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WO2025119569A1 - Variantes de protéase à stabilité de blanchiment améliorée - Google Patents

Variantes de protéase à stabilité de blanchiment améliorée Download PDF

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Publication number
WO2025119569A1
WO2025119569A1 PCT/EP2024/081351 EP2024081351W WO2025119569A1 WO 2025119569 A1 WO2025119569 A1 WO 2025119569A1 EP 2024081351 W EP2024081351 W EP 2024081351W WO 2025119569 A1 WO2025119569 A1 WO 2025119569A1
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WIPO (PCT)
Prior art keywords
amino acid
protease
positions
group
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/081351
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German (de)
English (en)
Inventor
Christian DEGERING
Shohana ISLAM
Margret VAN LIER
Antje Gebert-Schwarzwaelder
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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Filing date
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Publication of WO2025119569A1 publication Critical patent/WO2025119569A1/fr
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Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/39—Organic or inorganic per-compounds
    • C11D3/3902—Organic or inorganic per-compounds combined with specific additives
    • C11D3/3905—Bleach activators or bleach catalysts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • 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 with regard to automatic dishwashing detergents, in order to improve their stability, in particular their stability in the presence of bleach, 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 detergents and cleaning agents containing them, in particular detergents and cleaning agents containing bleach, preferably automatic dishwashing detergents, particularly preferably automatic dishwashing detergents containing bleach.
  • 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 break down 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 the 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.
  • subtilisin Enzymes edited by R. Bott and C. Betzel, New York, 1996.
  • Subtilases are naturally produced by microorganisms. Among them, the subtilisins, produced and secreted by Bacillus species, are particularly noteworthy as the most important group within the subtilases.
  • 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.
  • WO 2017/215925 discloses a protease from Bacillus gibsonii or variants thereof intended for use in detergents and cleaning agents.
  • proteases are suitable for use in preparations containing surfactants and/or bleaching agents. Many proteases do not show any sufficient catalytic performance or they are not sufficiently stable. For the use of proteases in washing and cleaning agents, high catalytic activity and stability under conditions such as those encountered during a washing or cleaning process are therefore particularly desirable. Consequently, prior art formulations containing proteases, surfactants, and/or bleaching agents have the disadvantage that the proteases they contain do not have satisfactory proteolytic activity and/or are not sufficiently stable under standard washing and/or cleaning conditions, and the formulations therefore do not demonstrate optimal cleaning performance on protease-sensitive soils.
  • Protease-sensitive soils are preferably selected from the group consisting of blood, egg (yolk), milk, and other protein-containing soils.
  • Protein-containing soils especially egg soils, and bleachable soils, especially tea soils, are stubborn stains that are often not satisfactorily removed.
  • Such soils, especially bleachable soils are typically addressed by adding bleaching agents to detergents and cleaning products.
  • bleaching agents are unstable in the presence of bleaching and/or oxidizing agents and lose their activity. This is especially true for liquid detergents and cleaning products. Therefore, the washing and cleaning performance of conventional detergents and cleaning products that combine enzymes and bleaching agents in one composition is greatly reduced.
  • a protease from Bacillus gibsonii or a sufficiently similar protease which, based on the numbering according to SEQ ID NO:1, has (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, at least one amino acid substitution, which is selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, is improved with regard to its stability
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid
  • a preferred subject of 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least two amino
  • a further preferred subject of 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one, preferably two of the positions corresponding to positions 211 and 212, at least one, preferably two amino acid substitution(s) selected from the group consisting
  • the invention further relates to a process for the preparation of a protease, comprising introducing (i) at least one amino acid substitution at at least one of the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one amino acid substitution at at least one of the positions which, based on the numbering according to SEQ ID NO:1, correspond to positions 29, 58, 89, 117 and 216, selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, into a starting molecule having an amino acid sequence which
  • 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 both to the protease itself and to the proteases produced by corresponding processes, as well as to the corresponding processes, in particular to the production processes of the protease.
  • proteases according to the invention relate to the nucleic acids encoding these proteases, proteases according to the invention or nucleic acids containing non-human host cells and washing and cleaning agents comprising proteases according to the invention, in particular washing and cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach, washing and cleaning methods, and uses of the proteases according to the invention in washing or cleaning agents, in particular Bleach-containing washing and cleaning agents, preferably automatic dishwashing detergents, particularly preferably bleach-containing automatic dishwashing detergents, for removing protease-sensitive soiling.
  • 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.
  • 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.
  • washing and cleaning agent or “washing or cleaning agent” as used herein is synonymous with the term “agent” or “composition” or “washing and/or cleaning agent composition” and refers to a composition for cleaning textiles and/or hard surfaces, in particular dishes, as explained in the description.
  • 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.
  • Liquid 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 that they 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 1,013 mbar.
  • a substance e.g., a composition or agent
  • Liquid also includes gel.
  • Variant refers to natural or artificially produced variations of a native protease that have a modified amino acid sequence compared to the reference form.
  • the present invention is based on the surprising discovery of the inventors that amino acid substitutions at the positions described herein result in improved stability of this modified protease in detergents and cleaning agents, in particular detergents and cleaning agents containing bleach, preferably automatic dishwashing detergents, particularly preferably automatic dishwashing detergents containing bleach.
  • the present invention is based in particular on the surprising discovery of the inventors that amino acid substitutions at the positions described herein result in improved stability of this modified protease in the presence of bleaching agents, in particular oxidizing agents.
  • the changes according to the invention (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, at least one amino acid substitution selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, leads to improved stability of this modified protease in washing and cleaning agents, in particular washing and cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach.
  • the changes according to the invention (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, at least one amino acid substitution selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, leads to improved stability of this modified protease in the presence of bleaching agents, in particular oxidizing agents.
  • the protease (i) according to the invention has at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, at least one amino acid substitution selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, wherein the combination of the at least one amino acid substitution from group (i) and the at least one amino acid substitution from group (ii) leads to improved stability of this modified protease in washing or cleaning agents, in particular washing and cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach.
  • the protease according to the invention has (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, at least one amino acid substitution selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, wherein the combination of the at least one amino acid substitution from group (i) and the at least one amino acid substitution from group (ii) leads to improved stability of this modified protease in the presence of bleaching agents, in particular oxid
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • the protease according to the invention is a protease which has proteolytic activity and comprises an amino acid sequence which corresponds to that shown in SEQ ID NO:1 specified amino acid sequence 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1 , (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L,
  • 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%, 98%, 98.5% or 99% identical, where the protease, each with reference to the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L, P212D
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, has (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO: 1, has (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, (i) an amino acid substitution or amino acid substitution combination selected from the group consisting of I43V, M122L-N154S-T156A, M211 N-P212D,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, (i) an amino acid substitution or amino acid substitution combination selected from the group consisting of I43V, M122L-N154S-T156A, M211 N-P212D,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of M211 L-P212D-A29G, M211 L-P212D-A29G-D58P,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-Y89A, I43V-A29G-M117A, I43V-A29G-M117F, I43V-A29G-M117I, I43V-A29G-M117L, I43V-A29G-M117V, I43V-A29G-M216A, I43V-A29G-M216F, I43V-A29G-M216L, I43V-A29G-D58P-Y89A, I43V-A29G-D58P-M117A, I43V-A29G-D58P-M117F, I43V-A29G-D58P-M1171, I43V-A29G-D58P-M117L, I43V-A29G-D58P-M117V, I43V-A
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of M211 L-P212D-A29G, M211 L-P212D-A29G-D58P, M211 L-P212D-A29G-Y89A, M211 L-P212D-A29G-M117A, M211 L-P212D-A29G-M117F, M211 L-P212D-A29G-M1171, M211 L-P212D-A29G-M117L, M211 L-P212D-A29G-M117V, M211 L- P212D-A29G-M216A, M211 L-P212D-A29G-M216F, M211 L-P212D-A29G-M216L, M211 L-P212D-A29G-M216L, M211 L-P212D-A29G-M216L, M211 L-P212D-A29G-D58P-Y89A, M211 L-P212D-A
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D, M117V-M211 L-P212D, A29G-M117V-M211 L-P212D, M117F-M211 L-P212D, M117I-M211 L-P212D, M117L-M211 L-P212D, M211 L-P212D-M216F, D58P-M211 L-P212D-M216A and M211 L-P212D-M216L, wherein the numbering is in each case based on the numbering according to SEQ ID NO:1 and wherein the Protease does not contain any further changes besides the amino acid substitutions mentioned.
  • the combinations of the amino acid substitutions according to the invention from groups (i) and (ii) described herein lead to improved cleaning performance of this modified protease in washing or cleaning agents, in particular bleach-containing washing and cleaning agents, preferably automatic dishwashing agents, particularly preferably bleach-containing automatic dishwashing agents, on at least one protease-sensitive soil.
  • Proteases according to the invention therefore enable improved removal of at least one, preferably several, protease-sensitive soils on textiles and/or hard surfaces, in particular 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 is present when the protease shows improved cleaning performance compared to a reference protease on at least one protease-sensitive soil, which is preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils.
  • the combinations of the amino acid substitutions from groups (i) and (ii) described herein lead to improved stability of this modified protease in washing or cleaning agents, in particular bleach-containing washing or cleaning agents, preferably automatic dishwashing agents, particularly preferably bleach-containing automatic dishwashing agents.
  • the combinations of the amino acid substitutions from groups (i) and (ii) described herein lead to improved stability of this modified protease in the presence of bleaching agents, in particular oxidizing agents.
  • Proteases according to the invention can therefore be used in washing or cleaning agents, in particular bleach-containing washing and cleaning agents, preferably automatic dishwashing agents, particularly preferably bleach-containing automatic dishwashing agents.
  • Proteases according to the invention can therefore be used in washing or cleaning agents, in particular bleach-containing washing and cleaning agents, preferably automatic dishwashing agents, particularly preferably bleach-containing automatic dishwashing agents, and exhibit a cleaning performance in such agents.
  • proteases according to the invention can improve the cleaning performance of bleach- and enzyme-containing, in particular protease-containing, washing and cleaning agents, in particular automatic dishwashing agents, particularly with regard to protease-sensitive soils.
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, two amino acid substitutions selected from the group consisting of M211N, M211L,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case with reference to the numbering according to SEQ ID NO:1, (i) an amino acid substitution or Amino acid substitution combination selected from the group consisting of 143V, M122L-N154S-T156A, M211 N-P212
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% is identical, and in each case based on the numbering according to SEQ ID NO:1, (i) an amino acid substitution or
  • Amino acid substitution combination selected from the group consisting of I43V, M122L-N154S-T156A, M211 N-P212D, M211 L-P212D, G160S, D127P-M211 L-P212D, P212H and Q12L-M122L-A222S, and (ii) an amino acid substitution or amino acid substitution combination selected from the group consisting of M117A, M117A-Y89L, M117V, M117V-A29G, M117F, M117I, M117L, M216F, D58P-M216A, M216L, wherein the protease has improved stability in washing or cleaning agents, in particular bleach-containing washing and cleaning agents, preferably Dishwashing detergents, particularly preferably dishwashing detergents containing bleach, the stability being determined as described in Example 3.
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of M211 L-P212D-A29G, M211 L-P212D-A29G-D58P,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-Y89A, I43V-A29G-M117A, I43V-A29G-M117F, I43V-A29G-M117I, I43V-A29G-M117L, I43V-A29G-M117V, I43V-A29G-M216A, I43V-A29G-M216F, I43V-A29G-M216L, I43V-A29G-D58P-Y89A, I43V-A29G-D58P-M117A, I43V-A29G-D58P-M117F, I43V-A29G-D58P-M1171, I43V-A29G-D58P-M117L, I43V-A29G-D58P-M117V, I43V-N-N
  • N154S-T156A-D58P-Y89A-M117I M122L-N154S-T156A-D58P-Y89A-M117L, M122L-N154S-T156A- D58P-Y89A-M117V, M122L-N154S-T156A-D58P-Y89A-M216A, M122L-N154S-T156A-D58P-Y89A-M216F, M122L-N154S-T156A-D58P-Y89A-M216L, M122L-N154S-T156A-D58P-M117A-M216A, M122L-N154S-T156A-D58P-M117A-M216F, M122L-N154S-T156A-D58P-M117A-M216L, M122L-N154S-T156A-D58P-M117A-M216L, M122L-N154S-T156A-D58P-M117A
  • the protease according to the invention comprises a M211 L-P212D-A29G, M211 L-P212D-A29G-D58P, M211 L-P212D-A29G-Y89A, M211 L-P212D-A29G-M117A, M211 L-P212D-A29G-M117F, M211 L-P212D-A29G-M117I, M211 L-P212D-A29G-M117L, M211 L-P212D-A29G-M117V, M211 L-P212D-A29G-M216A, M211 L-P212D-A29G-M216F, M211 L-P212D-A29G- M216L, M211 L-P212D-A29G-D58P-Y89A, M211 L-P212D-A29G-D58P-M117A, M211 L-P212D-A29G- D58P-M117F, M211 L-P212
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D, M117V-M211 L-P212D, A29G-M117V-M211 L-P212D, M117F-M211 L-P212D, M117I-M211 L-P212D, M117L-M211 L-P212D, M211 L-P212D-M216F, D58P-M211 L-P212D-M216A and M211 L-P212D-M216L, 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 has improved stability in washing or cleaning agents, in particular washing and cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach, the stability being determined as described
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least two of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 211 and 212, at least one amino acid substitution selected from the group consisting of M211N,
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, has two amino acid substitutions selected from the group consisting of M211N, M211L
  • 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%, 98%, 98.5% or 99% identical, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 211 and 212, the amino acid substitutions M211L and P212D, and (ii) at least
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, (i) an amino acid substitution or amino acid substitution combination selected from the group consisting of I43V, M122L-N154S-T156A, M211 N-P212D,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, (i) an amino acid substitution or amino acid substitution combination selected from the group consisting of I43V, M122L-N154S-T156A, M211 N-P212D,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO: 1, has an amino acid substitution combination which is selected from the group consisting of M211 L-P212D-A29G, M211 L-P212D-A29G-D58P,
  • 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 by at least 70% and increasingly preferably by at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5% or 99% identical, and in each case based on the numbering according to SEQ ID NO:1, has an amino acid substitution combination which is selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of I43V-A29G, I43V-A29G-D58P, I43V-A29G-Y89A, I43V-A29G-M117A, I43V-A29G-M117F, I43V-A29G-M117I, I43V-A29G-M117L, I43V-A29G-M117V, I43V-A29G-M216A, I43V-A29G-M216F, I43V-A29G-M216L, I43V-A29G-D58P-Y89A, I43V-A29G-D58P-M117A, I43V-A29G-D58P-M117F, I43V-A29G-D58P-M1171, I43V-A29G-D58P-M117L, I43V-A29G-D58P-M117V, I43V-N-N
  • the protease according to the invention comprises a M211 L-P212D-A29G, M211 L-P212D-A29G-D58P, M211 L-P212D-A29G-Y89A, M211 L-P212D-A29G-M117A, M211 L-P212D-A29G-M117F, M211 L-P212D-A29G-M117I, M211 L-P212D-A29G-M117L, M211 L-P212D-A29G-M117V, M211 L-P212D-A29G-M216A, M211 L-P212D-A29G-M216F, M211 L-P212D-A29G- M216L, M211 L-P212D-A29G-D58P-Y89A, M211 L-P212D-A29G-D58P-M117A, M211 L-P212D-A29G- D58P-M117F, M211 L-P212
  • the protease according to the invention comprises an amino acid substitution combination selected from the group consisting of M117A-M211 L-P212D, Y89A-M117A-M211 L-P212D, M117V-M211 L-P212D, A29G-M117V-M211 L-P212D, M117F-M211 L-P212D, M117I-M211 L-P212D, M117L-M211 L-P212D, M211 L-P212D-M216F, D58P-M211 L-P212D-M216A and M211 L-P212D-M216L, 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 has improved stability in the presence of bleaching agents, in particular oxidizing agents, the stability being determined as described in Example 3.
  • proteases according to the invention have increased catalytic activity in washing and cleaning agents, in particular washing and cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach.
  • the proteases according to the invention can have a proteolytic activity of at least 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, or more, based on the wild type (SEQ ID NO:1).
  • Such performance-enhanced proteases enable improved cleaning results on protease-sensitive soils.
  • Such performance-improved cleaning results on protease-sensitive soils can be achieved in various temperature ranges, e.g. in a range from about 0°C to about 100°C, preferably about 20°C to about 75°C, more preferably about 30°C to about 60°C, particularly preferably about 40°C to about 55°C.
  • proteases according to the invention have high stability in detergents and cleaning agents, in particular detergents and cleaning agents containing bleach, preferably automatic dishwashing detergents, particularly preferably automatic dishwashing detergents containing bleach.
  • proteases according to the invention also have high stability, e.g., towards surfactants and/or chelators, and/or towards temperature influences, in particular towards high temperatures, e.g., 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 (auto)proteolytic degradation and/or towards a change in redox ratios.
  • proteases according to the invention have enzymatic activity, ie they are capable of hydrolyzing peptides and proteins, particularly in washing and cleaning agents.
  • the protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide/peptide bonds in protein/peptide substrates and is thus able to cleave proteins or peptides.
  • 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.
  • 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).
  • a composition may contain proteases that form an interaction complex with other molecules or that contain a "coat.”
  • a single or multiple protease molecules may be separated from the other components of the composition by a surrounding structure.
  • a separating structure may be formed by, but is not limited to, vesicles, such as a micelle or a liposome.
  • the surrounding structure may also be a virus particle, a bacterial cell, or a eukaryotic cell.
  • an agent may contain cells of, for example, Bacillus pumilus or Bacillus subtilis or other expression strains expressing the proteases of the invention, or cell culture supernatants of such cells.
  • 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., Basic local alignment search tool, J. Mol. Biol., 1990, 215:403-410, and Altschul et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 1997, 25:3389-3402) and is essentially performed by matching similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences. A tabular assignment of the relevant positions is referred to as an alignment.
  • Sequence comparisons are created using computer programs. Frequently used programs are, for example, the Clustal series (see, for example, Chenna et al., Multiple sequence alignment with the Clustal series of programs, Nucleic Acid Res., 2003, 31:3497-3500), T-Coffee (see, for example, Notredame et al., T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol., 2000, 302:205-217) or programs based on these programs or algorithms.
  • Sequence comparisons are also possible using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified default parameters. Its AlignX module for sequence comparisons is based on ClustalW. Clone Manager 10 (using the BLOSUM 62 scoring matrix for sequence alignment at the amino acid level) or Clone Manager 10 (using the BLOSUM 62 scoring matrix for sequence alignment at the amino acid level) are also possible. Unless otherwise stated, the sequence identity stated herein is determined using the BLAST algorithm.
  • Such a comparison also allows a statement about the similarity of the compared sequences. This is usually expressed as percent identity, i.e. the proportion of identical Nucleotides or amino acid residues at the same or corresponding positions in an alignment are specified.
  • 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 specified 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 have identical functions.
  • nucleic acid or amino acid sequence can be small and consist of only a few nucleotides or amino acids. Such small regions often exert essential functions for the overall activity of the protein. It can therefore be useful to relate sequence similarities only to individual, possibly small regions. Unless otherwise stated, details of identity or homology in the present application refer to the total length of the respective nucleic acid or amino acid sequence indicated.
  • 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.
  • 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.
  • the change positions in a protease according to the invention are those that are assigned to precisely these positions in an alignment.
  • proteases according to the invention can have 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 (e.g., with regard to their catalytic activity, stability, etc.).
  • nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely new types of proteases or other polypeptides. The aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules in order, for example, to improve the cleaning performance of enzymes according to the invention.
  • the surface charges and/or the isoelectric point of the molecules and thus their interactions with the substrate can be modified.
  • the net charge of the enzymes can be modified in order to influence substrate binding, particularly for use in detergents and cleaning agents.
  • one or more corresponding mutations can increase the stability or catalytic activity of the protease and thereby improve its purification 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, e.g., with regard to its stability during storage and/or activity and/or their tolerance with respect to the substrate spectrum, can therefore be further developed within the scope of the invention.
  • amino acid exchanges amino acid exchanges
  • 130D/V thus means that position 130 is mutated to D or V.
  • additional amino acids are named after the sequence position.
  • deletions the missing amino acid is replaced by a symbol, e.g., an asterisk or a dash, or an A is indicated before the corresponding position.
  • P9T describes the substitution of proline at position 9 with threonine
  • P9TH the insertion of histidine after the amino acid threonine at position 9
  • P9* or AP9 the deletion of proline at position 9.
  • 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.
  • 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.
  • 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, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids, wherein the protease, in each case based on the numbering according to SEQ ID NO:1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222
  • 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 105%, more preferably at least 110%, even more preferably at least 120% or more, 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.
  • 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 therefore 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: 29A, 58D, 89Y, 117M, 216M.
  • an amino acid exchange in a specific position of the protease according to SEQ ID NO:1 is accompanied by a change in an enzymatic parameter, e.g., an increase in the KM value, and a corresponding change in the enzymatic parameter, e.g., 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 is to be seen as confirmation of the correct assignment.
  • a process according to the invention for producing a protease comprises introducing (i) at least one amino acid substitution at at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, based on the numbering according to SEQ ID NO:1, selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one amino acid substitution at at least one of the positions corresponding to positions 29, 58, 89, 117 and 216, based on the numbering according to SEQ ID NO:1, selected from the group consisting of A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F and M216L, into a starting molecule having an amino acid sequence which is at least 70% and increasingly preferably at
  • a method according to the invention may further comprise one or more of the following method steps:
  • 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, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268 or 269 contiguous amino acids, wherein the protease, in each case based on the numbering according to SEQ ID NO: 1, (i) at least one of the positions corresponding to positions 12, 43, 122, 127, 154, 156, 160, 211, 212 and 222, at least one amino acid substitution selected from the group consisting of Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H and A222S, and (ii) at least one of the positions corresponding to positions 29,
  • a previously described protease is stabilized, in particular by one or more mutations, e.g., substitutions, or by coupling to a polymer.
  • mutations e.g., substitutions
  • 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.
  • Preferred embodiments are those in which the enzyme is stabilized in several ways, since several stabilizing mutations act additively or synergistically.
  • the protease according to the invention can be reacted with at least one reversible inhibitor compound selected from the group consisting of peptide inhibitors, in particular peptide aldehydes, 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) wherein R is in each case selected from -COOH, C 1-6 alkyl-substituted or unsubstituted C 2-6 dicarboxylic acids, C 1-6 alkyl-substituted or unsubstituted C 2-6 carboxylic acids and -OOC-NR 2 2, wherein R 2 is the same or different and is selected from C 1-5 alkyl or H, and salts, esters or derivatives thereof
  • the protease is characterized in that its cleaning performance is not significantly reduced compared to the wild-type enzyme (SEQ ID NO:1) (reference protease), i.e. it has at least 80% of the reference washing or cleaning performance, preferably at least 100%, more preferably at least 110%, particularly preferably at least 120% or more.
  • SEQ ID NO:1 reference protease
  • Washing or cleaning performance refers to the ability of a washing or cleaning agent to partially or completely remove existing soiling.
  • 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 protease thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent.
  • washing or cleaning liquor refers to the working solution containing the washing or cleaning agent that acts on textiles or fabrics or hard surfaces, especially dishes, and thus comes into contact with the soiling present on the textiles or fabrics or hard surfaces, especially dishes.
  • the washing or cleaning liquor is usually created when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, e.g., in a washing machine, dishwasher, or other suitable container.
  • the cleaning performance can be determined in a washing system containing a detergent at a dosage of between 2.0 and 8.0 grams per liter of wash liquor and the protease.
  • the proteases to be compared are used at the same concentration (based on active protein).
  • the protease concentration 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.
  • a liquid reference detergent for such a washing system can, for example, be composed as follows (all data in percent by weight (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% glycerol, 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, e.g., 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.
  • a liquid reference detergent for such a washing system should be composed as follows (all information in percent by weight): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foam agent, 6-7% glycerin, 0.3-0.5% ethanol, 4-7% FAEOS (fatty alcohol ether sulfate), 5-15% non-ionic surfactants, 5-15% anionic surfactants (LAS), 1% boric acid, 1-4% sodium citrate (dihydrate), 2-4% soda, 2-6% coconut fatty acids, 0.5-2.5% HEDP (1-hydroxyethane-(1,1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.15% optical brightener, 0-0.001% dye, balance demineralized water.
  • the dosage of the liquid detergent is preferably between 3.5 and 6.0 grams per liter of wash liquor, e.g., 4.7, 4.9, or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.
  • a powdered reference detergent for such a washing system can be composed as follows (all data in percent by weight): 10% linear alkylbenzenesulfonate (sodium salt), 1.5% Ci2-18 fatty alcohol sulfate (sodium salt), 2.0% C s fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium hydrogen carbonate, 4.0% amorphous sodium disilicate, 17% sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1.0% carboxymethylcellulose, 1.0% phosphonate, 27% sodium sulfate, remainder: foam inhibitors, optical brightener, perfumes.
  • the dosage of the powdered detergent is preferably between 4.5 and 7.0 grams per liter of wash liquor, e.g., and particularly preferably, 4.7 grams per liter of wash liquor, or 5.5, 5.9, or 6.7 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 9 and pH 11.
  • the cleaning performance is determined for soiling on cotton by measuring the degree of cleaning of the washed textiles.
  • 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).
  • the cleaning performance is determined, for example, at 20°C or 40°C using a liquid detergent as specified herein, with the washing process preferably being carried out for 60 minutes at 600 rpm.
  • the degree of whiteness i.e., the lightening of soils, as a measure of cleaning performance, is determined using optical measuring 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.
  • the cleaning performance of a dishwashing detergent can be determined in a system containing an automatic dishwashing detergent in a dosage as stated herein and the protease according to the invention, wherein the proteases to be compared are used at the same concentration (based on active protein) and the cleaning performance against a soiling of tea, meat, spaghetti and/or crèmekosée is determined according to the IKW method (Recommendations for the Quality Assessment of the Cleaning Performance of Dishwasher Detergents (Part B, Update 2015), sofwjournal, 142, 06/16, 34-48) in a Miele GSL (program 45°C, 21 °dH).
  • the concentration of the protease in the detergent intended for this washing system is 0.001 to 0.1 wt.%, preferably 0.01 to 0.06 wt.%, based on active, purified protein.
  • Liquid dishwashing detergent two-component formulation
  • the respective enzymes By using the respective enzymes with the same activity, 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, such as the cleaning performance on specific soils, can be compared. Generally speaking, a low specific activity can be compensated by adding a larger amount of protein.
  • the enzymes to be tested can also be used in the same amount of substance or weight if the enzymes to be tested show a different affinity for the test substrate in an activity test.
  • equal weight refers to the use of the same weight of the enzymes being tested.
  • 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 20°C and about 75°C, preferably between about 30°C and about 60°C and particularly preferably between about 40°C and about 55°C.
  • 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).
  • pNA chromophore para-nitroaniline
  • 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 enzymatic activity (cf. Del Mar et al., 1979).
  • 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.
  • 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.
  • 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 achieved by titrating the active sites using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J. Am. Chem. Soc. 1966, 88, 24, 5890-5913).
  • the invention further relates to a protease as described herein, which is characterized in that it has at least one chemical modification.
  • a protease with such a modification is referred to as a derivative, i.e. the protease is derivatized.
  • Derivatives in the sense of the present application 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 expressing the protein. Coupling of low-molecular 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 Connection to the protein.
  • another Connection can also be another protein that is bound to a protein according to the invention, for example, via bifunctional chemical compounds.
  • Derivatization is also understood to mean covalent binding to a macromolecular carrier, or noncovalent inclusion in suitable macromolecular cage structures.
  • Derivatizations can, for example, influence the 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.
  • coupling with macromolecular compounds, e.g., polyethylene glycol can improve the protein in terms of stability and/or skin compatibility.
  • Derivatives of a protein according to the invention can, in the broadest sense, also be understood to include preparations of these proteins.
  • a protein can be combined with various other substances, e.g., from the culture of the producing microorganisms.
  • a protein can also be deliberately mixed with other substances, e.g., to increase its storage stability.
  • all preparations of a protein according to the invention are also according to the invention. This is independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. It can 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 joint preparation of proteases with specific inhibitors is possible in this regard.
  • proteases or protease variants and/or derivatives described herein those whose storage stability and/or catalytic activity and/or substrate tolerance and/or purification performance is improved compared to a reference protease are particularly preferred within the scope of the present invention, wherein the catalytic activity and/or purification performance is determined as described herein.
  • 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.
  • a nucleic acid encoding a protease according to the invention
  • 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.
  • the sequences of both complementary strands must be taken into account in all three possible reading frames.
  • 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.
  • 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 any doubt, since, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, the skilled person can Starting from an amino acid sequence, nucleic acids coding for this amino acid sequence can be easily determined.
  • 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, e.g. 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 on the nucleic acid in the organism are faced with a comparatively small number of loaded tRNA molecules. Although it encodes the same amino acid, this leads to a codon being translated less efficiently in the organism than a synonymous codon that encodes 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.
  • vectors are understood to be elements consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic 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 special plasmids, i.e. circular genetic elements, particularly when used in bacteria.
  • 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.
  • vectors are capable of establishing themselves as stable units in the respective host cells over several generations. They can exist extrachromosomally as independent units or integrate 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 therein. 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.
  • 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, e.g., by changing the cultivation conditions or upon reaching a certain cell density of the nucleic acid containing them.
  • 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).
  • IPTG galactose derivative isopropyl-ß-D-thiogalactopyranoside
  • lac operon lac operon
  • 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.
  • 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.
  • 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 a vector according to the invention, and the further components are then supplemented accordingly.
  • Methods for transforming cells are established in the prior art and sufficiently known to the person skilled in the art. In principle, all cells, i.e., prokaryotic or eukaryotic cells, are suitable as host cells.
  • Preferred host cells are those that are genetically advantageous to handle, for example, with regard to transformation with the nucleic acid or vector and its stable establishment, e.g., unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling.
  • Preferred host cells according to the invention secrete the (transgenically) expressed protein into the medium surrounding the host cells.
  • the proteases can be modified by the cells producing them after their production, e.g., by attaching sugar molecules, formylations, aminations, etc. Such post-translational modifications can influence the function of the protease.
  • 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, by 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.
  • 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 methods or production processes.
  • the specialist in bacteria in fermentation technology has a rich A wealth of experience.
  • either gram-negative or gram-positive bacteria may be suitable for a variety of reasons, which must be determined experimentally in each individual case, such as nutrient sources, product formation rate, time required, etc.
  • gram-negative bacteria such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes surrounding the cells. This can be advantageous for special applications.
  • Gram-negative bacteria can also be engineered so that they secrete the expressed proteins not only into the periplasmic space, but also into the medium surrounding the bacterium.
  • Gram-positive bacteria such as Bacilli or Actinomycetes or other members of the Actinomycetes family, on the other hand, do not have an outer membrane, so that 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 isolated directly from the medium or further processed.
  • 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 that 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 requirements for culture conditions, have different or additional selection markers, or express different or additional proteins.
  • they can also be host cells that transgenically express several proteins or enzymes.
  • the present invention is in principle applicable to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and leads to 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.
  • 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 carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Sten
  • the host cell can also be a eukaryotic cell, which is characterized by having a cell nucleus.
  • a further subject of the invention is therefore a host cell characterized by having a cell nucleus.
  • eukaryotic cells are capable of post-translationally modifying the protein 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, which enable such systems.
  • the modifications that eukaryotic systems carry out, particularly in connection with protein synthesis include the binding of low-molecular-weight compounds such as membrane anchors or oligosaccharides.
  • Such oligosaccharide modifications can, for example, be used to reduce allergenicity. of an expressed protein may be desirable. 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.
  • the host cells according to the invention are cultivated and fermented in a conventional manner, e.g., in discontinuous or continuous systems.
  • 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 formed protein can be simultaneously extracted from the medium.
  • 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.
  • This subject matter of the invention preferably comprises fermentation processes.
  • 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, e.g., 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. Here, the media components consumed by the ongoing cultivation are fed in. This allows significant increases in both cell density and cell mass or dry mass and/or, in particular, in the activity of the protease of interest to be achieved. Furthermore, the fermentation can also be designed such that unwanted 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., product preparation 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 to ensure that the host cells secrete the protease into the fermentation medium.
  • the protease can alternatively be isolated from the host cell, i.e., purified from the cell mass, e.g., by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
  • the invention further provides an agent characterized in that it contains a protease according to the invention as described herein.
  • the agent is preferably a washing and cleaning agent, in particular a washing and cleaning agent containing bleach, preferably a dishwasher detergent, particularly preferably a dishwasher detergent containing bleach.
  • proteases according to the invention are used in washing or cleaning agents, in particular dishwasher detergents, which contain at least one bleaching agent preparation.
  • the bleaching agent preparation comprises an oxygen bleaching agent selected from the group consisting of hydrogen peroxide, peroxomonosulfate salts, peroxodisulfate salts and phthalimidoperoxohexanoic acid (PAP), wherein the weight fraction of the oxygen bleaching agent in the total weight of the bleaching agent preparation is preferably between 0.1 and 50 wt.%, preferably between 0.2 and 35 wt.%, particularly preferably between 0.5 and 20 wt.% and in particular between 1.0 and 10 wt.%.
  • PAP phthalimidoperoxohexanoic acid
  • the bleaching agent preparation comprises an oxidizing agent.
  • the oxidizing agent is preferably selected from chlorine dioxide, calcium hypochlorite, potassium hypochlorite, sodium hypochlorite.
  • the bleaching agent preparation comprises sodium hypochlorite as oxidizing agent.
  • the bleaching agent composition contains the at least one oxidizing agent, preferably the alkali metal hypochlorite, in particular sodium hypochlorite, in amounts of 0.1 to 8.0 wt. %, preferably 0.15 to 3.0 wt. %, and more preferably 0.2 to 1.0 wt. %, based on the total weight of the bleaching agent preparation.
  • bleaching agents include peroxypyrophosphates, citrate perhydrates, and H2O2-yielding peracidic salts or peracids, such as perbenzoates, peroxophthalates, diperazelaic acid, phthaloiminoperacid, or diperdodecanedioic acid.
  • bleaching agents from the group of organic bleaching agents can also be used.
  • Typical organic bleaching agents are diacyl peroxides, such as dibenzoyl peroxide.
  • Other typical organic bleaching agents are peroxyacids, with alkyl peroxyacids and aryl peroxyacids being particularly mentioned as examples.
  • the washing or cleaning agent in particular automatic dishwashing agent, comprises at least one bleach catalyst from the group of transition metal catalysts.
  • These substances are preferably bleach-enhancing transition metal salts or transition metal complexes, such as Mn, Fe, Co, Ru, or Mo salen complexes or carbonyl complexes.
  • Mn, Fe, Co, Ru, Mo, Ti, V, and Cu complexes with N-containing tripod ligands, as well as Co, Fe, Cu, and Ru ammine complexes can also be used as bleach catalysts.
  • complexes of manganese in oxidation state II, III, IV, or IV which preferably contain one or more macrocyclic ligands with the donor functions N, NR, PR, O, and/or S.
  • bleach catalyst(s) in the agents according to the invention which contain 1,4,7-trimethyl-1,4,7-triazacyclononane (Me-TACN), 1,4,7-triazacyclononane (TACN), 1,5,9-trimethyl-1,5,9-triazacyclododecane (Me-TACD), 2-methyl-1,4,7-trimethyl-1,4,7-triazacyclononane (Me/Me-TACN) and/or 2-methyl-1,4,7-triazacyclononane (Me/TACN) as macromolecular ligands.
  • the weight fraction of the bleach catalyst in the total weight of the washing or cleaning agent is preferably 0.001 to 2.0 wt.%, preferably 0.001 to 1.0 wt.% and in particular 0.001 to 0.5 wt.%.
  • Dishwashing detergents according to the invention may further contain bleach activators, e.g., to achieve an improved bleaching effect when cleaning at temperatures of 60°C and below.
  • bleach activators e.g., to achieve an improved bleaching effect when cleaning at temperatures of 60°C and below.
  • Compounds that, under perhydrolysis conditions, yield aliphatic peroxocarboxylic acids having preferably 1 to 10 carbon atoms, in particular 2 to 4 carbon atoms, and/or optionally substituted perbenzoic acid can be used as bleach activators.
  • Suitable substances are those that carry O- and/or N-acyl groups with the stated number of carbon atoms and/or optionally substituted benzoyl groups.
  • Polyacylated alkylenediamines are preferred, with tetraacetylethylenediamine (TAED) having proven particularly suitable.
  • bleach activators in particular TAED, are preferably used in amounts of 0.1 to 10 wt.%, in particular 0.1 to 8 wt.%, particularly 2 to 8 wt.% and particularly preferably 2 to 6 wt.%, based on the total weight of the agent.
  • washing or cleaning agents in particular dishwasher detergents, which each comprise, based on the total weight of the agent:
  • (C) optionally at least one bleach catalyst, preferably in an amount of 0.001 to 2.0 wt.% and increasingly preferably from 0.001 to 1.0 wt.% or 0.001 to 0.5 wt.%, and
  • washing or cleaning agents in particular dishwasher detergents, which each comprise, based on the total weight of the agent:
  • washing or cleaning agents in particular dishwasher detergents, which each comprise, based on the total weight of the agent:
  • the protease according to the invention is used in agents that are essentially free of boron-containing compounds.
  • "Essentially 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.
  • the washing and cleaning agents according to the invention are free of boron-containing compounds, i.e., they contain, in particular, no boric acid and/or phenylboronic acid derivatives.
  • the protease according to the invention is used in agents that are essentially free of phosphonate-containing compounds.
  • Essentially free of phosphonate-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 phosphonate-containing compounds, based on the total weight of the agent. In particularly preferred embodiments, these agents are free of phosphonate-containing compounds.
  • the protease according to the invention is used in agents that are essentially free of phosphate-containing compounds.
  • "Essentially free of phosphate-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 phosphate-containing compounds, based on the total weight of the agent. In particularly preferred embodiments, these agents are free of phosphate-containing compounds.
  • 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 a washing machine or for hand washing or cleaning.
  • This includes, 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, i.e., in addition to manual and machine dishwashing detergents, scouring agents, glass cleaners, toilet air fresheners, etc.
  • 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 textile washing to achieve an additional effect.
  • cleaning agents also include textile pre- and post-treatment agents, i.e., agents with which the laundry is brought into contact before the actual washing process, e.g., to loosen stubborn dirt, and also agents that, in a step following the actual textile washing process, impart further desirable properties to the laundry, such as a pleasant feel, crease resistance, or low static charge.
  • Fabric softeners are considered to be among the latter agents.
  • the washing or cleaning agents according to the invention which can be in the form of powdered or granular solids, in compacted or recompacted particle form, as homogeneous solutions or suspensions, can 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 can contain, in particular, surfactants, builders, polymers, glass corrosion inhibitors, corrosion inhibitors, bleaching agents such as peroxygen compounds, bleach activators or bleach catalysts.
  • compositions according to the invention can 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, 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.
  • 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 preferably 50 pg to 10 mg per g of the agent.
  • 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.
  • An agent according to the invention increasingly preferably contains the protease in an amount of 1 x 10 -8 to 5 wt.%, from 0.0001 to 1 wt.%, from 0.0005 to 0.5 wt.%, from 0.001 to 0.1 wt.%, in each case based on active protein and based on the total weight of the detergent.
  • 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 several 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.
  • the agent may also be liquid, gel-like, or pasty, e.g., 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.
  • the agent may be in the form of a one-component system. Such agents consist of a Phase.
  • a remedy can also consist of several phases. Such a remedy is therefore divided into several components.
  • the proteases according to the invention are preferably used in automatic dishwashing detergents, in particular automatic dishwashing detergents containing bleach, for cleaning hard surfaces, in particular dishes.
  • 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.
  • 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.
  • each further enzyme is contained in agents according to the invention in an amount of 1 x 10 -7 to 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.%, 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 mutually support each other in their cleaning performance.
  • synergism very particularly preferably exists between the protease contained according to the invention and another enzyme in an agent according to the invention. 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.
  • Dishwashing detergents preferred according to the invention comprise at least one protease and at least one amylase.
  • Textile detergents preferred according to the invention comprise at least one protease and at least one amylase.
  • textile detergents comprise at least one protease and at least one cellulase.
  • textile detergents comprise at least one protease and at least one lipase.
  • textile detergents comprise at least one protease, at least one amylase, and at least one lipase.
  • textile detergents comprise at least one protease, at least one amylase, and at least one cellulase.
  • textile detergents comprise at least one protease, at least one amylase, at least one cellulase, and at least one lipase.
  • proteases are the subtilisins BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, subtilisin DY and the subtilases, but no longer the 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.
  • proteases are, for example, those sold under the trade names Durazym®, Relase®, Everlase®, Nafizym®, Kannase®, Progress Uno 101 L® and Ovozyme® by Novozymes, those sold under the trade names Purafect®, Purafect® OxP, Purafect® Prime, Excellase®, Properase®, Preferenz P100® and Preferenz P300® by Danisco/DuPont, those sold under the trade name Lavergy pro 104 LS® by BASF, those sold under the trade name Protosol® by Advanced Biochemicals Ltd., those sold under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., the enzymes 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.
  • 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 usable proteases are those that are naturally present in the microorganisms Stenotrophomonas maltophilia, in particular Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus.
  • amylases are the ⁇ -amylases from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus and, in particular, their further developments improved for use in detergents or 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 ⁇ -amylase are available under the trade names Duramyl® and Termamyl® ultra (both from Novozymes), Purastar® OxAm (Danisco/DuPont) and Keistase® (Daiwa Seiko Inc.).
  • the ⁇ -amylase from Bacillus amyloliquefaciens is marketed by Novozymes under the name BAN®, and derived variants of the ⁇ -amylase from Bacillus stearothermophilus under the names BSG® and Novamyl®, also from Novozymes.
  • Other suitable enzymes for this purpose include ⁇ -amylase from Bacillus sp. A 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948). Fusion products of all of these molecules can also be used.
  • ⁇ -amylase from Aspergillus niger and A are marketed by Novozymes under the name BAN®, and derived variants of the ⁇ -amylase from Bacillus stearothermophilus under the names BSG® and Novamyl®, also from Novozymes.
  • Other suitable enzymes for this purpose include ⁇ -
  • 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 AmplifyTM 12L, Amplify PrimeTM 120L, or Amplify PrimeTM 100L, the latter also from Novozymes, and the PREFERENZ S® series from Danisco/DuPont, including PREFERENZ S100®, PREFERENZ S1000®, or PREFERENZ S210®. Variants of these enzymes obtainable through point mutations can also be used according to the invention.
  • Suitable cellulases include those of bacterial or fungal origin.
  • 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.
  • cellulases examples include 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 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 with a sequence of at least 60% identity to positions 40 to 559 of SEQ ID NO:2 of WO 2001/062903.
  • a GH44 xyloglucanase e.g., a xyloglucanase enzyme with 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 CelluzymeTM, CarezymeTM, Carezyme PremiumTM, CellucleanTM (e.g.
  • CellucleanTM 5000L and CellucleanTM 4000T Celluclean ClassicTM, CellusoftTM, Endolase®, Renozyme® and WhitezymeTM (Novozymes A/S), ClazinaseTM and Puradax HATM (Genencor International Inc.), KAC-500(B)TM (Kao Corporation), RevitalenzTM 1000, RevitalenzTM 2000 and RevitalenzTM 3000 (DuPont), as well as Ecostone® and Biotouch® (AB Enzymes).
  • 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.
  • 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 LipolaseTM, LipexTM, LipolexTM and LipocleanTM (Novozymes A/S), Lumafast (Genencor/DuPont) and Lipomax (Gist-Brocades).
  • oxidoreductases e.g., oxidases, oxygenases, catalases, peroxidases, such as halo-, chloro-, bromo-, lignin-, glucose- or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases)
  • organic compounds, particularly aromatic ones, which interact with the enzymes are additionally added in order to increase the activity of the oxidoreductases in question (enhancers) or to Redox potentials between the oxidizing enzymes and the soils to ensure the flow of electrons (mediators).
  • the enzymes to be used can also be formulated together with accompanying substances, such as those from fermentation.
  • the enzymes are preferably used as liquid enzyme formulation(s).
  • 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.
  • the enzymes can be encapsulated for both solid and liquid dosage forms, e.g., by spray-drying or extrusion of the enzyme solution together with a preferably natural polymer, or in the form of capsules, e.g., those in which the enzymes are enclosed as if in a solidified gel, or in core-shell capsules, in which an enzyme-containing core is coated with a water-, air-, and/or chemical-impermeable protective layer.
  • Additional active ingredients e.g., stabilizers, emulsifiers, pigments, bleaching agents, or dyes, can be applied in superimposed layers.
  • Such capsules are applied using conventional methods, e.g., by shaking or rolling granulation or in fluid-bed processes.
  • Such granules, e.g., by applying polymeric film-forming agents are advantageously low in dust and, due to the coating, are storage-stable.
  • water-soluble films such as those used in the packaging of unit-dose detergents and cleaning agents.
  • Such a film allows the enzymes to be released upon contact with water.
  • water-soluble refers to a film structure that is preferably completely water-soluble.
  • a film consists of (fully or partially hydrolyzed) polyvinyl alcohol (PVA).
  • 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.
  • 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 75°C, more preferably about 30°C to about 60°C, and particularly preferably about 40°C to about 55°C.
  • Textile cleaning processes are generally characterized by the fact that various cleaning-active substances are applied to the fabric in several process steps. applied to the item to be cleaned and washed off after the exposure time, or that the item to be cleaned is treated in any other way with a detergent or a solution or dilution of this agent.
  • 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.
  • 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.
  • processes for textile raw materials, fibers, or textiles with natural components are preferred, and especially for those containing wool or silk.
  • Another subject of the invention relates to a method for cleaning textiles and/or hard surfaces, in particular dishes, wherein in at least one method step a washing or cleaning agent which contains a protease according to the invention as described herein and/or a protease according to the invention as described herein is used.
  • the invention also encompasses the use of proteases according to the invention, as described herein, in washing or cleaning agents, in particular washing or cleaning agents containing bleach, preferably automatic dishwashing agents, particularly preferably automatic dishwashing agents containing bleach, for cleaning textiles and/or hard surfaces, in particular dishes.
  • the present invention relates to an alkaline protease of the subtilisin type from Bacillus gibsonii.
  • Variants of this protease wild type: Bacillus gibsonii protease according to SEQ ID NO:1 were produced by random mutagenesis, which were then screened for, among other things, improved washing performance and/or improved enzyme stability.
  • the protease variants mentioned herein were generated from the above-mentioned wild-type protease (SEQ ID NO:1) by random mutagenesis and/or saturation mutagenesis. Furthermore, some mutants were produced by targeted generation of synthetic genes.
  • protease variants The recombinant expression of the protease variants according to the invention was carried out according to methods described in the literature (Vojcic et al., An efficient transformation method for Bacillus subtilis DB104, Appl Microbiol Biotechnol, 2012, 94 (2): 487-493).
  • the activity of the protease is determined by the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400).
  • AAPFpNA succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide
  • the measurement was carried out at a temperature of 25°C, at pH 8.6 and a wavelength of 410 nm.
  • the measurement time was 5 min with a measurement interval of 20 to 60 seconds.
  • a protease solution which causes an absorbance change of 0.500 OD under these conditions has, according to the current designation, an activity of 10 HPE per ml (cf. e.g. Schülein et al., Mol Gen Gent (1991) 227:137-143).
  • protease variants listed in Table 1 were added to a wash liquor containing various active chlorine concentrations and incubated for 30 min at 30°C. The reaction was stopped with 2% Na sulfite (40 ml / 2 ml) and the protease activity was determined using AAPF (see Example 2).
  • a 5,000 ppm active chlorine stock solution in detergent matrix was prepared according to Table 2 and the pH was adjusted to pH 8 with 2M HCl.
  • protease-containing samples and control samples were treated with various concentrations of active chlorine (0 ppm, 0.5 ppm, 1 ppm, 2 ppm, 4 ppm, 10 ppm).
  • the samples were stirred for 30 s and then incubated in a water bath for 30 min at 30°C. After incubation, 2% sodium sulfite (40 mg/2 ml) was added to inactivate the active chlorine.
  • the samples were mixed and stored on ice until analysis.
  • the protease activity (determined according to Example 2) of the respective proteases is shown in Table 3.
  • the activity of the respective protease after incubation for 30 min at 30°C without the addition of active chlorine was normalized to 100%.
  • protease variants according to the invention show significantly improved stability towards sodium hypochlorite compared to the wild type (P1).
  • proteases according to the invention exhibit improved cleaning performance compared to the wild type (P1).
  • proteases according to the invention can be used in various washing and cleaning compositions and realize their effect.

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Abstract

L'invention concerne des protéases qui présentent une activité protéolytique et qui comprennent une séquence d'acides aminés qui est identique à la séquence d'acides aminés indiquée dans SEQ ID NO : 1 sur sa longueur totale à raison d'au moins 70 % et, de préférence, de manière croissante, à raison d'au moins 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 % et 98 %, la protéase présentant, respectivement par rapport à la numérotation selon SEQ ID NO : 1, (i) à au moins une des positions qui correspondent aux positions 12, 43, 122, 127, 154, 156, 160, 211, 212 et 222, au moins une substitution d'acide aminé sélectionnée dans le groupe constitué par Q12L, I43V, M122L, D127P, N154S, T156A, G160S, M211N, M211L, P212D, P212H et A222S, et (ii) à au moins une des positions qui correspondent aux positions 29, 58, 89, 117 et 216, au moins une substitution d'acide aminé sélectionnée dans le groupe constitué par A29G, D58P, Y89A, M117A, M117F, M117I, M117L, M117V, M216A, M216F et M216L. En outre, l'invention concerne un produit de lavage ou de nettoyage, en particulier un produit de lavage ou de nettoyage à teneur en agent de blanchiment, de préférence un détergent pour lave-vaisselle, plus préférentiellement un détergent pour lave-vaisselle à teneur en agent de blanchiment, comprenant une protéase selon l'invention.
PCT/EP2024/081351 2023-12-07 2024-11-06 Variantes de protéase à stabilité de blanchiment améliorée Pending WO2025119569A1 (fr)

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