and / or containing stabilizers or other additives.

[0107] Alternatively, the enzymes can be encapsulated for both solid and liquid dosage forms, for example by spray-drying or extrusion of the enzyme solution together with a preferably natural polymer, or in the form of capsules, for example those in which the enzymes are enclosed as if in a solidified gel, or in those of the core-shell type, in which an enzyme-containing core is coated with a water-, air-, and / or chemical-impermeable protective layer. Additional active ingredients, such as stabilizers, emulsifiers, pigments, bleaching agents, or dyes, can be applied in superimposed layers. Such capsules are applied using methods known per se, for example by shake or roll granulation or in fluid-bed processes. Such granules, for example by applying polymeric film formers, are advantageously low in dust and, due to the coating, are stable during storage.

[0108] Furthermore, it is possible to package two or more enzymes together so that a single granulate has multiple enzyme activities.

[0109] The enzymes can also be incorporated into water-soluble films, such as those used, for example, in the packaging of unit-dose detergents and cleaning agents. Such a film allows the enzymes to be released upon contact with water. As used herein, "water-soluble" refers to a film structure that is preferably completely water-soluble. Preferably, such a film consists of (fully or partially hydrolyzed) polyvinyl alcohol (PVA).

[0110] The invention further relates to a method for cleaning textiles and / or hard surfaces, in particular tableware, which is characterized in that an agent according to the invention is used in at least one method step. In various embodiments, the described 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.

[0111] This includes both manual and mechanical processes, with mechanical processes being preferred due to their more precise controllability, for example, regarding the quantities used and contact times. Processes for cleaning textiles are generally characterized by the application of various cleaning agents to the items to be cleaned in several steps and their subsequent washing off after the contact time, or by the cleaning items being treated in some other way with a detergent or a solution or dilution of this agent.

[0112] Since proteases according to the invention naturally already possess hydrolytic activity and exhibit this activity even in media that otherwise have no cleaning power, such as in mere buffer, a single and / or the only step of such a process can consist of bringing a protease according to the invention into contact with the soil as the sole cleaning-active component, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.

[0113] Alternative embodiments of this subject matter of the invention also include processes for treating textile raw materials or for textile care, in which a protease according to the invention is activated in at least one process step. Among these, processes for textile raw materials, fibers, or textiles with natural components are preferred, and very particularly for those containing wool or silk.

[0114] Finally, the invention also covers the use of the proteases described herein in washing or cleaning agents, for example as described above, for the (improved) removal of peptide- or protein-containing soils, for example from textiles and / or hard surfaces.

[0115] In a further preferred embodiment, the invention relates to the use of a protease according to the invention described herein in a washing or cleaning agent for improving the cleaning performance on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the improvement in cleaning performance compared to a reference protease as described in Example 2 is determined, in particular in a temperature range of about 20°C to about 40°C.

[0116] In a further preferred embodiment, the invention relates to the use of a protease in a washing or cleaning agent, in particular liquid washing or cleaning agent, for improving the cleaning performance on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the improvement in cleaning performance compared to a reference protease, as described in Example 2, is determined, in particular in a temperature range from about 20°C to about 40°C, wherein the protease is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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% is identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one, and increasingly preferably at two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one, and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156H, G166A and K170R. All facts, objects and embodiments described for the protease according to the invention and agents containing it,are also applicable to this subject matter of the invention. Therefore, explicit reference is made here to the disclosure at the appropriate point, with the note that this disclosure also applies to the above-mentioned inventive methods and uses.

[0117] EXAMPLES

[0118] Table : Detergent matrix used

[0119] Table 2: Proteases used:

[0120] Example 1: Determination of protease activity

[0121] Protease activity was determined in a discontinuous assay using casein as a 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. Synthetic tap water is a solution of 0.029% (w / v) CaCh • 2 H2O, 0.014% (w / v) MgCh • 6 H2O, and 0.021% (w / v) NaHCCh with 15° dH (German hardness). The substrate solution was heated to 70°C and the pH adjusted to 8.5 at 50°C using 0.1 N NaOH. The protease solution was prepared by adding 2% (w / v) anhydrous pentasodium tripolyphosphate to synthetic tap water and adjusting to pH 8.5 with hydrochloric acid. 200 μl of the enzyme solution were added to 600 μl of the casein solution. The mixture was incubated at 50°C for 15 minutes. The reaction was terminated by adding 600 μl of 0.44 M trichloroacetic acid (TCA) and 0.22 M sodium acetate at 3% w / v.After a 15-minute cooling step on ice, the TCA-insoluble protein was removed by centrifugation. 900 μl of the remaining solution was mixed with 300 μl of 2 N NaOH, and the absorbance of this mixture, containing TCA-soluble proteins, was measured at 290 nm. Control values ​​were generated by adding 600 μl of TCA solution to 600 μl of casein solution, followed by the addition of 200 μl of enzyme solution. A protease solution that causes an absorbance change of 0.500 OD at 290 nm under these conditions has, according to the current designation, an activity of 10 HPE per ml.

[0122] Example 2: Determination of cleaning performance

[0123] Mini wash test

[0124] The cleaning performance was determined in mini-wash tests with Bacillus subtilis culture supernatants containing the expressed protease variants. The supernatants were used at an activity equivalent to the benchmark (1.15 pg / ml active enzyme protein in the wash liquor).

[0125] Conditions: 20°C and 40°C, 16° dH water, 1 h

[0126] Soiling:

[0127] 1 . CS38 (egg yolk / pigment (dried))

[0128] 2. 10N (Whole i / soot)

[0129] 3. C05 (Blood / Milk / Ink)

[0130] 4. E111 (Blood)

[0131] 5. PC10 (Milk / Oil)

[0132] Punched-out fabrics (diameter = 10 mm) were placed in microtiter plates, wash liquor pre-tempered to 20°C or 40°C, final concentration 3.17 g / L, lye and enzyme were added to the soil, incubated for 1 h at 20°C or 40°C and 600 rpm, then the soil was rinsed several times with clean water, allowed to dry, and the brightness was determined using a colorimeter. The brighter the fabric, the better the cleaning performance. The Y value = brightness is measured here; the higher the value, the brighter. All measured Y values ​​were corrected by the performance of the wash liquor alone (without protease) and by the performance of the wash liquor (Y variant - Y variant = AY variant). For each soil, AY variant was determined for each protease, and all AY variant values ​​of all soils per variant were summed to determine ZAY variant.To compare the cleaning performance of the variants according to the invention with the benchmark (= Protease P1), both AYpi (for each soil) and ZAYpt (overall cleaning performance) were normalized to 100% and the relative cleaning performance of the variants according to the invention was calculated. A >15% increase in overall performance is considered a significant performance improvement.

[0133] The results are summarized in Tables 3 and 4.

[0134] Table 3: Washing performance in % at 20°C:

[0135] Table 4: Washing performance in % at 40°C:

[0136] The proteases P2 to P4 according to the invention show a significantly improved washing performance at both 20°C and 40°C compared to the benchmark protease P1.

Claims

PATENT CLAIMS 1. A protease which has proteolytic activity and comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length to at least 70% and increasingly preferably to 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, whereby the protease, in each case based on the numbering according to SEQ ID NO:1 , (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R.

2. Protease according to claim 1, wherein the protease has one of the following amino acid substitution combinations, each based on the numbering according to SEQ ID NO:1: (a) P9T-S63Q-S89A-N130D-T133A-N144K-G166A-S189T-Y217M-S224A-N252T-Q271E; (b) P9T-S89A-N99H-N130D-T133A-N144K-K170R-S189T-Y217M-S224A-N252T-Q271 E; and (c) P9T-S89A-N130D-T133A-N144K-S156R-S189T-Y217M-S224A-N252T-Q271E.

3. Protease, characterized in that (a) it is obtainable from a protease according to claim 1 or 2 as starting molecule by single or multiple conservative amino acid substitution, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one, and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one, and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R; (b) it is obtainable from a protease according to claim 1 or 2 as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which corresponds to the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R.

4. Protease according to one of the preceding claims, wherein the protease shows improved cleaning performance compared to a reference protease on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, the cleaning performance being determined as described in Example 2.

5. A process for producing a protease, comprising introducing (i) 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, respectively, based on the numbering according to SEQ ID NO:1, and (ii) at least one and increasingly preferably two, three, four or five amino acid substitution(s) at at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, based on the numbering according to SEQ ID NO:1, selected from the group consisting of S63Q, N99H, S156R, G166A and K170R, in a starting molecule which has an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length.

6. The method according to claim 5, further comprising one or more of the following method steps: (a) introducing a single or multiple conservative amino acid substitution, wherein the protease, in each case based on the numbering according to SEQ ID NO:1 , (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one, and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one, and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R; (b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence which, over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids with the starting molecule, whereby the protease, in each case based on the numbering according to SEQ ID NO:1 , (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R.

7. Nucleic acid coding for a protease according to any one of claims 1 to 4 or coding for a protease obtained by a process of claims 5 to 6.

8. Vector containing a nucleic acid according to claim 7, in particular a cloning vector or an expression vector.

9. A non-human host cell comprising a nucleic acid according to claim 7 or a vector according to claim 8, or comprising a protease according to any one of claims 1 to 4, or comprising a protease obtained by a process of claims 5 to 6, in particular one which secretes the protease into the medium surrounding the host cell.

10. A process for producing a protease comprising a) culturing a host cell according to claim 9 and b) isolating the protease from the culture medium or from the host cell.

11. Agent, in particular washing or cleaning agent, in particular liquid washing or cleaning agent, characterized in that it contains at least one protease according to one of claims 1 to 4 or a protease obtained by a process of claims 5 to 6, wherein the agent is substantially free of boron-containing compounds and / or wherein the agent has a pH of about 8 to about 9.

12. A method for cleaning textiles or hard surfaces, characterized in that an agent according to claim 11 is used in at least one method step.

13. Use of a protease according to any one of claims 1 to 4 or a protease obtained by a process of claims 5 to 6 in a washing or cleaning agent, in particular liquid washing or cleaning agent, for removing peptide- or protein-containing soils.

14. Use of a protease according to any one of claims 1 to 4 or a protease obtained by a process according to claims 5 to 6 in a washing or cleaning agent, in particular liquid washing or cleaning agent for improving the cleaning performance on at least one and increasingly preferably on two, three, four or five protease-sensitive soil(s), which is / are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the improvement in cleaning performance compared to a reference protease is determined as described in Example 2, in particular in a temperature range of about 20°C to about 40°C.