[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO2024125930A1 - Détergents et agents de nettoyage contenant une protéase - Google Patents

Détergents et agents de nettoyage contenant une protéase Download PDF

Info

Publication number
WO2024125930A1
WO2024125930A1 PCT/EP2023/082002 EP2023082002W WO2024125930A1 WO 2024125930 A1 WO2024125930 A1 WO 2024125930A1 EP 2023082002 W EP2023082002 W EP 2023082002W WO 2024125930 A1 WO2024125930 A1 WO 2024125930A1
Authority
WO
WIPO (PCT)
Prior art keywords
protease
agent
washing
amino acid
cleaning
Prior art date
Application number
PCT/EP2023/082002
Other languages
German (de)
English (en)
Inventor
Christian DEGERING
Susanne Wieland
Shohana ISLAM
Claudia LINDNER
Johannes Bongaerts
Petra Siegert
Fabian Falkenberg
Original Assignee
Henkel Ag & Co. Kgaa
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Henkel Ag & Co. Kgaa filed Critical Henkel Ag & Co. Kgaa
Publication of WO2024125930A1 publication Critical patent/WO2024125930A1/fr

Links

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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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 is in the field of enzyme technology.
  • the invention relates to proteases which can be used in particular with regard to use in washing and cleaning agents, in particular with regard to liquid washing and cleaning agents, all sufficiently similar proteases with a correspondingly similar sequence to SEQ ID NO:1 and nucleic acids encoding them.
  • the invention further relates to their production and methods for using these proteases, their use as such and agents containing them, in particular washing and cleaning agents, in particular liquid washing and cleaning agents.
  • proteases are among the most technically important enzymes of all. They are the longest established enzymes for detergents and cleaning agents and are contained in practically all modern, high-performance detergents and cleaning agents. They break down protein-containing soiling on the items being cleaned.
  • proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important; they are serine proteases due to the catalytically active amino acids. They act as nonspecific endopeptidases and hydrolyze any acid amide bonds that are located inside peptides or proteins. Their pH optimum is usually in the clearly 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 these, the subtilisins produced and secreted by Bacillus species are particularly worthy of mention 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 proteases mentioned which have an amino acid sequence that is different from the original protease.
  • Proteases are modified in a targeted or random manner using methods known from the state of the art and are thus optimized for use in detergents and cleaning agents, for example. This includes point, deletion or insertion mutagenesis or fusion with other proteins or protein parts. For most of the proteases known from the state of the art, optimized variants are known.
  • proteases are suitable for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such preparations or they are not sufficiently stable. For the use of proteases in cleaning agents, high catalytic activity and stability under conditions such as those encountered during a washing process are therefore particularly desirable. Consequently, prior art liquid formulations containing proteases and surfactants have the disadvantage that the proteases they contain do not have satisfactory proteolytic activity under standard washing conditions (e.g. in a temperature range of about 20°C to about 40°C) and/or are not sufficiently stable in storage, and the formulations therefore do not show optimal cleaning performance on protease-sensitive stains.
  • standard washing conditions e.g. in a temperature range of about 20°C to about 40°C
  • Protease-sensitive stains are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing stains.
  • Stubborn stains are also usually addressed in common washing and cleaning agents using bleach and/or other cleaning-active substances in the washing and cleaning agent, such as phosphonates and/or phosphates.
  • the difficulties in removing stubborn stains and stains are particularly increased when formulating liquid washing and cleaning agents or when formulating detergents for colored textiles, since these usually do not contain bleach. Due to sustainability efforts and concerns about the environmental impact of phosphates and phosphonates in detergents and cleaning agents, more and more detergents and cleaning agents are being developed that contain little or no phosphate and/or phosphonate-containing compounds.
  • washing and cleaning agents in particular liquid washing and cleaning agents, in particular those without bleach and/or without phosphonates and/or without phosphates, in particular with regard to the cleaning performance on protease-sensitive soiling, in particular in a temperature range of about 20°C to about 40°C.
  • the invention therefore relates to a protease comprising an amino acid sequence which is at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length, or variants thereof.
  • the variants according to the invention are characterized in that they consist of a protease which has an amino acid sequence with at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length, are obtainable as a starting molecule by single or multiple conservative amino acid substitution.
  • the variants according to the invention are characterized in that characterized in that it consists of a protease which has an amino acid sequence with at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length, is obtainable as the starting molecule by fragmentation, deletion, insertion or
  • Another object of the invention is a process for producing a protease, comprising providing a starting protease which contains at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity to the amino acid sequence given in SEQ ID NO:1 over its entire length or variants of the parent protease, where the variants are as defined above.
  • a protease within the meaning of the present patent application therefore includes both the protease as such and a protease produced using a method according to the invention. All statements on the protease therefore refer both to the protease as such and to the proteases produced using corresponding methods, as well as to the corresponding methods, in particular production methods of the protease.
  • proteases relate to the nucleic acids encoding these proteases, non-human host cells containing proteases according to the invention or nucleic acids, and agents comprising proteases according to the invention, in particular washing and cleaning agents, in particular liquid washing and cleaning agents, washing and cleaning processes, and uses of the proteases according to the invention in washing or cleaning agents for removing protease-sensitive stains, which are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing stains.
  • washing and cleaning agents in particular liquid washing and cleaning agents, washing and cleaning processes, and uses of the proteases according to the invention in washing or cleaning agents for removing protease-sensitive stains, which are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing stains.
  • 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.
  • agent and cleaning agent or “detergent or cleaning agent” as used herein is synonymous with the term “agent” and refers to a composition for cleaning textiles and/or hard surfaces, in particular dishes, as explained in the description.
  • 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 as used herein includes liquids and gels as well as pasty compositions. It is preferred that the liquid compositions be flowable and pourable at room temperature, but it is also possible 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 1013 mbar.
  • a substance e.g. a composition or an agent, is liquid according to the definition of the invention if it is in the liquid state at 25°C and 1013 mbar. Liquid also includes gel form.
  • Variant refers to natural or artificially generated 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 finding of the inventors that a protease according to the invention from Metabacillus, in particular Metabacillus indicus, which has an amino acid sequence which corresponds to the one given in SEQ ID NO:1 to at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical amino acid sequence, which removes protease
  • 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 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%
  • Protease variants according to the invention are obtainable from a protease with the stated sequence identity as starting molecule by single or multiple conservative amino acid substitution.
  • the variants used are obtainable from a protease with the specified sequence identity as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprise an amino acid sequence that corresponds to the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids.
  • proteases according to the invention in a washing or cleaning agent, preferably a liquid washing or cleaning agent, leads to an improved cleaning performance of these agents on at least one and increasingly preferably two, three, four, five or six protease-sensitive soiling(s), which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soilings.
  • protease-sensitive soiling(s) which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soilings.
  • protease-sensitive soiling(s) which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soilings.
  • protease-sensitive soiling(s) which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soilings.
  • protease-sensitive soiling(s) include, for example, blood, egg (yol
  • An improvement in cleaning performance according to the invention in particular in proteolytic cleaning performance, is present when the protease shows an improved cleaning performance compared to an agent without protease and/or an improved cleaning performance compared to a reference protease on at least one and increasingly preferably two, three, four, five or six protease-sensitive soil(s), which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, as described herein.
  • 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 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical, wherein the protease is present on at least one and increasingly preferably two,
  • Such performance-improved washing results on protease-sensitive soiling 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 60°C, in particular about 20°C to about 40°C.
  • the proteases according to the invention have a high stability in washing or cleaning agents, for example against surfactants and/or bleaching agents and/or chelators and/or against temperature influences, in particular against high temperatures, for example between about 50°C and about 65°C, in particular about 60°C, and/or against acidic or alkaline conditions and/or against pH changes and/or against denaturing or oxidizing agents and/or against (auto)proteolytic degradation and/or against a change in the redox ratios.
  • Particularly preferred embodiments of the invention therefore provide performance-improved protease variants.
  • Such advantageous embodiments of proteases according to the invention consequently enable improved washing results on protease-sensitive stains, which are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing stains.
  • cleaning performance is understood to mean the lightening performance on one or more stains, in particular on laundry or dishes.
  • both the washing or cleaning agent which comprises the protease or the washing or cleaning liquor formed by this agent and the protease itself have a respective cleaning performance.
  • the cleaning performance of the enzyme thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent.
  • the cleaning performance is preferably determined as stated below.
  • wash liquor refers to the 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 dirt present on textiles or fabrics or hard surfaces.
  • the wash liquor is usually created when the washing or cleaning process begins and the washing or cleaning agent is diluted with water, for example in a dishwasher, a washing machine or in another suitable container.
  • the proteases according to the invention have enzymatic activity, ie they are capable of hydrolyzing peptides and proteins, in particular in a washing or cleaning agent.
  • a protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide/peptide bonds in protein/peptide substrates and is thus able to cleave proteins or peptides.
  • the washing and cleaning agent according to the invention advantageously has improved cleaning performance, in particular when removing protease-sensitive stains, which are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing stains.
  • the washing and cleaning agent according to the invention is particularly preferably suitable for removing protease-sensitive stains which are selected from the group consisting of blood, egg (yolk), milk and other protein-containing soiling.
  • 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 given also refer to mature (processed) enzymes.
  • the protease is a free enzyme. This means that the protease can interact directly with all components of an agent and, if the agent is a liquid agent, that the protease is in direct contact with the agent's solvent (e.g. water).
  • an agent can contain proteases that form an interaction complex with other molecules or that contain a "coating".
  • a single or multiple protease molecule(s) can be separated from the other components of the agent by a structure surrounding them.
  • a separating structure can be created by, but is not limited to, vesicles, such as a micelle or a liposome.
  • the surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell.
  • an agent can contain cells of, for example, Bacillus pumilus or Bacillus subtilis or other expression strains that express the protease according to the invention, or cell culture supernatants of such cells.
  • nucleic acid or amino acid sequences is determined by comparing sequences. This sequence comparison is based on the BLAST algorithm, which is established and commonly used in the state of the art (see, for example, Altschul et al. (1990) Basic local alignment search tool, J. Mol. Biol., 215:403-410, and Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 25:3389-3402) and is basically done by matching similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another. A tabular assignment of the relevant positions is called an alignment.
  • Sequence comparisons are created using computer programs.
  • Commonly used programs include the Clustal series (see e.g. Chenna et al. (2003) Multiple sequence alignment with the Clustal series of programs, Nucleic Acid Res., 31:3497-3500), T-Coffee (see e.g. Notredame et al. (2000) T-Coffee: A novel method for multiple sequence alignments, J. Mol. Biol., 302:205-217) or programs based on these programs or algorithms.
  • Sequence comparisons can also be carried out using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW, or Clone Manager 10 (use of the BLOSUM 62 scoring matrix for sequence alignment at the amino acid level). Unless otherwise stated, sequence identity reported herein is determined using the BLAST algorithm.
  • Such a comparison also allows a statement to be made about the similarity of the sequences being compared. This is usually expressed as percent identity, i.e. the proportion of identical Nucleotides or amino acid residues are specified at the same positions or at positions corresponding to one another in an alignment.
  • the broader term homology includes conserved amino acid exchanges in amino acid sequences, i.e. amino acids with similar chemical activity, since these usually perform 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 information can be provided for entire polypeptides or genes or only 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. They can be small and contain only a few nucleotides or amino acids. Such small regions often perform essential functions for the overall activity of the protein. It can therefore be useful to refer sequence matches only to individual, possibly small regions. Unless otherwise stated, identity or homology information in the present application refers to the total length of the nucleic acid or amino acid sequence specified in each case.
  • an amino acid position corresponds to a numerically designated position in SEQ ID NO:1 means that the corresponding position is assigned to the numerically designated position in SEQ ID NO:1 in an alignment as defined above. Furthermore, the assignment of the positions is based on the mature protein. This assignment is also to be used in particular if the amino acid sequence of a protease comprises a higher number of amino acid residues than the protease according to SEQ ID NO:1.
  • proteases according to the invention can have amino acid changes, 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 certain applications or with regard to special properties (for example 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 enzymes 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 changed.
  • the net charge of the enzymes can be changed in order to influence substrate binding, in particular for use in washing and cleaning agents.
  • the stability or catalytic activity of the protease can be increased by one or more corresponding mutations and thereby its cleaning performance can be improved.
  • Advantageous properties of individual mutations, eg individual substitutions, can complement each other.
  • a protease that has already been optimized with regard to certain properties, eg with regard to its activity and/or its tolerance with regard 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, for example an asterisk or a dash, or an A is given in front of the corresponding position.
  • P9T describes the substitution of proline at position 9 by threonine
  • P9TH the insertion of histidine after the amino acid threonine at position 9
  • P9* or AP9 the deletion of proline at position 9.
  • a further subject of the invention is therefore 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.
  • conservative amino acid substitution means the exchange (substitution) of an amino acid residue for another amino acid residue, whereby 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 a non-polar amino acid residue for another non-polar amino acid residue.
  • the protease may comprise an amino acid sequence before and/or after the conservative amino acid substitution which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length by at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% is identical.
  • the protease is characterized in that it is obtainable from a protease according to the invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which corresponds to the starting molecule over a length of at least 190, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274 or 275 contiguous amino acids.
  • the protease may comprise, before and/or after fragmentation, deletion, insertion or substitution mutagenesis, an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length by at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% is identical.
  • the enzymes retain their catalytic activity even after mutagenesis, ie their catalytic activity corresponds at least to that of the starting enzyme, ie in a preferred embodiment the catalytic activity is at least 80%, preferably at least 90%, more preferably at least 100% of the activity of the starting enzyme.
  • Other substitutions can also have advantageous effects. Both individual and several connected amino acids can be exchanged for other amino acids.
  • an amino acid exchange in a certain position of the protease according to SEQ ID NO:1 is accompanied by a change in an enzymatic parameter, for example with an increase in the KM value, and a corresponding change in the enzymatic parameter, for example also an increase in the KM value, is observed in a protease variant according to the invention, the amino acid exchange of which was achieved by the same introduced amino acid, this is to be seen as confirmation of the correct assignment.
  • the invention also covers fragments of the protease as defined herein, in particular those according to SEQ ID NO:1, which are shortened at the N- and/or C-terminus such that one or more amino acids of the protease, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, are no longer contained.
  • Variants of these shortened fragments can also be used in various embodiments of the invention, which correspond to the form shortened by (each) 1 to 10 N-terminal and/or C-terminal amino acids starting from the amino acid sequence given in SEQ ID NO:1, over the total length to at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% are identical.
  • the invention also covers proteases which have an amino acid sequence which, via the protease, comprises an amino acid sequence which is at least 75% and increasingly preferably 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%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length or the variants thereof described herein, without losing or reducing the catalytic activity.
  • such proteases are those which are N- and/or C-terminally additional amino acids, for example the signal peptide or fragments of the signal peptide, whereby the signal peptide or the fragments of the signal peptide are formed during the production of the protease.
  • a method according to the invention further comprises one or more of the following method steps:
  • a previously described protease is stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.
  • Increasing stability during storage and/or during use, for example during the washing process means that the enzymatic activity lasts longer and thus the cleaning performance is improved.
  • all stabilization options described in the prior art and/or expedient are possible. Preference is given to those stabilizations that are achieved via mutations of the enzyme itself, since such stabilizations do not require any further work steps after the enzyme has been obtained. Examples of sequence changes suitable for this purpose are mentioned above. Other suitable sequence changes are known from the prior art.
  • metal ions in particular the calcium binding sites, for example by exchanging one or more of the amino acids involved in calcium binding for one or more negatively charged amino acids and/or by introducing sequence changes in at least one of the sequences of the two amino acids arginine/glycine;
  • Preferred embodiments are those in which the enzyme is stabilized in multiple ways, since multiple stabilizing mutations act additively or synergistically.
  • the protease is characterized in that its cleaning performance is not significantly reduced compared to the wild-type enzyme (SEQ ID NO:1), i.e. it has at least 70% and increasingly preferably 75%, 80%, 85%, 90% or 95% of the reference washing performance, and more preferably at least 100%, even more preferably at least 110%, particularly preferably at least 120% or more.
  • the cleaning performance can be determined in a washing system that contains a detergent in a dosage of between 2.0 and 8.0 grams per liter of washing liquor and the enzyme.
  • the enzymes to be compared are used in the same concentration (based on active protein). Using the same activity of the respective enzymes ensures that even if there is a discrepancy in the ratio of active substance to total protein (the values of the specific activity), the respective enzymatic properties, e.g. the cleaning performance on certain stains, are compared. In general, 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 have a different affinity for the test substrate in an activity test.
  • the expression "same amount of substance” in this context refers to using the same mole of the enzymes to be tested.
  • the expression “same weight amount” refers to using the same weight of the enzymes to be tested.
  • the concentration of protease in the detergent intended for such a washing system is 0.001 to 0.1 wt.%, preferably 0.01 to 0.06 wt.%, based on active protein.
  • Washing or cleaning performance is understood to mean the ability of a washing or cleaning agent to partially or completely remove an existing stain, in particular the lightening performance of one or more stains on textiles, in particular cotton textiles, polyester textiles and/or cotton-polyester blended textiles.
  • stains are blood on cotton or chocolate-milk/soot on cotton, cocoa on cotton, egg yolk on cotton, milk/soot on cotton or porridge on cotton, etc.
  • Further examples include the aforementioned stains on cotton-polyester blended textiles or polyester-containing textiles or other blended textiles.
  • both the washing or cleaning agent which comprises the protease, or the washing or cleaning liquor formed by this agent, and the protease itself have a respective cleaning performance. The cleaning performance of the protease thus contributes to the cleaning performance of the agent or the washing or cleaning liquor formed by the agent.
  • washing or cleaning liquor refers to the solution containing the washing or cleaning agent that acts on the textiles or hard surfaces, especially dishes, and thus comes into contact with the dirt present on the textiles or hard surfaces.
  • 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.
  • a liquid reference detergent for such a washing system can, for example, be composed as follows (all information 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% defoamer, 2% glycerin, 0.08% preservatives, 1% ethanol, the rest demineralized water.
  • the dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of washing liquor, for example 4.7, 4.9 or 5.9 grams per liter of washing liquor. Washing is preferably carried out in a pH range between pH 7 and pH 10.5, preferably between pH 8 and pH 9.
  • the cleaning performance is determined for soiling 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 about 20°C or about 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 such as the one specified above, with the washing process preferably taking place for 60 minutes at 600 rpm.
  • the degree of whiteness i.e. the lightening of the soiling, as a measure of the cleaning performance is determined using optical measuring methods, preferably photometric.
  • One suitable device for this is the Minolta CM508d spectrometer.
  • the devices used for the measurement are usually calibrated beforehand with a white standard, preferably a supplied white standard.
  • Preferred embodiments of proteases according to the invention achieve such advantageous cleaning performance even at low temperatures, in particular in the temperature ranges between about 10°C and about 60°C, preferably between about 15°C and about 50°C and particularly preferably between about 20°C and about 40°C.
  • 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 the absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979).
  • the measurement is carried out at a temperature of 25°C, at pH 8.6, and a wavelength of 410 nm.
  • the measurement time is 5 minutes and the measurement interval is 20 s to 60 s.
  • the protease activity is usually given in protease units (PU). Suitable protease activities are, for example, 2.25, 5 or 10 PU per ml of wash liquor. However, the protease activity is not zero.
  • An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measuring method, preferably a photometric method.
  • a suitable test involves the protease-dependent cleavage of the substrate protein casein. This is split by the protease into a large number of smaller partial products. All of these partial products have an increased absorption at 290 nm compared to non-cleaved casein. This increased absorption can be determined using a photometer and thus a conclusion can 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 active protein concentration can be determined by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (cf. Bender et al., J. Am. Chem. Soc. 88, 24 (1966): 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 change is referred to as a derivative, i.e. the protease is derivatized.
  • derivatives are understood to mean proteins whose pure amino acid chain has been chemically modified.
  • derivatizations can, for example, take place in vivo by the host cell that expresses the protein.
  • couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy.
  • Derivatizations can also be carried out in vitro, for example by chemically converting a side chain of an amino acid or by covalently binding another compound to the protein. For example, coupling amines to carboxyl groups of an enzyme is possible to change the isoelectric point.
  • Another compound can also be another protein that is bound to a protein according to the invention, for example via bifunctional chemical compounds.
  • Derivatization also means covalent binding to a macromolecular carrier or non-covalent inclusion in suitable macromolecular cage structures. Derivatizations can, for example, influence the substrate specificity or the binding strength to the substrate or cause a temporary blocking of the enzymatic activity if the coupled substance is an inhibitor. This can be useful for the storage period, for example.
  • Such modifications can also influence the stability or the 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, such as polyethylene glycol can improve the protein in terms of stability and/or skin compatibility.
  • Derivatives of a protein according to the invention can also be understood in the broadest sense as preparations of these proteins.
  • a protein can be combined with various other substances, for example from the culture of the producing microorganisms.
  • a protein can also be deliberately mixed with other substances, for example to increase its storage stability. All preparations of a protein according to the invention are therefore also in accordance with the invention.
  • proteases or protease variants and/or derivatives described herein those are particularly preferred within the scope of the present invention whose storage stability and/or catalytic activity and/or substrate tolerance and/or cleaning performance corresponds to the protease according to SEQ ID NO:1 and/or is improved compared to the protease according to SEQ ID NO:1, wherein the catalytic activity and/or cleaning performance is determined as described herein.
  • proteases according to the invention and those that can be used in the detergents according to the invention are obtainable from plants, fungi and/or bacteria.
  • the protease comprising SEQ ID NO:1 can be obtained from Metabacillus indicus.
  • proteases are often very low. It may therefore be useful to increase production by expressing protease genes in foreign production hosts.
  • vectors containing a nucleic acid encoding a protease according to the invention are generally used.
  • the invention further relates to a nucleic acid that codes for a protease according to the invention, as well as a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.
  • a nucleic acid that codes for 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. It must also be taken into account that different codons, i.e. base triplets, can code for the same amino acids, so that a certain amino acid sequence can be coded by several different nucleic acids.
  • nucleic acid sequences that can code for one of the proteases described above are included in this subject matter of the invention.
  • the person skilled in the art is able to determine these nucleic acid sequences without a doubt, since despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, starting from an amino acid sequence, the person skilled in the art can easily determine nucleic acids coding for this amino acid sequence.
  • 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.
  • each organism for example a host cell of a production strain, has a specific codon usage.
  • Codon usage is understood to mean the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons on the nucleic acid in the organism are faced with a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this leads to a codon being translated less efficiently in the organism than a synonymous codon that codes for the same amino acid. Due to the presence of a higher number of tRNA Molecules for the synonymous codon can be translated more efficiently in the organism.
  • a specialist can use commonly known methods such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and/or protein chemical methods to produce the corresponding nucleic acids, including complete genes, based on known DNA and/or amino acid sequences.
  • PCR polymerase chain reaction
  • Such methods are known, for example, from Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press.
  • vectors are understood to mean elements consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic nucleic acid region. They are able to establish this as a stable genetic element in a species or a 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 able to establish themselves as stable units in the host cells concerned over several generations. They can be present extrachromosomally as separate units or can be integrated into a chromosome or chromosomal DNA.
  • Expression vectors comprise nucleic acid sequences that enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a contained nucleic acid there. Expression is influenced in particular by the promoter(s) that regulate transcription. In principle, expression can occur through the natural promoter originally located in front 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 is provided for the expression of a nucleic acid according to the invention and used for its expression.
  • Expression vectors can also be regulated, for example by changing the cultivation conditions or when a certain cell density of the host cells containing them is reached or by adding certain substances, in particular activators of gene expression.
  • An example of such a substance is the galactose derivative isopropyl-ß-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon).
  • IPTG galactose derivative isopropyl-ß-D-thiogalactopyranoside
  • lac operon lac operon
  • the invention further relates to a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention, or which contains 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 host cell.
  • 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 other components are then supplemented accordingly.
  • Methods for transforming cells are established in the prior art and are well known to the person skilled in the art. In principle, all cells are suitable as host cells, i.e. prokaryotic or eukaryotic cells. Preference is given to host cells that can be handled genetically in a favorable manner, for example with regard to transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handleability.
  • 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 they have been produced, for example 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 on the vector, for example, but can also be present in these cells from the outset. These can be stimulated to express, for example, by controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions, or when a certain cell density is reached. This enables economical production of the proteins according to 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 makes it possible to establish cost-effective cultivation methods or production processes.
  • the expert in fermentation technology has a wealth of experience with bacteria.
  • gram-negative or gram-positive bacteria can be suitable for a variety of reasons that can be determined experimentally in each individual case, such as nutrient sources, product formation rate, time required, etc.
  • gram-negative bacteria such as Escherichia co//
  • a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications.
  • Gram-negative bacteria can also be designed in such a way that they secrete the expressed proteins not only into the periplasmic space, but also into the medium surrounding the bacteria.
  • Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales, on the other hand, do not have an outer membrane, so that secreted proteins immediately enter the bacteria surrounding medium, 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 organisms of origin 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 the culture conditions, have other or additional selection markers or express other or additional proteins. In particular, 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 means that proteins according to the invention can be produced by using 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, which is characterized by having a cell nucleus.
  • eukaryotic cells are able to modify the protein formed post-translationally. 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.
  • modifications that eukaryotic systems carry out, particularly in connection with protein synthesis include, for example, the binding of low-molecular compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications can be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can 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 the usual way, for example 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 grow back and at the same time the protein formed can be removed from the medium.
  • Host cells according to the invention are preferably used to produce proteases according to the invention.
  • the invention therefore further relates to a method 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 of the product produced, for example the proteases according to the invention. All fermentation processes that are based on a corresponding process for producing a protease according to the invention represent embodiments of this subject matter of the invention. Fermentation processes that are characterized in that the fermentation is carried out using a feed strategy are particularly suitable. In this case, the media components that are consumed by the ongoing cultivation are fed in. This can achieve considerable increases in both the cell density and the cell mass or dry mass and/or in particular in the activity of the protease of interest. Furthermore, the fermentation can also be designed in such a way that undesirable metabolic products are filtered out or neutralized by adding buffer or appropriate counterions.
  • the protease produced can be harvested from the fermentation medium. Such a fermentation process is preferred over isolation of 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 of one or more suitable secretion markers or mechanisms and/or transport systems so that the host cells secrete the protease into the fermentation medium. Without secretion, the protease can alternatively be isolated from the host cell, i.e. purified from the cell mass, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.
  • the invention further relates to an agent which is characterized in that it contains a protease according to the invention as described herein.
  • the agent is preferably a washing or cleaning agent, more preferably a liquid washing or cleaning agent, particularly preferably a liquid washing agent.
  • the washing and cleaning agent is particularly preferably essentially free of boron-containing compounds.
  • the protease according to the invention is used in agents or compositions that are essentially free of boron-containing compounds.
  • "Essentially free of boron-containing compounds" in this context means that the agents according to the invention contain less than 2% by weight, preferably less than 1% by weight, of further 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 from boron-containing compounds, ie they contain in particular no boric acid and/or phenylboronic acid derivatives.
  • the protease according to the invention is used in agents or compositions that are essentially free of phosphonate-containing compounds.
  • "Essentially free of phosphonate-containing compounds” in this context means that the corresponding agents or compositions 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/composition. In particularly preferred embodiments, these agents/compositions are free of phosphonate-containing compounds.
  • the protease according to the invention is used in agents or compositions that are essentially free of phosphate-containing compounds.
  • "Essentially free of phosphate-containing compounds" in this context means that the corresponding agents or compositions 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/composition. In particularly preferred embodiments, these agents/compositions are free of phosphate-containing compounds.
  • a washing or cleaning agent is understood to mean all conceivable types of washing or cleaning agent, both concentrates and agents that can be used undiluted, for use on a commercial scale, in the washing machine or for hand washing or cleaning.
  • washing agents for textiles, carpets or natural fibers, for which the term washing agent is used.
  • 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.
  • 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 in order to achieve an additional effect.
  • washing and cleaning agents within the scope of the invention also include textile pre- and post-treatment agents, i.e. agents with which the item of laundry is brought into contact before the actual washing, for example to dissolve stubborn dirt, and also agents which, in a step following the actual textile washing, give the laundry other desirable properties such as a pleasant feel, freedom from creases or low static charge.
  • the latter agents include fabric softeners, among others.
  • the washing or cleaning agents according to the invention which can be in the form of powdered or granular solids, in compacted or re-compacted particle form, as homogeneous solutions or suspensions, can contain, in addition to a protease according to the invention, all known ingredients that are 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.
  • agents according to the invention can also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, sequestering agents, electrolytes, pH regulators and/or other auxiliary substances such as optical brighteners, graying inhibitors, color transfer inhibitors, foam regulators and dyes and fragrances and combinations thereof.
  • Advantageous ingredients of agents 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. Express reference is made to this disclosure 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 very particularly 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% by weight, preferably 0.0001 or 0.001 to 0.1% by weight, based on the total weight of the agent or composition.
  • An agent according to the invention contains the protease increasingly preferably in an amount of from 1 x 10 -8 to 5% by weight, from 0.0001 to 1% by weight, from 0.0005 to 0.5% by weight, from 0.001 to 0.1% by weight, in each case based on active protein and based on the total weight of the detergent.
  • the embodiments of the present invention include all solid, powdery, liquid, gel-like or pasty dosage forms of agents according to the invention, which may optionally also consist of several phases and may be in compressed or non-compressed 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.
  • the solid dosage forms of the agent also include extrudates, granules, tablets or pouches.
  • the agent may also be liquid, gel-like or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste.
  • Liquid agents are generally preferred.
  • the agent may be in the form of a one-component system. Such agents consist of one phase. Alternatively, an agent may also consist of several phases. Such a remedy is therefore divided into several components.
  • proteases according to the invention are preferably used in liquid detergents for cleaning textiles, particularly preferably in liquid detergents with a pH of about 8 to about 9.
  • Detergents or cleaning agents according to the invention can contain only one protease according to the invention. Alternatively, they can also contain other hydrolytic enzymes or other enzymes in a concentration suitable for the effectiveness of the agent.
  • a further embodiment of the invention thus represents agents which further comprise one or more other enzymes.
  • All enzymes which can develop a catalytic activity in the agent according to the invention can preferably be used as further enzymes, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ß-glucosidase, pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase or a protease which is different from the protease according to the invention, as well as mixtures thereof.
  • enzymes are advantageously contained in the agent in an amount of 1 x 10 -8 to 5% by weight, based on active protein.
  • Each additional enzyme is increasingly preferably contained in agents according to the invention in an amount of 1 x 10 -7 to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight, based on active protein.
  • the enzymes particularly preferably show synergistic cleaning performance against certain soilings or stains, ie the enzymes contained in the agent support each other in their cleaning performance.
  • Such synergism is very particularly preferably present 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.
  • Textile detergents preferred according to the invention contain at least one protease and at least one amylase.
  • textile detergents contain at least one protease and at least one cellulase.
  • textile detergents contain at least one protease and at least one lipase.
  • textile detergents contain at least one protease, at least one amylase and at least one lipase.
  • textile detergents contain at least one protease, at least one amylase and at least one cellulase.
  • textile detergents contain at least one protease, at least one amylase, at least one cellulase and at least one lipase.
  • Textile detergents which contain 3 to 10 different enzymes are particularly preferred, whereby textile detergents which contain 3 to 10 different types of enzyme can be particularly preferred with regard to cleaning performance over a very wide range of stains.
  • 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 enzymes thermitase, proteinase K and the proteases TW3 and TW7, which are classified as subtilases but no longer as subtilisins in the narrower sense.
  • Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from the company Novozymes.
  • subtilisins 147 and 309 are sold under the trade names Esperase® and Savinase® by the company Novozymes.
  • 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®, Natalase®, Kannase®, Progress Uno 101 L® and Ovozyme® by the company Novozymes, those sold under the trade names Purafect®, Purafect® OxP, Purafect® Prime, Excellase®, Properase®, Preferenz P100® and Preferenz P300® by the company Danisco/DuPont, those sold under the trade name Lavergy pro 104 LS® by the company BASF, those sold under the trade name Protosol® by the company Advanced Biochemicals Ltd., those sold under the trade name Wuxi® by the company Wuxi Snyder Bioproducts Ltd., those sold under the trade names Proleather® and Protease P® by the company Amano Pharmaceuticals Ltd., and those sold under the name Proteinase K-16 from the company 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 proteases that can be used are those that are naturally present in the microorganisms Stenotrophomonas maltophilia, in particular Stenotrophomonas maltophilia K279a, Bacillus intermedius and Bacillus sphaericus.
  • amylases are the a-amylases from Bacillus licheniformis, Bacillus amyloliquefaciens or Bacillus stearothermophilus and, in particular, their further developments that have been improved for use in washing or cleaning products.
  • the enzyme from Bacillus licheniformis is available from Novozymes under the name Termamyl® and from Danisco/DuPont under the name Purastar® ST. Further development products of this a-amylase are available under the trade names Duramyl® and Termamyl® ultra (both from Novozymes), Purastar® OxAm (Danisco/DuPont) and Keistase® (Daiwa Seiko Inc.).
  • the a-amylase from Bacillus amyloliquefaciens is sold by Novozymes under the name BAN®, and derived variants of the a-amylase from Bacillus stearothermophilus under the names BSG® and Novamyl®, also from Novozymes.
  • Other notable examples for this purpose are the a-amylase from Bacillus sp. A 7-7 (DSM 12368) and the cyclodextrin glucanotransferase (CGTase) from Bacillus agaradherens (DSM 9948). Fusion products of all of the molecules mentioned can also be used.
  • oryzae available under the trade name Fungamyl® from the company 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 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 that can be obtained by point mutations can also be used according to the invention.
  • Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Suitable cellulases are cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum, which are described in US 4435307, US 5648263, US 5691178, US 5776757 and WO 89/09259 Particularly suitable cellulases are the 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 include a GH44 xyloglucanase, e.g.
  • a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40 to 559 of SEQ ID NO:2 of WO 2001/062903.
  • Commercially available cellulases include 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 generated 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 are now renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes, P. cepacia, P. sp. Strain SD705, P.
  • Preferred lipases include, for example, the lipases originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or developed further therefrom, in particular those with one or more of the following amino acid substitutions starting from the lipase mentioned in 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, particularly preferably aromatic, compounds that interact with the enzymes are also advantageously added in order to increase the activity of the oxidoreductases in question (enhancers) or to ensure the flow of electrons when the redox potentials between the oxidizing enzymes and the soiling differ greatly (mediators).
  • the enzymes to be used can also be formulated together with accompanying substances, for example from fermentation.
  • the enzymes are preferably used as enzyme liquid 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, in particular in the case of liquid or gel-like agents, solutions of the enzymes, advantageously as concentrated as possible, with little water and/or mixed with stabilizers or other additives.
  • the enzymes can be encapsulated for both the solid and liquid dosage forms, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are enclosed as if in a solidified gel or in those of the core-shell type, in which an enzyme-containing core is coated with a protective layer that is impermeable to water, air and/or chemicals.
  • active ingredients such as stabilizers, emulsifiers, pigments, bleaching agents or dyes, can be applied in superimposed layers.
  • Such capsules are applied using methods known per se, for example by shaking or rolling granulation or in fluid bed processes. Such granules are advantageously low in dust, for example by applying polymeric film formers, and are stable in storage due to the coating.
  • water-soluble films such as those used in the packaging of detergents and cleaning agents in unit dosage form.
  • Such a film enables the release of the enzymes after 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).
  • Another subject of the invention is a method for cleaning textiles and/or hard surfaces, in particular dishes, which is characterized in that an agent according to the invention is used in at least one method step.
  • the method described is characterized in that the protease is used at a temperature of about 0°C to about 100°C, preferably about 20°C to about 60°C and more preferably about 20°C to about 40°C. 20°C represents low washing temperature, while 40°C is the preferred/average washing temperature in Europe.
  • Processes for cleaning textiles are generally characterized by the fact that various cleaning-active substances are applied to the 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 method can consist in bringing a protease according to the invention into contact with the soiling as the only 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 represent 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 very particularly for those with wool or silk.
  • the invention also covers the use of the proteases described herein in washing or cleaning agents, for example as described above, for the (improved) removal of protease-sensitive soils, which are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils.
  • the invention relates to the use of a protease according to the invention described herein in a washing or cleaning agent for improving the cleaning performance of such a protease-containing washing or cleaning agent, in particular a liquid washing or cleaning agent, on at least one and increasingly preferably two, three, four, five or six protease-sensitive soil(s), which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the improvement in the cleaning performance of an agent with protease according to the invention is determined compared to an agent without protease and/or compared to an agent which contains a reference protease, as described in Example 2, in particular in a temperature range of about 20°C to about 40°C.
  • the invention relates to the use of a protease according to the invention described herein in a washing or cleaning agent for improving the cleaning performance of such a protease-containing washing or cleaning agent, in particular a liquid washing or cleaning agent, on at least one and increasingly preferably two, three, four, five or six protease-sensitive soil(s), which is/are preferably selected from the group consisting of blood, egg (yolk), milk and other protein-containing soils, wherein the improvement in the cleaning performance of an agent with a protease according to the invention is determined compared to an agent without a protease and/or compared to an agent which contains a reference protease, as described in Example 2, in particular in a temperature range of about 20°C to about 40°C, wherein the protease is a protease which has proteolytic activity and comprises an amino acid sequence which is 75% and increasingly preferably 76%, 77%, 78%, 79%, 80%, 81%, 82%,
  • Protease activity was determined in a discontinuous assay using casein as a substrate.
  • the final concentration of the substrate solution was 12 mg/ml casein (prepared according to Hammarsten; Merck, Darmstadt, #2242) and 30 mM Tris in synthetic tap water.
  • Synthetic tap water is a solution of 0.029% (w/v) CaCL • 2 H2O, 0.014% (w/v) MgCL • 6 H2O and 0.021% (w/v) NaHCCh with 15° dH (German hardness).
  • the substrate solution was heated to 70°C and the pH adjusted to 8.5 at 50°C using 0.1 N NaOH.
  • the protease solution was prepared by adding 2% (w/v) anhydrous pentasodium tripolyphosphate to synthetic tap water and adjusting to pH 8.5 with hydrochloric acid. To 600 ⁇ l of the casein solution, 200 ⁇ l of the enzyme solution was added. The mixture was incubated at 50°C for 15 minutes. The reaction was terminated by adding 600 ⁇ l of 0.44 M trichloroacetic acid (TCA), 0.22 M sodium acetate in 3% (w/v). After a cooling step of 15 minutes on ice, the TCA insoluble protein was removed by centrifugation.
  • TCA trichloroacetic acid
  • the cleaning performance was determined in mini-wash tests with Bacillus subtilis culture supernatants containing the expressed protease according to SEQ ID NO:1.
  • the supernatants were used with the same activity as the benchmark (6 HPE/ml active enzyme protein in the wash solution).
  • the protease according to the invention shows a comparable or significantly improved washing performance on various types of soiling compared to two conventional proteases that are used commercially. This is particularly remarkable because the protease according to SEQ ID NO:1 is a newly isolated wild-type enzyme that has not previously been isolated by protein engineering. modified and yet shows comparable or improved cleaning performance to commercially available proteases for detergents and cleaning agents.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Detergent Compositions (AREA)

Abstract

L'invention se rapporte au domaine de la technologie enzymatique. L'invention concerne des protéases de Metabacillus indicus qui sont particulièrement appropriées pour être utilisées dans des détergents et des agents de nettoyage, toutes les protéases suffisamment similaires ayant une séquence similaire de manière correspondante à SEQ ID N :1, et des acides nucléiques codant pour lesdites protéases. L'invention concerne en outre la production desdites protéases, un procédé d'utilisation desdites protéases, leur utilisation en tant que telles, et des agents contenant lesdites protéases, en particulier des détergents et des agents de nettoyage.
PCT/EP2023/082002 2022-12-13 2023-11-16 Détergents et agents de nettoyage contenant une protéase WO2024125930A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102022213538.5A DE102022213538A1 (de) 2022-12-13 2022-12-13 Wasch- und reinigungsmittel enthaltend protease
DE102022213538.5 2022-12-13

Publications (1)

Publication Number Publication Date
WO2024125930A1 true WO2024125930A1 (fr) 2024-06-20

Family

ID=88837381

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2023/082002 WO2024125930A1 (fr) 2022-12-13 2023-11-16 Détergents et agents de nettoyage contenant une protéase

Country Status (2)

Country Link
DE (1) DE102022213538A1 (fr)
WO (1) WO2024125930A1 (fr)

Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992021760A1 (fr) 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO2001062903A1 (fr) 2000-02-24 2001-08-30 Novozymes A/S Xyloglucanases appartenant a la famille 44
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
WO2007131656A1 (fr) 2006-05-11 2007-11-22 Henkel Ag & Co. Kgaa Subtilisine dérivée de bacillus pumilus et produits détergents et nettoyants contenant cette nouvelle subtilisine
WO2008086916A1 (fr) 2007-01-16 2008-07-24 Henkel Ag & Co. Kgaa Nouvelle protéase alcaline issue de bacillus gibsonii et détergents et nettoyants contenant cette nouvelle protéase alcaline
WO2009121725A1 (fr) 2008-04-02 2009-10-08 Henkel Ag & Co. Kgaa Agents de lavage et de nettoyage contenant des protéases sécrétées par xanthomonas
WO2013060621A1 (fr) 2011-10-28 2013-05-02 Henkel Ag & Co. Kgaa Variants de protéase à performance améliorée et thermiquement stables
WO2015038792A1 (fr) * 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2016145428A1 (fr) * 2015-03-12 2016-09-15 Danisco Us Inc Compositions et procédés comprenant des variants de protéase lg12-clade
WO2017215925A1 (fr) 2016-06-15 2017-12-21 Henkel Ag & Co. Kgaa Protéase de bacillus gibsonii et variantes de celle-ci
EP3660151A1 (fr) 2018-11-29 2020-06-03 Henkel AG & Co. KGaA Variantes de prothèse stables au stockage et à la performance améliorée
WO2021175696A1 (fr) 2020-03-03 2021-09-10 Henkel Ag & Co. Kgaa Variants de protéase vi à stabilité améliorée
WO2021175697A1 (fr) 2020-03-03 2021-09-10 Henkel Ag & Co. Kgaa Variantes de protéase à performance améliorée vii

Patent Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4435307A (en) 1980-04-30 1984-03-06 Novo Industri A/S Detergent cellulase
EP0258068A2 (fr) 1986-08-29 1988-03-02 Novo Nordisk A/S Additif enzymatique pour détergent
EP0305216A1 (fr) 1987-08-28 1989-03-01 Novo Nordisk A/S Lipase recombinante de humicola et procédé de production de lipases recombinantes de humicola
US5691178A (en) 1988-03-22 1997-11-25 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase
US5648263A (en) 1988-03-24 1997-07-15 Novo Nordisk A/S Methods for reducing the harshness of a cotton-containing fabric
WO1989009259A1 (fr) 1988-03-24 1989-10-05 Novo-Nordisk A/S Preparation de cellulase
US5776757A (en) 1988-03-24 1998-07-07 Novo Nordisk A/S Fungal cellulase composition containing alkaline CMC-endoglucanase and essentially no cellobiohydrolase and method of making thereof
EP0531372A1 (fr) 1990-05-09 1993-03-17 Novo Nordisk As Preparation de cellulase comprenant un enzyme d'endoglucanase.
EP0495257A1 (fr) 1991-01-16 1992-07-22 The Procter & Gamble Company Compositions de détergent compactes contenant de la cellulase de haute activité
WO1992021760A1 (fr) 1991-05-29 1992-12-10 Cognis, Inc. Enzymes proteolytiques mutantes tirees de bacillus
WO1995023221A1 (fr) 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO1996011262A1 (fr) 1994-10-06 1996-04-18 Novo Nordisk A/S Enzyme et preparation enzymatique presentant une activite endoglucanase
WO1996013580A1 (fr) 1994-10-26 1996-05-09 Novo Nordisk A/S Enzyme a activite lipolytique
WO1996029397A1 (fr) 1995-03-17 1996-09-26 Novo Nordisk A/S Nouvelles endoglucanases
WO1998008940A1 (fr) 1996-08-26 1998-03-05 Novo Nordisk A/S Nouvelle endoglucanase
WO2001062903A1 (fr) 2000-02-24 2001-08-30 Novozymes A/S Xyloglucanases appartenant a la famille 44
WO2002099091A2 (fr) 2001-06-06 2002-12-12 Novozymes A/S Endo-beta-1,4-glucanase
WO2007131656A1 (fr) 2006-05-11 2007-11-22 Henkel Ag & Co. Kgaa Subtilisine dérivée de bacillus pumilus et produits détergents et nettoyants contenant cette nouvelle subtilisine
WO2008086916A1 (fr) 2007-01-16 2008-07-24 Henkel Ag & Co. Kgaa Nouvelle protéase alcaline issue de bacillus gibsonii et détergents et nettoyants contenant cette nouvelle protéase alcaline
WO2009121725A1 (fr) 2008-04-02 2009-10-08 Henkel Ag & Co. Kgaa Agents de lavage et de nettoyage contenant des protéases sécrétées par xanthomonas
WO2013060621A1 (fr) 2011-10-28 2013-05-02 Henkel Ag & Co. Kgaa Variants de protéase à performance améliorée et thermiquement stables
WO2015038792A1 (fr) * 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2016145428A1 (fr) * 2015-03-12 2016-09-15 Danisco Us Inc Compositions et procédés comprenant des variants de protéase lg12-clade
WO2017215925A1 (fr) 2016-06-15 2017-12-21 Henkel Ag & Co. Kgaa Protéase de bacillus gibsonii et variantes de celle-ci
EP3660151A1 (fr) 2018-11-29 2020-06-03 Henkel AG & Co. KGaA Variantes de prothèse stables au stockage et à la performance améliorée
WO2021175696A1 (fr) 2020-03-03 2021-09-10 Henkel Ag & Co. Kgaa Variants de protéase vi à stabilité améliorée
WO2021175697A1 (fr) 2020-03-03 2021-09-10 Henkel Ag & Co. Kgaa Variantes de protéase à performance améliorée vii

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ALTSCHUL ET AL.: "Basic local alignment search tool", J. MOL. BIOL., vol. 215, 1990, pages 403 - 410, XP002949123, DOI: 10.1006/jmbi.1990.9999
ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: 10.1093/nar/25.17.3389
BAND, vol. 7, 1970, pages 125 - 132
BENDER ET AL., J. AM. CHEM. SOC., vol. 88, no. 24, 1966, pages 5890 - 5913
CHENNA ET AL.: "Multiple sequence alignment with the Clustal series of programs", NUCLEIC ACID RES., vol. 31, 2003, pages 3497 - 3500, XP002316493, DOI: 10.1093/nar/gkg500
DATABASE UniProt [online] 29 October 2014 (2014-10-29), "SubName: Full=Peptidase S8 {ECO:0000313|EMBL:KEZ53851.1};", XP002810938, retrieved from EBI accession no. UNIPROT:A0A084H2N9 Database accession no. A0A084H2N9 *
GORNALL ET AL., J. BIOL. CHEM., vol. 177, 1948, pages 751 - 766
S. J. STROPKO ET AL: "Genome-based reclassification of Bacillus cibi as a later heterotypic synonym of Bacillus indicus and emended description of Bacillus indicus", INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, vol. 64, no. Pt 11, 20 August 2014 (2014-08-20), GB, pages 3804 - 3809, XP055408818, ISSN: 1466-5026, DOI: 10.1099/ijs.0.068205-0 *
SAMBROOK, J.FRITSCH, E.FMANIATIS, T.: "Molecular cloning: a laboratory manual", 2001, COLD SPRING LABORATORY PRESS
SCHÜLEIN ET AL., MOL GEN GENT, vol. 227, 1991, pages 137 - 143
T-COFFEE ET AL.: "T-Coffee: A novel method for multiple sequence alignments", J. MOL. BIOL., vol. 302, 2000, pages 205 - 217, XP004469125, DOI: 10.1006/jmbi.2000.4042

Also Published As

Publication number Publication date
DE102022213538A1 (de) 2024-06-13

Similar Documents

Publication Publication Date Title
WO2021175697A1 (fr) Variantes de protéase à performance améliorée vii
EP3679133B1 (fr) Variantes de protéases à performances améliorées
WO2021175696A1 (fr) Variants de protéase vi à stabilité améliorée
EP4253511A2 (fr) Variants de protéases présentant une performance ameliorée et stabilité au stockage
EP3963065A1 (fr) Protéases présentant une stabilité enzymatique améliorée dans des détergents et produits de nettoyage iii
EP3679131B1 (fr) Variantes de protéases à performances améliorées
WO2020221578A1 (fr) Performance de nettoyage améliorée contre des salissures vi sensibles aux protéines
EP3660146B1 (fr) Variantes de prothèse stables au stockage et à la performance améliorée
WO2020221580A1 (fr) Performance de nettoyage améliorée contre des salissures v sensibles à des protéines
EP3679132B1 (fr) Variantes de protéase à performances améliorées
EP3433359B1 (fr) Performance de nettoyage améliorée sur des salissures sensibles aux protéines
EP3458583B1 (fr) Protéases à performances améliorées
DE102016204815A1 (de) Proteasen mit verbesserte Enzymstabilität in Waschmittel
DE102018004207A1 (de) Leistungsverbesserte Varianten der alkalischen Protease aus B. lentus
WO2024125930A1 (fr) Détergents et agents de nettoyage contenant une protéase
WO2024125929A1 (fr) Détergents et agents de nettoyage contenant une protéase
WO2024037685A1 (fr) Variants ix de protéase à performance améliorée
WO2024037686A1 (fr) Variants x de protéase à performance améliorée
DE102022208890A1 (de) Leistungsverbesserte protease-varianten ix
DE102022208891A1 (de) Leistungsverbesserte protease-varianten x
DE102022205594A1 (de) Leistungsverbesserte und lagerstabile protease-varianten
WO2024227591A1 (fr) Variantes de protéase aux performances améliorées
DE102022209245A1 (de) Wasch- und reinigungsmittel enthaltend tannase i
DE102022209246A1 (de) Wasch- und reinigungsmittel enthaltend tannase ii
EP3440203B1 (fr) Nouvelle protéase à performance de lavage améliorée

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23806281

Country of ref document: EP

Kind code of ref document: A1