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WO2009037258A1 - Protéases à performances améliorées et détergents et produits de lavage contenant ces protéases - Google Patents

Protéases à performances améliorées et détergents et produits de lavage contenant ces protéases Download PDF

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Publication number
WO2009037258A1
WO2009037258A1 PCT/EP2008/062309 EP2008062309W WO2009037258A1 WO 2009037258 A1 WO2009037258 A1 WO 2009037258A1 EP 2008062309 W EP2008062309 W EP 2008062309W WO 2009037258 A1 WO2009037258 A1 WO 2009037258A1
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Prior art keywords
protease
amino acid
seq
proteases
bacillus
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German (de)
English (en)
Inventor
Petra Siegert
Susanne Wieland
Angrit Weber
Karl-Heinz Maurer
Cornelius Bessler
Marion Merkel
Stefan Evers
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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    • 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

Definitions

  • the present application relates to proteases whose proteolytic performance has been improved by changing the amino acid sequence, in particular with regard to their use in detergents and cleaners, all sufficiently similar proteases with a comparable change and nucleic acids and technical applications for these proteases, especially their use in washing - And cleaning agents and appropriate means themselves.
  • proteases of the subtilisin type are particularly important, which are attributed to the serine proteases due to the catalytically active amino acids. They act as nonspecific endopeptidases, that is, they hydrolyze any acid amide linkages that are internal to peptides or proteins. Their pH optimum is usually in the clearly alkaline range.
  • Subtilases become natural formed by microorganisms; Of these, in particular, the subtilisins formed and secreted by Bacillus species are to be mentioned as the most important group within the subtilases.
  • Proteases are, in addition to other enzymes, established active ingredients of detergents and cleaners. They cause the breakdown of protein-containing stains on the items to be cleaned. At best, there are synergies between the enzymes and the remaining components of the funds concerned.
  • the detergent and detergent proteases subtilases occupy an outstanding position due to their favorable enzymatic properties such as stability or pH optimum. They are also suitable for a variety of other technical uses, for example as components of cosmetics or in the organic-chemical synthesis.
  • subtilisin-type proteases preferably used in detergents and cleaners are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and those the subtilases, but no longer Enzymes Thermitase, Proteinase K, and Proteases TW3 and TW7, which can be classified as subtilisins in the narrower sense.
  • subtilisin BPN ' which is derived from Bacillus amyloliquefaciens, or B. subtilis, is known from the work of Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 and JA Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925.
  • Subtilisin BPN ' is used in particular with regard to the numbering of the positions as reference enzyme of Subtilisins.
  • Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes A / S, Bagsvaerd, Denmark. It is described in the publications of EL Smith et al.
  • protease PB92 is naturally derived from the alkaliphilic bacterium Bacillus nov. spec. 92 and was available under the trade name Maxacal® from Gist-Brocades, Delft, The Netherlands. In its original sequence, it is described in the patent application EP 283075 A2.
  • the subtilisins 147 and 309 are sold under the trade names Esperase®, and Savinase® by the company Novozymes.
  • Bacillus strains which are disclosed in the application GB 1243784 A.
  • the protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) is derived from the variants described under the name BLAP®, which are described in particular in WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2 and WO 03 / 038082 A2.
  • Subtilisin DY is originally from Nedkov et al. Chem., 1985, Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 421-430.
  • proteases are, for example, those under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase®, Kannase® and Ovozyme® from Novozymes, which are available under the trade names, Purafect®, Purafect® OxP, Purafect® Prime and Properase ® from Genencor, sold under the trade name Protosol® by Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi® by Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® and Protease P From Amano Pharmaceuticals Ltd., Nagoya, Japan, and that available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan.
  • Proteases are selectively or randomly modified by methods known from the prior art and thus optimized, for example, for use in detergents and cleaners. These include point mutagenesis, deletion or insertion mutagenesis or fusion with other proteins or protein parts or other modifications. Thus, correspondingly optimized variants are known for most proteases known from the prior art.
  • proteases there is still a high demand for proteases in various technical applications.
  • their usability in detergents and cleaners even for the family of subtilisin proteases established in the prior art there is still a need for optimization concerning, for example, their catalytic activity, the reaction conditions, the stability or the substrate specificity.
  • a protease in detergents and cleaning agents it is generally not possible to conclude on the basis of the measurable or calculable enzymatic properties of the enzyme per se on its behavior in washing or cleaning formulations.
  • other factors such as stability to oxidizing agents, denaturation by surfactants, folding effects or synergies with other ingredients play a role, which influence the behavior of the enzyme or enzymes in detergents or in the resulting washing or cleaning solutions.
  • the object of the present invention is therefore to further develop an alkaline protease of the alkaline protease type from Bacillus gibsonii DSM 14391 and to obtain performance-improved variants which show improved performance in industrial applications.
  • those should be found whose performance improves an increased cleaning performance of detergents and / or detergents.
  • those protease variants should be generated which are one of the starting proteases improved wash performance with respect to at least one soiling, preferably with respect to multiple soils.
  • proteases in particular of the subtilisin type, which have improved stability with respect to the prior art to temperature influences, pH fluctuations, denaturing or oxidizing agents, proteolytic degradation, high temperatures, acidic or alkaline conditions or to a change in the redox ratios. Further objects may be seen in the provision of proteases with reduced immunogenicity or decreased allergenic activity.
  • an alkaline protease alkaline protease from Bacillus gibsonii DSM 14391 should be further developed such that the resulting protease variant has improved detergency relative to the parent protease with respect to at least one soiling, preferably multiple soils.
  • proteases Other subtasks have been to provide nucleic acids encoding such proteases, and to provide vectors, host cells, and methods of production that can be used to obtain such proteases. Furthermore, appropriate means, in particular washing and cleaning agents, appropriate washing and cleaning methods and corresponding uses for such proteases should be made available. Finally, technical applications for the proteases found should be defined.
  • An object of the invention thus forms a protease which comprises an amino acid sequence which is at least 78.5% identical to the amino acid sequence given in SEQ ID NO. 2, and which is characterized in that it comprises in the counting method according to SEQ ID NO. 2 at position 21 1 or position 212 or at positions 211 and 212 has an amino acid modified in comparison to the amino acid indicated in SEQ ID NO.
  • protease modified according to the invention as described above differs markedly from other subtilisins, such as, for example, subtilisin 309, PB92, the alkaline protease from Bacillus lentus DSM 5483, BPN ' , proteinase K-16, etc.
  • the protease is characterized by comprising an amino acid sequence identical to that shown in SEQ ID NO. 2, more preferably at least 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 93.5%, 94%, 94.5%, 95% , 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, and most preferably 99.25%.
  • a further subject of the invention is therefore a protease, which is characterized in that the protease is selected from a) a protease, which in the counting manner according to SEQ ID NO.
  • amino acid 2 at position 211 has an amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan , Tyrosine and valine b) a protease, which in the counting manner according to SEQ ID NO.
  • alanine arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan , Tyrosine and valine c) a protease which is in the counting manner according to SEQ ID NO. 2 at position 211 has an amino acid according to feature a) and has at position 212 an amino acid according to feature b).
  • protease is characterized in that the protease is selected from a) a protease which is in the counting manner according to SEQ ID NO. 2 at position 21 1 has a serine residue b) a protease which is in the counting manner according to SEQ ID NO. 2 at position 212 has an asparagine residue c) a protease which is in the counting manner according to SEQ ID NO. 2 at position 21 1 has a serine residue and at position 212 an asparagine residue.
  • the likewise preferred variants according to the invention therefore have the amino acid changes M21 1S (the amino acid methionine at position 211 has been replaced by the amino acid serine) or P212N (the amino acid proline at position 212 has been replaced by the amino acid Asparagine) alone or in combination.
  • proteases of the invention may have other amino acid changes, especially amino acid substitutions, insertions or deletions.
  • nucleic acids With all the subject invention mentioned above are as further subjects of the invention the associated nucleic acids, corresponding host cells, suitable methods for their identification, in particular based on the nucleic acids molecular biological methods and process elements and agents, detergents and cleaners, washing and cleaning processes and on the relevant proteases identified uses connected. Due to the provided nucleic acids, an additional optimization of this enzyme is possible, for example via further point mutations. Furthermore, this DNA can be incorporated into shuffling approaches and thus used to generate completely new proteases.
  • an enzyme is to be understood as meaning a protein which has a specific biocatalytic function.
  • protease is understood as meaning an enzyme which catalyzes the hydrolysis of peptide bonds and is thereby able to cleave peptides or proteins.
  • a protein is a largely linear composition composed of the natural amino acids and usually performs one of its functions three-dimensional structure accepting polypeptide.
  • a peptide consists of amino acids that are covalently linked to each other via peptide bonds.
  • polypeptide clarifies in this regard the fact that this peptide chain usually consists of many amino acids, which are connected to each other via peptide bonds.
  • Amino acids may be in an L and a D configuration, with the amino acids that make up proteins in the L configuration. They are called proteinogenic amino acids.
  • the proteinogenic, naturally occurring L-amino acids are designated by the internationally used 1- and 3-letter codes.
  • pre-proteins ie together with a signal peptide.
  • the N-terminal part of the protein the function of which is usually to ensure the discharge of the protein formed from the producing cell into the periplasm or the surrounding medium and / or its correct folding.
  • the signal peptide is cleaved under natural conditions by a signal peptidase from the rest of the protein, so that this exerts its actual catalytic activity without the initially present N-terminal amino acids.
  • Pro-proteins are inactive precursors of proteins. Their signal sequence precursors are referred to as pre-pro proteins.
  • the mature, ie mature, peptides, ie the enzymes processed after their preparation are preferred over the preproteins.
  • the proteins may be modified by the cells producing them after production of the polypeptide chain, for example, by attachment of sugar molecules, formylations, aminations, etc. Such modifications are referred to as post-translational modifications. These post-translational modifications may or may not have an effect on the function of the protein.
  • the enzymatic activity of a considered enzyme can be deduced from the amino acid or nucleotide sequence. This can be qualitatively or quantitatively modified by other regions of the protein that are not involved in the actual reaction. This could, for example, relate to enzyme stability, activity, reaction conditions or substrate specificity.
  • a sequence comparison is done by assigning similar sequences in the nucleotide sequences or the amino acid sequences to each other. This is called homologization.
  • a The tabular assignment of the relevant positions is referred to as alignment.
  • alignments are created using computer programs, such as the algorithms FASTA or BLAST; This procedure is described, for example, by DJ Lipman and WR Pearson (1985) in Science, Vol. 227, pp. 1435-1441.
  • a summary of all matching positions in the compared sequences is called a consensus sequence.
  • Such a comparison also allows a statement about the similarity or homology of the compared sequences to each other. This is represented in percent identity, that is the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions.
  • a broader concept of homology involves conserved amino acid substitutions in amino acid sequences in this value. It then speaks of percent similarity. Such statements can be made about whole proteins or genes or only over individual areas.
  • Homologous or identical regions of different nucleic acid or amino acid sequences are defined by matches in the sequences. These can also be identified by identical function. It goes as far as complete identities in the smallest areas, so-called boxes, which comprise only a few nucleotides or amino acids and which usually perform essential functions for the overall activity of the nucleic acid or amino acid sequence. Examples of such functions may be the interaction with specific molecules in the case of nucleic acids, for example the interaction with specific transcription factors in the case of promoter sequences. In the case of amino acid sequences, such functions can also be understood as meaning the smallest subfunctions of the function carried out by the entire protein, such as the formation of individual hydrogen bonds for complexing a substrate or transition complex.
  • the protease is characterized in that its washing performance corresponds at least to that of a protease which comprises an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 2, wherein the washing performance is determined in a washing system containing a detergent in a dosage between 4.5 and 7.0 grams per liter of wash liquor and the protease, wherein the proteases to be compared are used in the same activity and the washing performance against one or more of the stains blood milk / ink on cotton, VoII- egg / pigment (whole / soot) on cotton, chocolate milk / ink on cotton, peanut oil pigment / ink on polyester / cotton, grass on cotton and Cocoa on cotton, in particular against one or more of the stains
  • Full egg / pigment (whole egg / carbon black) on cotton Product No. 1ON available from the company wfk Testgewebe GmbH; Brüggen-Bracht, Germany, or product CS-37 available from CFT (Center For Testmaterials) B.V. Viaardingen, Netherlands
  • whole egg / carbon black or whole egg / pigment are to be regarded as equivalent and mutually corresponding with regard to soiling.
  • a preferred liquid detergent for such a washing system is composed as follows (all figures in weight percent): 0.3- 0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3-0.5% ethanol, 4-7% FAEOS (fatty alcohol ether sulfate), 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut Fatty acids, 0.5% HEDP (1-hydroxyethane- (1, 1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.05% optical brightener, 0-0.001% dye, Rest demineralized water.
  • the dosage of the liquid detergent is between 4.5 and 6.0 grams per liter of wash liquor, for example, 4.7, 4.9 or 5.9 grams per liter of wash liquor. Preference is given to washing in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.
  • a preferred powdered detergent for such a washing system is composed as follows (all figures in weight percent): 10% linear alkylbenzenesulfonate (sodium salt), 1.5% C12-C18 fatty alcohol sulfate (sodium salt), 2.0% C12-C18 fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium bicarbonate, 4.0% amorphous sodium disilicate, 17 % Sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1, 0% carboxymethylcellulose, 1, 0% phosphonate, 25% sodium sulfate, remainder: optional foam inhibitors, optical brightener, fragrances and, if necessary, water ad 100%.
  • the dosage of the powdered detergent is between 5.5 and 7.0 grams per liter of wash liquor, for example, 5.6, 5.9 or 6.7 grams per liter of wash liquor.
  • the degree of whiteness i. the brightening of the stains, is preferably determined by optical measuring methods, preferably photometrically.
  • a suitable device for this purpose is for example the spectrometer Minolta CM508d.
  • the devices used for the measurement are previously calibrated with a white standard, preferably a supplied white standard.
  • the activity-like use ensures that, even if the ratio of active substance to total protein (the values of the specific activity) diverge, the respective enzymatic properties, for example the washing performance of certain soils, are compared. In general, a low specific activity can be compensated by adding a larger amount of protein.
  • Methods for the determination of the protease activities are familiar to the expert in the field of enzyme technology and are routinely used by him. For example, such methods are disclosed in Tenside, Vol. 7 (1970), pp. 125-132.
  • the protease activity is preferably indicated in PE (protease units).
  • suitable protease activities are 5 or 10 PE (protease units) per ml wash liquor. However, the protease activity is not equal to zero.
  • Proteins can also be grouped into groups of immunologically related proteins by reaction with an antiserum or antibody.
  • the members of a group are characterized by having the same antigenic determinant recognized by an antibody.
  • a further subject of the invention therefore proteases, which are characterized in that they have at least one and increasingly preferably two, three or four matching antigenic determinants with a protease according to the invention.
  • Proteases or enzymes in general can be further developed by various methods, for example targeted genetic modification by mutagenesis methods, and optimized for specific purposes or with regard to specific properties, for example catalytic activity, stability, etc.
  • Changes in the nucleotide sequence as can be brought about, for example, by molecular biological methods known per se, are accordingly termed mutations.
  • mutations are accordingly termed mutations.
  • the associated organisms are called mutants.
  • the proteins derived from mutant nucleic acids are called variants.
  • deletion, insertion, substitution mutations or fusions lead to deletion, insertion, substitution mutated or fusion genes and at the protein level to corresponding deletion, insertion or substitution variants or fusion proteins.
  • the strategy of introducing targeted point mutations into the known molecules for example to improve the washing performance of proteases, for example subtilisins, is also referred to as rational protein design.
  • a similar performance improvement strategy is to change the surface charges and / or the isoelectric point of the molecules and, above that, their interactions with the substrate.
  • the net charge of the subtilisins can be changed via point mutations in order to influence the substrate binding, in particular for use in detergents and cleaners.
  • Another, and in particular complementary, strategy is to increase the stability of the proteases in question and thereby increase their effectiveness. Such stabilization can be carried out, for example, via coupling to a polymer or, in particular for detergents and cleaners, by point mutations.
  • chimeras or hybrid proteins are to be understood as meaning those proteins whose sequence comprises the sequences or partial sequences of at least two starting proteins.
  • the source proteins may be derived from different or from the same organism.
  • Chimeric or hybrid proteins may be obtained, for example, by recombinant mutagenesis.
  • the purpose of such recombination may be to induce or modify a particular enzymatic function using the fused protein portion.
  • it is irrelevant whether such a chimeric protein consists of a single polypeptide chain or several subunits on which different functions can be distributed.
  • proteins obtained by insertion mutation are meant those variants obtained by inserting a protein fragment into the starting sequences. They are due to their principle similarity to the chimeric proteins. They differ from those only in the size ratio of the unchanged protein part to the size of the entire protein. In such insertionsmut elected proteins, the proportion of foreign protein is lower than in chimeric proteins.
  • Inversion mutagenesis ie a partial sequence reversal
  • a further subject of the invention is a protease which is characterized in that it is obtainable from a protease according to the invention as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is over a length of at least 266 and increasingly preferred at least 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60 and 50 contiguous amino acid positions matches the parent molecule.
  • Insertions and substitutions can also give positive results. In principle, this also includes single substitutions of amino acids, but it can also be several contiguous amino acids exchanged for others. This also includes recombinations of larger enzyme sections with other proteases or proteins of other functions.
  • proteins according to the invention can also be linked with amylases or cellulases, for example, in order to perform a dual function.
  • a further subject of the invention is a protease, which is characterized in that it is obtainable from a protease according to the invention as the starting molecule and has one or more amino acid substitutions in positions which correspond to the positions 3, 4, 36, 42, 47, 56, 61 , 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 224, 229, 236, 237, 242 , 243, 255 and 268 of the Bacillus lentus alkaline protease according to SEQ ID NO. 3 in an alignment.
  • amino acid positions are hereby assigned by an alignment of the amino acid sequence of a protease according to the invention with the amino acid sequence of the alkaline protease from Bacillus lentus, as indicated in SEQ ID NO. In this way, so-called homologous positions are determined.
  • An example of such an alignment is given in FIG.
  • substitutions 3T, 4I, 61A, 99G, 99A, 99S, 154D, 154E, 211D, 211G and 211E are, for example, substitutions 3T, 4I, 61A, 99G, 99A, 99S, 154D, 154E, 211D, 211G and 211E, provided that the correspondingly homologous positions are not naturally taken up by one of these preferred amino acids.
  • the Bacillus lentus alkaline protease wildtype molecule has the following amino acid residues: S3, V4, S36, N42, A47, T56, G61, T69, E87, A96, R99, A101, 1102, S104, N114, H118, A120 , S130, S139, T141, S142, S154, S157, A188, V193, V199, G205, L211, A224, K229, S236, N237, N242, H243, N255 and T268, respectively.
  • derivatives are understood as meaning those proteins whose pure amino acid chain has been chemically modified. It is preferably a covalent modification, but may also be given a non-covalent modification. Such modifications may affect, for example, stability, substrate specificity, or binding strength to the substrate or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, increase its skin compatibility.
  • modifications may be made biologically in the context of protein biosynthesis by the host cell.
  • couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy.
  • molecular biological methods can be used for this purpose.
  • modifications may also be made chemically, such as by the chemical transformation of a side chain of an amino acid or by covalent attachment of another compound to the protein, for example the coupling of amines to carboxyl groups of an enzyme to alter the isoelectric point.
  • a compound can also be, for example, macromolecules, including other proteins, which are bound, for example via bifunctional chemical compounds (so-called "linkers"), to proteins according to the invention.
  • a protein of the invention with a specific binding domain.
  • Such derivatives are particularly suitable for use in detergents or cleaners.
  • protease inhibitors optionally via linkers, in particular amino acid linkers, can also be bound to a protein according to the invention. This can be useful, for example, for the period of storage. Couplings with other macromolecular compounds, such as polyethylene glycol, can also improve a protein of the invention for other properties, such as stability or skin tolerance.
  • derivatization is understood to mean covalent attachment to a macromolecular carrier, as well as noncovalent inclusion in suitable macromolecular cage structures.
  • a derivative can also be understood in a broader sense to mean a preparation of the protein.
  • a protein may be associated with various other substances, for example from the culture of the producing microorganisms.
  • a protein may also have been deliberately added to certain other substances, for example to increase its storage stability. Therefore, all preparations of a protein according to the invention are also according to the invention. This is also independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. Because it can be desired be that it has little or no activity during storage, and only at the time of use unfolds its proteolytic function. This can be controlled, for example, via appropriate accompanying substances.
  • the joint preparation of proteases with protease inhibitors is advantageous.
  • a further preferred embodiment of the invention is a protease described above, which is additionally stabilized, in particular by coupling to a polymer.
  • those stabilizations are also suitable which are possible by means of point mutagenesis of the molecule itself (and because of the sequence differences already fall under the embodiments described above). Because these stabilizations require after the protein recovery no further steps.
  • Some point mutations suitable for this purpose are known per se from the prior art. For example, proteases can also be stabilized by replacing certain tyrosine residues with others.
  • Changing the binding of metal ions, in particular the calcium binding sites for example by exchanging one or more of the amino acids involved in the calcium binding for one or more negatively charged amino acids and / or introducing point mutations in at least one of the sequences of the two amino acids arginine / glycine;
  • Preferred embodiments are those in which the molecule is stabilized in several ways. Because it can be assumed that several stabilizing mutations act additive or synergistic.
  • the solution of a partial task and thus an independent subject of the invention form nucleic acid molecules which code for a protease according to the invention as well as vectors containing such nucleic acid molecules.
  • nucleic acids are understood to mean the molecules which are naturally constructed from nucleotides and serve as information carriers, which code for the linear amino acid sequence in proteins or enzymes. They can be present as a single strand, as a single strand that is complementary to this single strand, or as a double strand. As the naturally more durable information carrier, the nucleic acid DNA is preferred for molecular biology work. In contrast, for the realization of the invention in natural environment, such as in a nucleic acid expressing cell, an RNA is formed, which is why corresponding RNA molecules are also embodiments of the present invention.
  • the information unit corresponding to a protein is also referred to as gene within the meaning of the present application.
  • a person skilled in the art will be able to produce the corresponding nucleic acids by methods known today, such as, for example, chemical synthesis or polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences.
  • methods known today such as, for example, chemical synthesis or polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences.
  • PCR polymerase chain reaction
  • the nucleic acid molecule which codes for a protease according to the invention is characterized in that it is increasingly preferably at least 78.5%, 80%, 82.5%, 85% to the nucleic acid sequence indicated in SEQ ID NO %, 87.5%, 90%, 91%, 92%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97% , 97.5%, 98%, 98.5%, 99%, and most preferably 99.25%.
  • This aspect relates to the heterologous expression of the proteases in question.
  • every organism in particular every production strain, has a certain codon usage. This can lead to bottlenecks in protein biosynthesis, if the lying on the transgenic nucleic acid codons in the host cell of a relatively small number of loaded tRNAs face. Synonymous codons encode the same amino acids and can be better translated depending on the host. This possibly necessary rewriting thus depends on the choice of the expression system. Especially with samples from unknown, possibly non-cultivable organisms, a corresponding adaptation may be necessary.
  • the present invention involves the production of recombinant proteins.
  • these are to be understood as meaning all genetic engineering or microbiological processes which are based on the genes for the proteins of interest being introduced into a suitable host cell for the production and being transcribed and translated by it.
  • the introduction of the relevant genes via vectors, in particular expression vectors; but also those that cause the gene of interest in the host cell to be inserted into an already existing genetic element, such as the chromosome or other vectors.
  • the functional unit of gene and promoter and any other genetic elements is called an expression cassette. However, it does not necessarily have to exist as a physical entity.
  • the nucleic acid is suitably cloned into a vector.
  • Another molecular biological aspect of the invention thus consists in vectors with the genes for the corresponding proteins. These may include, for example, those derived from 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 relevant host cells over several generations. It is irrelevant in the context of the invention whether they establish themselves as extrachomosomal units or integrate into a chromosome or chromosomal DNA. Which of the numerous systems known from the prior art is chosen depends on the individual case. Decisive factors may be, for example, the achievable copy number, the selection systems available, in particular antibiotic resistances, or the cultivability of the host cells capable of accepting the vectors.
  • the vectors form suitable starting points for molecular biological and biochemical investigations of the relevant gene or protein and for further developments according to the invention and ultimately for the amplification and production of proteins according to the invention.
  • Expression vectors are chemically similar to the cloning vectors, but differ in those partial sequences that enable them to be used for the production of proteins provided host cells or host organisms to replicate and there to bring the gene contained expression, ie to realize the genetic information of the gene of interest in the host cell as a protein.
  • Preferred embodiments are expression vectors which themselves carry the genetic elements necessary for expression.
  • the expression is influenced, for example, by promoters which regulate the transcription of the gene.
  • expression may be by the natural promoter originally located upstream of this gene, but also by genetic engineering, both by a host cell promoter provided on the expression vector and by a modified or completely different promoter from another organism or host cell.
  • a further embodiment represents expression systems in which additional genes, for example those which are provided on other vectors, influence the production of proteins according to the invention. These may be modifying gene products or those which are to be purified together with the protein according to the invention, for example in order to influence its enzymatic function. These may be, for example, other proteins or enzymes, inhibitors or elements that influence the interaction with various substrates.
  • Alternative embodiments of the present invention may also be cell-free expression systems in which protein biosynthesis is understood in vitro. Such expression systems are also established in the art.
  • a further subject of the invention is a non-human host cell which contains a protease according to the invention or which can be excited to produce it, preferably using an expression vector.
  • the in vivo synthesis of an enzyme according to the invention ie by living cells, requires the transfer of the associated gene into a host cell, the so-called transformation thereof.
  • all cells that is to say prokaryotic or eukaryotic cells, are suitable as host cells.
  • preferred host cells are characterized by good microbiological and biotechnological handling.
  • Preferred embodiments represent such host cells, which are regulatable in their activity due to genetic regulatory elements which are provided, for example, on the expression vector, but may also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the culture conditions or when reaching a specific cell density, these can be excited for expression. This allows a very economical production of the proteins of interest.
  • Preferred host cells are prokaryotic or bacterial cells.
  • bacteria are distinguished from eukaryotes by shorter generation times and lower demands on culturing conditions.
  • cost-effective methods for obtaining proteins according to the invention can be established.
  • Gram-negative bacteria such as Escherichia coli (E. coli)
  • E. coli Escherichia coli
  • a large number of proteins are secreted into the periplasmic space, ie into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications.
  • Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of Actinomycetales
  • have no outer membrane so that secreted proteins are released immediately into the nutrient medium surrounding the cells, from which, according to a further preferred embodiment, the expressed proteins according to the invention can be purified directly.
  • a further embodiment of the invention thus represents host cells which are characterized in that they secrete a protease according to the invention into the medium surrounding the host cell.
  • the host cell according to the invention is characterized in that it is a bacterium, in particular one which is selected from the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas.
  • the host cell is a bacterium which is selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii gibsonii, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.
  • the host cells may be altered in their culture conditions requirements, have different or additional selection markers, or express other or additional proteins.
  • these may be those host cells which, in addition to the protein produced according to the invention, also express further, in particular economically interesting, proteins.
  • the host cell may also be a eukaryotic cell, which is characterized in that it has a cell nucleus.
  • a further subject of the invention therefore represents a host cell, which is characterized in that it has a cell nucleus.
  • eukaryotic cells are capable of post-translationally modifying the protein formed.
  • fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces.
  • yeasts such as Saccharomyces or Kluyveromyces.
  • Modifications that eukaryotic systems perform, especially in connection with protein synthesis include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides.
  • Oligosaccharide modifications may be desirable, for example, to reduce allergenicity.
  • coexpression with the enzymes naturally produced by such cells, such as cellulases may be advantageous.
  • thermophilic fungal expression systems may be particularly suitable for the expression of temperature-resistant variants.
  • the host cells according to the invention are cultured and fermented in a manner known per se, 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 an experimentally determined period of time.
  • Continuous fermentations are characterized by achieving a flow equilibrium in which over a relatively long period of time cells partly die off but also regrow and at the same time product can be removed from the medium.
  • 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 recombinant protein. All fermentation processes which are based on one of the above-described processes for the preparation of the recombinant proteins represent correspondingly preferred embodiments of this subject matter of the invention.
  • the optimum conditions for the production processes used, for the host cells and / or the proteins to be produced must be experimentally determined on the basis of the previously optimized culture conditions of the relevant strains according to the knowledge of the person skilled in the art, for example regarding fermentation volume, media composition, oxygen supply or stirrer speed.
  • Fermentation processes which are characterized in that the fermentation is carried out via a feed strategy, are also contemplated.
  • the media components consumed by the ongoing cultivation are fed;
  • considerable increases in both the cell density and in the dry biomass and / or especially the activity of the protein of interest can be achieved.
  • the fermentation can also be designed so that unwanted metabolic products are filtered out or neutralized by the addition of buffer or matching counterions.
  • the produced protein can be harvested subsequently from the fermentation medium. This fermentation process is preferred over dry matter product processing, but requires the provision of suitable secretion markers and transport systems. Without secretion, it may be necessary to purify the protein from the cell mass, and various methods are known, such as precipitation, for example, by ammonium sulfate or ethanol, or chromatographic purification, if necessary, to homogeneity. However, the majority of the described technical methods should manage with an enriched, stabilized preparation.
  • An independent subject matter of the invention thus also provides methods for producing a protease according to the invention.
  • This includes any method which is suitable for producing a protease according to the invention described above or which makes it possible to obtain a protease according to the invention. These include, for example, chemical synthesis methods.
  • nucleotide sequence has been adapted in one, preferably a plurality of codons, to the codon usage of the host strain.
  • An inventive subject matter represents an agent which is characterized in that it contains at least one protease according to the invention as described above.
  • compositions especially mixtures, formulations, solutions, etc., the utility of which is improved by addition of a protein of the invention described above, within the scope of the present invention.
  • these may be, for example, solid mixtures, for example powders with freeze-dried or encapsulated proteins, or gel or liquid agents.
  • Preferred formulations contain, for example, buffer substances, stabilizers, reaction partners and / or cofactors of the proteases and / or other ingredients synergistic with the proteases.
  • this appropriation is to be understood as the areas of application set out below. Further fields of application emerge from the prior art and are described, for example, in the manual "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998.
  • a combination of a protease according to the invention with one or more further ingredients of the compositions proves to be advantageous, since such an agent has an improved cleaning performance by resulting synergisms, in particular between the protease and the further ingredient.
  • the agent effects an improved removal of stains, for example proteinaceous stains, in comparison with an agent which either contains only one of the two components or also in comparison with the expected cleaning performance of an agent with both components due to the mere addition of respective individual contributions of these two components to the cleaning performance of the agent.
  • the combination of a protease according to the invention with one of the surfactants and / or builders and / or bleaches described below achieves such a synergism.
  • compositions according to the invention may comprise one or more surfactants, in particular anionic surfactants, nonionic surfactants and mixtures thereof, but also cationic, zwitterionic and amphoteric surfactants.
  • Suitable nonionic surfactants are in particular alkyl glycosides and ethoxylation and / or propoxylation of alkyl glycosides or linear or branched alcohols each having 12 to 18 carbon atoms in the alkyl moiety and 3 to 20, preferably 4 to 10 alkyl ether groups. Furthermore, corresponding ethoxylation and / or propoxylation of N-alkyl-amines, vicinal diols, fatty acid esters and fatty acid amides, which correspond to said long-chain alcohol derivatives with respect to the alkyl moiety, and of alkylphenols having 5 to 12 carbon atoms in the alkyl radical.
  • the nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, especially primary alcohols having preferably 8 to 18 carbon atoms and an average of 1 to 12 moles of ethylene oxide (EO) per mole of alcohol, in which the alcohol radical is linear or preferably 2-position may be methyl-branched or may contain linear and methyl-branched radicals in the mixture, as they are usually present in Oxoalkoholresten.
  • EO ethylene oxide
  • alcohol ethoxylates with linear radicals of alcohols of natural origin having 12 to 18 carbon atoms, for example of coconut, palm, tallow or oleyl alcohol, and on average 2 to 8 EO per mole of alcohol are preferred.
  • Preferred alcohol ethoxylates have a narrow homolog distribution (narrow rank ethoxylates, NRE).
  • fatty alcohols with more than 12 EO can also be used. Examples of these are (TaIg) fatty alcohols with 14 EO, 16 EO, 20 EO, 25 EO, 30 EO or 40 EO.
  • agents for use in mechanical processes usually extremely low-foam compounds are used. These include, preferably, C 12 -C 18 -alkyl polyethylene glycol polypropylene glycol ethers, each containing up to 8 moles of ethylene oxide and propylene oxide units in the molecule.
  • the nonionic surfactants also include alkyl glycosides of the general formula RO (G) x in which R is a primary straight-chain or methyl-branched, in particular 2-methyl-branched aliphatic radical having 8 to 22, preferably 12 to 18 carbon atoms and G represents a glycose unit having 5 or 6 C atoms, preferably glucose.
  • the degree of oligomerization x which indicates the distribution of monoglycosides and oligoglycosides, is an arbitrary number - which, as a variable to be determined analytically, may also assume fractional values - between 1 and 10; preferably x is 1, 2 to 1, 4.
  • polyhydroxy fatty acid amides of the formula (III) in which R 1 CO is an aliphatic acyl radical having 6 to 22 carbon atoms, R 2 is hydrogen, an alkyl or hydroxyalkyl radical having 1 to 4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radical having 3 to 10 carbon atoms and 3 to 10 hydroxyl groups:
  • nonionic surfactants used either as the sole nonionic surfactant or in combination with other nonionic surfactants, in particular together with alkoxylated fatty alcohols and / or alkyl glycosides, are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably from 1 to 4 carbon atoms in the alkyl chain, especially fatty acid methyl ester.
  • Nonionic surfactants of the amine oxide type for example N-cocoalkyl-N, N-dimethylamine oxide and N-tallowalkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamides may also be suitable.
  • the anionic surfactants may be in the form of their sodium, potassium or ammonium salts and as soluble salts of organic bases, such as mono-, di- or triethanolamine.
  • the anionic surfactants are preferably present in the form of their sodium or potassium salts, in particular in the form of the sodium salts.
  • organic builder substances may be present in amounts of up to 40% by weight, in particular up to 25% by weight and preferably from 1% by weight to 8% by weight. Quantities close to the stated upper limit are preferably used in paste-form or liquid, in particular water-containing, agents according to the invention.
  • Suitable water-soluble inorganic builder materials are, in particular, alkali metal silicates, alkali metal carbonates and alkali metal phosphates, which may be in the form of their alkaline, neutral or acidic sodium or potassium salts.
  • detergent grade crystalline sodium aluminosilicates particularly zeolite A, P and optionally X, alone or in mixtures, for example in the form of a cocrystal of zeolites A and X (Vegobond® AX, a commercial product of Condea Augusta SpA)
  • zeolites A and X a cocrystal of zeolites A and X
  • Amounts near the above upper limit are preferably used in solid, particulate agents.
  • suitable aluminosilicates have no particles with a particle size greater than 30 .mu.m and preferably consist of at least 80% by weight of particles having a size of less than 10 .mu.m.
  • Their calcium binding capacity which can be determined according to the specifications of the German patent DE 24 12 837, is generally in the range of 100 to 200 mg CaO per gram.
  • amorphous alkali silicates can be used in inventive compositions.
  • a crystalline sodium layer silicate with a modulus of 2 to 3 is used, as can be prepared from sand and soda.
  • Crystalline sodium silicates with a modulus in the range of 1.9 to 3.5 are used in a further preferred embodiment of compositions according to the invention.
  • Crystalline layered silicates of the above Formula (I) are sold by the company.
  • Na-SKS Clariant GmbH under the trade name Na-SKS, for example Na-SKS-1 (Na 2 Si 22 O 45 XH 2 O, kenyaite), Na-SKS-2 (Na 2 Si 14 O 29 XH 2 O, magadiite), Na-SKS-3 (Na 2 Si 8 O 17 XH 2 O) or Na-SKS-4 (Na 2 Si 4 O 9 XH 2 O, makatite).
  • Na-SKS-5 OC-Na 2 Si 2 O 5
  • Na-SKS-7 ⁇ -Na 2 Si 2 0 5 , natrosilite
  • Na-SKS-9 NaHSi 2 O 5 3H 2 O
  • Na-SKS-10 NaHSi 2 O 5 3H 2 O, kanemite
  • Na-SKS-11 t-Na 2 Si 2 0 5
  • Na-SKS-13 NaHSi 2 O 5
  • Na-SKS-6 5-Na 2 Si 2 O 5 .
  • composition according to the invention a granular compound of crystalline phyllosilicate and citrate, of crystalline phyllosilicate and of the above-mentioned (co-) polymeric polycarboxylic acid, or of alkali silicate and alkali metal carbonate, such as, for example, commercially available under the name Nabion® 15, is used ,
  • an agent according to the invention contains peroxygen compounds, they are present in amounts of preferably up to 50% by weight, in particular from 5% by weight to 30% by weight.
  • bleach stabilizers such as phosphonates, borates or metaborates and metasilicates and magnesium salts such as magnesium sulfate may be useful.
  • bleach activators it is possible to use compounds which, under perhydrolysis conditions, give aliphatic peroxycarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid.
  • Suitable substances are those which carry O- and / or N-acyl groups of the stated C atom number and / or optionally substituted benzoyl groups.
  • hydrophilic substituted acyl acetals and the acyl lactams are also preferably used.
  • Combinations of conventional bleach activators can also be used.
  • Such bleach activators can, in particular in the presence of the abovementioned hydrogen peroxide-supplied bleach, in the usual amount range, preferably in amounts of from 0.5 wt .-% to 10 wt .-%, in particular 1 wt .-% to 8 wt .-%, based on However, total agent, be included, missing when using percarboxylic acid as the sole bleach, preferably completely.
  • sulfone imines and / or bleach-enhancing transition metal salts or transition metal complexes may also be present as so-called bleach catalysts.
  • organic solvents which can be used in addition to water include alcohols having 1 to 4 C atoms, in particular methanol, ethanol, isopropanol and tert-butanol, diols having 2 to 4 C -Ato- men, in particular ethylene glycol and propylene glycol, and mixtures thereof and derived from the said classes of compounds ethers.
  • Such water-miscible solvents are preferably present in the compositions according to the invention in amounts of not more than 30% by weight, in particular from 6% by weight to 20% by weight.
  • Graying inhibitors have the task of keeping suspended from the textile fiber dirt suspended in the fleet.
  • Water-soluble colloids of mostly organic nature are suitable for this purpose, for example starch, glue, gelatin, salts of ether carboxylic acids or ether sulfonic acids of starch or of cellulose or salts of acidic sulfuric acid esters of cellulose or starch.
  • water-soluble polyamides containing acidic groups are suitable for this purpose.
  • starch derivatives can be used, for example aldehyde starches.
  • cellulose ethers such as carboxymethylcellulose (Na salt), Methylcellulose, hydroxyalkylcellulose and mixed ethers, such as methylhydroxyethylcellulose, methylhydroxypropylcellulose, methylcarboxymethylcellulose and mixtures thereof, for example in amounts of 0.1 to 5 wt .-%, based on the means used.
  • Detergents according to the invention may contain, for example, derivatives of diaminostilbenedisulfonic acid or their alkali metal salts as optical brighteners, although they are preferably free of optical brighteners for use as color detergents.
  • optical brighteners for use as color detergents.
  • salts of 4,4'-bis (2-anilino-4-morpholino-1, 3,5-triazinyl-6-amino) stilbene-2,2'-disulphonic acid or compounds of similar construction which are used instead of the morpholino Group carry a diethanolamino group, a methylamino group, an anilino group or a 2-methoxyethylamino group.
  • brighteners of the substituted diphenylstyrene type may be present, for example, the alkali salts of 4,4'-bis (2-sulfostyryl) -diphenyl, 4,4'-bis (4-chloro-3-sulfostyryl) -diphenyl, or 4 - (4-chlorostyryl) -4 '- (2-sulfostyryl).
  • Mixtures of the aforementioned optical brightener can be used.
  • foam inhibitors are, for example, soaps of natural or synthetic origin, which have a high proportion of C 18 -C 24 fatty acids.
  • Suitable non-surfactant foam inhibitors are, for example, organopolysiloxanes and mixtures thereof with microfine, optionally silanized silica and paraffins, waxes, microcrystalline waxes and mixtures thereof with silanated silica or bis-fatty acid alkylene diamides. It is also advantageous to use mixtures of various foam inhibitors, for example those of silicones, paraffins or waxes.
  • the foam inhibitors in particular silicone and / or paraffin-containing foam inhibitors, are bound to a granular, water-soluble or dispersible carrier substance.
  • a granular, water-soluble or dispersible carrier substance In particular, mixtures of paraffins and bistearylethylenediamide are preferred.
  • the ingredients to be selected as well as the conditions under which the agent is used, such as temperature, pH, ionic strength, redox ratios or mechanical influences, should be optimized for the particular cleaning problem.
  • usual temperatures for detergents and cleaning agents are present in areas of 1O 0 C for manual compositions over 4O 0 C and 6O 0 C to 95 ° for machine agents or industrial applications. Since the temperature is usually infinitely adjustable in modern washing machines and dishwashers, all intermediate stages of the temperature are included.
  • the ingredients of the respective agents are coordinated. Synergies in terms of cleaning performance are preferred.
  • an agent according to the invention in particular a washing or cleaning agent, further comprises
  • grayness inhibitor 0.01 to 5% by weight of grayness inhibitor and / or
  • the agent may further comprise optical brighteners, preferably from 0.01% to 5% by weight.
  • compositions according to the invention presents no difficulties and can be carried out in a known manner, for example by spray-drying or granulation, enzymes and possibly other thermally sensitive ingredients such as, for example, bleaching agents optionally being added separately later.
  • inventive compositions having an increased bulk density in particular in the range from 650 g / l to 950 g / l, a process comprising an extrusion step is preferred.
  • compositions according to the invention in tablet form, which may be monophasic or multiphase, monochromatic or multicolor and in particular consist of one or more layers, in particular two layers
  • the procedure is preferably such that all constituents - if appropriate one per layer - in one Mixer mixed together and the mixture by means of conventional tablet presses, such as eccentric or rotary presses, pressed with compressive forces in the range of about 50 to 100 kN, preferably at 60 to 70 kN.
  • a tablet produced in this way has a weight of 10 g to 50 g, in particular 15 g up to 40 g.
  • the spatial form of the tablets is arbitrary and can be round, oval or angular, with intermediate forms are also possible. Corners and edges are advantageously rounded.
  • round Tablets preferably have a diameter of 30 mm to 40 mm.
  • the size of rectangular or cuboid-shaped tablets, which are introduced predominantly via the metering device, for example the dishwasher is dependent on the geometry and the volume of this metering device.
  • Exemplary preferred embodiments have a base area of (20 to 30 mm) x (34 to 40 mm), in particular of 26x36 mm or 24x38 mm.
  • Liquid or pasty compositions according to the invention in the form of customary solvent-containing solutions are generally prepared by simply mixing the ingredients, which can be added in bulk or as a solution in an automatic mixer.
  • Embodiments of the present invention thus comprise all such solid, powdered, liquid, gelatinous or paste-like administration forms of the agents, which if appropriate can also consist of several phases and can be present in compressed or uncompressed form.
  • a further embodiment of the invention therefore represents agents which are characterized in that they are present as a one-component system. Such means preferably consist of one phase. Of course, means according to the invention may also consist of several phases.
  • the washing or cleaning agent is therefore characterized in that it is divided into several components.
  • the solid dosage forms according to the invention also include extrudates, granules, tablets or pouches, which may be present both in large packages and in portions.
  • the agent is present as 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.
  • agents according to the invention may also be liquid, gelatinous or pasty.
  • a further embodiment of the invention is therefore characterized in that the washing or cleaning agent is in liquid, gel or pasty form, in particular 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.
  • an agent according to the invention is characterized in that it contains the protease in an amount of from 2 ⁇ g to 20 mg, preferably from 5 ⁇ g to 17.5 mg, more preferably from 20 ⁇ g to 15 mg and most preferably from 50 ⁇ g to 10 mg contains per g of the agent.
  • the washing or cleaning agent according to the invention may be packaged in a container, preferably an air-permeable container, from which it is ready for use shortly before use or during the Washing process is released.
  • the protease contained in the composition and / or other ingredients of the composition may further be coated with a substance which is impermeable to the enzyme at room temperature or in the absence of water, which becomes permeable to the enzyme under conditions of use of the agent.
  • 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.
  • compositions according to the invention may contain only one protease. Alternatively, they may also contain other proteases or other enzymes in a concentration effective for the effectiveness of the agent.
  • a further subject of the invention thus represents agents which further comprise one or more further enzymes, wherein in principle all enzymes established in the prior art for these purposes can be used.
  • Preferred enzymes which can be used as enzymes are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular proteases, amylases, cellulases, hemicellulases, mannanases, tannases, xylanases, xanthanases, .beta.-glucosidases, carrageenases, oxidases, oxidoreductases or lipases, and preferably their mixtures.
  • These enzymes are basically of natural origin; Starting from the natural molecules, improved variants are available for use in detergents and cleaners, which are preferably used accordingly.
  • compositions according to the invention preferably contain enzymes in total amounts of 1 ⁇ 10 -8 to 5 percent by weight, based on active protein.
  • the enzymes are from 0.001 to 5% by weight, more preferably from 0.01 to 5% by weight, even more preferably from 0.05 to 4% by weight and most preferably from 0.075 to 3.5% by weight.
  • the protein concentration can be determined by known methods, for example, the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766).
  • the further enzymes particularly preferably support the effect of the agent, for example the cleaning performance of a washing or cleaning agent, with regard to certain stains or stains.
  • the enzymes show synergistic effects with respect to their action against certain stains or stains, ie the enzymes contained in the middle composition mutually support each other in their cleaning performance.
  • the agent according to the invention is therefore characterized in that it contains at least one further enzyme which comprises a protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, ⁇ -glucosidase, carrageenase, oxidase, oxidoreductase or a lipase.
  • the enzymes used in agents of the invention are either originally from microorganisms, such as the genera Bacillus, Streptomyces, Humicola, or Pseudomonas, and / or are produced by biotechnological methods known per se by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or by filamentous fungi ,
  • protease activity in such agents can be determined by the method described in Tenside, Vol. 7 (1970), pp. 125-132. It is given in PE (protease units).
  • a separate subject of the invention is the use of an above-described agent according to the invention for the removal of protease-sensitive stains on textiles or hard surfaces, i. for cleaning textiles or hard surfaces.
  • agents according to the invention can be used, in particular in accordance with the properties described above, to remove proteinaceous impurities from textiles or from hard surfaces.
  • Embodiments include, for example, hand washing, manual removal of stains from fabrics or hard surfaces, or use in conjunction with a machine process.
  • the relevant agents according to the invention preferably detergents or cleaning agents, are provided according to one of the embodiments set forth above.
  • a further subject of the invention are processes for cleaning textiles or hard surfaces, in which at least one of the process steps involves an inventive appropriate means is used.
  • the process for the cleaning of textiles or hard surfaces is accordingly characterized in that an agent according to the invention is used in at least one process step.
  • Methods for cleaning textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned in a plurality of process steps and washed off after the action time, or that the items to be cleaned are otherwise treated with a detergent or a solution of this agent.
  • a single substep of such a process for the mechanical cleaning of textiles may consist in optionally adding, in addition to stabilizing compounds, Salts or buffer substances is applied as the only cleaning-active component of an enzyme according to the invention.
  • Another object of the invention are methods for the purification of textiles or hard surfaces, which are characterized in that in at least one method step, a protease according to the invention is proteolytically active, in particular such that the protease in an amount of 40 micrograms to 4 g, preferably from 50 ⁇ g to 3 g, more preferably from 100 ⁇ g to 2 g and most preferably from 200 ⁇ g to 1 g per application.
  • Alternative embodiments of this subject matter of the invention are also processes for the treatment of textile raw materials or for textile care in which a protease according to the invention becomes active in at least one of the process steps.
  • methods for textile raw materials, fibers or textiles with natural components are preferred, and especially for those with wool or silk.
  • These may be, for example, processes in which materials for processing in textiles are prepared, for example for anti-fungal finishing, or, for example, for processes which enrich the cleaning of worn textiles with a nourishing component.
  • processes in which materials for processing in textiles are prepared for example for anti-fungal finishing, or, for example, for processes which enrich the cleaning of worn textiles with a nourishing component.
  • they are processes for the treatment of textile raw materials, fibers or textiles with natural constituents, in particular with wool or silk.
  • enzymes according to the invention are advantageously usable in agents according to the invention, in particular detergents and cleaners, and processes, in particular washing and cleaning processes. They can be used to remove proteinaceous contaminants from textiles or hard surfaces. Embodiments include, for example, hand washing, manual removal of stains from fabrics or hard surfaces, or use in conjunction with a machine process.
  • Another object of the invention is therefore the use of a protease according to the invention, as described above for the cleaning of textiles or hard surfaces.
  • the protease is used in an amount of from 40 ⁇ g to 4 g, preferably from 50 ⁇ g to 3 g, more preferably from 100 ⁇ g to 2 g and most preferably from 200 ⁇ g to 1 g per application.
  • the relevant enzymes according to the invention are provided within the scope of an agent according to the invention, preferably a washing or cleaning agent according to the invention.
  • Another embodiment of this subject invention is the use of a protease according to the invention for activating or deactivating ingredients of detergents or cleaners.
  • proteolysis activates another component, such as when it is a hybrid protein from the actual enzyme and the corresponding inhibitor.
  • Another example of such regulation is that in which an active component is used to protect or control its activity in encapsulated in a material which is attacked by proteolysis.
  • Proteases according to the invention can thus be used for inactivation, activation or release reactions, in particular in multiphase agents.
  • protease for the recovery or treatment of raw materials or intermediates in textile production, in particular for the removal of protective layers on fabrics;
  • the present invention is also realized in the form of such a protease-containing agent of the present invention, which are cosmetics. This is understood to mean all types of cleansing and conditioning agents for human skin or hair, in particular cleansing agents.
  • proteases are also used as bioactive components in skin care agents to aid in the breakdown of desmosome structures that are increased in dry skin.
  • proteases according to the invention can be developed further, for example via amino acid substitutions and / or point mutations.
  • proteases according to the invention in particular those which are controlled in their activity, for example after mutagenesis or by addition of corresponding substances interacting with them, are also suitable as active components in skin or hair cleansing or care preparations.
  • Particularly preferred are those preparations of these enzymes, which are stabilized as described above, for example by coupling to macromolecular carrier and / or derivatized by point mutations at highly allergenic positions, so that they have a higher skin compatibility for humans.
  • subtilases In addition to the use in detergents and cleaners and cosmetics numerous applications of proteases, in particular subtilases are established in the prior art. a For example, the handbook “Industrial Enzymes and their Applications” by H. Uhlig, Wiley-Verlag, New York, 1998 provides an overview of this. All of these techniques can be enriched by proteases according to the invention.
  • protease for biochemical analysis or for the synthesis of low molecular weight compounds or of proteins
  • protease for the preparation, purification or synthesis of natural substances or biological valuable substances, preferably in the context of corresponding agents or processes;
  • protease for the synthesis of proteins or other low molecular weight chemical compounds
  • protease for the treatment of natural raw materials, in particular for surface treatment, more particularly in a process for the treatment of leather, preferably in the context of appropriate agents or processes;
  • proteases according to the invention in all other fields of technology is included in the scope of protection of the present application, for which it has proven to be suitable.
  • protease variants were prepared by site-directed mutagenesis of the alkaline
  • Amino acid position 211 (nucleotides 631-633) in the counting method according to SEQ ID NO. 2 of ATG changed, for example, to TCT, so that an exchange of the amino acid methionine (M) takes place against the amino acid serine (S).
  • Counting according to SEQ ID NO. 2 changed from CCT, for example, after AAT, so that a
  • the nucleic acid coding for the particular protease is cloned in the vector pAWA22.
  • This is a pBC16-derived expression vector for use in Bacillus species (Bernhard et al., (1978) J. Bacteriol., Vol. 133 (2), pp. 897-903).
  • This vector was transformed into the host strain Bacillus subtilis DB 104 (Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444) by standard methods.
  • the transformants were first on DM3 medium (8 g / l agar, 0.5 M succinic acid, 3.5 g / l K 2 HPO 4 , 1, 5 g / l KH 2 PO 4 , 20 mM MgCl 2 , 5 g / l casiaminoacids, 5 g / l yeast extract, 6 g / l glucose, 0.1 g / l BSA) and then on TBY Skimmilk plates (10 g / l peptone, 10 g / l milk powder (see above), 5 g / l yeast, 5 g / l NaCl, 15 g / l agar).
  • Proteolytically active clones were identified by their lysis sites. From the resulting proteolytically active clones, one was selected, the plasmid isolated and the insert sequenced by standard methods to check for correctness. The desired protease activity is contained in culture supernatants according to verified clones and can be further processed therefrom if necessary.
  • Example 3 Determination of the Washing Performance When Used in a Commercially Available Powdered Detergent
  • Testmaterialien AG (St. Gallen, Switzerland), wfk Testgewebe GmbH (Brüggen-Bracht,
  • the control detergent used was a detergent base formulation of the following composition (all figures in percent by weight): 10% linear alkylbenzenesulfonate (sodium salt), 1.5% C12-C18 fatty alcohol sulfate (sodium salt), 2.0 % C12-C18 fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium bicarbonate, 4.0% amorphous sodium disilicate, 17% sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1.0% carboxymethylcellulose , 1, 0% phosphonate, 25% sodium sulfate, balance: foam inhibitors, optical brightener, fragrances.
  • the detergent base formulation was treated with the same activity for the different test series with the following proteases: protease according to SEQ ID NO. 2 (WO 03/054185), protease according to SEQ ID NO. 2 with the amino acid substitution P212N and the alkaline protease from Bacillus lentus variant F49 (WO 95/23221).
  • the protease variant shows an improved performance compared to the starting molecule according to SEQ ID NO.2 on different soils, in particular on the soils B and D, and in comparison with the Bacillus lentus protease established in the prior art.
  • the control detergent used was a detergent base formulation of the following composition (all figures in percent by weight): 0.3- 0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0 , 3-0.5% ethanol, 4-7% FAEOS, 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0 , 5% HEDP, 0-0.4% PVP, 0-0.05% optical brightener, 0-0.001% dye, balance demineralized water.
  • the detergent base formulation was treated with the same activity for the different test series with the following proteases: protease according to SEQ ID NO.
  • the protease variant shows markedly improved performance in comparison to the starting molecule according to SEQ ID NO.2 and in comparison with the protease established in the prior art ("reference") on various soils, in particular on soils A, B and D.
  • Test Materials AG St. Gallen, Switzerland
  • CFT Center For Testmaterials
  • the dosage of the detergent was 5.6 g / l (solid) or 4.7 g / l (liquid) at a water hardness of 16 ° German hardness.
  • the respective detergent formulations without enzymes represent the basis and thus the control value.
  • Proteases according to the invention were used as indicated in the tables below and a performance-improved variant of the alkaline protease from Bacillus lentus, which is disclosed as SEQ ID NO. 16 in EP 0701605 (hereinafter referred to as "reference")
  • the enzymes were used in the same activity (1080 PE / 200 ml in solid detergents, 2300 PE / 200 ml in liquid detergents).
  • FIG. 1 Alignment of the mature starting protease according to the invention (SEQ ID NO).
  • SEQ ID NO. 2 Starting protease according to the invention according to SEQ ID NO.
  • BLAP Alkaline protease from Bacillus lentus DSM 5483
  • Subtilisin 309 Subtilisin 309 according to SEQ ID NO.4

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Abstract

L'invention concerne des protéases dont la puissance protéolytique est améliorée par modification de la séquence aminoacide, en particulier, pour ce qui concerne leur utilisation dans des détergents et des produits de lavage. L'invention concerne également toutes les protéases suffisamment semblables ayant une modification comparable, et des acides nucléiques, ainsi que des possibilités d'utilisation techniques de ces protéases, principalement leur utilisation dans des détergents et des produits de lavage, et les agents correspondants eux-mêmes.
PCT/EP2008/062309 2007-09-17 2008-09-16 Protéases à performances améliorées et détergents et produits de lavage contenant ces protéases Ceased WO2009037258A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102010002762A1 (de) * 2010-03-11 2012-03-08 Henkel Ag & Co. Kgaa Leistungsverbesserte Proteasevariante
WO2013120948A1 (fr) * 2012-02-17 2013-08-22 Novozymes A/S Variants de subtilisine et polynucléotides codant ces derniers
WO2016205755A1 (fr) * 2015-06-17 2016-12-22 Danisco Us Inc. Protéases à sérines du clade du bacillus gibsonii
EP3472312A1 (fr) * 2016-06-15 2019-04-24 Henkel AG & Co. KGaA Protéase de bacillus gibsonii et variantes de celle-ci
WO2021013686A1 (fr) * 2019-07-22 2021-01-28 Henkel Ag & Co. Kgaa Produit de lavage comprenant une protéase permettant un dosage automatique
WO2021013687A1 (fr) * 2019-07-22 2021-01-28 Henkel Ag & Co. Kgaa Détergents comprenant une enzyme
US10913919B2 (en) 2018-06-19 2021-02-09 The Procter & Gamble Company Automatic dishwashing detergent composition
EP3799601A1 (fr) * 2018-06-19 2021-04-07 Danisco US Inc. Variants de subtilisine
US11220656B2 (en) 2018-06-19 2022-01-11 The Procter & Gamble Company Automatic dishwashing detergent composition

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102016221849A1 (de) * 2016-11-08 2018-05-09 Henkel Ag & Co. Kgaa Tensidzusammensetzung enthaltend eine Amylase

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023221A1 (fr) * 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO2003054185A1 (fr) * 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline issue de bacillus gibsonii (dsm 14391) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2003054184A1 (fr) * 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline extraite de bacillus gibsonii (dsm 14393) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2004099401A1 (fr) * 2003-05-07 2004-11-18 Novozymes A/S Enzymes de subtilisine variant (subtilases)

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1234445A (fr) 1967-10-03 1971-06-03
EP0283075B1 (fr) 1987-02-27 1999-05-12 Genencor International, Inc. Clonage moléculaire et expression de gènes codant pour des enzymes protéolytiques
PT89702B (pt) 1988-02-11 1994-04-29 Gist Brocades Nv Processo para a preparacao de novos enzimas proteoliticos e de detergentes que os contem
EP0493398B1 (fr) 1989-08-25 1999-12-08 Henkel Research Corporation Enzyme proteolytique alcaline et procede de production
US5340735A (en) 1991-05-29 1994-08-23 Cognis, Inc. Bacillus lentus alkaline protease variants with increased stability
ATE376061T1 (de) 1992-07-17 2007-11-15 Genencor Int Hochalkalische serinproteasen
AU7870394A (en) 1993-09-15 1995-04-03 Procter & Gamble Company, The Subtilisin bpn' variants with decreased adsorption and increased hydrolysis
ES2364776T3 (es) 1994-02-24 2011-09-14 HENKEL AG & CO. KGAA Enzimas mejoradas y detergentes que las contienen.
US5691295A (en) 1995-01-17 1997-11-25 Cognis Gesellschaft Fuer Biotechnologie Mbh Detergent compositions
ZA952220B (en) 1994-05-02 1995-12-14 Procter & Gamble Bpn' variants having decreased adsorption and increased hydrolysis wherein one or more loop regions are substituted
US6599730B1 (en) 1994-05-02 2003-07-29 Procter & Gamble Company Subtilisin 309 variants having decreased adsorption and increased hydrolysis
US6455295B1 (en) 1995-03-08 2002-09-24 The Procter & Gamble Company Subtilisin Carlsberg variants having decreased adsorption and increased hydrolysis
FR2782323B1 (fr) 1998-08-12 2002-01-11 Proteus Procede de production in vitro de sequences polynucleotidiques recombinees, banques de sequences et sequences ainsi obtenues
DE10121463A1 (de) 2001-05-02 2003-02-27 Henkel Kgaa Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten
DE10153792A1 (de) 2001-10-31 2003-05-22 Henkel Kgaa Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023221A1 (fr) * 1994-02-24 1995-08-31 Cognis, Inc. Enzymes ameliorees et detergents les contenant
WO2003054185A1 (fr) * 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline issue de bacillus gibsonii (dsm 14391) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2003054184A1 (fr) * 2001-12-20 2003-07-03 Henkel Kommanditgesellschaft Auf Aktien Nouvelle protease alcaline extraite de bacillus gibsonii (dsm 14393) et agents de lavage et de nettoyage contenant cette nouvelle protease alcaline
WO2004099401A1 (fr) * 2003-05-07 2004-11-18 Novozymes A/S Enzymes de subtilisine variant (subtilases)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE Geneseq [online] 11 April 1996 (1996-04-11), "BLAP M131 (R99X).", XP002503615, retrieved from EBI accession no. GSP:AAR80418 Database accession no. AAR80418 *
YAMAGATA Y ET AL: "The structure of subtilisin ALP I from alkalophilic Bacillus sp. NKS-21.", CURRENT MICROBIOLOGY APR 1995, vol. 30, no. 4, April 1995 (1995-04-01), pages 201 - 209, XP008098562, ISSN: 0343-8651 *
YAMANE TAKASHI ET AL: "Structure of a new alkaline serine protease (M-protease) from Bacillus sp. KSM-K16", ACTA CRYSTALLOGRAPHICA SECTION D BIOLOGICAL CRYSTALLOGRAPHY, vol. 51, no. 2, 1995, pages 199 - 206, XP002503614, ISSN: 0907-4449 *

Cited By (18)

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DE102010002762A1 (de) * 2010-03-11 2012-03-08 Henkel Ag & Co. Kgaa Leistungsverbesserte Proteasevariante
WO2013120948A1 (fr) * 2012-02-17 2013-08-22 Novozymes A/S Variants de subtilisine et polynucléotides codant ces derniers
US10093911B2 (en) 2012-02-17 2018-10-09 Novozymes A/S Subtilisin variants and polynucleotides encoding same
JP7015695B2 (ja) 2015-06-17 2022-02-03 ダニスコ・ユーエス・インク バチルス・ギブソニイ（Ｂａｃｉｌｌｕｓ ｇｉｂｓｏｎｉｉ）クレードセリンプロテアーゼ
WO2016205755A1 (fr) * 2015-06-17 2016-12-22 Danisco Us Inc. Protéases à sérines du clade du bacillus gibsonii
JP2018521646A (ja) * 2015-06-17 2018-08-09 ダニスコ・ユーエス・インク バチルス・ギブソニイ（Ｂａｃｉｌｌｕｓ ｇｉｂｓｏｎｉｉ）クレードセリンプロテアーゼ
EP4234693A3 (fr) * 2015-06-17 2023-11-01 Danisco US Inc Protéases à sérines du clade du bacillus gibsonii
US11499146B2 (en) 2015-06-17 2022-11-15 Danisco Us Inc. Bacillus gibsonii-clade serine proteases
EP3472312A1 (fr) * 2016-06-15 2019-04-24 Henkel AG & Co. KGaA Protéase de bacillus gibsonii et variantes de celle-ci
EP4600337A3 (fr) * 2016-06-15 2025-11-12 Henkel AG & Co. KGaA Protease de bacillus gibsonii et ses variants
EP3472312B1 (fr) * 2016-06-15 2025-08-20 Henkel AG & Co. KGaA Protéase de bacillus gibsonii et variantes de cette protéase
US10913919B2 (en) 2018-06-19 2021-02-09 The Procter & Gamble Company Automatic dishwashing detergent composition
US11220656B2 (en) 2018-06-19 2022-01-11 The Procter & Gamble Company Automatic dishwashing detergent composition
EP3799601A1 (fr) * 2018-06-19 2021-04-07 Danisco US Inc. Variants de subtilisine
CN114127244A (zh) * 2019-07-22 2022-03-01 汉高股份有限及两合公司 用于自动计量的包含蛋白酶的清洁剂
CN114144507A (zh) * 2019-07-22 2022-03-04 汉高股份有限及两合公司 包含酶的清洁剂
WO2021013687A1 (fr) * 2019-07-22 2021-01-28 Henkel Ag & Co. Kgaa Détergents comprenant une enzyme
WO2021013686A1 (fr) * 2019-07-22 2021-01-28 Henkel Ag & Co. Kgaa Produit de lavage comprenant une protéase permettant un dosage automatique

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