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EP4114933A1 - Variantes de protéase à performance améliorée vii - Google Patents

Variantes de protéase à performance améliorée vii

Info

Publication number
EP4114933A1
EP4114933A1 EP21708613.1A EP21708613A EP4114933A1 EP 4114933 A1 EP4114933 A1 EP 4114933A1 EP 21708613 A EP21708613 A EP 21708613A EP 4114933 A1 EP4114933 A1 EP 4114933A1
Authority
EP
European Patent Office
Prior art keywords
amino acid
protease
positions
acid substitutions
correspond
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21708613.1A
Other languages
German (de)
English (en)
Inventor
Christian DEGERING
Susanne Wieland
Nina Mussmann
Inken Prueser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and 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 and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP4114933A1 publication Critical patent/EP4114933A1/fr
Pending legal-status Critical Current

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
    • 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 washing process can take place for 60 minutes at a temperature of 40 ° C and the water has a water hardness between 15.5 and 16.5 ° (German hardness).
  • the concentration of the protease in the detergent intended for this washing system is 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active, purified protein.
  • a liquid reference detergent for such a washing system can be composed as follows (all data in percent by weight): 4.4% alkylbenzenesulfonic acid, 5.6% other anionic surfactants, 2.4% C12-C18 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% glycerine, 0.08% preservatives, 1% ethanol, the remainder demineralized Water.
  • the dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH range between pH 7 and pH 10.5, preferably between pH 7.5 and pH 8.5.
  • the cleaning performance is determined, for example, at 20 ° C. or 40 ° C. using a liquid detergent as indicated above, 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 photometrically.
  • a suitable device for this is, for example, the Minolta CM508d spectrometer.
  • the devices used for the measurement are usually calibrated beforehand with a white standard, preferably a supplied white standard.
  • the use of the respective protease for the same level of activity ensures that the respective enzymatic properties, e.g. the cleaning performance on certain soiling, are compared even if there is a gap in the ratio of active substance to total protein (the values of the specific activity). In general, a low specific activity can be compensated for by adding a larger amount of protein.
  • the 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 the pNA causes an increase in the absorbance at 410 nm, whose the time course 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 specified 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.
  • proteases according to the invention can have further amino acid changes, in particular amino acid substitutions, insertions or deletions.
  • Such proteases are further developed, for example, through targeted genetic modification, i.e. through mutagenesis processes, and optimized for specific purposes 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 batches and thus used to generate completely new proteases or other polypeptides.
  • amino acid substitutions first, the naturally present amino acid is designated in the form of the internationally common single-letter code, then the associated sequence position and finally the inserted amino acid. Multiple or alternative exchanges within the same polypeptide chain are separated from one another by slashes. “130D / V” therefore means that the remainder at position 130 can be D or V. In the case of insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, for example an asterisk or a dash, or a D is given in front of the corresponding position.
  • the invention therefore also provides a protease which is characterized in that it can be obtained from a protease as described above as the starting molecule by single or multiple conservative amino acid substitutions, the protease being those described above in the number according to SEQ ID NO: 1 Has amino acid substitutions.
  • 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 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 enzymes advantageously retain their proteolytic activity even after the mutagenesis, ie their proteolytic activity corresponds at least to that of the starting enzyme, ie in a preferred embodiment the proteolytic activity is at least 80%, preferably at least 90%, more preferably at least 100% of the activity of the starting enzyme. Further substitutions can also have advantageous effects. Both single and several connected amino acids can be exchanged for other amino acids.
  • the protease or the protease produced with a method according to the invention is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92 , 5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% or 97%, identical to the amino acid sequence given in SEQ ID NO: 1 over their total length.
  • the protease or the protease produced using a method according to the invention is still at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5 or 99% identical to the amino acid sequence given in SEQ ID NO: 2 over its entire length.
  • the protease or the protease produced with a method according to the invention has the amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T and 271 E at the positions corresponding to positions 9, 130, 133, 144, 224, 252 and correspond to 271; and at least one, preferably at least two, further amino acid substitution (s) in at least one of the positions which correspond to positions 89, 131 or 189, each based on the numbering according to SEQ ID NO: 1.
  • Examples include the following amino acid substitution variants: P9T, N130D, T133A, N144K, Y217M, S224A, N252T and Q271 E combined with one of (i) S89A and S189T; (ii) S89A and G131 H, and (iii) S89A, G131 H and S189T.
  • examples are those with P9T, N130V, T133A, N144K, Y217M, S224A, N252T and Q271 E combined with S89A, G131 H and S189T, the numbering in each case being based on the numbering according to SEQ ID NO: 1 and those in the examples described variants.
  • any of those described herein Proteases in addition to 9T, 130D / V, 133A, 144K, 217M, 224A, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 224, 252 and 271, at least the substitution at position 89, in particular S89A.
  • the invention also relates to a protease described above which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.
  • An increase in the 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 and / or expedient in the prior art come into consideration. Stabilizations that are achieved via mutations of the enzyme itself are preferred, since such stabilizations do not require any further work steps following the recovery of the enzyme. Examples of sequence changes suitable for this are mentioned above. Further suitable sequence changes are known from the prior art.
  • Changing the binding of metal ions, in particular the calcium binding sites for example by exchanging one or more of the amino acid (s) involved in calcium binding for one or more negatively charged amino acids and / or by introducing sequence changes in at least one of the consequences of the two amino acids arginine / glycine;
  • Preferred embodiments are those in which the enzyme is stabilized in several ways, since several stabilizing mutations act additively or synergistically.
  • Another object of the invention is a protease as described above, which is characterized in that it has at least one chemical modification.
  • a protease with such a change is called a derivative, i.e. the protease is derivatized.
  • derivatives are accordingly understood to mean those proteins whose pure amino acid chain has been chemically modified.
  • derivatizations can take place, for example, in vivo by the host cell which expresses the protein.
  • couplings of low molecular weight compounds such as lipids or oligosaccharides should be particularly emphasized.
  • derivatizations can also be carried out in vitro, for example by chemical conversion of a side chain of an amino acid or by covalent ones Binding of another compound to the protein.
  • Such a different compound can also be a further protein which is bound to a protein according to the invention, for example via bifunctional chemical compounds.
  • derivatization is to be understood as the covalent bond to a macromolecular carrier, or also a non-covalent inclusion in suitable macromolecular cage structures.
  • Derivatizations can, for example, influence the substrate specificity or the strength of the binding 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 period of storage, for example.
  • Such modifications can also affect the stability or the enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, to increase its skin tolerance.
  • couplings with macromolecular compounds for example polyethylene glycol, can improve the protein with regard to stability and / or skin tolerance.
  • derivatives of a protein according to the invention can also be understood to mean 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 have been specifically 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. This is also independent of whether it actually displays this enzymatic activity in a particular preparation or not. This is because it may be desired that it has little or no activity during storage and that it only develops its enzymatic function at the time of use. This can be controlled, for example, via corresponding accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.
  • proteases or protease variants and / or derivatives described above particularly preferred in the context of the present invention are those whose storage stability corresponds to at least one of that of the proteases according to SEQ ID NO: 2 or the variants tested in the examples, and / or their cleaning performance at least corresponds to one of those of the proteases according to SEQ ID NO: 2 or the variants tested in the examples, the cleaning performance being determined in a washing system as described above.
  • nucleic acid which 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. In the case of DNA molecules in particular, the sequences of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codons, ie 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 are included in this subject matter of the invention which can code for one of the proteases described above.
  • the person skilled in the art is able to determine these nucleic acid sequences unequivocally because, despite the degeneracy of the genetic code, defined amino acids have to be assigned to individual codons. The person skilled in the art can therefore easily determine nucleic acids coding for this amino acid sequence on the basis of an 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.
  • 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 are compared to a comparatively small number of loaded tRNA molecules in the organism. Although coding for the same amino acid, this means that a codon is translated less efficiently in the organism than a synonymous codon which codes for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, this can be translated more efficiently in the organism.
  • a person skilled in the art is able to use known DNA and / or amino acid sequences to use known DNA and / or amino acid sequences to identify the corresponding nucleic acids up to complete genes using methods that are generally known nowadays, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or protein chemical methods to manufacture.
  • PCR polymerase chain reaction
  • Such methods are for example from Sambrook, J., Fritsch, E.F. 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 the characterizing 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, especially when used in bacteria.
  • a nucleic acid according to the invention is cloned into a vector.
  • the vectors include, for example, those whose origin is bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the host cells concerned over several generations. They can exist extrachromosomally as separate units or can be integrated into a chromosome or chromosomal DNA.
  • Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and to express a nucleic acid contained there.
  • the expression is influenced in particular by the promoter or promoters that regulate transcription.
  • expression can take place through the natural promoter originally located in front of the nucleic acid to be expressed, but also through a promoter of the word cell provided on the expression vector or also through a modified or a completely different promoter from another organism or another word cell.
  • at least one promoter is made available for the expression of a nucleic acid according to the invention and used for its expression.
  • Expression vectors can also be regulatable, for example by changing the cultivation conditions or when a certain cell density of the word cells they contain has been reached or by adding certain substances, in particular activators of gene expression.
  • An example of such a substance is the galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon).
  • IPTG galactose derivative isopropyl- ⁇ -D-thiogalactopyranoside
  • lac operon lac operon
  • the invention also 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 preferably transformed into a microorganism which then represents a word cell according to the invention.
  • individual components, ie nucleic acid parts or fragments of a nucleic acid according to the invention can also be introduced into a word cell in such a way that the then resulting word cell contains a nucleic acid according to the invention or a vector according to the invention.
  • This procedure is particularly suitable when the word cell already contains one or more components of a nucleic acid according to the invention or a vector according to the invention and the further components are then supplemented accordingly.
  • Methods for transforming cells are established in the prior art and are sufficiently known to the person skilled in the art. In principle, all cells, that is, prokaryotic or eukaryotic cells, are suitable as host cells. Preference is given to those word cells which can be manipulated in a genetically advantageous manner, for example with regard to the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred word cells are characterized by good microbiological and biotechnological manageability.
  • 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 functionally influence the protease.
  • Further preferred embodiments are those host cells whose activity can be regulated on the basis of genetic regulatory elements which are provided, for example, on the vector, but can also be present in these cells from the outset. For example, by the controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached, these can be stimulated to express. This enables the proteins according to the invention to be produced economically.
  • An example of such a connection is IPTG as described above.
  • Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on the cultivation conditions. In this way, inexpensive cultivation processes or manufacturing processes can be established. In addition, the specialist in bacteria in fermentation technology has a wealth of experience. Gram-negative or Gram-positive bacteria may be suitable for a special production for a wide variety of reasons, to be determined experimentally in individual cases, such as nutrient sources, product formation rate, time required, etc.
  • Host cells according to the invention can be changed with regard to their requirements for the culture conditions, have different or additional selection markers or still different or express additional proteins.
  • these host cells can also be those which transgenically express several proteins or enzymes.
  • the present invention can in principle be applied to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and leads to the fact that proteins according to the invention can be produced by using such microorganisms. Such microorganisms then represent word cells within the meaning of the invention.
  • the word 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 which is selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausumacterii, Bacillus halodurium, Bacillus paphumlocus, Bacillus paphumlocus Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas malt
  • the host cell can, however, also be a eukaryotic cell, which is characterized in that it has a cell nucleus.
  • a further subject matter of the invention therefore represents a word cell which is characterized in that it has a cell nucleus.
  • eukaryotic cells are able to modify the protein produced post-translationally. Examples 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 make such systems possible.
  • Host cells according to the invention are preferably used to produce proteases according to the invention.
  • the invention therefore also 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.
  • the detergents 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 laundry in order to achieve a further effect.
  • laundry detergents and cleaning agents in the context of the invention also include textile pretreatment and aftertreatment agents, i.e. those agents with which the item of laundry is brought into contact before the actual washing, for example to loosen stubborn dirt, and also those agents that are in one of the actual Textile washing, the subsequent step, give the laundry further desirable properties such as a pleasant grip, crease resistance or low static charge.
  • Fabric softeners, among others, are included in the latter means.
  • the detergents or cleaning agents according to the invention which can be present as pulverulent solids, in 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 in particular contain surfactants, builders, peroxygen compounds or bleach activators. Furthermore, they can contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, graying inhibitors, foam regulators and colorants and fragrances and combinations thereof.
  • a combination of a protease according to the invention with one or more further ingredient (s) of the agent is advantageous, since such an agent in preferred embodiments according to the invention has an improved cleaning performance due to the resulting synergies.
  • Such a synergism can be achieved in particular by combining a protease according to the invention with a surfactant and / or a builder and / or a peroxygen compound and / or a bleach activator.
  • the agent according to the invention cannot contain any boric acid.
  • Advantageous ingredients of agents according to the invention are disclosed in the international patent application WO 2009/121725, beginning there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. Reference is expressly made to this disclosure and the content of the disclosure therein is incorporated into the present patent application.
  • An agent according to the invention advantageously contains the protease in an amount of 2 ⁇ g to 20 mg, preferably 5 ⁇ g to 17.5 mg, particularly preferably 20 ⁇ g to 15 mg and very particularly preferably 50 ⁇ g to 10 mg per g of the agent.
  • the concentration of the protease (active enzyme) described herein 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 the composition.
  • the protease contained in the agent and / or other ingredients of the agent can be coated with a substance which is impermeable to the enzyme at room temperature or in the absence of water and which becomes permeable to the enzyme under the conditions in which the agent is used.
  • Such an embodiment of the invention is thus characterized in that the protease is coated with a substance which is impermeable to the protease at room temperature or in the absence of water.
  • the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.
  • the agent is characterized in that it
  • (a) is in solid form, in particular as a free-flowing powder with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or
  • (b) is present in pasty or liquid form, and / or
  • inventions of the present invention include all solid, powdery, liquid, gel-like or pasty dosage forms of agents according to the invention, which can optionally also consist of several phases and can be in compressed or uncompressed form.
  • the agent can 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 can 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 funds are generally preferred.
  • the agent can be in the form of a one-component system. Such means consist of a phase. Alternatively, a remedy can also consist of several phases. Such a means is therefore divided into several components. Washing or cleaning agents according to the invention can exclusively contain a protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in an appropriate concentration for the effectiveness of the agent. Agents which further comprise one or more further enzymes thus represent a further embodiment of the invention.
  • enzymes that can preferably be used are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, ⁇ -glucosidase, pectinase, carrageenase, perhydrolase, Oxidase, oxidoreductase or other proteases - distinguishable from the proteases according to the invention - and mixtures thereof.
  • Further enzymes are advantageously contained in the agent in an amount of 1 ⁇ 10 -8 to 5 percent by weight based on active protein.
  • Each further enzyme is increasingly preferred in an amount of 1 ⁇ 10 -7 to 3% by weight, from 0.00001 to 1% by weight, from 0.00005 to 0.5% by weight, from 0.0001 up to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight in agents according to the invention, based on active protein.
  • the enzymes particularly preferably show synergistic cleaning performance with respect to certain soiling or stains, ie the enzymes contained in the agent composition mutually support one another in their cleaning performance.
  • Such a synergism is very particularly preferably present between the protease contained according to the invention and a further enzyme of an agent according to the invention, including in particular between said protease and an amylase and / or a lipase and / or a mannanase and / or a cellulase and / or a pectinase .
  • Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients of the agent according to the invention.
  • the enzymes to be used can also be packaged together with accompanying substances, for example from fermentation.
  • the enzymes are preferably used as liquid enzyme formulation (s).
  • the enzymes are not provided in the form of the pure protein, but rather in the form of stabilized, storable and transportable preparations.
  • These ready-made preparations include, for example, the 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 with stabilizers or other auxiliaries.
  • the enzymes can be encapsulated both for the solid and for the liquid dosage form, 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 in a solidified gel or in those of the core-shell type, in which an enzyme-containing core with a water, air and / or chemical impermeable protective layer is coated.
  • Additional active ingredients for example stabilizers, emulsifiers, pigments, bleaches or dyes, can also be applied in superimposed layers.
  • Such capsules are applied by methods known per se, for example by pouring or rolling granulation or in fluid-bed processes. Such granules are advantageously low in dust, for example due to the application of polymeric film formers, and due to the coating are stable in storage.
  • water-soluble films such as those used, for example, in the formulation of detergents and cleaning agents in unit dose form.
  • Such a film enables the enzymes to be released after contact with water.
  • water soluble refers to a film structure that is preferably completely water soluble.
  • Such a film preferably consists of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).
  • the following table shows the detergent matrix (commercially available, without enzymes, optical brighteners, perfume and dyes) that was used for the wash festival:

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  • Detergent Compositions (AREA)

Abstract

L'invention concerne des protéases comprenant une séquence d'acides aminés partageant au moins 70 % d'identité de séquence avec la séquence d'acides aminés donnée dans SEQ ID NO : 1, sur toute leur longueur, et, par rapport à la numérotation selon SEQ ID NO : 1 : (a) comporte des substitutions d'acides aminés 9T, 133A, 224A, 252T et 271E à des positions correspondant aux positions 9T, 130D/V, 133A, 144K, 217M, 224A, 252T et 271 ; et (b) présente au moins une substitution supplémentaire d'acide aminé au moins à l'une des positions correspondant aux positions 89, 131 et 189. L'invention concerne également la production et l'utilisation desdites protéases. Les protéases de ce type présentent une très bonne performance de nettoyage.
EP21708613.1A 2020-03-03 2021-02-25 Variantes de protéase à performance améliorée vii Pending EP4114933A1 (fr)

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DE102020105721.0A DE102020105721A1 (de) 2020-03-03 2020-03-03 Leistungsverbesserte Proteasevarianten VII
PCT/EP2021/054689 WO2021175697A1 (fr) 2020-03-03 2021-02-25 Variantes de protéase à performance améliorée vii

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EP4114933A1 true EP4114933A1 (fr) 2023-01-11

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US (1) US20230159907A1 (fr)
EP (1) EP4114933A1 (fr)
KR (1) KR20220148187A (fr)
CN (1) CN115516088A (fr)
DE (1) DE102020105721A1 (fr)
WO (1) WO2021175697A1 (fr)

Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4525615A2 (fr) 2022-05-14 2025-03-26 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes
DE102022205594A1 (de) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa Leistungsverbesserte und lagerstabile protease-varianten
DE102022205588A1 (de) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit verbesserter enzymstabilität
DE102022205591A1 (de) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit verbesserter enzymstabilität
DE102022205593A1 (de) 2022-06-01 2023-12-07 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit verbesserter enzymstabilität
EP4573195A1 (fr) 2022-08-16 2025-06-25 Henkel AG & Co. KGaA Variants x de protéase à performance améliorée
DE102022208891A1 (de) 2022-08-16 2024-02-22 Henkel Ag & Co. Kgaa Leistungsverbesserte protease-varianten x
DE102022208890A1 (de) 2022-08-16 2024-02-22 Henkel Ag & Co. Kgaa Leistungsverbesserte protease-varianten ix
EP4573194A1 (fr) 2022-08-16 2025-06-25 Henkel AG & Co. KGaA Variants ix de protéase à performance améliorée
EP4324900A1 (fr) 2022-08-17 2024-02-21 Henkel AG & Co. KGaA Composition détergente comprenant des enzymes
JP2025529133A (ja) 2022-09-02 2025-09-04 ダニスコ・ユーエス・インク サブチリシン変異体及びそれに関連する方法
DE102022209245A1 (de) 2022-09-06 2024-03-07 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel enthaltend tannase i
DE102022209246A1 (de) 2022-09-06 2024-03-07 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel enthaltend tannase ii
DE102022131732A1 (de) 2022-11-30 2024-06-06 Henkel Ag & Co. Kgaa Verbesserte Waschleistung durch den Einsatz einer Protease fusioniert mit speziellem Adhäsionsvermittlerpeptid
DE102022213537A1 (de) 2022-12-13 2024-06-13 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel enthaltend protease
DE102022213538A1 (de) 2022-12-13 2024-06-13 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel enthaltend protease
DE102023200106A1 (de) 2023-01-10 2024-07-11 Henkel Ag & Co. Kgaa Enzymhaltiges wasch- und reinigungsmittel
DE102023201692A1 (de) 2023-02-24 2024-08-29 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit dispersin und duftstoff
DE102023201695A1 (de) 2023-02-24 2024-08-29 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit dispersin
DE102023201696A1 (de) 2023-02-24 2024-08-29 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel mit dispersin
DE102023204055A1 (de) 2023-05-03 2024-11-07 Henkel Ag & Co. Kgaa Leistungsverbesserte protease-varianten
DE102023205632A1 (de) 2023-06-15 2024-12-19 Henkel Ag & Co. Kgaa Peptide mit schmutzablösender wirkung für wasch- und reinigungsmittel
DE102023211308A1 (de) 2023-11-14 2025-05-15 Henkel Ag & Co. Kgaa Reinigungsmittel enthaltend protease
DE102023211746A1 (de) 2023-11-24 2025-05-28 Henkel Ag & Co. Kgaa Wasch- und reinigungsmittel enthaltend antimikrobielles peptid
WO2025113890A1 (fr) 2023-11-28 2025-06-05 Henkel Ag & Co. Kgaa Composition de lavage et de nettoyage avec une protéase
WO2025113889A1 (fr) 2023-11-28 2025-06-05 Henkel Ag & Co. Kgaa Composition de lavage et de nettoyage avec une protéase
DE102023212361A1 (de) 2023-12-07 2025-06-12 Henkel Ag & Co. Kgaa Protease-varianten mit verbesserter bleichestabilität
DE102024202187A1 (de) 2024-03-08 2025-09-11 Henkel Ag & Co. Kgaa Verfahren zur Entfernung von Fett und/oder fett- und/oder ölhaltigen Anschmutzungen
WO2025186246A2 (fr) 2024-03-08 2025-09-12 Henkel Ag & Co. Kgaa Agents de lavage et de nettoyage présentant une performance de nettoyage améliorée sur des salissures contenant des graisses
WO2025186242A2 (fr) 2024-03-08 2025-09-12 Henkel Ag & Co. Kgaa Agents de lavage et de nettoyage présentant une performance de nettoyage améliorée sur des salissures contenant de la graisse

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102006022224A1 (de) 2006-05-11 2007-11-15 Henkel Kgaa Subtilisin aus Bacillus pumilus und Wasch- und Reinigungsmittel enthaltend dieses neue Subtilisin
DE102008017103A1 (de) 2008-04-02 2009-10-08 Henkel Ag & Co. Kgaa Wasch- und Reinigungsmittel enthaltend Proteasen aus Xanthomonas
DE102018208778A1 (de) 2018-06-05 2019-12-05 Henkel Ag & Co. Kgaa Leistungsverbesserte Proteasevarianten IV
WO2019048495A1 (fr) * 2017-09-05 2019-03-14 Henkel Ag & Co. Kgaa Variantes de protéases à performances améliorées
EP3679131B1 (fr) * 2017-09-05 2024-09-04 Henkel AG & Co. KGaA Variantes de protéases à performances améliorées
DE102019111057A1 (de) 2019-04-29 2020-10-29 Henkel Ag & Co. Kgaa Proteasen mit verbesserter Enzymstabilität in Wasch- und Reinigungsmitteln III

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CN115516088A (zh) 2022-12-23
DE102020105721A1 (de) 2021-09-09
WO2021175697A1 (fr) 2021-09-10
US20230159907A1 (en) 2023-05-25
KR20220148187A (ko) 2022-11-04

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