DE102007044415A1 - Performance-enhanced proteases and detergents and cleaners containing these proteases - Google Patents

Abstract

The present application relates to proteases whose proteolytic performance was 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.

Description

The The present application relates to proteases whose proteolytic activity by changing the amino acid sequence in particular with regard to their use in detergents and cleaners has been improved, all sufficiently similar proteases with a comparable Change and nucleic acids as well as technical Possible uses for these proteases, above especially their use in detergents and cleaners.

Proteases are among the most technically important enzymes of all. Of these, in turn, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) 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. For an overview of this family, for example, the article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 , Subtilases are naturally produced by microorganisms; 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 In addition to other enzymes, active ingredients of washing and detergents. They cause the breakdown proteinaceous Stains on the items to be cleaned. Conveniently result synergies between the enzymes and the rest Components of the funds concerned. Among the detergent and cleaner proteases take subtilases because of their favorable enzymatic Properties such as stability or pH optimum are outstanding Position. They are also suitable for a variety other technical uses, for example as components of cosmetics or in organic-chemical Synthesis. Regarding the use of proteases in washing and Detergents are known to improve the levels of proteases Washing or cleaning performance together with others Enzymes, for example amylases, cellulases, hemicellulases, mannanases, β-glucosidases, Oxidases, oxidoreductases or lipases can be used. Likewise, the use of proteases in detergents in combination with other active ingredients such as bleaches or soil release agents known to the skilled person.

Examples for those preferably used in detergents and cleaners Subtilisin-type proteases are the subtilisins BPN 'and Carlsberg, the protease P692, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and those subtilases, not but more to the subtilisins in the narrower sense attributable enzymes Thermitase, proteinase K and the proteases TW3 and TW7.

proteases are targeted by methods known in the art or changed randomly and so for example optimized use in detergents and cleaners. These include point mutagenesis, Deletion or insertion mutagenesis or fusion with other proteins or protein parts or other modifications. So are known to most of the prior art Proteases correspondingly optimized variants known.

The subtilisin BPN ', which is derived from Bacillus amyloliquefaciens, or B. subtilis, is from the work of Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 and from JA Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925 known. Subtilisin BPN 'serves as a reference enzyme of the subtilisins, in particular with regard to the numbering of the positions. The international patent application WO 95/07991 A2 discloses, for example, double mutants in which the amino acid in position 217 is mutated to 15 other amino acids, including L too. Detergents with such BPN 'variants are disclosed in the patent application WO 95/29979 A1 disclosed.

Subtilisin Carlsberg in a developed form under the trade names Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark. She will be in the Publications by EL Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191 , and from Jacobs et al. (1985) in Nucl. Acids Res., Vol. 13, pp. 8913-8926 and is naturally produced by Bacillus licheniformis. Variants obtainable by point mutations in the loop regions of this molecule are from the application WO 96/28566 A2 known.

The protease PB92 is naturally derived from the alkaliphilic bacterium Bacillus nov. spec. 92 and Gist-Brocades, Delft, The Netherlands, available under the trade name was Maxacal ^® by the company.. In its original sequence, it is in the patent application EP 283075 A2 described. By Punktmu tion obtained variants of this enzyme for use in detergents and cleaning agents, for example, in the applications WO 94/02618 A1 and EP 328229 A1 disclosed. Exchanges emerge from both in position 211, for example L211E and 1211Y respectively, but only alone or in combination with other point mutations that are not relevant here.

The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® from Novozymes. They are originally from Bacillus strains with the application GB 1243784 A be revealed. Variants of these enzymes further developed by point mutagenesis with regard to the use in detergents and cleaners are described, for example, in the applications WO 94/02618 A1 (see above) and WO 95/30011 A2 disclosed. Out WO 95/30011 A2 Mutations occur in the loop regions of this protease, including at position 211.

From the protease from Bacillus lentus DSM 5483 ( WO 91/02792 A1 ) derive from the name under the name BLAP ^® variants, which in particular in WO 92/21760 A1 . WO 95/23221 A1 . WO 02/088340 A2 and WO 03/038082 A2 to be discribed. From the registration WO 95/23221 A1 or the associated US patents US 5691295 . US 5801039 and US 5855625 In addition, performance-enhanced B. lentus alkaline protease variants are produced by targeted point mutagenesis for use in detergents and cleaners. As a relevant substitution to the wild-type enzyme from B. lentus DSM 5483 is also the position 211 called, in particular 211D and 211E. The associated strategy, namely to specifically change the charge ratios near the substrate binding pocket, illustrates the patent US 6197589 B1 ,

Other proteases are, for example, under the trade names Durazym ^®, relase ^®, Everlase® ^®, Nafizym, Natalase ^®, Kannase® ^®, Shinezyme ^® and Ovozyme ^® from Novozymes, the ^® under the trade names Purafect ^®, Purafect ^® OxP, Purafect Prime and Properase.RTM ^® from Genencor, that under the trade name Protosol® ^® from Advanced Biochemicals Ltd., Thane, India, under the trade name PRODET P by the company Rich Core India, Bangalore, India, that under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China, under the trade name ^® Proleather and protease P ^® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and sold under the name proteinase K-16 from Kao Corp., Tokyo, Japan , available enzymes.

A special feature in the international patent application WO 03/054185 disclosed alkaline proteases from Bacillus gibsonii (DSM 14391). This strain is under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms of April 28, 1977 at the German Collection of Microorganisms and Tent Cultures GmbH, Inhoffenstraße 7B, 38124 Braunschweig ( https://www.dsmz.de ) under the designation ID 01-192 and the accession number DSM 14391. Compared to the proteases mentioned above, these proteases have considerable differences in the amino acid sequence, so that an identity comparison of the amino acid sequences results in identity values that are below 80% identity. Table 1 below illustrates this fact with concrete examples: Table 1: protease Identity of the alkaline proteases from Bacillus gibsonii DSM 14391 (in%) Subtilisin 309 78.4 Alkaline protease from Bacillus lentus DSM 5483 77.7 BPN ' 55.3 PB92 78.1

For the alkaline proteases from Bacillus gibsonii (DSM 14391) none improved for use in detergents and cleaners Protease variants known in the art, in particular none Variants that involve a change at positions 211 and / or 212 in the counting method of Bacillus alkaline proteases gibsonii (DSM 14391) based on the mature enzyme.

The proteases used in the compositions according to the invention are either originally derived from microorganisms, for example from microorganisms of 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 through filamentous fungi. The latter applies in particular to protease variants which have been changed starting from a starting molecule and have thus been optimized, for example, for their use in detergents and cleaners.

It It is also known that some for use in detergents established proteases also for cosmetic purposes or for the organic-chemical synthesis are suitable.

It there is still a high demand for proteases in various technical Applications. In particular with regard to their usability in detergents and cleaners exists even for the family of subtilisin proteases established in the art still a need for optimization concerning, for example, their catalytic activity, reaction conditions, stability or the substrate specificity. Especially in terms of use However, it is a protease in detergents and cleaners in usually not possible due to the measurable or calculable enzymatic properties of the enzyme per se on its behavior to conclude in washing or cleaning formulations. In this regard, other factors such as stability play towards oxidizing agents, denaturation by surfactants, Folding effects or synergies with other ingredients, the behavior of the enzyme or enzymes in detergents or cleaning agents or in the resulting washing or cleaning solutions influence.

Of the The present invention is therefore based on the object, an alkaline Protease of the type of alkaline proteases from Bacillus gibsonii To further develop DSM 14391 and performance-enhanced variants to obtain improved performance in technical applications demonstrate. In particular, those should be found whose performance improvement an increased cleaning performance of detergents and / or cleaning agents causes. In this regard, such protease variants should which has been improved with respect to the parent protease Washing performance with respect to at least one soiling, preferably in relation to multiple soils.

Further Objects of the present invention can be seen therein to provide proteases, in particular of the subtilisin type, which compared to the prior art, an improved stability against temperature influences, pH fluctuations, denaturing or oxidizing agents, proteolytic degradation, high temperatures, acidic or alkaline conditions or opposite have a change in the redox ratios. Other tasks may result in decreased immunogenicity or reduced allergenic effects.

A Another particular object of the present invention was proteases to locate at temperatures of 20 to 60 ° C. a good washing performance, preferably an improved washing performance in the Compared to the proteases disclosed in the prior art.

Further Partial tasks consisted of nucleic acids which code for such proteases and vectors, To provide host cells and manufacturing processes, which can be used to obtain such proteases. Furthermore, appropriate means, in particular washing and cleaning agents, appropriate washing and cleaning procedures and corresponding Uses for such proteases to provide. Finally, should technical applications for the found Proteases are defined.

A Solution for the above object results according to the invention from the teaching of the first Claim. An object of the invention thus forms a protease, which comprises an amino acid sequence corresponding to that shown in SEQ ID NO. 2 specified amino acid sequence at least 78.5% is identical, and characterized in that it is in the Counting according to SEQ ID NO. 2 at position 211 or 212 or at the positions 211 and 212 an im Comparison to that shown in SEQ ID NO. 2 amino acid changed Has amino acid.

Surprisingly, it has been found that a change of the positions 211 and / or 212 in a protease which corresponds to the one shown in SEQ ID NO. 2 amino acid sequence indicated at least 78.5% identical amino acid sequence, an improved performance of this modified protease in detergents and cleaners causes compared to a corresponding protease that does not have these changes. This is particularly surprising since the 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. It was therefore unlikely that performance-enhanced protease variants would be obtained for alkaline proteases from Bacillus gibsonii (DSM 14391) for use in detergents and cleaners by a change tion at positions 211 and / or 212 in the counting method of the alkaline proteases from Bacillus gibsonii (DSM 14391), based on the mature enzyme. Unexpectedly, these very changes in these enzymes lead to a performance improvement that is different from the classical, established subtilisin-type proteases.

In a preferred embodiment of the invention is the Protease characterized in that it has an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 2 indicated amino acid sequence increasingly 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% identical is.

• a) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 211 eine Aminosäure aufweist, die ausgewählt ist aus der Gruppe bestehend aus: Alanin, Arginin, Asparagin, Asparaginsäure, Cystein, Glutamin, Glutaminsäure, Glycin, Histidin, Isoleucin, Leucin, Lysin, Phenylalanin, Prolin, Serin, Threonin, Tryptophan, Tyrosin und Valin
• b) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 212 eine Aminosäure aufweist, die ausgewählt ist aus der Gruppe bestehend aus: Alanin, Arginin, Asparagin, Asparaginsäure, Cystein, Glutamin, Glutaminsäure, Glycin, Histidin, Isoleucin, Leucin, Lysin, Methionin, Phenylalanin, Serin, Threonin, Tryptophan, Tyrosin und Valin
• c) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 211 eine Aminosäure gemäß Merkmal a) aufweist und an Position 212 eine Aminosäure gemäß Merkmal b) aufweist.

Surprisingly, it has also been found that there are several ways to alter the amino acid present at positions 211 and / or 212 to obtain performance enhancement of the resulting protease. In principle, it is important that the protease at any one of these positions is altered at all, ie that the amino acid present at the respective position has been replaced by another proteinogenic amino acid. Alternatively, both positions can be changed in this way. A further subject of the invention therefore forms a protease, which is characterized in that the protease is selected from

• a) a protease which in the counting method according to SEQ ID NO. 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 is in the counting manner according to SEQ ID NO. 2 at position 212 has an amino acid selected from the group consisting of: 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 in the counting method 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).

• a) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 211 einen Serinrest aufweist
• b) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 212 einen Asparaginrest aufweist
• c) einer Protease, die in der Zählweise gemäß SEQ ID NO. 2 an Position 211 einen Serinrest und an Position 212 einen Asparaginrest aufweist.

In a preferred embodiment of the invention, protease is characterized in that the protease is selected from

• a) a protease which in the counting method according to SEQ ID NO. 2 at position 211 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 in the counting method according to SEQ ID NO. 2 at position 211 has a serine residue and at position 212 an asparagine residue.

Because for the above substitutions, a Particularly advantageous performance improvement found in a Use of these protease variants in detergents and cleaners, especially in detergent formulations. Outgoing and in the Counting of SEQ ID NO. 2 show the inventive Variants therefore the amino acid changes M211S (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.

additionally to the above-explained amino acid changes Proteases of the invention may be further Amino acid changes, especially amino acid substitutions, Insertions or deletions.

With All of the above subject invention are as further subject of the invention the associated Nucleic acids, corresponding natural cells, appropriate methods for their identification, in particular the Nucleic acid-based molecular biology method and process elements and agents, detergents and cleaning agents, Washing and cleaning procedures and the concerned Proteases identified uses connected. Due to the provided nucleic acids is an additional optimization of this enzyme, for example via further point mutations possible. Furthermore, this DNA introduced into shuffling approaches and thus to the generation completely new types of proteases are used.

Under For the purposes of the present application, an enzyme is a protein to understand that has a certain biocatalytic function. Protease in the sense of the present application is an enzyme which catalyzes the hydrolysis of peptide bonds and thereby be able to cleave peptides or proteins.

One Protein is one of the natural within the meaning of the present application Amino acids composed, largely linearly structured, to perform his function usually a three-dimensional structure accepting polypeptide. A peptide consists of amino acids, covalently linked to each other via peptide bonds are. The term polypeptide clarifies in this regard The fact that this peptide chain usually consists of many Amino acids exist that via peptide bonds connected to each other. Amino acids can in an L and a D configuration, with the amino acids, that make up proteins that are in the L configuration. she are called proteinogenic amino acids. In the present application, the proteinogens, of course occurring L-amino acids with the internationally used 1 and 3 letter codes. Numerous proteins will be as so-called pre-proteins, ie together with a signal peptide educated. Below that is the N-terminal part of the protein too understand whose function is usually the discharge of the formed protein from the producing cell into the periplasm or to ensure the surrounding medium and / or its correct folding. Subsequently, the signal peptide under natural Conditions due to a signal peptidase from the remaining protein split off, giving this its actual catalytic activity without the initially existing N-terminal amino acids exercises. Pro-proteins are inactive precursors of proteins. their Precursors with signal sequence are called pre-pro-proteins designated. For technical applications are due to their enzymatic activity the maturen, d. H. mature peptides, d. H. the enzymes processed after their preparation the preproteins are preferred. The proteins can from the cells producing them after the production of the polypeptide chain be modified, for example by attachment of sugar molecules, Formylations, aminations, etc. Such modifications will become referred to as post-translational modifications. This post translational Modifications can, but do not have to be Exert influence on the function of the protein.

in the For the purposes of the present invention, all enzymes, proteins, fragments and derivatives, unless explicitly addressed as such to be summarized under the generic term proteins.

By Comparison with known enzymes, for example, in general accessible databases From the amino acid or nucleotide sequence the enzymatic Activity of a considered enzyme infer. This can through other areas of the protein that are not at the actual Reaction involved, qualitatively or quantitatively modified become. This could be, for example, the enzyme stability, the activity, the reaction conditions or the substrate specificity affect.

Such a comparison is accomplished by associating similar sequences in the nucleotide or amino acid sequences of the proteins of interest. This is called homologization. A tabular assignment of the respective positions is referred to as alignment. In the analysis of nucleotide sequences, in turn, both complementary strands and all three possible reading frames are to be taken into account; as well as the degeneracy of the genetic code and the organism-specific use of codons (codon usage). Meanwhile, alignments are created using computer programs, such as the algorithms FASTA or BLAST; This procedure is for example by DJ Lipman and WR Pearson (1985) in Science, Vol. 227, pp. 1435-1441 described.

A Compilation of all matching in the compared sequences Positions 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 one will in percent identity, that is the proportion of identical nucleotides or amino acid residues on the same or in an alignment corresponding positions reproduced. One more broad homology term refers to the conserved Amino acid substitutions in this value. It is then from percent similarity the speech. Such statements can over entire proteins or genes or only over individual areas to be hit.

homologous Areas of different proteins are by matches defined in the amino acid sequence. these can also be characterized by identical function. She goes up to complete identities in the smallest areas, so-called boxes, which comprise only a few amino acids and mostly essential for overall activity Exercise functions. Among the functions of homologues Areas are the smallest subfunctions of the whole protein To understand function, such as the training of individual Hydrogen bonds for complexation of a substrate or transitional complex.

proteins can also talk about the reaction with an antiserum or a particular antibody to groups immunologically related proteins are summarized. The members A group is characterized by being the same, by one Antibodies recognized to have antigenic determinant.

a Another subject of the invention therefore proteases, characterized are that they have at least one and increasingly two, three or four matching antigenic determinants with one having protease of the invention.

proteases or enzymes in general can be prepared by various methods, z. B. targeted genetic modification by mutagenesis, evolved and for specific purposes or in terms of specific properties, such as catalytic activity, Stability, etc., to be optimized. amendments the nucleotide sequence, as known for example by per se molecular biological methods can be brought about, are therefore called mutations. Depending on the type of change knows for example, deletion, insertion or substitution mutations or those where different genes or parts of genes are related to each other to be shuffled; these are gene mutations. The associated Organisms are called mutants. Those of mutated nucleic acids derived proteins are referred to as variants. To lead for example, deletion, insertion, substitution mutations or fusions to deletions-, insertions-, substitutionsmutierten or fusion genes and at the protein level to corresponding deletion, Insertion or substitution variants, or fusion proteins. The Strategy, in the known molecules targeted point mutations introduce, for example, the washing performance of the proteases, for example Subtilisins, to improve, is also called rational protein design designated. A similar strategy of performance improvement consists of the surface charges and / or the isoelectric Point of the molecules and above their interactions to change with the substrate. For example, about Point mutations changed the net charge of the subtilisins in order to discuss substrate binding in particular for to influence the use in detergents and cleaners. A further, and in particular complementary, strategy is increase the stability of the proteases in question and thus to increase their effectiveness, for example, over Coupling to one or more similar or diverse Polymers. Especially for cosmetic applications such a coupling in an improved skin compatibility result. In particular for detergents and cleaners While stabilizations by point mutations are more common, corresponding couplings to polymers but still possible.

A modern direction of enzyme development is to combine elements from known, related proteins via statistical methods into new enzymes with previously unattainable properties. Such methods are also summarized under the generic term Directed Evolution. These include, for example, the following methods: The StEP method (Zhao et al., 1998, Nat. Biotechnol., Vol. 16, pp. 258-261) . Random priming recombination (Shao et al., (1998), Nucleic Acids Res., Vol. 26, pp. 681-683) . DNA shuffling (Stemmer, WPC (1994), Nature, Vol. 370, pp. 389-391) or RACHITT (Coco, WM et al. (2001) Nat. Biotechnol., Vol. 19, pp. 354-359) , Another shuffling method called recombining ligation reaction (RLR) is described in US Pat WO 00/09679 A1 described.

For the description of point mutations that exactly one amino acid position affect (amino acid exchanges), the following convention applied: first, the natural existing amino acid in the form of the internationally used Denotes one-letter codes, then follows the associated sequence position and finally the inserted amino acid. Several exchanges within the same polypeptide chain are performed Slashes separated.

It It is also well known in the art that advantageous properties of individual mutations, eg. B. individual Point mutations, can complement. One regarding certain properties already optimized protease, for example in terms of their stability to surfactants or other components, according to the invention additionally be further developed.

Under Fragments are understood to mean all proteins or peptides that are smaller are as natural proteins and, for example, also synthetic can be obtained. Due to their amino acid sequences can they are assigned to the relevant complete proteins become. For example, you can assume the same structures or proteolytic activities or partial activities exercise, such as the complexation of a substrate. Fragments and deletion variants of starting proteins are in principle similar; while fragments are rather smaller fragments lacking deletion mutants tend to lack only short regions, and thus only individual subfunctions.

For the purposes of the present application, chimeric or hybrid proteins are proteins whose sequence comprises the sequences or partial sequences of at least two parent 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. For example, the purpose of such recombination may be to induce or modify a particular enzymatic function using the fused protein portion. For the purposes of the present invention, it is irrelevant whether such a chimeric protein consists of a single polypeptide chain or several subunits to which different functions can be distributed.

Under proteins obtained by insertion mutation are such variants to understand that by inserting a protein fragment were obtained in the starting sequences. They are their principal similarity because of the chimeric proteins. They differ of them only in the size ratio of unchanged protein part to size of the entire protein. In such insertionsmutierten proteins the proportion of foreign protein is lower than in chimeric Proteins.

Inversion, So a partial sequence reversal, as a special form both the deletion, as well as the insertion. The same thing applies to one of the original amino acid sequence differing regrouping of different parts of the molecule. It can be used both as deletion variant, as insertion variant, as also as a shuffling variant of the original protein be considered.

Under Derivatives are for the purposes of the present application, such proteins understood, the pure amino acid chain chemically modified has been. Such derivatizations can be, for example biological in the context of protein biosynthesis by the Host cell done. For this purpose, molecular biological Methods are used. You can also do it chemically be carried out, for example by the chemical transformation a side chain of an amino acid or by covalent Binding of another compound to the protein. With such a Compound can also be, for example, other proteins, for example, bifunctional chemical compounds be bound to proteins of the invention. Such modifications may include, for example, substrate specificity or affect the bond strength to the substrate or a temporary block of enzymatic activity cause, if it is at the coupled substance is an inhibitor. This can be for example the period of storage be useful. Likewise, under derivatization covalent attachment to a macromolecular carrier as well as a noncovalent inclusion in suitable macromolecular cage structures.

a Another object of the invention thus represents a protease, which is characterized in that it consists of a Protease is obtainable as a parent molecule by fragmentation, deletion, Insertion or substitution mutagenesis and an amino acid sequence which covers a length of at least 266 and increasingly preferably 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.

So For example, it is possible at the terms or in the Loops of the enzyme to delete single amino acids, without that thereby the proteolytic activity is lost. Furthermore, by such deletions, the allergenicity decreased proteases and thus their overall applicability can be improved. The fragmentation comes as follows performed aspect of insertion or substitution mutagenesis and / or also a possible fusion with other enzymes benefit. Regarding the intended use of these enzymes it is particularly preferred if they also after fragmentation or Deletion mutagenesis have a proteolytic activity.

numerous Prior art documents disclose beneficial effects of insertions and substitutions in subtilases. In principle belong this also individual exchanges of amino acids, but it can also several contiguous amino acids against others are exchanged. This also includes new combinations of larger enzyme sections, d. H. the combination of fragments, with other proteases or proteins of other function or again fragments of the same. That's the way it is, for example possible, a protein according to the invention or parts thereof via peptidic linkers or directly as a fusion protein with binding domains from other proteins, such as the cellulose-binding domain, to provide and thereby the hydrolysis of the substrate more effectively shape. Likewise, proteins according to the invention for example, also linked to amylases or cellulases be used 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 are assigned in an alignment. The amino acid positions are determined here 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 described in SEQ ID NO. 3 is assigned. In this way, so-called homologous positions are determined. An example of such an alignment is in 1 specified.

A final confirmation in the assignment homologous Positions, d. H. in particular their functional equivalent, can ultimately only provide comparative experiments, according to which the two are assigned to each other on the basis of an alignment Positions in both compared proteases on the same way point mutated and is observed, whether in both changed the enzymatic activity in the same way becomes. For example, goes an amino acid exchange in one certain position of B. lentus alkaline protease (BLAP) with an increase of KM or another enzymatic Parameters, and will tend to be the same shift of the KM value in a protease variant according to the invention, their amino acid exchange introduced by the same Amino acid is reached, this is the confirmation to see this aspect of the invention.

In the said positions are in the wild-type molecule of the For example, lentus alkaline protease has the following amino acid residues: S3, V4, S36, N42, A47, T56, G61, T69, E87, A96, R99, A101, I102, S104, N114, H118, A120, S130, S139, T141, S142, S154, S157, A188, V193, V199, G205, A224, K229, S236, N237, N242, H243, N255 and T268.

There in addition to the alkaline protease from Bacillus licheniformis the B. lentus alkaline protease in the art is an important reference molecule to describe new proteases and point mutations and the new proteases described herein, and thus their sequence hitherto unknown, it seems advantageous in the assignment of the point mutations on the count of the alkaline protease Bacillus lentus reference. Furthermore, the count depends generally after the mature (mature) protein. Advantageous positions for sequence changes, for example by Point mutations, the alkaline protease from Bacillus lentus, that transmit to homologous positions of the proteases of the invention are of importance and the protease advantageous functional Lend properties are given below.

From the registration WO 92/21760 A1 single and multiple variants of the subtilisin from Bacillus lentus DSM 5483 are evident in the following positions: 3, 4, 36, 42, 47, 56, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139 , 141, 142, 157, 188, 193, 199, 205, 224, 229, 236, 237, 242, 243, 255 and 268. The application WO 95/23221 A1 in addition to a change in position 211, additionally discloses exchanges of this molecule in positions 99 and 154, in particular R99G, R99A, R99S, S154D and S154E. The registration WO 02/088340 discloses, in addition to a possible change at position 211, further possible amino acid changes in one or more of positions 3, 4 and 199, the exchanges at position 3 (for example S3T) and at position 4 (for example V4I) presumably having a stabilizing effect on the molecule lead to an improvement in its contribution to the washing performance of a washing or cleaning agent.

One Another object of the invention is a protease described above, which is additionally stabilized, in particular by coupling to a polymer.

Because an increase in stability during storage and / or during use, for example during the washing process causes their activity to last longer continues and is thus reinforced in effect. As stabilization options are all in the state of Technique described and appropriate strategies in Consider, for example, the covalent coupling to a polymer. alternative For this purpose, such stabilizations are suitable over Point mutagenesis of the molecule itself are possible (and due to the sequence differences already below those described above Embodiments fall). Because these require stabilization following the protein recovery no further steps. Some point mutations suitable for this purpose are in themselves known in the art. For example, proteases can be stabilized by one or more tyrosine residues exchanged with other proteinogenic amino acids.

Another way to stabilize enzymes is changing the bin, for example tion of metal ions, in particular the binding of calcium ions, by changing the corresponding binding sites, in particular the calcium binding sites. For this purpose, for example, one or more involved in the calcium bond or metal ion binding amino acids exchanged for negatively charged amino acids become. Alternatively or additionally, for the stabilization via the calcium binding, at least one of the sequences of the two amino acid residues arginine / glycine can be used to introduce point mutations. As a further possibility for stabilization proteins can be protected by mutations on the surface of the protein against the influence of denaturing agents such as surfactants, in particular ionic surfactants. In particular, hydrophobic amino acid residues can be replaced by more hydrophilic amino acid residues and / or polar uncharged amino acid residues can be replaced by sterically more demanding amino acid residues, especially those having longer hydrophilic side chains than the original amino acid, and / or ionic amino acids can be replaced by uncharged hydrophilic amino acid residues or by Amino acid residues whose charge is not contrary to the net charge of the denaturing agent, so for example, the ionic surfactant.

A another way to stabilize elevated temperature and the action of surfactants through the exchange of amino acids near the N-terminus, against such amino acids, probably over non-covalent interactions with the rest of the molecule make contact and thus contribute to the maintenance to afford the globular structure.

preferred Embodiments are those in which the molecule is stabilized in several ways. Because usually several act stabilizing mutations additive or synergistic on overall stability of the enzyme. Particularly preferred are therefore combinations of stabilizing Enzyme changes that interact synergistically and complement each other in their stabilizing effect, that the combinatorial stabilization is the bare one Sum of individual stabilization effects exceeds.

a Another object of the invention provides a protease as above described, which is characterized in that they at least has a chemical modification. A protease with such a Change is also called derivative, i. H. the Protease is derivatized.

Under Derivatives are therefore understood as meaning those proteins which are replaced by a additional modification from the executed Proteins are obtained. This is preferably a chemical modification of the invention Proteins. Such modifications may be, for example stability, substrate specificity or binding strength to the substrate or the enzymatic activity. They can also serve to increase the allergenicity and / or to reduce immunogenicity of the protein and for example, to increase its skin tolerance.

Such Derivatizations, in particular chemical modifications, can for example, by biological means, such as in context with protein biosynthesis by the producing host organism. Here are couplings of low molecular weight compounds such as lipids or oligosaccharides.

derivatizations but can also be done by chemical means be, for example, by the chemical transformation of a side chain or by covalent bonding of another, for example macromolecular Connection to the protein. For example, the coupling of Amines on carboxyl groups of an enzyme a change effect of the isoelectric point of the protein. Furthermore, can for example, other macromolecules such as proteins, such as over bifunctional chemical compounds, to inventive Proteins are bound. For example, it is possible a protein according to the invention also via a Nonprotein linker with a specific binding domain to provide. Such derivatives are particularly suitable for Use in detergents or cleaners. Analog can for example, protease inhibitors via linkers, in particular Amino acid linker to the invention Proteins are bound. Couplings with other macromolecular Compounds such as polyethylene glycol improve the molecule in terms of other properties such as stability or Skin compatibility as already explained above.

Derivatives of proteins according to the invention can in the broadest sense also be understood to mean preparations of these enzymes. Depending on the extraction, processing or preparation, 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, according to the invention, all preparations of a protein according to the invention are independent of whether in a particular preparation actually the intended enzymatic Activity unfolded or not. Because it may be desired that it has no or only low activity during storage, and unfolds its enzymatic function only at the time of use. This can be controlled, for example, via appropriate accompanying substances. In particular, the joint preparation of proteases with protease inhibitors is advantageous and known from the prior art.

The Solution of a subtask and thus an independent task Subject of the invention form nucleic acid molecules, that for a protease according to the invention encode as well as vectors containing such a nucleic acid.

Under Nucleic acids are within the meaning of the present application naturally constructed from nucleotides as information carriers serving for the linear molecules Code amino acid sequence in proteins or enzymes. she can be as a single strand, as one to this single strand complementary single strand or as a double strand. As the naturally more permanent information carrier the nucleic acid DNA for molecular biological Works preferred. In contrast, for the Realization of the invention in natural environment, such as for example, in an expressing cell, an RNA is formed, which is why essential RNA molecules also embodiments of the present invention.

at DNA is the sequence of both complementary strands to be considered in all three possible reading frames. It should also be noted that different codon triplets can code for the same amino acids, so that a certain amino acid sequence of several different and possibly low identity containing nucleotide sequences can be derived, what as Degeneracy of the genetic code is called. Furthermore Different organisms have differences in the use of these codons on. For these reasons, both amino acid sequences must as well as nucleotide sequences in the consideration of the scope be included. Therefore, all nucleotide sequences are included in the invention, one of the above-described Enzyme can encode. The expert is able to do this Nucleotide sequences to determine beyond doubt, because despite the degeneracy of the genetic code of individual codons defined amino acids are assigned. Therefore, those skilled in the art can start from one each only as an exemplary coding for a particular one To look at amino acid sequence.

The a protein corresponding information unit is also in the sense referred to as the gene of the present application.

A person skilled in the art can use well-known methods such as chemical synthesis or the polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences, the corresponding nucleic acids to complete genes manufacture. Such methods are for example Sambrook, J., Fritsch, EF and Maniatis, T. 2001: Molecular Cloning: A Laboratory Manual, 3rd Edition (Cold Spring Laboratory Press) known.

amendments the nucleotide sequence, as known for example by per se molecular biological methods can be brought about, are called mutations. Depending on the type of change knows for example, deletion, insertion or substitution mutations or those where different genes or parts of genes are related to each other fused or recombined; these are gene mutations. The associated organisms are called mutants. The proteins derived from mutant nucleic acids become referred to as variants. For example, deletion, Insertion substitution mutations or fusions to deletion, insertion-substitution-mutated or fusion genes and at the protein level to corresponding deletion, insertion or substitution variants, or fusion proteins.

In a preferred embodiment of the invention is that Nucleic acid molecule, which is responsible for a Protease encoded, characterized in that it corresponds to the in SEQ ID NO. 1 indicated nucleic acid sequence increasingly preferred at least 78.5%, 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% identical.

Prefers are such nucleic acids that are mature for mature Encode proteins, and increasingly preferred are those for increasingly active variants of the invention Encode proteins.

Farther preferred are those nucleic acids of which one or preferably several codons have been replaced by synonymous codons are.

This Aspect refers to the heterologous expression of the subject Proteases. So every organism, especially every production strain, has over a certain codon usage. This can lead to bottlenecks come in protein biosynthesis when acting on the transgenic nucleic acid lying codons in the host cell of a comparatively small Face number of loaded tRNAs. Synonyms Codons encode, however, for the same amino acids and can be better translated depending on the host. This possibly necessary rewriting thus depends of the choice of the expression system. Especially for samples from unknown, possibly non-cultivable organisms can have a appropriate adjustment necessary.

The The present invention involves the production of recombinant proteins. These include all genetic engineering according to the invention or microbiological processes based on that the genes for the proteins of interest in a introduced for production suitable host cell and be transcribed and translated by this. suitably the introduction of the relevant genes takes place via vectors, in particular expression vectors; but also about those which cause the gene of interest in the host cell in an already existing genetic element like the chromosome or other vectors can be inserted. The functional Unit of gene and promoter and any other genetic Elements according to the invention as an expression cassette designated. You do not necessarily have to also exist as a physical entity.

Under For the purposes of the present invention, vectors are made from nucleic acids existing elements understood as the characteristic nucleic acid region contain a gene of interest. You can do this in one species or cell line over several generations or to establish cell divisions as a stable genetic element. Vectors, especially when used in bacteria, are special plasmids, so circular genetic elements. There is a difference in genetic engineering on the one hand between such vectors, the storage and thus to a certain extent also serve the genetic engineering work, the so-called cloning vectors, and on the other hand those which the Function, the gene of interest in the host cell to realize, that is, the expression of the relevant Enable protein. These vectors are called expression vectors designated.

in the The present invention relates to the nucleic acid suitably cloned into a vector. The molecular biological Dimension of the invention thus consists in vectors with the genes for the corresponding proteins. This can, for example those that are different from bacterial plasmids, from viruses or derived from bacteriophages, or predominantly synthetic Vectors or plasmids with elements of various origins. With the other genetic elements that are available Vectors, in the respective host cells over several Generations as stable units. It is thereby irrelevant in the context of the invention, whether they are extrachromosomal establish as own units or integrate into a chromosome. Which of the numerous known from the prior art systems is chosen, depends on the individual case. decisive For example, the achievable copy number used for Available selection systems, including above all Antibiotic resistance, or the cultivability of the for admission the vectors are capable host cells.

The Vectors form suitable starting points for molecular biology and biochemical studies of the gene or its associated Proteins and for further developments of the invention and ultimately for amplification and production according to the invention Proteins. They represent insofar embodiments of the present Invention, as the sequences of the invention contained Nucleic acid regions each within the above closer designated homology regions are.

a Another object of the invention thus represent vectors that contain at least one nucleic acid molecule, which for a protease according to the invention encodes, in particular a nucleic acid molecule, as described above. In a further embodiment invention, the vector is characterized in that the vector a cloning vector or an expression vector. cloning vectors are in addition to storage, biological amplification or Selection of the gene of interest for characterization of the gene in question, such as the creation of a restriction map or the sequencing. Cloning vectors are also preferred Embodiments of the present invention because they a transportable and storable form of the claimed Represent DNA. They are also preferred starting points for molecular biology techniques that are not bound to cells, such as for example, the polymerase chain reaction.

expression vectors are chemically similar to cloning vectors but in those subsequences that enable them in the host cells optimized for the production of proteins or To replicate host organisms and there the gene for expression bring to. 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. So the expression by the natural, originally before this Gene localized promoter done, but also after genetic engineering Fusion by both provided on the expression vector Promoter of the host cell as well as by a modified or a completely different promoter of another organism or another host cell.

preferred Embodiments are those expression vectors that undergo changes the culture conditions or addition of certain compounds, such as for example cell density or special factors, regulatable are. Expression vectors allow the associated Protein heterologous, so in another cell or host cell as the one from which it is naturally derived can, is produced. The cells can be quite belonging to different organisms or of different ones Come from organisms. Also a homologous protein recovery from a the gene naturally expressing host cell an appropriate vector is within the scope of the present invention Invention. This may have the advantage that natural, translation related transformations be performed on the resulting protein as well as they would naturally occur.

A further embodiment represent expression systems, in which additional genes, such as those that be provided on other vectors that Influence production of proteins according to the invention. These may be modifying gene products or to those with the protein of the invention be cleaned together, about its enzymatic function to influence. These may be, for example, other proteins or enzymes to inhibitors or to act on such elements influence the interaction with different substrates.

alternative Embodiments of the present invention may be also be cell-free expression systems involving protein biosynthesis understood in vitro. Such expression systems are available the technique also established.

One Another object of the invention is a non-human host cell, a protease of the invention or a fragment of the same or which are encouraged to produce them can, preferably using an expression vector. The in vivo synthesis an enzyme according to the invention, ie by living cells, requires the transfer of the associated Gene into a host cell, their so-called transformation. As host cells In principle, all cells are suitable, that is prokaryotic or eukaryotic cells. Preferred are such host cells, the be genetically advantageous to handle what, for example the transformation with the expression vector and its stable Establishment, for example, unicellular fungi or bacteria. In addition, preferred host cells are characterized by a good microbiological and biotechnological manageability. That concerns for example easy cultivability, high growth rates, low requirements on fermentation media and good production and secretion rates for foreign proteins. Often need to out The abundance of different according to the state of the art available standing systems the optimal expression systems for determine the individual case experimentally. Each invention Protein can in this way from a multiplicity of host cells be won. Also, such host cells are preferred by the that after transformation with one of them have been obtained as described above. It can For example, be cloning vectors for storage and / or modification, for example in any bacterial strain or another host cell according to the invention introduced have been. Such steps are in storage and further development genetic elements. Because of these Host cells transfer the relevant genetic elements into subsequent, immediately transferred to the expression of suitable host cells can be, are the same with the previous ones Transformation products to realizations of the subject invention.

preferred Embodiments illustrate such host cells that are due to genetic regulatory elements, for example, on the expression vector be made available, but also from the outset may be present in these cells, in their activity are adjustable. For example, by controlled addition of chemical compounds that serve as activators, by amendment the cultivation conditions or when a certain Cell density, these can be excited for expression. This allows a very economical production of proteins of interest.

preferred Host cells are prokaryotic or bacterial cells. bacteria are usually distinguished from eukaryotes shorter generation times and lower demands to the cultivation conditions. This can be cost effective Method for obtaining proteins according to the invention established become. In Gram-negative bacteria, such as Escherichia coli (E. coli), are a variety of proteins in the periplasmic Space secreted, so in the compartment between the two Cells enclosing membranes. This can be for special applications be beneficial. Gram-positive bacteria, such as Bacilli or Actinomycetes or other representatives the Actinomycetales, in contrast, have no external Membrane so that secreted proteins readily enter the cells surrounding nutrient medium are released from which according to a further preferred embodiment, the expressed purify proteins according to the invention directly to let.

preferred dimensions the host cell is therefore characterized by being the protein of the invention or a fragment or derivative thereof secreted into the surrounding medium. A further subject of the invention thus provide host cells which are characterized in that they are the protease or a Fragment of the same secrete into the host cell surrounding medium.

In Another preferred embodiment is the invention Host cell characterized in that it is a bacterium, in particular one which is selected from the group of genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas.

Especially Preferably, 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 can meet their requirements the cultural conditions have changed, others or additional Have selection markers or other or additional Express proteins. It may in particular be such host cells acting in addition to the protein produced according to the invention even more, especially economically interesting proteins express.

The However, the host cell can also be a eukaryotic cell that is characterized characterized in that it has a nucleus. Another one The invention therefore provides a host cell which is characterized characterized in that it has a nucleus.

in the Unlike prokaryotic cells are eukaryotic cells able to posttranslationally modify the protein formed. Examples are fungi such as Actinomycetes or yeasts like Saccharomyces or Kluyveromyces. This can be, for example be particularly beneficial if the proteins associated with to undergo specific modifications of their synthesis, the like Enable systems. Among the modifications, the eukaryotic Perform systems especially in the context of protein synthesis, For example, include the binding of low molecular weight compounds like membrane anchors or oligosaccharides. Such oligosaccharide modifications For example, to reduce the allergenicity be desirable. Also a coexpression with the naturally enzymes formed by such cells, such as cellulases, can be beneficial. Thermophilic fungal expression systems For example, they are particularly suitable for the expression of temperature-resistant Enzymes or enzyme variants.

The Host cells according to the invention are in themselves cultivated and fermented in a known manner, for example in discontinuous or continuous systems. In the first case will be a suitable one Nutrient medium inoculated with the host cells and the product after an experimentally determined period of time from the medium harvested. Continuous fermentations are characterized by reaching a steady state in which over a relatively long-term cells partly die off but regrow and simultaneously remove product from the medium can be.

fermentation process are known per se from the prior art and represent the actual large-scale technical Production step, usually followed by a suitable one Purification method of the product produced, for example of the recombinant protein. All fermentation methods based on one of the above-mentioned processes for the preparation of based recombinant proteins, make corresponding preferred Embodiments of this subject invention is.

in this connection must be used for the manufacturing processes used, for the host cells and / or the proteins to be produced each optimal conditions based on the previously optimized culture conditions of the strains concerned, according to the knowledge of the person skilled in the art, for example, fermentation volume, media composition, Oxygen supply or stirrer speed experimental be determined.

Fermentation process, which are characterized in that the fermentation over An inflow strategy is also implemented into consideration. Here are the media components, by the continuous cultivation are consumed, fed; One also speaks of a feeding strategy. hereby can bring significant increases in both the Cell density, as well as in the dry biomass and / or especially the Activity of the protein of interest can be achieved.

Analogous In addition, the fermentation can also be designed so that unwanted Metabolites filtered out or by adding buffer or each appropriate counterions are neutralized.

The produced protein can be retrofitted from the fermentation medium be harvested. This fermentation process is opposite the product preparation from the dry mass preferably requires however, the provision of suitable secretory markers and transport systems. Without secretion is possible the purification of the protein from the cell mass required, too For this purpose, various methods are known, such as precipitation to Example by ammonium sulfate or ethanol, or the chromatographic purification, if necessary to homogeneity. The majority of However, the technical procedure described is likely with a enriched, stabilized preparation manage.

All Already executed above elements can be used to process be combined to proteins of the invention manufacture. It is for each invention Protein a variety of possible combinations Process steps conceivable. The optimal procedure must be for be determined experimentally for each specific case.

By Expression or cloning can be the invention Proteases in the required for technical use Amount to be made available.

a independent subject of the invention thus also provide Process for the preparation of an inventive Protease

To Every method that comes to making a top belongs described protease invention suitable or that is the receipt of an inventive Protease allows. These include, for example also chemical synthesis methods.

In contrast, however, all of those known in the art are preferred above in some aspects already mentioned molecular biological, microbiological or biotechnological production processes, the invention referred to above Build up proteins or nucleic acids. Therefor can according to the above, for example, in the sequence listing under SEQ ID NO. 1 specified nucleic acid or from this obtained mutants or partial sequences are used.

Preferably these are procedures that are made using a previously designated nucleic acid, preferably using a as described above, and more preferably using a previously designated, advantageously genetically modified host cell done. Because this is the corresponding preferred genetic information in microbiologically utilizable Form provided.

embodiments The present invention can be described on the basis of associated nucleic acid sequences also cell-free Expression systems in which protein biosynthesis in vitro is traced. All elements already listed above can also be combined to new methods to inventive Produce proteins. It is for each invention Protein a variety of possible combinations Process steps conceivable, so that optimal process for must be determined experimentally for each specific case.

Corresponding the above, such methods are still among the methods mentioned preferred in which the nucleotide sequence in one, preferably several codons have been adapted to the codon usage of the host strain is.

a own subject of the invention represents a means by that characterized in that it is at least one inventive Protease as described above contains.

This includes all types of 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. Depending on the field of application, 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. In particular, 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 shown.

In preferred embodiments of the invention is an inventive Agent characterized in that it contains a detergent, hand detergent, Dishwashing liquid, hand dishwashing detergent, dishwasher detergent, Cleaning agents, denture or contact lens care products, Rinsing agent, disinfectant, cosmetic, pharmaceutical agent or a means of treating filter media, Textiles, furs, paper, skins or leather, is.

this Subject of the invention are as preferred embodiment Attributed to funds that are detergents or cleaners is.

Because as in the embodiments of the present application was shown, could use for detergents and cleaners a preferred protease according to the invention a Performance increase over a protease-free agent be noted and a very good cleaning performance in terms on protease-sensitive stains can be achieved.

To this invention includes all conceivable types of detergents, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or at the hand laundry or cleaning. This includes For example, detergents for textiles, carpets, or Natural fibers for which according to the present invention Label detergent is used. These include, for example also dishwashing detergents for dishwashers or manual dishwashing detergent or cleaner for hard surfaces such as metal, glass, porcelain, ceramics, Tiles, stone, painted surfaces, plastics, wood or leather; for such is according to the present invention the term used cleaning agent.

One Means according to the invention can be both an agent for bulk consumers or technical users as also be a product for the private consumer, whereby all types of detergents and cleaning agents established in the prior art also embodiments of the present invention represent. These include, for example, concentrates and undiluted agent for use in commercial Scale, in the washing machine or in the hand wash or cleaning. Likewise, for example, include detergents for textiles, carpets, or natural fibers, for the term detergent according to the present invention is used. Furthermore, this includes, for example Dishwashing liquid for dishwashers or manual dishwashing detergent or cleaner for hard surfaces such as metal, glass, porcelain, ceramics, Tiles, stone, painted surfaces, plastics, wood or leather; for such is according to the present invention the term used cleaning agent.

embodiments The present invention includes all established and / or all appropriate dosage forms. These include For example, solid, powdered, liquid, gelatinous or pasty agents, if necessary can also consist of several phases as well as in compressed or uncompressed form. Another one The subject of the invention therefore represents means which are characterized are that they are present as a one-component system. Such means preferably consist of one phase. Of course However, agents according to the invention can also consist of several phases. Another subject of the invention form therefore means characterized by being in several Components are split. To the invention Dosage forms also include extrudates, granules, Tablets or pouches, both in bulk containers as may also be packaged in portions. Prefers an agent according to the invention is characterized that it contains the protease in an amount of 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, more preferably from 20 μg to 15 mg and most preferably from 50 μg to 10 mg per g of the agent.

invention Agents may be in solid form. Another one The invention therefore relates to agents which are characterized are that they are in solid form. In a preferred embodiment the agent is a free-flowing powder with a bulk density from 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l, before.

alternative agents according to the invention can also be liquid or be pasty. Another object of the invention the agent is therefore characterized in that it is pasty or liquid form, in particular in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of a aqueous liquid detergent or a hydrous paste.

The Composition according to the invention, in particular washing or Detergent may be in a container, preferably an air-permeable container, be packed, from it just before use or during the washing process is released, in particular, the protease and / or further Ingredients of the agent with a room temperature or at Absence of water impermeable to the enzyme Substance be wrapped, which under application conditions the agent becomes permeable to the enzyme. The Means is thus characterized in that the protease with a at room temperature or in the absence of water for the protease impermeable substance envelops is.

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. Other enzymes which can be used as further enzymes are all enzymes which can display 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. Preferably, 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. % contained in agents according to the invention, wherein each enzyme contained can be present in the stated proportions.

The Protein concentration can be determined using known methods, for example the BCA process (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method (A.G. Gornall, C.S. Bardawill and M. M. David, J. Biol. Chem., 177 (1948), pp. 751-766) become. Particularly preferably, the further enzymes assist the effect of the agent, such as the cleaning performance of a Washing or cleaning agent, with regard to certain soiling or stains. Most preferably, the enzymes show synergistic Effects in terms of their effect on certain Stains or stains, d. H. those in the middle composition contained enzymes assist in their cleaning performance each other. Synergistic effects can not only be between different enzymes, but also between one or more Enzymes and other ingredients of the invention By means of occur. In a further preferred embodiment The invention is the agent according to the invention thus characterized by having at least one further enzyme containing a protease, amylase, cellulase, hemicellulase, Mannanase, tannase, xylanase, xanthanase, β-glucosidase, Carrageenase, oxidase, oxidoreductase or a lipase is.

The enzymes used in agents according to the invention are either originally from microorganisms, about genera Bacillus, Streptomyces, Humicola, or Pseudomonas, and / or are carried out by biotechnological methods known per se produces suitable microorganisms, for example by transgenic Expression hosts of the genera Bacillus or by filamentous Fungi.

In agents according to the invention, the protease according to the invention is combined, for example, with one or more of the following ingredients: nonionic, anionic and / or cationic surfactants, (optionally further) bleaching agents, bleach activators, bleach catalysts, builders and / or cobuilders, acids, alkaline substances, hydrotropes, solvents , Thickeners, sequestering agents, electrolytes, optical brighteners, grayness inhibitors, corrosion inhibitors, in particular silver protectants (silver corrosion inhibitors), soil release agents, color transfer (or transfer) inhibitors, foam inhibitors, abrasives, dyes, perfumes, perfumes, antimicrobial agents, UV- Protective agents or absorbents, An tistatics, pearlescers and skin protection agents, enzymes such as protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase or a lipase, stabilizers, especially enzyme stabilizers, and other components derived from the State of the art are known. In a further embodiment of the invention, an agent according to the invention is therefore characterized in that it contains at least one further component selected from the group consisting of surfactants, builders, acids, alkaline substances, hydrotropes, solvents, thickeners, bleaching agents, dyes, perfumes, corrosion inhibitors , Sequestering agents, electrolytes, optical brighteners, grayness inhibitors, silver corrosion inhibitors, color transfer inhibitors, foam inhibitors, abrasives, UV absorbers, solvents, antistatic agents, pearlescers and skin protection agents.

The to select ingredients as well as the conditions under which the agent is used, such as temperature, pH, ionic strength, redox ratios or mechanical influences, should be for the respective Cleaning problem to be optimized. So are usual temperatures for detergents and cleaners in the range of 10 ° C with manual means over 40 ° C and 60 ° C up to 95 ° for mechanical means or technical Applications. As in modern washing and dishwasher the Temperature is usually infinitely adjustable, are also all intermediate stages the temperature included. Preferably, the ingredients become of the funds concerned. Synergies are preferred in terms of cleaning performance. Especially preferred in this regard are synergies that are in a temperature range between 20 ° C and 60 ° C are present, as well as in the inventive Agents contained in this temperature range catalytically is active.

• – 5 Gew.-% bis 70 Gew.-%, insbesondere 5 Gew.-% bis 30 Gew.-% Tenside und/oder
• – 10 Gew.-% bis 65 Gew.-%, insbesondere 12 Gew.-% bis 60 Gew.-% wasserlösliches oder wasserdispergierbares anorganisches Buildermaterial und/oder
• – 0,5 Gew.-% bis 10 Gew.-%, insbesondere 1 Gew.-% bis 8 Gew.-%, wasserlösliche organische Buildersubstanzen und/oder
• – 0,01 bis 15 Gew.-% feste anorganische und/oder organische Säuren beziehungsweise saure Salze und/oder
• – 0,01 bis 5 Gew.-% Komplexbildner für Schwermetalle und/oder
• – 0,01 bis 5 Gew.-% Vergrauungsinhibitor und/oder
• – 0,01 bis 5 Gew.-% Farbübertragungsinhibitor und/oder
• – 0,01 bis 5 Gew.-% Schauminhibitor.

In a further preferred embodiment, an agent according to the invention, in particular a washing or cleaning agent, furthermore comprises

• - 5 wt .-% to 70 wt .-%, in particular 5 wt .-% to 30 wt .-% of surfactants and / or
• From 10% by weight to 65% by weight, in particular from 12% by weight to 60% by weight, of water-soluble or water-dispersible inorganic builder material and / or
• From 0.5% by weight to 10% by weight, in particular from 1% by weight to 8% by weight, of water-soluble organic builders and / or
• 0.01 to 15% by weight of solid inorganic and / or organic acids or acid salts and / or
• 0.01 to 5% by weight complexing agent for heavy metals and / or
• 0.01 to 5% by weight of grayness inhibitor and / or
• 0.01 to 5% by weight of color transfer inhibitor and / or
• - 0.01 to 5 wt .-% foam inhibitor.

optional the agent may further comprise optical brighteners. The optical Brighteners are preferably used from 0.01 to 5 wt .-%.

The Protease activity in such agents may occur after in Surfactants, Vol. 7 (1970), pp. 125-132 be determined. It is accordingly in PE (protease units) specified.

at comparing the performance of two detergent enzymes, such as in the examples of the present application, must between distinguished from the same and the same activity become. Especially when genetically engineered, largely free of side activity Preparations the same protein use is appropriate. Because that is a statement about whether it is possible the same amount of protein - as a measure of the yield of fermentative production - comparable Results. Gap the respective conditions from active substance to total protein (the values of the specific activity) apart, so is an activity-same comparison too recommend because this is the respective enzymatic properties be compared. Generally speaking, a low specific Activity by adding a larger one Protein amount can be compensated. This is ultimately for an economic consideration.

a own subject invention represents the use of an above-described agent according to the invention for the removal of protease-sensitive Soiling on textiles or hard surfaces, d. H. for cleaning textiles or hard surfaces, represents.

For 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 ma Schinelle method.

In a preferred embodiment of this use the relevant agents according to the invention, preferably Washing or cleaning agent, according to one of the above Embodiment provided.

a another own subject of the invention provide methods for cleaning textiles or hard surfaces in which at least in one of the method steps an inventive Medium is used. The procedure for cleaning textiles or hard surfaces is thus characterized that in at least one method step, an inventive Medium is applied.

this includes fall both manual and mechanical procedures, whereby machine Method because of their more precise controllability, what For example, the amounts used and exposure times, preferably are.

method for the cleaning of textiles are generally characterized from that in several process steps different cleaning active Substances applied to the items to be cleaned and after the exposure time be washed off, or that the items to be cleaned in any other way with a detergent or a solution of this agent is treated. The same applies to procedures for cleaning of all materials other than textiles, which are covered by the term hard surfaces are summarized. All conceivable Washing or cleaning methods can be used in at least one the process steps to an inventive Means are enriched, and then provide embodiments of the present invention.

In Another preferred embodiment of such methods become the respective enzymes of the invention in the context of one of the above recipes for Composition according to the invention, preferably according to the invention Detergents or cleaning agents provided.

There preferred enzymes of the invention naturally already have a protein-dissolving activity and they also unfold in media, which otherwise no cleaning power own, such as in bare buffer, can single sub-step of such a method for machine Cleaning of textiles is that, if desired in addition to stabilizing compounds, salts or buffer substances as only cleaning-active component an inventive Enzyme is applied. This constitutes a particularly preferred embodiment of the present invention.

In Another embodiment of the invention is the method for cleaning textiles or hard surfaces therefore characterized in that in at least one method step a protease according to any one of claims 1 to 11 proteolytically is active. The protease is preferably used in an amount of 40 μg to 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g, and most preferably from 200 μg used up to 1 g per application.

preferred Embodiments of this subject invention provide Process for the treatment of textile raw materials or for textile care in which in at least one of the method steps an inventive Alkaline protease becomes active.

this includes are processes for textile raw materials, fibers or textiles preferred with natural ingredients, and especially for those with wool or silk.

It This may be, for example, processes in which materials prepared for processing in textiles, such as anti-fungal finishing, or, for example, methods which carried the cleaning Enrich textiles with a nourishing component. Because of the above described effect of proteases on natural, protein-containing Raw materials are in preferred embodiments to processes for the treatment of textile raw materials, fibers or textiles with natural ingredients, especially with wool or Silk.

invention Enzymes are as described above advantageous in agents according to the invention, in particular Detergents and cleaners, and processes, in particular washing and cleaning Cleaning method, can be used. You can use this be proteinaceous of textiles or hard surfaces Eliminate impurities. Provide embodiments for example, hand washing, manual removal of patches of textiles or of hard surfaces or the use in connection with a machine process represents.

a Another object of the invention is therefore the use of a protease according to the invention, as described above has been described for cleaning textiles or hard surfaces. Preferably, the protease is added in an amount of 40 μg 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g, and most preferably from 200 μg used up to 1 g per application.

In a preferred embodiment of this use the respective inventive alkaline Proteases in the context of one of the above formulations for agents according to the invention, preferably Detergents or cleaning agents provided.

A further embodiment of this subject invention provides the use of an alkaline according to the invention Protease for activating or deactivating ingredients of Detergents or cleaners.

Because As is known, protein components of washing or detergents inactivated by the action of a protease become. This otherwise rather undesirable effect targeted is also an object of the present invention. Likewise, as described above, it is possible that by proteolysis another component is first activated, such as when it is a hybrid protein from the actual enzyme and the appropriate inhibitor. Another example of such regulation is, in which an active component to protect or control his Activity encapsulated in a material is present through Proteolysis is attacked. invention Proteins can thus be converted into inactivation, activation or release reactions, especially in multiphase Means.

• – Die Verwendung einer erfindungsgemäßen Protease zur Gewinnung oder Behandlung von Rohmaterialien oder Zwischenprodukten in der Textilherstellung, insbesondere zum Entfernen von Schutzschichten auf Geweben;
• – die Verwendung einer erfindungsgemäßen Protease zur Behandlung von Textilrohstoffen oder zur Textilpflege und hierunter bevorzugt
• – die entsprechende Verwendung für Textilrohstoffe, Fasern oder Textilien mit natürlichen Bestandteilen und ganz besonders für solche mit Wolle oder Seide.

In accordance with the foregoing, the following uses are also embodiments of the present invention:

• The use of a protease according to the invention for the recovery or treatment of raw materials or intermediates in textile production, in particular for the removal of protective layers on fabrics;
• The use of a protease according to the invention for the treatment of textile raw materials or for textile care and is preferred among these
• - the corresponding use for textile raw materials, fibers or textiles with natural components and especially for those with wool or silk.

The The present invention is also in the form of such an inventive Alkaline protease-containing agents realized in which it is cosmetics. Below are all types of cleansing and nourishing agents for human skin or hair, especially cleansers.

Because proteases also play a crucial role in the cell renewal process of human skin (desquamation) ( T. Egelrud et al., Acta Derm. Venerol., Vol. 71 (1991), pp. 471-474 ). Accordingly, 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. In this regard, it is possible, for example, the use of subtilisin proteases, for example, variants of the alkaline protease from Bacillus lentus with amino acid substitutions in the positions R99G / A / S, S154D / E and / or L211D / E, for cosmetic purposes. As explained above, proteases according to the invention can be further developed via the corresponding point mutations. Thus, 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 modified by point mutations at highly allergenic positions so that they have a higher skin compatibility.

Accordingly also appropriate cosmetic cleaning and care procedures and the use of such proteolytic enzymes in cosmetic Purposes included in this subject of the invention, in particular in appropriate means, such as shampoos, soaps or washing lotions, or in care products, for example, in the form of creams offered become. Also use in a peeling drug or for its production is included in this article.

• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur biochemischen Analyse oder zur Synthese von niedermolekularen Verbindungen oder von Proteinen;
• – darunter bevorzugt die Verwendung zur Endgruppenbestimmung im Rahmen einer Peptid-Sequenzanalyse;
• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur Präparation, Reinigung oder Synthese von Naturstoffen oder biologischen Wertstoffen, vorzugsweise im Rahmen entsprechender Mittel oder Verfahren;
• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur Synthese von Proteinen oder anderen niedermolekularen chemischen Verbindungen;
• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur Behandlung von natürlichen Rohstoffen, insbesondere zur Oberflächenbehandlung, ganz besonders in einem Verfahren zur Behandlung von Leder, vorzugsweise im Rahmen entsprechender Mittel oder Verfahren;
• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur Behandlung von photographischen Filmen, insbesondere zur Entfernung von gelatinhaltigen oder ähnlichen Schutzschichten; und
• – die Verwendung einer erfindungsgemäßen Alkalischen Proteasen zur Herstellung von Lebensmitteln oder von Futtermitteln.

In addition to the use in detergents and cleaners and cosmetics numerous applications of proteases, in particular subtilases are established in the prior art. An overview here For example, ber offers the manual "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 , All of these techniques can be enriched according to the invention alkaline proteases. Should it turn out that they can be further developed by the use of proteases according to the invention, they are included in the scope of protection of the present invention. These include in particular the following fields of application:

• The use of an alkaline protease according to the invention for the biochemical analysis or for the synthesis of low molecular weight compounds or of proteins;
• - including preferred use for end-group determination in a peptide sequence analysis;
• The use of an alkaline protease according to the invention for the preparation, purification or synthesis of natural substances or biological valuable substances, preferably in the context of appropriate agents or processes;
• The use of an alkaline protease according to the invention for the synthesis of proteins or other low-molecular-weight chemical compounds;
• The use of an alkaline protease according to the invention 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;
• The use of an alkaline protease according to the invention for the treatment of photographic films, in particular for the removal of gelatin-containing or similar protective layers; and
• The use of an alkaline protease according to the invention for the production of food or feed.

in principle becomes the use of alkaline according to the invention Proteases in all other technical fields in the protection area included in the present application for which it is turns out to be suitable.

The previously described inventive agent or those according to the invention described above Process prepared means may contain further Ingredients such as other enzymes, enzyme stabilizers, surfactants, z. Nonionic, anionic and / or amphoteric surfactants, and / or bleaches, and / or builder, and optionally further customary Ingredients. In particular, combinations of the invention Enzymes with selected other ingredients of the agents prove to be advantageous and are therefore particularly preferred. Particularly advantageous effects are shown when the inventive Enzymes with builders (hereinafter also referred to as Builder or builders), surfactants, bleaches or be combined with other enzymes. Such advantageous combinations, the improved cleaning performance by resulting synergisms will be described below.

Combination inventive Enzymes with builders

By the combination with selected builders, which will be described in more detail below, becomes a synergistic one Cleaning performance of the agent achieved. This means that Means an improved removal of stains, for example proteinaceous soils, compared with agents, either only one of the two components, ie enzymes or builders, include, or even compared to the expected cleaning performance a means with both components due to the mere Addition of the respective individual contributions of these two components for cleaning performance of the agent. Thus, the selected represents Combination of enzymes according to the invention the builders an essential aspect of the invention which in the agents according to the invention a synergistic Have effect.

To In this regard, advantageous builders include in particular the zeolites, silicates, carbonates, organic cobuilders and - where no ecological prejudices against her Use exist - also the phosphates.

The finely crystalline, synthetic and bound water-containing zeolite used is preferably zeolite A and / or P. As zeolite P, zeolite ^MAP® (commercial product from Crosfield) is particularly preferred. Also suitable, however, are zeolite X and mixtures of A, X and / or P. Commercially available and preferably usable in the context of the present invention is, for example, a cocrystal of zeolite X and zeolite A (about 80% by weight of zeolite X) ), by the formula nNa ₂ O · (1 - n) K ₂ O · Al ₂ O ₃ · (2 - 2.5) SiO ₂ · (3.5-5.5) H ₂ O can be described. The zeolite can be used both as a builder in a granular compound and for a kind of "powdering" of a granular mixture, preferably a mixture to be compressed, whereby usually both ways of incorporating the zeolite into the premix are used an average particle size of less than 10 microns (volume distribution, measuring method: Coulter Counter) and preferably contain 18 to 22 wt .-%, in particular 20 to 22 wt .-% of bound water.

Preference is also given to using crystalline layer-form silicates of the general formula NaMSi _x O _{2x + 1} .yH ₂ O, in which M represents sodium or hydrogen, x represents from 1.9 to 22, preferably from 1.9 to 4, with particularly preferred Values for x are 2, 3 or 4, and y is a number from 0 to 33, preferably from 0 to 20. The crystalline layer-form silicates of the formula NaMSi _x O _{2x + 1} .yH ₂ O are for example sold by Clariant GmbH (Germany) under the trade name Na-SKS. Examples of these silicates are 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).

Particularly suitable for the purposes of the present invention are crystalline phyllosilicates of the formula NaMSi _x O _{2x + 1} .yH ₂ O, in which x is 2. In particular, both β- and δ-sodium disilicates are Na ₂ Si ₂ O ₅ .yH ₂ O and furthermore especially Na-SKS-5 (α-Na ₂ Si ₂ O ₅ ), Na-SKS-7 (β-Na ₂ Si ₂ O _5, natrosilite), Na-SKS-9 (NaHSi ₂ O ₅ · H ₂ O), Na-SKS-10 (NaHSi ₂ O ₅ · 3H ₂ O, kanemite), Na-SKS-11 (t- Na ₂ Si ₂ O ₅ ) and Na-SKS-13 (NaHSi ₂ O ₅ ), but especially Na-SKS-6 (δ-Na ₂ Si ₂ O ₅ ).

Detergents or cleaning agents preferably contain a weight fraction of the crystalline layered silicate of the formula NaMSi _x O _{2x + 1} · yH ₂ O of from 0.1 to 20% by weight, preferably from 0.2 to 15% by weight and in particular of 0 , 4 to 10 wt .-%, each based on the total weight of these agents.

It is also possible to use amorphous sodium silicates with a Na ₂ O: SiO ₂ modulus of from 1: 2 to 1: 3.3, preferably from 1: 2 to 1: 2.8 and in particular from 1: 2 to 1: 2.6, which preferably delayed release and have secondary washing properties. The dissolution delay compared with conventional amorphous sodium silicates may have been caused in various ways, for example by surface treatment, compounding, compaction / densification or by overdrying. In the context of this invention, the term "amorphous" is understood to mean that the silicates do not yield sharp X-ray reflections typical of crystalline substances in X-ray diffraction experiments, but at most one or more maxima of the scattered X-rays having a width of several degrees of diffraction angle , cause.

alternative or in combination with the aforementioned amorphous sodium silicates X-ray amorphous silicates are used whose silicate particles fuzzy or even sharp in electron diffraction experiments Provide diffraction maxima. This is to be interpreted as meaning that Products microcrystalline size ten up to several hundred nanometers (nm), where values up to max. 50 nm and in particular up to max. 20 nm are preferred. such X-ray amorphous silicates also have a dissolution delay compared to conventional water glasses on. Particularly preferred are compacted / compacted amorphous Silicates, compounded amorphous silicates and overdried X-ray amorphous silicates.

in the Within the scope of the present invention, it is preferred that this (s) Silicate (s), preferably alkali metal silicates, particularly preferably crystalline or amorphous alkali disilicates, in detergents or cleaners in amounts of from 3 to 60% by weight, preferably from 8 to 50% by weight and in particular from 20 to 40 wt .-%, each based on the weight of the washing or cleaning agent are included.

Of course is also a use of the well-known phosphates as builders (Builders) possible, if such use not be avoided for environmental reasons should. Among the variety of commercially available Phosphates are the alkali metal phosphates with particular preference of pentasodium or pentapotassium triphosphate (sodium or potassium tripolyphosphate) preferred according to the invention.

Alkali metal phosphates is the summary term for the alkali metal (especially sodium and potassium) salts of various phosphoric acids, in which one can distinguish metaphosphoric acids (HPO ₃ ), and orthophosphoric H ₃ PO _{4 in} addition to high molecular weight representatives. The phosphates combine several advantages: they act as alkali carriers, prevent lime deposits on machine parts or lime incrustations in fabrics and contribute to the cleaning performance.

Particularly important phosphates are the pentasodium triphosphate, Na ₅ P ₃ O ₁₀ (sodium tripolyphosphate) and the corresponding potassium salt pentapotassium triphosphate, K ₅ P ₃ O ₁₀ (potassium tripolyphosphate). The sodium potassium tripolyphosphates are also preferably used according to the invention.

Become in the context of the present application phosphates as washing or cleaning active Substances used in detergents or cleaners, so included preferred agents are said phosphate (s), preferably alkali metal phosphate (s), particularly preferably pentasodium or pentapotassium triphosphate (sodium or potassium tripolyphosphate), in amounts of 5 to 80 wt .-%, preferably from 15 to 75% by weight, in particular, from 20 to 70% by weight, respectively based on the weight of the washing or cleaning agent.

Further builders usable according to the invention are the alkali carriers. To be considered as alkali carriers for example, alkali metal hydroxides, alkali metal carbonates, alkali metal bicarbonates, Alkali metal sesquicarbonates, the aforesaid alkali silicates, alkali metal silicates, and mixtures of the aforementioned substances, wherein for the purposes of this invention preferably the alkali metal carbonates, in particular sodium carbonate, sodium bicarbonate or sodium sesquicarbonate. Especially preferred is a builder system containing a mixture of tripolyphosphate and sodium carbonate. Because of their compared with other builders low chemical compatibility with the rest Ingredients of detergents or cleaning agents, the alkali metal hydroxides preferably only in small amounts, preferably in amounts below 10 Wt .-%, preferably below 6 wt .-%, more preferably below 4 wt .-% and in particular below 2 wt .-%, each based on the total weight of the detergent or cleaning agent used. Especially preferred are agents which are less based on their total weight contain as 0.5 wt .-% and in particular no alkali metal hydroxides.

Especially preference is given to the use of carbonate (s) and / or bicarbonate (s), preferably alkali metal carbonate (s), more preferably sodium carbonate, in amounts of from 2 to 50% by weight, preferably from 5 to 40% by weight and in particular from 7.5 to 30 wt .-%, each based on the Weight of washing or cleaning agent. Particularly preferred Means, based on the weight of the detergent or cleaning agent less than 20% by weight, preferably less than 17% by weight, is preferred less than 13% by weight and in particular less than 9% by weight of carbonate (s) and / or bicarbonate (s), preferably alkali carbonate (s), especially preferably contain sodium carbonate.

When Organic co-builders are in particular polycarboxylates / polycarboxylic acids, polymeric polycarboxylates, aspartic acid, polyacetals, Dextrins, other organic cobuilders (see below) and phosphonates to call. These classes of substances are described below.

Further Organic builders usable according to the invention are, for example, in the form of the free acid and / or their sodium salts usable polycarboxylic acids, wherein Under polycarboxylic acids such carboxylic acids understood which carry more than one acidity function. For example these are citric acid, adipic acid, succinic acid, Glutaric, malic, tartaric, maleic, Fumaric acid, sugar acids, aminocarboxylic acids, Nitrilotriacetic acid (NTA), if such use For ecological reasons not to complain about is, as well as mixtures of these. Own the free acids In addition to their builder effect typically also the property of a Acid component and thus also serve for adjustment a lower and milder pH of detergents or cleaners. In particular, here are citric acid, succinic acid, Glutaric acid, adipic acid, gluconic acid and to name any mixtures of these.

When Builders are further suitable polymeric polycarboxylates, These are, for example, the alkali metal salts of polyacrylic acid or polymethacrylic acid, for example those with a molecular weight of 500 to 70000 g / mol.

For the purposes of this document, the molecular weights stated for polymeric polycarboxylates are weight-average molar masses M _{w of} the particular acid form, which were determined in principle by means of gel permeation chromatography (GPC), a UV detector being used. The measurement was carried out against an external polyacrylic acid standard, which provides realistic molecular weight values due to its structural relationship with the polymers investigated. These data differ significantly from the molecular weight data, in which polystyrene sulfonic acids are used as standard. The molar masses measured against polystyrenesulfonic acids are generally significantly higher than the molecular weights specified in this document.

Suitable polymers are in particular polyacrylates, which preferably have a molecular weight of 2,000 to 20,000 g / mol. Because of their superior solubility, the short-chain polyacrylates, which have molar masses of from 2000 to 10000 g / mol, and particularly preferably from 3000 to 5000 g / mol, may again be preferred from this group.

Suitable are furthermore copolymeric polycarboxylates, especially those of Acrylic acid with methacrylic acid and acrylic acid or methacrylic acid with maleic acid. As special suitable copolymers of acrylic acid with maleic acid proven, the 50 to 90 wt .-% acrylic acid and 50 to 10 Wt .-% maleic acid. Your molecular weight, based on free acids, is generally 2000 to 70000 g / mol, preferably 20,000 to 50,000 g / mol and in particular 30,000 to 40,000 g / mol.

The (co) polymeric polycarboxylates can be used either as a powder or used as an aqueous solution. The content of detergents or cleaning agents on (co) polymeric polycarboxylates is preferably 0.5 to 20 wt .-%, in particular 3 to 10% by weight.

to The water solubility can be improved Polymers also allylsulfonic acids such as allyloxybenzenesulfonic acid and methallylsulfonic acid, as a monomer.

Especially Biodegradable polymers of more than two are also preferred various monomer units, for example, those as monomers Salts of acrylic acid and maleic acid and vinyl alcohol or vinyl alcohol derivatives or the salts of acrylic acid as monomers and the 2-alkylallylsulfonic acid and sugar derivatives.

Further preferred copolymers are those which are used as monomers acrolein and Acrylic acid / acrylic acid salts or acrolein and vinyl acetate exhibit.

As well as further preferred builder substances are polymeric aminodicarboxylic acids, to mention their salts or their precursors. Especially Polyaspartic acids or their salts are preferred.

Further Suitable builders are polyacetals, which by reaction of dialdehydes with polyolcarboxylic acids, which are 5 to 7 C atoms and at least 3 hydroxyl groups can be obtained. Preferred polyacetals are selected from dialdehydes such as glyoxal, glutaraldehyde, Terephthalaldehyde and mixtures thereof and from Polyolcarbonsäuren such as gluconic acid and / or glucoheptonic acid.

Further suitable organic builders are dextrins, for example Oligomers or polymers of carbohydrates by partial Hydrolysis of starches can be obtained. The hydrolysis can be carried out by customary, for example acidic or enzyme-catalyzed processes. Preferably, they are hydrolysis products with middle Molar masses in the range of 400 to 500,000 g / mol. This is a polysaccharide with a dextrose equivalent (DE) in the range of 0.5 to 40, in particular from 2 to 30 preferred, DE being a common Measure of the reducing effect of a polysaccharide compared to dextrose, which has a DE of 100. Useful are both maltodextrins with a DE between 3 and 20 and dry glucose syrups with a DE between 20 and 37 as well as so-called yellow dextrins and white dextrins with higher molecular weights in the range from 2000 to 30,000 g / mol.

at the oxidized derivatives of such dextrins are their reaction products with oxidants, which are able are at least one alcohol function of the saccharide ring to the carboxylic acid function to oxidize.

Also Oxydisuccinates and other derivatives of disuccinates, preferably Ethylenediamine disuccinate are other suitable cobuilders. there ethylenediamine-N, N'-disuccinate (EDDS) is preferably used in the form of its sodium or magnesium salts used. Further preferred are in this Also related glycerol disuccinates and glycerol trisuccinates. Suitable amounts are zeolithhaltigen and / or silicate-containing Formulations at 3 to 15 wt .-%.

Further useful organic cobuilders are, for example, acetylated Hydroxycarboxylic acids or their salts, which optionally may also be present in Lactonform and which at least 4 carbon atoms and at least one hydroxy group and maximum contain two acid groups.

About that In addition, all connections that are able to Complexes with alkaline earth ions form, in addition as Builders are used.

Combination inventive Enzymes with surfactants

Also by combining with selected surfactants which will be described in more detail below, will be a synergistic Cleaning performance of the agent achieved. Also by the combination of enzymes according to the invention with such surfactants It is achieved that the agent improved removal of stains causes compared with funds that either only one of the two Components, ie enzymes or surfactants, include, or in the Comparison to the expected cleaning performance of one agent with both Components due to the mere addition of each Individual contributions of these two components to the cleaning performance of the agent. Thus, also represents the selected combination of enzymes according to the invention with the surfactants an essential aspect of the invention, which in inventive Agents have a synergistic effect.

following the surfactants suitable for this purpose are described in more detail, subdivided into nonionic, anionic, cationic and amphoteric Surfactants.

Suitable nonionic surfactants are 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 is the symbol which 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 any number between 1 and 10; preferably x is 1.2 to 1.4.

A another class of preferred nonionic surfactants to be used, either as the sole nonionic surfactant or in combination with other nonionic surfactants are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably having 1 to 4 carbon atoms in the alkyl chain.

Also nonionic surfactants of the amine oxide type, for example N-cocoalkyl-N, N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamide may be suitable. The amount of these nonionic surfactants is preferably not more than that of the ethoxylated Fatty alcohols, especially not more than half of them.

in der R für einen aliphatischen Acylrest mit 6 bis 22 Kohlenstoffatomen, R¹ für Wasserstoff, einen Alkyl- oder Hydroxyalkylrest mit 1 bis 4 Kohlenstoffatomen und [Z] für einen linearen oder verzweigten Polyhydroxyalkylrest mit 3 bis 10 Kohlenstoffatomen und 3 bis 10 Hydroxylgruppen steht. Bei den Polyhydroxyfettsäureamiden handelt es sich um bekannte Stoffe, die üblicherweise durch reduktive Aminierung eines reduzierenden Zuckers mit Ammoniak, einem Alkylamin oder einem Alkanolamin und nachfolgende Acylierung mit einer Fettsäure, einem Fettsäurealkylester oder einem Fettsäurechlorid erhalten werden können.Further suitable surfactants are polyhydroxy fatty acid amides of the formula

wherein R is an aliphatic acyl radical having 6 to 22 carbon atoms, R ^{1 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. The polyhydroxy fatty acid amides are known substances which can usually be obtained by reductive amination of a reducing sugar with ammonia, an alkylamine or an alkanolamine and subsequent acylation with a fatty acid, a fatty acid alkyl ester or a fatty acid chloride.

in der R für einen linearen oder verzweigten Alkyl- oder Alkenylrest mit 7 bis 12 Kohlenstoffatomen, R¹ für einen linearen, verzweigten oder zyklischen Alkylrest oder einen Arylrest mit 2 bis 8 Kohlenstoffatomen und R² für einen linearen, verzweigten oder zyklischen Alkylrest oder einen Arylrest oder einen Oxy-Alkylrest mit 1 bis 8 Kohlenstoffatomen steht, wobei C_1-4-Alkyl- oder Phenylreste bevorzugt sind und [Z] für einen linearen Polyhydroxyalkylrest steht, dessen Alkylkette mit mindestens zwei Hydroxylgruppen substituiert ist, oder alkoxylierte, vorzugsweise ethoxylierte oder propxylierte Derivate dieses Restes.The group of polyhydroxy fatty acid amides also includes compounds of the formula

R is a linear or branched alkyl or alkenyl radical having 7 to 12 carbon atoms, R ^{1 is} a linear, branched or cyclic alkyl radical or an aryl radical having 2 to 8 carbon atoms and R ^{2 is} a linear, branched or cyclic alkyl radical or an aryl radical or an oxyalkyl radical having 1 to 8 carbon atoms, with C _1-4 alkyl or phenyl radicals being preferred and [Z] being a linear polyhydroxyalkyl radical whose alkyl chain is substituted by at least two hydroxyl groups, or alkoxylated, preferably ethoxylated or propoxylated Derivatives of this residue.

[Z] is preferably by reductive amination of a reduced sugar obtained, for example, glucose, fructose, maltose, lactose, galactose, Mannose or xylose. The N-alkoxy or N-aryloxy-substituted Compounds can be prepared by reaction with fatty acid methyl esters in the presence of an alkoxide as a catalyst in the desired Polyhydroxyfatty acid amides are transferred.

Also preferred surfactants are nonionic surfactants from the group of alkoxylated alcohols. These are preferably alkoxylated, advantageously ethoxylated, in particular 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 can be linear or preferably methyl-branched in the 2-position or linear and methyl-branched radicals in the mixture can contain, as they are usually present in Oxoalkoholresten. In particular, however, alcohol ethoxylates with linear radicals of alcohols of native origin having 12 to 18 carbon atoms, for. From coconut, palm, tallow or oleyl alcohol, and on average from 2 to 8 moles of EO per mole of alcohol. The preferred ethoxylated alcohols include, for example, C _12-14 alcohols with 3 EO or 4 EO, C _9-11 alcohols with 7 EO, C _13-15 alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C _12-18 alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of C _12-14 -alcohol with 3 EO and C _12-18 -alcohol with 5 EO. The stated degrees of ethoxylation represent statistical averages, which may correspond to a particular product of an integer or a fractional number. Preferred alcohol ethoxylates have a narrow range of ethoxylates (NREs) In addition to these nonionic surfactants, it is also possible to use fatty alcohols containing more than 12 EO, examples of which are tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO.

Of particular preference are ethoxylated nonionic surfactants selected from C _6-20 monohydroxyalkanols or C _6-20 alkylphenols or C _16-20 fatty alcohols and more than 12 moles, preferably more than 15 moles and especially more than 20 moles of ethylene oxide per mole of alcohol were used. A particularly preferred nonionic surfactant is obtained from a straight-chain fatty alcohol having 16 to 20 carbon atoms (C _16-20 alcohol), preferably a C ₁₈ -alcohol and at least 12 mol, preferably at least 15 mol and especially at least 20 mol of ethylene oxide. Of these, the so-called "narrow range ethoxylates" are particularly preferred.

With Particular preference continues to be combinations of one or several tallow fatty alcohols with 20 to 30 EO and silicone defoamers used.

Especially preferred are nonionic surfactants which have a melting point above Have room temperature. Nonionic surfactant (s) having a melting point above 20 ° C, preferably above 25 ° C, more preferably between 25 and 60 ° C and in particular between 26.6 and 43.3 ° C, is / are particularly preferred.

suitable nonionic surfactants, the melting or softening points in said Have temperature range, for example, low-foaming nonionic surfactants that are solid or highly viscous at room temperature could be. Nonionic surfactants are used at room temperature are highly viscous, it is preferred that these have a viscosity above 20 Pa · s, preferably above 35 Pa · s and in particular above 40 Pa · s. Also nonionic surfactants, which have waxy consistency at room temperature are in Depending on their application preferred.

nonionic surfactants from the group of alkoxylated alcohols, particularly preferably from the group of mixed alkoxylated alcohols and in particular from the group of EO-AO-EO-Niotenside, are also with special Preference used.

The nonionic surfactant solid at room temperature preferably has propylene oxide units in the molecule. Preferably, such propylene oxide units up to 25% by weight, more preferably up to 20% by weight and especially up to 15% by weight of the total molecular weight of the nonionic surfactant out. Particularly preferred nonionic surfactants are ethoxylated Monohydroxyalkanols or alkylphenols which additionally contain polyoxyethylene-polyoxypropylene Have block copolymer units. The alcohol or alkylphenol content such nonionic surfactant molecules preferably makes more as 30 wt .-%, more preferably more than 50 wt .-% and in particular more than 70 wt .-% of the total molecular weight of such nonionic surfactants. Preferred agents are characterized by being ethoxylated and propoxylated nonionic surfactants containing the propylene oxide units in the molecule up to 25% by weight, preferably up to 20% by weight and in particular up to 15 wt .-% of the total molecular weight of the nonionic Make up surfactants.

Prefers Surfactants to be used come from the groups of the alkoxylated Nonionic surfactants, in particular the ethoxylated primary alcohols and mixtures of these surfactants structurally complicated Surfactants such as polyoxypropylene / polyoxyethylene / polyoxypropylene ((PO / EO / PO) surfactants). Such (PO / EO / PO) nonionic surfactants are also outstanding by good foam control.

Further particularly preferably used nonionic surfactants with melting points above room temperature 40 to 70% of a polyoxypropylene / polyoxyethylene / polyoxypropylene block polymer blend containing 75% by weight of a reverse block copolymer of polyoxyethylene and polyoxypropylene with 17 moles of ethylene oxide and 44 moles of propylene oxide and 25% by weight of a block copolymer of polyoxyethylene and polyoxypropylene initiated with trimethylolpropane and containing 24 moles of ethylene oxide and 99 moles of propylene oxide per mole of trimethylolpropane.

bevorzugt, in der R¹ für einen geradkettigen oder verzweigten, gesättigten oder ein- bzw. mehrfach ungesättigten C_6-24-Alkyl- oder -Alkenylrest steht; jede Gruppe R² bzw. R³ unabhängig voneinander ausgewählt ist aus -CH₃, -CH₂CH₃, -CH₂CH₂-CH₃, CH(CH₃)₂ und die Indizes w, x, y, z unabhängig voneinander für ganze Zahlen von 1 bis 6 stehen.In the context of the present invention, particularly preferred nonionic surfactants have been low foaming nonionic surfactants which have alternating ethylene oxide and alkylene oxide units. Among these, in turn, surfactants with EO-AO-EO-AO blocks are preferred, wherein in each case one to ten EO or AO groups are bonded to each other before a block of the other groups follows. Here are nichionic surfactants of the general formula

in which R ^{1 is} a straight-chain or branched, saturated or mono- or polyunsaturated C _6-24 alkyl or alkenyl radical; each group R ² or R ^{3 is} independently selected from -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ -CH ₃ , CH (CH ₃ ) ₂ and the indices w, x, y, z independently stand for integers from 1 to 6.

The preferred nonionic surfactants of the above formula can be prepared by known methods from the corresponding alcohols R ¹ -OH and ethylene or alkylene oxide. The radical R ¹ in the above formula may vary depending on the origin of the alcohol. When native sources are used, the radical R ^{1 has} an even number of carbon atoms and is usually unbranched, the linear radicals being selected from alcohols of native origin having 12 to 18 C atoms, eg. B. from coconut, palm, tallow or oleyl alcohol, are preferred. Alcohols which are accessible from synthetic sources are, for example, the Guerbet alcohols or methyl-branched or linear and methyl-branched radicals in the 2-position, as they are usually present in oxo alcohol radicals. Irrespective of the type of alcohol used to prepare the nonionic surfactants contained in the compositions, preference is given to nonionic surfactants in which R ¹ in the above formula is an alkyl radical having 6 to 24, preferably 8 to 20, particularly preferably 9 to 15 and in particular 9 to 11 Carbon atoms.

As the alkylene oxide unit which is contained in the preferred nonionic surfactants in alternation with the ethylene oxide unit, in particular butylene oxide is considered in addition to propylene oxide. But also other alkylene oxides in which R ² or R ^{3 are} independently selected from -CH ₂ CH ₂ -CH ₃ or CH (CH ₃ ) ₂ are suitable. Preference is given to using nonionic surfactants of the above formula in which R ² or R ^{3 is} a radical -CH ₃ , w and x independently of one another for values of 3 or 4 and y and z independently of one another are values of 1 or 2.

In summary, particularly preferred are nonionic surfactants having a C _9-15 alkyl group having 1 to 4 ethylene oxide units followed by 1 to 4 propylene oxide units followed by 1 to 4 ethylene oxide units followed by 1 to 4 propylene oxide units. These surfactants have the required low viscosity in aqueous solution and can be used according to the invention with particular preference, in particular as described above by their synergistic combination with enzymes according to the invention.

Surfactants of the general formula R ¹ -CH (OH) CH ₂ O- (AO) _w - (A'O) _x - (A''O) _y - (A '''O) _z -R ² in which R ¹ and R ² independently represent a straight-chain or branched, saturated or mono- or polyunsaturated C _2-40 alkyl or alkenyl radical; A, A ', A''andA''' are independently of one another a radical from the group -CH ₂ CH ₂ , -CH ₂ CH ₂ -CH ₂ , -CH ₂ -CH (CH ₃ ), -CH ₂ - CH _{2 is} -CH ₂ -CH ₂ , -CH ₂ -CH (CH ₃ ) -CH ₂ -, -CH ₂ -CH (CH ₂ -CH ₃ ); and w, x, y and z are values between 0.5 and 90, where x, y and / or z can also be 0 are preferred according to the invention.

Preference is given in particular to those end-capped poly (oxyalkylated) nonionic surfactants which are of the formula R ¹ O [CH ₂ CH ₂ O] _x CH ₂ CH (OH) R ² in addition to a radical R ¹ , which is linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon radicals having 2 to 30 carbon atoms, preferably having 4 to 22 carbon atoms, further having a linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon radical R2 Have from 1 to 30 carbon atoms, wherein x stands for values between 1 and 90, preferably for values between 40 and 80 and in particular for values between 40 and 60.

Particularly preferred are surfactants of the formula R ¹ O [CH ₂ CH (CH ₃ ) O] _x [CH ₂ CH ₂ O] _y CH ₂ CH (OH) R ² , in which R ^{1 is} a linear or branched aliphatic hydrocarbon radical having 4 to 18 carbon atoms or mixtures thereof, R ² denotes a linear or branched hydrocarbon radical having 2 to 26 carbon atoms or mixtures thereof and x for values between 0.5 and 1.5 and y is a value of at least 15.

Particular preference is furthermore given to those end-capped poly (oxyalkylated) nonionic surfactants of the formula R ¹ O [CH ₂ CH ₂ O] _x [CH ₂ CH (R ³ ) O] _y CH ₂ CH (OH) R ² , in which R ¹ and R ² independently of one another are a linear or branched, saturated or mono- or polyunsaturated hydrocarbon radical having 2 to 26 carbon atoms, R ^{3 is} independently selected from -CH ₃ , -CH ₂ CH ₃ , -CH ₂ CH ₂ -CH ₃ , CH (CH ₃ ) ₂ , but preferably represents -CH ₃ , and x and y independently of one another are values between 1 and 32, wherein nonionic surfactants with R ³ = -CH ₃ and values for x of 15 to 32 and y of 0.5 and 1.5 are very particularly preferred.

Other preferred nonionic surfactants are the end-capped poly (oxyalkylated) nonionic surfactants of the formula R ¹ O [CH ₂ CH (R ³ ) O] _x [CH ₂ ] _k CH (OH) [CH ₂ ] _j OR ² , in which R ¹ and R ^{2 are} linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon radicals having 1 to 30 carbon atoms, R ^{3 is} H or a methyl, ethyl, n-propyl, iso-propyl, n- Butyl, 2-butyl or 2-methyl-2-butyl radical, x are values between 1 and 30, k and j are values between 1 and 12, preferably between 1 and 5. When the value x ≥ 2, each R ³ in the above formula R ¹ O [CH ₂ CH (R ³ ) O] [CH ₂ ] _k CH (OH) [CH ₂ ] _j OR ^{2 may be} different. R ¹ and R ² are preferably linear or branched, saturated or unsaturated, aliphatic or aromatic hydrocarbon radicals having 6 to 22 carbon atoms, with radicals having 8 to 18 carbon atoms being particularly preferred. For the radical R ³ , H, -CH ₃ or -CH ₂ CH _{3 are} particularly preferred. Particularly preferred values for x are in the range from 1 to 20, in particular from 6 to 15.

As described above, each R ³ in the above formula may be different if x ≥ 2. As a result, the alkylene oxide unit in the square bracket can be varied. For example, when x is 3, the radical R ³ can be selected to form ethylene oxide (R ³ = H) or propylene oxide (R ³ = CH ₃ ) units which may be joined in any order, for example (EO) ( PO) (EO), (EO) (EO) (PO), (EO) (EO) (EO), (PO) (EO) (PO), (PO) (PO) (EO) and (PO) ( PO) (PO). The value 3 for x has been selected here by way of example and may well be greater, with the variation width increasing with increasing x values and including, for example, a large number (EO) groups combined with a small number (PO) groups, or vice versa ,

Particularly preferred end-capped poly (oxyalkylated) alcohols of the above formula have values of k = 1 and j = 1, so that the above formula zu R ¹ O [CH ₂ CH (R ³ ) O] _x CH ₂ CH (OH) CH ₂ OR ² simplified. In the latter formula, R ¹ , R ² and R ^{3 are} as defined above and x is from 1 to 30, preferably from 1 to 20 and in particular from 6 to 18. Particularly preferred are surfactants in which the radicals R ¹ and R ^{2 has} 9 to 14 C atoms, R ^{3 is} H and x assumes values of 6 to 15.

The specified C chain lengths and degrees of ethoxylation or alkoxylation of pre Nonionic surfactants are statistical averages that can be a whole or a fractional number for a particular product. Due to the manufacturing process, commercial products of the formulas mentioned are usually not made of an individual representative, but of mixtures, which may result in mean values for the C chain lengths as well as for the degrees of ethoxylation or degrees of alkoxylation and subsequently broken numbers.

Of course Not only can the aforementioned nonionic surfactants as individual substances, but also as surfactant mixtures of two, three, four or more surfactants are used. As surfactant mixtures are not mixtures of nonionic surfactants, in their entirety under one of the above general formulas but rather such mixtures, the two, three, four or contain more nonionic surfactants that are different by the above general formulas can be described.

As anionic surfactants, for example, those of the sulfonate and sulfates type can be used according to the invention. The surfactants of the sulfonate type are preferably C _9-13 -alkylbenzenesulfonates, olefinsulfonates, ie mixtures of alkene and hydroxyalkanesulfonates and disulfonates, as are obtained, for example, from C _12-18 -monoolefins having terminal or internal double bonds by sulfonation with gaseous sulfur trioxide and subsequent alkaline or acid hydrolysis of the sulfonation products into consideration. Also suitable are alkanesulfonates which are obtained from C _12-18 alkanes, for example by sulfochlorination or sulfoxidation with subsequent hydrolysis or neutralization. Likewise, the esters of α-sulfo fatty acids (ester sulfonates), z. As the α-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids suitable.

Further Suitable anionic surfactants are sulfated fatty acid glycerol esters. Among fatty acid glycerol esters are the mono-, di- and triesters As well as their mixtures to understand, as in the production through Esterification of a monoglycerine with 1 to 3 moles of fatty acid or in the transesterification of triglycerides with 0.3 to 2 moles of glycerol to be obtained. Preferred sulfated fatty acid glycerol esters are the sulfonation of saturated fatty acids with 6 to 22 carbon atoms, for example caproic acid, Caprylic acid, capric acid, myristic acid, Lauric acid, palmitic acid, stearic acid or behenic acid.

Alk (en) ylsulfates are the alkali metal salts and in particular the sodium salts of the sulfuric monoesters of C ₁₂ -C ₁₈ fatty alcohols, for example coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol or the C ₁₀ -C ₂₀ oxo alcohols and those half-esters of secondary alcohols of these chain lengths are preferred. Also preferred are alk (en) ylsulfates of said chain length, which contain a synthetic, produced on a petrochemical basis straight-chain alkyl radical, which have an analogous degradation behavior as the adequate compounds based on oleochemical raw materials. Of washing technology interest, the C ₁₂ -C ₁₆ alkyl sulfates and C ₁₂ -C ₁₅ alkyl sulfates and C ₁₄ -C ₁₅ alkyl sulfates are preferred. Also 2,3-alkyl sulfates, which can be obtained as commercial products of Shell Oil Company under the name DAN ^® , are suitable anionic surfactants.

The sulfuric acid monoesters of straight-chain or branched C _7-21 -alcohols ethoxylated with from 1 to 6 mol of ethylene oxide, such as 2-methyl-branched C _9-11- alcohols having on average 3.5 mol of ethylene oxide (EO) or C _12-18 . Fatty alcohols with 1 to 4 EO are suitable. Due to their high foaming behavior they are used only in relatively small amounts, for example in amounts of 1 to 5 wt .-%.

Further suitable anionic surfactants are also the salts of alkylsulfosuccinic acid, which are also referred to as sulfosuccinates or as sulfosuccinic acid esters and which are monoesters and / or diesters of sulfosuccinic acid with alcohols, preferably fatty alcohols and in particular ethoxylated fatty alcohols. Preferred sulfosuccinates contain C _8-18 fatty alcohol residues or mixtures of these. Particularly preferred sulfosuccinates contain a fatty alcohol residue derived from ethoxylated fatty alcohols, which by themselves are nonionic surfactants. Sulfosuccinates, whose fatty alcohol residues are derived from ethoxylated fatty alcohols with a narrow homolog distribution, are again particularly preferred. Likewise, it is also possible to use alk (en) ylsuccinic acid having preferably 8 to 18 carbon atoms in the alk (en) yl chain or salts thereof.

When other anionic surfactants are especially soaps into consideration. Suitable are saturated fatty acid soaps, such as the salts of lauric acid, myristic acid, palmitic acid, Stearic acid, hydrogenated erucic acid and behenic acid and in particular from natural fatty acids, z. As coconut, palm kernel or tallow, derived Soap mixtures.

The anionic surfactants including soaps can in the form of their sodium, potassium or ammonium salts and as soluble Salts of organic bases, such as mono-, di- or triethanolamine, are present. The anionic surfactants are preferably in the form of their sodium or potassium salts, especially in the form of the sodium salts.

At Position of said surfactants or in conjunction with them In addition, cationic and / or amphoteric surfactants are used.

worin jede Gruppe R¹ unabhängig voneinander ausgewählt ist aus C_1-6-Alkyl-, -Alkenyl- oder -Hydroxyalkylgruppen; jede Gruppe R² unabhängig voneinander ausgewählt ist aus C_8-28-Alkyl- oder -Alkenylgruppen; R³ = R¹ oder (CH₂)_n-T-R²; R⁴ = R¹ oder R² oder (CH₂)_n-T-R²; T = -CH₂-, -O-CO- oder -CO-O- und n eine ganze Zahl von 0 bis 5 ist.As such cationic active substances, for example, cationic compounds of the following formulas can be used:

wherein each R ^{1 group is} independently selected from C _1-6 alkyl, alkenyl or hydroxyalkyl groups; each R ^{2 group is} independently selected from C _8-28 alkyl or alkenyl groups; R ³ = R ¹ or (CH ₂ ) _n -TR ² ; R ⁴ = R ¹ or R ² or (CH ₂ ) _n -TR ² ; T = -CH ₂ -, -O-CO- or -CO-O- and n is an integer from 0 to 5.

In automatic dishwashing detergents, is the Content of cationic and / or amphoteric surfactants preferably less than 6% by weight, preferably less than 4% by weight, very particularly preferably less than 2% by weight and in particular less than 1% by weight. Machine dishwashing detergents which are not cationic or amphoteric surfactants are particularly preferred.

Combination inventive Enzymes with bleaches

Farther is due to the combination of enzymes according to the invention with selected bleaching agents, which are closer below be described, a synergistic cleaning performance of the agent reached. Such means provides improved removal of soiling compared with means that either only one of the two components, ie enzymes or bleaches, or compared to the expected cleaning performance of an agent with both components due to the mere addition of each Individual contributions of these two components to the cleaning performance of the agent. Thus, also represents the selected combination of enzymes according to the invention with such bleaching agents an essential aspect of the invention, which in inventive Agents have a synergistic effect.

The bleaching agents suitable for this purpose are described in more detail below. Among the compounds serving as bleaches in water H ₂ O ₂ , sodium percarbonate, sodium perborate tetrahydrate and sodium perborate monohydrate are of particular importance. Other bleaches which can be used according to the invention are, for example, peroxypyrophosphates, citrate perhydrates and peroxygenic salts or peracids which yield H ₂ O ₂ , such as perbenzoates, peroxophthalates, diperazelaic acid, phthaloimino peracid or diperdodecanedioic acid.

In addition, bleaching agents from the group of organic bleaching agents can be used. Typical organic bleaching agents are the diacyl peroxides, such as. B. dibenzoyl peroxide. Other typical organi bleaching agents are the peroxyacids, in particular the alkyl peroxyacids and the aryl peroxyacids are used. Preferred representatives are (a) the peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, (b) the aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid [phthaliminoperoxyhexanoic acid (PAP)] , o-Carboxybenzamidoperoxycaproic acid, N-Nonenylamidoperadipic Acid and N-Nonenylamidopersuccinate, and (c) aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-Diperoxycarboxylic acid, 1,9-Diperoxyazelaic acid, Diperocysebacic acid, Diperoxybrassic acid, the Diperoxyphthalic acids, 2-Decyldiperoxybutan-1,4- diacid, N, N-terephthaloyl-di (6-aminopercapronate).

Further Make used according to the invention bleach enzymatic and chemical enzymatic bleaching systems a suitable substrate is reacted by a corresponding enzyme, so that hydrogen peroxide is formed. This can then be enzymatic or chemically activated. Conversely, chemically released Hydrogen peroxide is transferred by an enzymatic system in an activated form become.

When Bleaching agents can also chlorine or bromine releasing substances be used. Among the suitable chlorine or bromine releasing Materials include, for example, heterocyclic N-bromo- and N-chloroamides, for example, trichloroisocyanuric acid, tribromoisocyanuric acid, Dibromoisocyanuric acid and / or dichloroisocyanuric acid (DICH) and / or their salts with cations such as potassium and sodium into consideration. Hydantoin compounds, such as 1,3-dichloro-5,5-dimethylhydanthoin are also suitable.

According to the invention Washing or cleaning preparations, in particular automatic dishwashing detergents, preferably 1 to 35% by weight, preferably 2.5 to 30% by weight, particularly preferably from 3.5 to 20% by weight and in particular from 5 to 15 Wt .-% bleach, preferably sodium percarbonate.

Of the Active oxygen content of detergents or cleaners, in particular the automatic dishwashing detergent, in each case based on the total weight of the composition, preferably between 0.4 and 10 wt .-%, particularly preferably between 0.5 and 8 wt .-% and in particular between 0.6 and 5 wt .-%. Especially preferred Means have an active oxygen content above 0.3 wt .-%, preferably above 0.7 wt .-%, more preferably above 0.8 wt .-% and in particular above 1.0% by weight.

According to the invention preferred bleaches which may be used further include bleach activators include, for example, when cleaning at temperatures of 60 ° C. and below to achieve a further improved bleaching effect. Surprisingly It was found that a combination of inventive Enzymes with certain, described in more detail below Bleach activators, synergistic cleaning effects of a corresponding By means causes. Such bleach activators are compounds which under perhydrolysis conditions, aliphatic peroxycarboxylic acids with preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Suitable substances are the O- and / or N-acyl groups of the mentioned C atom number and / or optionally substituted benzoyl groups wear. Preference is given to polyacylated alkylenediamines, in particular Tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated Glycolurils, in particular tetraacetylglycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular n-nonanoyl- or isononanoyloxybenzenesulfonate (n- or iso-NOBS), carboxylic acid anhydrides, especially phthalic anhydride, acylated polyhydric alcohols, especially triacetin, ethylene glycol diacetate and 2,5-diacetoxy-2,5-dihydrofuran, n-methyl-morpholinium-acetonitrile-methylsulfate (MMA) and acetylated sorbitol and mannitol or their Mixtures (SORMAN), acylated sugar derivatives, in particular pentaacetylglucose (PAG), Pentaacetylfruktose, Tetraacetylxylose and Octaacetyllactose as well as acetylated, optionally N-alkylated glucamine and gluconolactone, and / or N-acylated lactams, for example N-benzoyl-caprolactam. Hydrophilic substituted acyl acetals and acyl lactams are also used preferably used. Also combinations of conventional bleach activators can be used.

These Bleach activators are preferably used in amounts of up to 10% by weight, in particular 0.1% by weight to 8% by weight, especially 2 to 8% by weight, and especially preferably 2 to 6 wt .-%, each based on the total weight the bleach activator-containing agent used.

in der R¹ für -H, -CH₃, einen C_2-24-Alkyl- oder -Alkenylrest, einen substituierten C_2-24-Alkyl- oder -Alkenylrest mit mindestens einem Substituenten aus der Gruppe -Cl, -Br, -OH, -NH₂, -CN, einen Alkyl- oder Alkenylarylrest mit einer C_1-24-Alkylgruppe, oder für einen substituierten Alkyl- oder Alkenylarylrest mit einer C_1-24-Alkylgruppe und mindestens einem weiteren Substituenten am aromatischen Ring steht, R² und R³ unabhängig voneinander ausgewählt sind aus -CH₂-CN, -CH₃, -CH₂-CH₃, -CH₂-CH₂-CH₃, -CH(CH₃)-CH₃, -CH₂-OH, -CH₂-CH₂-OH, -CH(OH)-CH₃, -CH₂-CH₂-CH₂-OH, -CH₂-CH(OH)-CH₃, -CH(OH)-CH₂-CH₃, -(CH₂CH₂-O)_nH mit n = 1, 2, 3, 4, 5 oder 6 und X ein Anion ist.Further bleach activators preferably used in the context of the present application are compounds from the group of cationic nitriles, in particular cationic nitriles of the formula

in the R ^{1 is} -H, -CH ₃ , a C _2-24 alkyl or alkenyl radical, a substituted C _2-24 alkyl or alkenyl radical having at least one substituent from the group -Cl, -Br, - OH, -NH ₂ , -CN, an alkyl or alkenylaryl radical having a C _1-24 -alkyl group, or represents a substituted alkyl or alkenylaryl radical having a C _1-24 -alkyl group and at least one further substituent on the aromatic ring, R ² and R ^{3 are} independently selected from -CH ₂ -CN, -CH ₃ , -CH ₂ -CH ₃ , -CH ₂ -CH ₂ -CH ₃ , -CH (CH ₃ ) -CH ₃ , -CH ₂ - OH, -CH ₂ -CH ₂ -OH, -CH (OH) -CH ₃ , -CH ₂ -CH ₂ -CH ₂ -OH, -CH ₂ -CH (OH) -CH ₃ , -CH (OH) - CH ₂ -CH ₃ , - (CH ₂ CH ₂ -O) _n H where n = 1, 2, 3, 4, 5 or 6 and X is an anion.

in der R⁴, R⁵ und R⁶ unabhängig voneinander ausgewählt sind aus -CH₃, -CH₂-CH₃, -CH₂-CH₂-CH₃, -CH(CH₃)-CH₃, wobei R⁴ zusätzlich auch -H sein kann und X ein Anion ist, wobei vorzugsweise R⁵ = R⁶ = -CH₃ und insbesondere R⁴ = R⁵ = R⁶ = -CH₃ gilt und Verbindungen der Formeln (CH₃)₃N⁽⁺⁾CH₂-CN X^–, (CH₃CH₂)₃N⁽⁺⁾CH₂-CN X^–, (CH₃CH₂CH₂)₃N⁽⁺⁾CH₂-CN X^–, (CH₃CH(CH₃))₃N⁽⁺⁾CH₂-CN X^–, oder (HO-CH₂-CH₂)₃N⁽⁺⁾CH₂-CN X^– besonders bevorzugt sind, wobei aus der Gruppe dieser Substanzen wiederum das kationische Nitril der Formel (CH₃)₃N⁽⁺⁾CH₂-CN X^–, in welcher X^– für ein Anion steht, das aus der Gruppe Chlorid, Bromid, Iodid, Hydrogensulfat, Methosulfat, p-Toluolsulfonat (Tosylat) oder Xylolsulfonat ausgewählt ist, besonders bevorzugt wird.Particularly preferred is a cationic nitrile of the formula

in which R ⁴ , R ⁵ and R ^{6 are} independently selected from -CH ₃ , -CH ₂ -CH ₃ , -CH ₂ -CH ₂ -CH ₃ , -CH (CH ₃ ) -CH ₃ , wherein R ⁴ additionally also -H may be and X is an anion, preferably R ⁵ = R ⁶ = -CH ₃ and in particular R ⁴ = R ⁵ = R ⁶ = -CH ₃ and compounds of the formulas (CH ₃ ) ₃ N ⁽⁺⁾ CH ₂ -CN X ^- , (CH ₃ CH ₂ ) ₃ N ⁽⁺⁾ CH ₂ -CN X ^- , (CH ₃ CH ₂ CH ₂ ) ₃ N ⁽⁺⁾ CH ₂ -CN X ^- , (CH ₃ CH ( CH ₃ )) ₃ N ⁽⁺⁾ CH ₂ -CN X ^- , or (HO-CH ₂ -CH ₂ ) ₃ N ⁽⁺⁾ CH ₂ -CN X ^{- are} particularly preferred, wherein from the group of these substances turn the cationic Nitrile of the formula (CH ₃ ) ₃ N ⁽⁺⁾ CH ₂ -CN X ^- in which X ^{- represents} an anion selected from the group consisting of chloride, bromide, iodide, hydrogensulfate, methosulfate, p-toluenesulfonate (tosylate) or xylenesulfonate is particularly preferred.

According to the invention preferred Bleaches that can be used in addition to bleach activators or bleach catalysts instead include. These substances are bleach-enhancing transition metal salts or transition metal complexes such as Mn, Fe, Co, Ru or Mo saline complexes or carbonyl complexes. Also Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexes with N-containing tripod ligands and Co, Fe, Cu and Ru ammine complexes are used as bleach catalysts usable.

Bleach-enhancing transition metal complexes, in particular with the central atoms Mn, Fe, Co, Cu, Mo, V, Ti and / or Ru, preferably selected from the group of manganese and / or Cobalt salts and / or complexes, more preferably the cobalt (ammin) complexes, the cobalt (acetate) complexes, the cobalt (carbonyl) complexes, the Chlorides of cobalt or manganese, manganese sulfate are used in common Quantities, preferably in an amount up to 5 wt .-%, in particular from 0.0025 wt.% to 1 wt.%, and more preferably from 0.01 Wt .-% to 0.25 wt .-%, each based on the total weight of inventive agent used. In special cases however, more bleach activator can also be used.

It is particularly preferred to use complexes of manganese in the oxidation state II, III, IV or IV, which preferably contain one or more macrocyclic ligands with the donor functions N, NR, PR, O and / or S. Preferably, ligands are used which have nitrogen donor functions. It is particularly preferred to use bleach catalyst (s) in the compositions of the invention, which as macromolecular ligands 1,4,7-trimethyl-1,4,7-triazacyclononan (Me-TACN), 1,4,7-triazacyclononane (TACN ), 1,5,9-trimethyl-1,5,9-triazacyclododecane (Me-TACD), 2-methyl-1,4,7-trimethyl-1,4,7-triazacyclononane (Me / Me-TACN) and or 2-methyl-1,4,7-triazacyclononane (Me / TACN). Suitable manganese complexes are, for example, [Mn ^III ₂ (μ-O) ₁ (μ-OAc) ₂ (TACN) ₂ ] (ClO ₄ ) ₂ , [Mn ^III Mn ^IV (μ-O) ₂ (μ-OAc) ₁ (TACN ) ₂ ] (BPh ₄ ) ₂ , [Mn ^IV (μ-O) ₆ (TACN) ₄ ] (ClO ₄ ) ₄ , [Mn ^III ₂ (μ-O) ₁ (μ-OAc) ₂ (Me-TACN)] ₂ ] (ClO ₄ ) ₂ , [Mn ^III Mn ^IV (μ-O) ₁ (μ-OAc) ₂ (Me-TACN) ₂ ] (ClO ₄ ) ₃ , [Mn ^IV ₂ (μ-O) ₃ (Me -TACN) ₂ ] (PF ₆ ) ₂ and [Mn ^IV ₂ (μ-O) ₃ (Me / Me-TACN) ₂ ] (PF ₆ ) ₂ (OAc = OC (O) CH ₃ ).

Combination inventive Enzymes with other enzymes

The combination of enzymes of the invention with other enzymes, which are described in more detail below, causes a synergistic cleaning performance of the agent. For agents containing selected enzyme combinations, improved stain removal was noted as compared to agents containing either only one of the enzymes or even compared to the expected pure performance of an agent with both components due to the mere addition of the respective individual contributions of these two components to the cleaning performance of the agent. The different enzymes thus support each other in their work. Thus, the selected combination of enzymes according to the invention with other enzymes also represents an essential aspect of the invention.

These include in particular enzymes which are selected from the group consisting of protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, carrageenase, oxidase, oxidoreductase, pectin degrading enzyme or lipase. Also, enzyme mixtures of several different types of enzymes, for example enzymes of the invention in combination with amylases and lipases, enzymes of the invention in combination with amylases and cellulases, enzymes of the invention in combination with amylases and hemicellulases, in particular mannanases or xylanases, enzymes of the invention in combination with amylases and xanthanases and inventive Enzymes in combination with amylases and carrageenases preferably have synergistic cleaning performance. These enzymes are basically of natural origin; However, starting from the natural molecules, improved variants are often available for use in detergents and cleaners, which are preferably used accordingly. Detergents or cleaning agents contain enzymes preferably in total amounts of 1 × 10 ^-6 to 5 wt .-% based on active protein. The protein concentration can be determined by known methods, for example the BCA method or the biuret method.

Under Proteases are preferred to those of the subtilisin type. Examples these are the subtilisins BPN 'and Carlsberg and their more advanced forms, the protease P692, the subtilisins 147 and 309, the Bacillus lentus alkaline protease, subtilisin Dy and the subtilases, but not the subtilisins in the In the narrower sense, enzymes thermitase, proteinase K and the proteases TW3 and TW7.

Examples for amylases usable according to the invention are the α-amylases from Bacillus licheniformis, from B. amyloliquefaciens, from B. stearothermophilus, from Aspergillus niger and A. oryzae as well as those for use in detergents and cleaners improved developments of the aforementioned amylases. Furthermore For this purpose Bacillus α-amylase is used sp. A 7-7 (DSM 12368) and cyclodextrin glucanotransferase (CGTase) from B. agaradherens (DSM 9948).

Usable according to the invention are still lipases or cutinases, especially because of their Triglyceride-cleaving activities, but also to make suitable Precursors generate in situ peracids. These include for example, originally from Humicola lanuginosa (Thermomyces lanuginosus) available, respectively further developed lipases, in particular those with the amino acid exchange D96L. Furthermore, for example, the cutinases can be used, originally from Fusarium solani pisi and Humicola insolens have been isolated. Also usable are lipases, or cutinases whose initial enzymes originally from Pseudomonas mendocina and Fusarium solanii have been isolated.

Farther Preferably used are enzymes which are termed hemicellulases be summarized. These include mannanases, Xanthan lyases, pectin lyases (= pectinases), pectin esterases, pectate lyases, Xyloglucanases (= xylanases), pullulanases and β-glucanases. Further usable enzymes are carrageenases and tannases. As well can in the inventive compositions Perhydrolases are used.

to Increasing the bleaching effect can further according to the invention Oxidoreductases, for example further oxidases, oxygenases, catalases, Peroxidases such as halo, chloro, bromo, lignin, glucose or Manganese peroxidases, dioxygenases or laccases (phenol oxidases, Polyphenoloxidasen) can be used. Advantageously additionally preferably organic, more preferably added aromatic compounds interacting with the enzymes, to enhance the activity of the oxidoreductases in question (Enhancer) or at very different redox potentials between the oxidizing enzymes and the soiling the electron flow to ensure (mediators).

Particularly preferred according to the invention are enzyme combinations which bring about a synergistic cleaning performance of the composition comprising them, comprising enzymes according to the invention in combination with amylases, enzymes according to the invention in combination with lipases, enzymes according to the invention in combination with cellulases, enzymes according to the invention in combination with hemicellulases, in particular mannanases or xylanases , enzymes according to the invention in combination with xanthanases and enzymes of the invention in combination with carrageenases. Very particular preference is given to enzymes according to the invention in a synergistic manner with lipases, hemicellulases, xanthanases or carrageenans combined.

The Enzymes can be established in any of the prior art Form are used. These include, for example, the obtained by granulation, extrusion or lyophilization solid Preparations or, in particular in liquid or gel agents, solutions of enzymes, advantageously as concentrated as possible, low in water and / or mixed with stabilizers.

alternative The enzymes can be used both for the solid as well be encapsulated 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 included 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. In superimposed layers In addition, other active ingredients, for example Stabilizers, emulsifiers, pigments, bleaching or dyes applied become. Such capsules are prepared by methods known per se, for example, by shaking or rolling granulation or applied in fluid-bed processes. Advantageously, such Granules, for example by applying polymeric film formers, low in dust and storage-stable due to the coating.

Farther is it possible to assemble two or more enzymes together, so that a single granule has multiple enzyme activities having.

The or the enzymes can protect against damage such for example, inactivation, denaturation or decay, such as by physical influences, oxidation or proteolytic Splitting, to be protected. For microbial extraction of the proteins and / or enzymes is an inhibition of proteolysis particularly preferred, especially if the agents proteases contain. Detergents or cleaners can be added to this Purpose additionally contain stabilizers that are sufficient are known in the art. invention Means can therefore in principle all known enzyme stabilizers contain.

The The following examples further illustrate the invention, but without restricting it.

Examples

All molecular biology work steps follow standard methods, as for example in the manual of Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 , or similar relevant works. Enzymes and kits were used according to the instructions of the respective manufacturers.

Example 1: Site-directed mutagenesis

The Protease variants were prepared by site-directed Mutagenesis of the alkaline protease from Bacillus gibsonii DSM 14391 produced. A standard mutagenesis method was used using the "quick change site-directed mutagenesis Kit "from Stratagene (catalog number 200518, Stratagene, La Jolla, Ca, USA). It became the codon for the amino acid position 211 (nucleotides 631-633) in the counting manner according to SEQ ID NO. 2 changed from ATG to TCT, allowing an exchange the amino acid methionine (M) against the amino acid Serine (S) takes place. Alternatively or in addition, the codon for position 212 (nucleotides 634-636) in the Counting according to SEQ ID NO. 2 from CCT changed to AAT, leaving an exchange of the amino acid Proline (P) takes place against the amino acid asparagine (N).

Example 2: Expression of the protease variants

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 inserted into the host strain Bacillus subtilis DB 104 ( Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444 ) transformed with standard methods.

Transformants were initially digested on DM3 medium (8 g / l agar, 0.5 M succinic acid, 3.5 g / l K2HPO4, 1.5 g / l KH2PO4, 20 mM MgCl2, 5 g / l casiaminoacids, 5 g / 1 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 The proteolytically active clones obtained were selected, their 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 powdery laundry detergent

• A Gras auf Baumwolle, EMPA 164
• B Schokomilch/Ruß auf Baumwolle, C-03
• C Kakao, EMPA 112
• D Blut/Milch auf Baumwolle, C-5 (044).

For this example, standardized contaminated textiles were used, which had been purchased from the Eidgenössische Material-Prüfungs- und -Versuchsanstalt, St. Gallen, Switzerland (EMPA), or the Laundry Research Institute, Krefeld. The following stains and textiles were used:

• A grass on cotton, EMPA 164
• B chocolate milk / soot on cotton, C-03
• C Cocoa, EMPA 112
• D blood / milk on cotton, C-5 (044).

With This test material was based on various detergent formulations their washing performance examined. That's what the approaches became washed for 60 minutes at a temperature of 40 ° C. The dosage was 5.9 g of the detergent per liter of wash liquor. It became German with city water with a water hardness of approximately 16 ° Hardness washed.

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 ).

After washing, the whiteness of the washed fabrics was measured. The measurement was performed on a Minolta CM508d spectrometer, illuminant D65, 10. The instrument was previously calibrated with a supplied white standard. The results obtained are the differential remissions between a wash with a detergent containing a protease and a parallel control wash with a detergent without protease. The results are summarized in Table 2 below. They allow a direct conclusion on the contribution of each contained enzyme to the washing performance of the agent used. Table 2: Washing results with a powdered detergent at 40 ° C. soiling SEQ ID NO. 2 SEQ ID NO. 2 P212N Lentus A 3.7 4.6 1.2 B 8.6 11.1 3.6 C 6.2 6.5 4.1 D 14.5 17.7 8.1

The Protease variant shows on various stains, in particular at stains B and D, an improved performance in comparison to the starting molecule according to SEQ ID NO. 2 and in comparison with the prior art established protease from Bacillus lentus.

Example 4: Determination of the Washing Performance when used in a commercial liquid laundry detergent

• A Gras auf Baumwolle, EMPA 164
• B Ganzei/Ruß auf Baumwolle, 10N
• C Schokomilch/Ruß auf Baumwolle, C-03
• D Blut/Milch auf Baumwolle, C-5 (044).

The experiment was carried out essentially as described in Example 3. The following stains and textiles were used:

• A grass on cotton, EMPA 164
• Whole egg / soot on cotton, 10N
• C chocolate milk / soot on cotton, C-03
• D blood / milk on cotton, C-5 (044).

With This test material was based on various detergent formulations their washing performance examined. That's what the approaches became washed for 60 minutes at a temperature of 40 ° C. The dosage was 5.9 g of the detergent per liter of wash liquor. It became German with city water with a water hardness of approximately 16 ° Hardness washed.

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% FASOS, 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. 2 ( WO 03/054185 ), Protease according to SEQ ID NO. 2 with the amino acid substitution M211S and the alkaline protease which is sold under the trade names Purafect Prime ^® by the company Genencor International.

After washing, the whiteness of the washed fabrics was measured. The measurement was performed on a Minolta CM508d spectrometer, illuminant D65, 10. The instrument was previously calibrated with a supplied white standard. The results obtained are the differential remissions between a wash with a detergent containing a protease and a parallel control wash with a detergent without protease. The results are summarized in Table 3 below. They allow a direct conclusion on the contribution of each contained enzyme to the washing performance of the agent used. Table 3: Wash results with a liquid detergent at 40 ° C soiling SEQ ID NO. 2 SEQ ID NO. 2 M211S Purafect Prime A 2.7 5.5 3.2 B 8.6 11.1 3.0 C 4.1 7.9 8.1 D 16.6 26.1 15.2

The Protease variant shows on various stains, in particular on Soils A, B and D, a significantly improved performance in comparison to the starting molecule according to SEQ ID NO. 2 and compared with that established in the prior art Protease Purafect Prime.

Description of the figures

1 : Alignment of the mature starting protease according to the invention (SEQ ID NO: 2) with proteases from the prior art, calculated using the program Vector NTI Suite Ver. 7 (InforMax, Inc. Bethesda, USA) under standard parameters (the mature enzyme is indicated in each case). In this mean: SEQ ID NO. 2: Inventive starting protease according to SEQ ID NO. 2 (mature enzyme) BLAP: Alkaline protease from Bacillus lentus DSM 5483 ( WO 92/21760 A1 ) according to SEQ ID NO. 3 Subtilisin 309: Subtilisin 309 according to SEQ ID NO. 4

It follows Sequence listing according to WIPO St. 25. This can of the official publication platform of the DPMA become.

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• WO 95/07991 A2 [0006]
• WO 95/29979 A1 [0006]
• WO 96/28566 A2 [0007]
• - EP 283075 A2 [0008]
• WO 94/02618 A1 [0008, 0009]
• - EP 328229 A1 [0008]
• - GB 1243784 A [0009]
• WO 95/30011 A2 [0009, 0009]
• WO 91/02792 A1 [0010]
• WO 92/21760 A1 [0010, 0055, 0265]
• WO 95/23221 A1 [0010, 0010, 0055]
• WO 02/088340 A2 [0010]
• WO 03/038082 A2 [0010]
• US 5691295 [0010]
• US 5801039 [0010]
• US 5855625 [0010]
• - US 6197589 B1 [0010]
• WO 03/054185 [0012, 0257, 0262]
• WO 00/09679 A1 [0040]
• WO 02/088340 [0055]
• WO 95/23221 [0257]

Cited non-patent literature

• - "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 [0002]
• - Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 [0006]
• - JA Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911-7925 [0006]
• - Publications by EL Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191 [0007]
• - Jacobs et al. (1985) in Nucl. Acids Res., Vol. 13, pp. 8913-8926 [0007]
• - https://www.dsmz.de [0012]
• - DJ Lipman and WR Pearson (1985) in Science, Vol. 227, pp. 1435-1441 [0033]
• StEP method (Zhao et al., 1998, Nat. Biotechnol., Vol. 16, pp. 258-261). [0040]
• Random priming recombination (Shao et al., (1998), Nucleic Acids Res., Vol. 26, pp. 681-683). [0040]
• DNA shuffling (Stemmer, WPC (1994), Nature, Vol. 370, pp. 389-391) [0040]
• RACHITT (Coco, WM et al., (2001) Nat. Biotechnol., Vol. 19, pp. 354-359). [0040]
• Sambrook, J., Fritsch, EF and Maniatis, T. 2001: Molecular cloning: a laboratory manual, 3rd Edition (Cold Spring Laboratory Press). [0070]
• - "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 [0109]
• T. Egelrud et al., Acta Derm. Venerol., Vol. 71 (1991), pp. 471-474 [0147]
• - "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 [0149]
• Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 [0251]
• Bernhard et al. (1978), J. Bacteriol., Vol. 133 (2), pp. 897-903 [0253]
• Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444 [0253]

Claims (42)

A protease comprising an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 2 is at least 78.5% identical, characterized in that it is expressed in the manner of SEQ ID NO. 2 at position 211 or position 212 or at positions 211 and 212 in comparison to that shown in SEQ ID NO. 2 indicated amino acid modified amino acid. Protease according to claim 1, characterized in that that it comprises an amino acid sequence identical to that described in SEQ ID NO. 2 indicated amino acid sequence increasingly preferred 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 is equal to 99.25% identical. Protease according to claim 1 or 2, characterized that the protease is selected from d) a protease, in the counting method according to SEQ ID NO. 2 at position 211 has an amino acid selected is 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 e) a protease that is in the counting mode according to SEQ ID NO. 2 at position 212 an amino acid which is selected from the group consisting of: Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, Glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, Serine, threonine, tryptophan, tyrosine and valine f) a protease, in the counting method according to SEQ ID NO. 2 at position 211 an amino acid according to feature a) and at position 212 an amino acid according to feature b). Protease according to one of claims 1 to 3, characterized in that the protease is selected out a) a protease, which in the counting manner according to SEQ ID NO. 2 at position 211 has a serine residue b) one Protease, which in the counting manner according to SEQ ID NO. 2 at position 212 has an asparagine residue c) one Protease, which in the counting manner according to SEQ ID NO. 2 at position 211 a serine residue and at position 212 a Has asparagine residue. Protease according to one of claims 1 to 4, characterized in that they at least one and increasingly preferably two, three or four matching antigens Determinants having one of the designated in claims 1 to 4 Protease has. Protease, characterized in that it consists of a Protease according to one of claims 1 to 5 as starting molecule is obtainable by fragmentation, deletion, insertion or Includes substitution mutagenesis and an amino acid sequence, those over a length of at least 266 and increasingly preferably 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, respectively Amino acid positions coincides with the parent molecule. Protease, characterized in that it consists of a Protease according to one of claims 1 to 6 as starting molecule is obtainable and one or more further amino acid exchanges in positions corresponding to 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 alkaline protease from Bacillus lentus according to SEQ ID NO. 3 are assigned in an alignment. Protease according to one of claims 1 to 7, characterized in that it additionally stabilized is, preferably by covalent coupling to a polymer. Protease according to one of claims 1 to 8, characterized in that it has at least one chemical modification having. Nucleic acid molecule, encoding for a protease according to any one of claims 1 to 9th Nucleic acid molecule according to claim 10, characterized in that it corresponds to the in SEQ ID NO. 1 indicated Nucleic acid sequence increasingly preferably at least 78.5%, 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 more especially preferably equal to 99.25%. Vector containing a nucleic acid according to one of claims 10 or 11. Vector according to Claim 12, characterized it is a cloning vector or an expression vector. Non-human host cell that is a protease according to any one of claims 1 to 9 or a fragment thereof contains or which can be stimulated to produce them, preferably using a vector according to claim 12 or 13. Host cell according to claim 14, characterized in that that they are the protease or a fragment of them into the host cell surrounding medium secreted. Host cell according to claim 14 or 15, characterized that she is a bacterium. Host cell according to claim 16, characterized in that that the bacterium is selected from the group of genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas. Host cell according to claim 16 or 17, characterized that the bacterium 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. Host cell according to claim 14 or 15, characterized that it has a nucleus. Process for the preparation of a protease after a of claims 1 to 9. The method of claim 20 using a nucleic acid according to one of claims 10 or 11, preferably below Use of a vector according to one of claims 12 or 13, more preferably using a host cell after a of claims 14 to 19. Means, characterized in that it is at least a protease according to any one of claims 1 to 9. Agent according to claim 22, characterized that it is a detergent, hand wash, dishwashing detergent, hand dishwashing detergent, Machine dishwashing detergents, cleaning agents, dentures or contact lens care products, rinsing agents, disinfectants, cosmetic, pharmaceutical or other means of Treatment of filter media, textiles, furs, paper, skins or Leather, is. Agent according to claim 22 or 23, characterized that it is a laundry detergent or dishwashing detergent is. Composition according to one of claims 22 to 24, characterized in that it is present as a one-component system. Composition according to one of claims 22 to 24, characterized in that it is divided into several components is. Composition according to one of claims 22 to 26, characterized in that it contains the protease in an amount from 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, more preferably from 20 μg to 15 mg and whole more preferably from 50 μg to 10 mg per g of the agent contains. Composition according to one of claims 22 to 27, characterized in that it is in solid form. Agent according to claim 28, characterized that it is a free-flowing powder with a bulk density from 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l. Composition according to one of claims 22 to 27, characterized in that it is in pasty or liquid form is present. Composition according to one of claims 22 to 30, characterized in that the protease with a at room temperature or impermeable to the protease in the absence of water Substance is sheathed. Composition according to one of claims 22 to 31, further comprising one or more further enzymes. Agent according to claim 32, characterized that at least one other enzyme is a protease, amylase, cellulase, Hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, Carrageenase, oxidase, oxidoreductase or a lipase is. Composition according to one of claims 22 to 33, characterized in that there is at least one further component contains, selected from the group consisting of Surfactants, builders, acids, alkaline substances, hydrotropes, Solvents, thickeners, bleaches, dyes, Perfumes, corrosion inhibitors, sequestering agents, electrolytes, optical brighteners, grayness inhibitors, silver corrosion inhibitors, Color transfer inhibitors, foam inhibitors, abrasives, UV absorbers, solvents, antistatic agents, pearlescing agents and skin protection products. Composition according to one of claims 22 to 34, further comprising - 5 wt .-% to 70 wt .-%, in particular 5 wt .-% to 30 wt .-% surfactants and / or 10% by weight to 65 wt .-%, in particular 12 wt .-% to 60 wt .-% water-soluble or water-dispersible inorganic builder material and / or - 0.5 Wt .-% to 10 wt .-%, in particular 1 wt .-% to 8 wt .-%, water-soluble organic builders and / or - 0.01 to 15 Wt .-% solid inorganic and / or organic acids or acid salts and / or 0.01 to 5% by weight of complexing agent for heavy metals and / or 0.01 to 5% by weight Grayness inhibitor and / or 0.01 to 5% by weight Color transfer inhibitor and / or - 0,01 to 5% by weight foam inhibitor The agent of claim 35, further comprising 0.01 to 5 wt .-% optical brightener. Use of an agent according to one of the claims 22 to 36 for removal of protease-sensitive stains on textiles or hard surfaces. Process for cleaning textiles or hard Surfaces, characterized in that in at least a method step, an agent according to one of the claims 22 to 36 is applied. Process for cleaning textiles or hard Surfaces, characterized in that in at least a method step, a protease according to one of the claims 1 to 10 is proteolytically active. Method according to claim 39, characterized that the protease in an amount of 40 micrograms to 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g and most preferably from 200 μg to 1 g is used per application. Use of a protease according to any one of the claims 1 to 10 for cleaning textiles or hard surfaces. Use according to claim 41, wherein the protease in an amount of 40 μg to 4 g, preferably 50 μg to 3 g, more preferably from 100 μg to 2 g and whole more preferably used from 200 μg to 1 g per application is.