EP2016175A1

EP2016175A1 - Subtilisin from bacillus pumilus and washing and cleaning agents containing said novel subtilisin

Info

Publication number: EP2016175A1
Application number: EP07724922A
Authority: EP
Inventors: Marion Merkel; Petra Siegert; Susanne Wieland; Karl-Heinz Maurer; Cornelius Bessler
Original assignee: Henkel AG and Co KGaA
Current assignee: Henkel AG and Co KGaA
Priority date: 2006-05-11
Filing date: 2007-05-07
Publication date: 2009-01-21
Also published as: DE102006022224A1; US20090170745A1; CN101506361A; WO2007131656A1; JP2009536630A

Abstract

The invention relates to a novel subtilisin-type alkaline protease from Bacillus pumilus and to sufficiently related proteins and the derivatives thereof. The invention also relates to washing and cleaning agents comprising the novel subtilisin-type alkaline protease, to the sufficiently related proteins and the derivatives thereof, to corresponding washing and cleaning methods and to their use in washing and cleaning agents and to other possible technical uses.

Description

"Subtilisin from Bacillus pumilus and detergents and cleaners containing this new subtilisin"

The present invention relates to a novel subtilisin-type alkaline protease from Bacillus pumilus and to sufficiently related proteins and their derivatives. It also relates to detergents and cleaners with this new Subtilisin-type alkaline protease, sufficiently related proteins and their derivatives, corresponding washing and cleaning processes and their use in detergents and cleaners, and other technical uses.

Enzymes are well-established active ingredients in detergents and cleaners. Proteases cause the degradation of protein-containing stains on the cleaning material, such as textiles or hard surfaces. At best, there are synergies between the enzymes and the remaining components of the funds concerned. The development of detergent proteases is based on naturally, preferably microbially formed enzymes. Such are optimized by per se known mutagenesis method, for example, point mutagenesis, deletion, insertion or fusion with other proteins or protein parts or other modifications for use in detergents and cleaners. Among the detergent and detergent proteases subtilisins occupy an outstanding position due to their favorable enzymatic properties such as stability or pH optimum.

Proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62), in particular subtilisins are attributed to the serine proteases due to the catalytically active amino acids. They are naturally produced and secreted by microorganisms, especially by Bac / 7 / us species. 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. An overview of this Family provides, 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. Subtilisins are useful for a variety of technical uses, as constituents of cosmetics and in particular as active ingredients of detergents or cleaners.

In the following, the most important subtilisins and the most important strategies for their technical development are listed.

The subtilisin BPN ', which is derived from Bacillus amyloliqυefaciens or B. subtilis, is known from the work of Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811-819 and by J.A. Wells et al. (1983) in Nucleic Acids Research, Volume H, pp. 7911-7925. Subtilisin BPN 'serves as a reference enzyme of the subtilisins, in particular with regard to the numbering of the positions.

The protease subtilisin Carlsberg is described in the publications of EL Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184-2191, and Jacobs et al. (1985) in Nucl. Acids Res., Vol. 13, pp. 8913-8926. It is naturally produced by Bacillus licheniformis and under the trade name Maxatase ^® from Genencor International Inc., Rochester, New York, USA, and under the trade name Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark.

The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® from Novozymes. They are originally from Bac / 7 / us strains, which are disclosed in application GB 1243784.

The subtilisin DY is originally from Nedkov et al. Chem., 1985, in Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 421-430. Other subtilisin-type proteases isolated from Bacillus strains are described in the recent patent applications WO03 / 054184 and WO03 / 054185.

A strategy to improve the washing performance of subtilisins is to substitute randomly or selectively in the known molecules individual amino acids against others and to check the variants obtained for their contributions to the washing performance. The enzymes can also be improved with regard to their allergenicity with certain amino acid exchanges or deletions.

To improve the washing performance of subtilisins, the strategy of inserting additional amino acids into the active loops was followed. This strategy should, in principle, be applicable to all subtilisins belonging to one of subgroups I-S1 (true subtilisins) or I-S2 (highly alkaline subtilisins).

Another strategy of performance improvement is to change the surface charges and / or the isoelectric point of the molecules and thereby their interactions with the substrate. Furthermore, point mutants with reduced pH dependent molecular charge variation have been described. From this principle, a method was also derived for the identification of variants, which should be suitable for use in detergents and cleaners; All disclosed variants have at least one exchange in position 103. Generally, variations in the literature are very often described as having an exchange at position 103, optionally combined with a variety of other possible substitutions. An alternative way of improving the performance of detergents and cleaners is to increase the hydrophobicity of the molecules, which may affect the stability of the enzyme.

Another way to modulate the performance of proteases is by forming fusion proteins. Thus, for example, fusion proteins from proteases and an inhibitor such as the Streptomyces subtilisin inhibitor are disclosed in the literature. Another possibility is for example the coupling to the cellulase-derived cellulose binding domain (CBD) to increase the concentration of active enzyme in close proximity to the substrate or the coupling of a peptide linker and therefrom of polymers to reduce allergenicity or immunogenicity.

For example, methods for generating statistical amino acid replacements can be based on the phage display. A modern direction of enzyme development is to combine elements from known, related proteins via statistical methods into new enzymes that have previously unattainable properties. Such methods are also summarized under the generic term recombination. 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. 37O, pp. 389-391) or recursive sequence recombination (RSR; WO 98/27230, WO 97/20078 , WO 95/22625) or the method RACHITT (Coco, WM et al. (2001), Nat. Biotechnol., Volume Ij), pp. 354-359). An overview of such methods is also given in the article "Judicious Evolution and Biocatalysis" by Powell et al. (2001), Angew. Chem., Vol. V \ 3, pp. 4068-4080.

Another, and in particular complementary, strategy is to increase the stability of the proteases in question and thereby increase their effectiveness. Stabilization via coupling to a polymer has been described, for example, for proteases used in cosmetics; As a result, a better skin compatibility could be achieved. In contrast, stabilizers by point mutations are more common, especially for detergents and cleaners. Thus, proteases can be stabilized by this, for example, in particular with regard to the use at elevated temperatures, that one exchanges certain tyrosine residues for other amino acid residues. Other described possibilities for stabilization via point mutagenesis are, for example:

- exchange of certain amino acid residues for proline;

Introduction of more polar or charged groups on the surface of the molecule; Enhancing the binding of metal ions, in particular via mutagenesis of the calcium binding sites;

Blockage of autolysis by modification or mutagenesis;

- Determination of the positions relevant for stabilization via an analysis of the three-dimensional structure.

It is known that proteases can be used to improve the washing or cleaning performance together with α-amylases and other detergent enzymes, in particular lipases. Likewise, the use of proteases in detergents in combination with other active ingredients such as bleaching agents or soil release agents is known in the art.

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

The various technical fields of application described here by way of example require proteases with different properties, such as the reaction conditions, the stability or the substrate specificity. Conversely, the technical utility of the proteases, for example in the context of a detergent formulation, depends on other factors such as stability of the enzyme to high temperatures, oxidizing agents, denaturation by surfactants, folding effects, or desired synergies with other ingredients.

Thus, there is still a high demand for technically useful proteases, which cover a wide range of properties due to the variety of their applications in their entirety to very subtle differences in performance.

The basis for this is extended by new proteases, which in turn can be further developed specifically for specific applications. The present invention therefore an object of the invention to find another, not yet known protease. The wild-type enzyme should preferably be characterized by being at least close to the enzymes established for this purpose when used in an appropriate means. In particular, the contribution to the performance of a detergent or cleaning agent was of interest.

Further objects of the present invention can be seen to provide proteases, in particular of the subtilisin type, which have improved stability with respect to the prior art to temperature influences, pH fluctuations, denaturing or oxidizing agents, proteolytic degradation, high temperatures, acidic or alkaline conditions or to a change in the redox ratios. Other tasks can be seen in a reduced immunogenicity or reduced allergenic effect.

Another particular object of the present invention was to find proteases which at temperatures of 20 to 60 ⁰ C, a good washing performance, preferably an improved washing performance in comparison to the disclosed in the prior art proteases, in particular those of the subtilisin type have ,

Other subtasks have been to provide nucleic acids encoding such proteases and provide vectors, host cells, and methods of preparation that can be used to obtain such proteases. Furthermore, appropriate means, in particular washing and cleaning agents, appropriate washing and cleaning methods and corresponding uses for such proteases should be made available. Finally, technical applications for the proteases found should be defined.

The object is achieved by subtilisin-type alkaline proteases having amino acid sequences which are as described in the sequence listing under SEQ ID NO. 2 amino acid sequence given from position 109 to 383 at least 98.5% are identical and / or differ with respect to this amino acid sequence in a maximum of 4 amino acid positions.

Increasingly preferred are those each having an increasing degree of identity with the novel Bacillus pumilus alkaline protease, ie those which differ only in 3 or 2 amino acid positions, preferably only in one amino acid position, and very particular preference is given to the Bacillus pumilus alkaline protease even.

Further solutions to the problem, or solutions of the subtasks and thus in each case separate inventive objects exist in nucleic acids whose sequences to the in SEQ ID NO. 1 nucleotide sequence are sufficiently similar or encode proteases according to the invention, in corresponding vectors, cells, or host cells and production methods. Furthermore, appropriate means, in particular washing and cleaning agents, appropriate washing and cleaning methods and corresponding uses for such proteases are provided. Finally, technical applications for the proteases found are defined.

The use of alkaline proteases from Bacillus pumilus in detergents and cleaners is already known to the person skilled in the art. For example, EP0572992 describes the use of alkaline proteases from Bacillus pumilus in detergents and cleaners. The protein sequence of the enzymes described there is not specified.

As the closest prior art for the subject of the present invention, the publications of Pan et al. (Current Microbiology 49 (2004), 165-169), Aoyama et al. (Microbiol Immunol 44 (5) (2000), 389-393), Huang et al. (Current Microbiology 46 (2003), 169-173), Aoyama et al. (Appl. Microbiol Biotechnol., 53 (2000), 390-395), Yasuda et al. (Appl. Microbiol Biotechnol 51 (1999), 474-479) and Miyaji et al. (Letters in Applied Microbiology 42 (2006), 242-247). These publications describe the use of Bacillus pumilus proteases with the sequences Q6SIX5 (Swiss-Prot), Q9KWR4 (Swiss-Prot), Q5XPN0 (Swiss-Prot) and Q2HXI3 (Swiss-Prot), which confer very high homology the invention Protease have. As a use for some of these proteases, the depilation of leather is described as these proteases are largely inactive against collagen and can be used for the depilation of leather, without damaging it in the treatment. Another possibility is the enzymatic treatment of soymilk and soy milk products. A possible use of the protease Q2HXI3 is the degradation of zein, the main constituent of the protein in the seed of maize. However, the use of these enzymes in detergents and cleaners is not disclosed in these documents.

The naturally formed subtilisin-type alkaline protease on which the present invention is based can be obtained from the culture supernatant of a new Bacillus pumilus slime obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures) as such, as can be understood from the examples has been identified. For the purpose of reworkability, according to the Budapest Treaty a plasmid containing the nucleic acid sequence of the enzyme according to the invention was deposited with the DSMZ (German Collection for Microorganisms and Cell Cultures, Braunschweig) with the accession number DSM 18097.

The present patent application has pursued the strategy to find from a natural habitat a protease-producing microorganism and thus a naturally formed enzyme that meets the requirements as fully as possible.

Such an enzyme could be found as described in the examples of the present application with the Bacillus pumilus alkaline protease.

As can be ascertained beyond the biochemical characterization employed by the German Collection of Microorganisms and Cell Cultures GmbH, this strain secretes a proteolytic activity. It has a molecular weight of 27 kD according to SDS polyacrylamide gel electrophoresis, with an isoelectric point of more than 8.5 determined according to isoelectric focusing. The nucleotide sequence of the novel alkaline protease from Bacillus pumilus according to the invention is shown in the sequence listing of the present application under SEQ ID NO. 1 indicated. It includes 1152 bp. The deduced amino acid sequence is shown in SEQ ID NO. 2 indicated. It comprises 383 amino acids, followed by a stop codon. Of these, the first 108 amino acids are unlikely to be present in the mature protein, so the mature protein is expected to be 275 amino acids in length.

These sequences have been synthesized using commonly available databases Swiss-Prot (Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland, http://www.genebio.com/sprot.html) and GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA) to determine the proteins with the greatest homology.

The measure of homology is a percentage of identity, such as can be determined, for example, according to the method given by D.J. Lipman and W.R. Pearson in Science 22Z (1985), pp. 1435-1441. This indication may refer to the entire protein or to the respective region to be assigned. A broad homology term, similarity, also includes conserved variations, that is, amino acids with similar chemical activity, as these usually perform similar chemical activities within the protein. With nucleic acids one knows only the percentage of identity.

At the level of DNA, the following genes were identified as closest to the entire gene: (1.) Sequence Q5XPN0 from Bacillus pumilus (Swiss-Prot) with 94% identity, (2.) Sequence Q6SIX5 from Bacillus pumilus (Swiss-Prot) with 91% identity, (3.) sequence Q9KWR4 from Bacillus pumilus (Swiss-Prot) with 91% identity, (4.) sequence Q2HXI3 from Bacillus pumilus (Swiss-Prot) with 91% identity.

At the level of the DNA coding for the mature protein: (1.) Sequence Q5XPN0 from Bacillus pumilus (Swiss-Prot) with 95% identity, (2.) Sequence Q2HXI3 from Bacillus pumilus (Swiss-Prot) with 91% identity, (3.) Sequence Q6SIX5 from Bacillus pumilus (Swiss-Prot) with 90% identity, (4.) Sequence Q9KWR4 from Bacillus pumilus (Swiss-Prot) with 90% identity.

At the level of the amino acids for the entire preproprotein, the following were identified as nearest similar: (1.) sequence Q2HXI3 from Bacillus pumilus (Swiss-Prot) with 98% identity or deviations in 7 amino acid positions, (2.) sequence Q9KWR4 from Bacillus pumilus (Swiss Protein) with 98% identity or deviations in 9 amino acid positions, (3.) sequence Q6SIX5 from Bacillus pumilus (Swiss-Prot) with 97% identity or deviations in 10 amino acid positions, (4.) sequence Q5XPN0 from Bacillus pumilus (Swiss -Prot) with 97% identity or deviations in 11 amino acid positions.

At the level of the amino acids for the mature protein, the following were identified as closest: (1.) Sequence Q2HXI3 from Bacillus pumilus (Swiss-Prot) with 98% identity or deviations in 5 amino acid positions, (2.) Sequence Q6SIX5 from Bacillus pumilus (Swiss Protein) with 98% identity or deviations in 5 amino acid positions, (3.) sequence Q9KWR4 from Bacillus pumilus (Swiss-Prot) with 98% identity or deviations in 6 amino acid positions, (4.) sequence Q5XPN0 from Bacillus pumilus (Swiss -Prot) with 97% identity or deviations in 7 amino acid positions.

Due to the correspondence to be recognized and the relationship to the other specified subtilisins, this alkaline protease is to be regarded as a subtilisin.

Accordingly, an object of the present invention is any polypeptide, in particular any hydrolase, in particular any subtilisin-type alkaline protease having an amino acid sequence which is at least 98.5% identical to the amino acid sequence given in SEQ ID NO: 2 and / or with respect to the amino acid sequence given in SEQ ID NO: 2 deviates at most in 6 amino acid positions.

Among these, those polypeptides whose amino acid sequence to the amino acid sequence indicated in SEQ ID NO: 2 is at least 99% is in particular at least 99.5% identical and / or those which differ in relation to the amino acid sequence given in SEQ ID NO: 2 at most in 5 or 4, in particular at most in 3 or 2 amino acid positions, more preferably at most in an amino acid position. Very particular preference is given to a protein having an amino acid sequence according to SEQ ID NO: 2.

For it is to be expected that their properties are increasingly similar to those of the alkaline protease according to the invention from S. pumilus.

As already mentioned, due to a comparison of the N-terminal sequences, amino acids 1 to 108 are presumably to be regarded as leader peptide, amino acids 1 to 51 presumably representing the signal peptide, and the mature protein is expected to extend from positions 109 to 383 according to SEQ ID NO: 2. The position 384 is therefore occupied by a stop codon, so actually corresponds to no amino acid. However, since the information about the end of a coding region can also be regarded as an important constituent of an amino acid sequence, this position is included according to the invention in the region corresponding to the mature protein.

A further subject of the present invention is thus any polypeptide, in particular any hydrolase, in particular any subtilisin-type alkaline protease having an amino acid sequence which is at least 98 apart from the amino acid sequence given in SEQ ID NO: 2 from position 109 to position 383, 5% identical and / or deviates in relation to this amino acid sequence at most in 4 amino acid positions.

Among these, those polypeptides whose amino acid sequence to the amino acid sequence given in SEQ ID NO: 2 from position 109 to position 383 is at least 99%, more preferably at least 99.5%, are more preferred and / or those whose amino acid sequence is related to the amino acid sequence given in SEQ ID NO: 2 deviates at most in 3, in particular at most in 2 amino acid positions, more preferably at most in an amino acid position. Very particular preference is given to a protein having an amino acid sequence from position 109 to position 383 according to SEQ ID NO: 2. Should it turn out, for example by an N-terminal sequencing of the in vivo released by Bacillus pumilutes proteolytic protein that the cleavage site is not between the 108th and the 109th amino acid according to SEQ ID NO: 2 but at another location, so relate this information on the actual cleavage site or on the actual mature protein.

Another object of the present invention are also fragments, in particular the mature protein, provided that they are new compared to the prior art.

The present invention therefore also provides polypeptides which have an amino acid sequence with at least 100, preferably at least 110, 120, 130 or 140, particularly preferably at least 150, 175 or 200, in particular at least 225 or 250, consecutive amino acids of the amino acid sequence of position 109 to 383 according to SEQ ID NO. 2 include.

Another object of the present invention are therefore also polypeptides having an amino acid sequence with at least 185, preferably at least 190, 200 or 210, especially at least 220, 230 or 250 consecutive amino acids from position 109 to 383 according to SEQ ID NO. 2 or at most differ in one amino acid position thereof.

Another object of the present invention are therefore also polypeptides having an amino acid sequence having at least 240, preferably at least 245, 250 or 255, especially at least 260, 265 or 270 consecutive amino acids from position 109 to 383 according to SEQ ID NO. 2 or at most in two amino acid positions, preferably at most in one amino acid position thereof.

Another object of the present invention are therefore also polypeptides having an amino acid sequence with at least 245, preferably at least 250 or 255, more preferably at least 260 or 270 consecutive amino acids from position 109 to 383 of SEQ ID NO. 2 or sequence specified at most in three, preferably at most in two, more preferably at most in one position, deviate therefrom.

Another object of the present invention are therefore also polypeptides having an amino acid sequence from position 207 to position 378 of SEQ ID NO. 2 or at most in four, preferably at most in three, more preferably at most in two positions, especially at most in one position, deviate therefrom.

Since also the signal peptide and the propeptide represent units which are as such of interest according to the invention, a further subject of the present invention are those peptides that are homologous to these polypeptides, if they are new. As said, amino acids 1 to 108 are likely to be the leader peptide, with amino acids 1 to 51 believed to represent the signal peptide and amino acids 52 to 108 corresponding to the propeptide. Another object of the present invention are therefore polypeptides having an amino acid sequence from position 1 to 51 and from position 1 to 108 according to SEQ ID NO: 2 as well as polypeptides which differ from these amino acid sequences in an amino acid position.

Another object of the present invention are polypeptides which are encoded by the polynucleotides according to the invention, mentioned below.

Among them, more preferred are those polypeptides which are derived from a nucleotide sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 nucleotide sequence is as similar as possible, in particular over the portion of the positions 109 to 384 of the polypeptide according to SEQ ID NO. 2 corresponds.

For it is to be expected that these nucleic acids encode proteins whose properties are increasingly similar to those of the alkaline protease from B. pumilus according to the invention, in particular the mature protein. Again, as for all subsequent embodiments applies again that this information on the refer to actual mature protein, if it should turn out that the cleavage site of the protein is located at a different location than stated above.

The most preferred embodiment of this subject invention is any subtilisin-type alkaline protease whose amino acid sequence is identical to that shown in SEQ ID NO. 2, preferably in positions 109 to 383, and / or their amino acid sequence is different from the amino acid sequence shown in SEQ ID NO. 1 nucleotide sequence, preferably from positions 325 to 1152, can be derived.

For such is the case of the newly found and provided with the present application alkaline protease from Bacillus pumilus.

This is a protease not yet known in the prior art. It is, as indicated in the examples, isolatable, manufacturable and usable. It is also characterized, as also documented in the examples, by the fact that, when used in an appropriate agent, it at least approximates, or even exceeds, the performance of enzymes established for this purpose.

The polypeptides according to the invention are preferably enzymes, particularly preferably hydrolases, in particular proteases, particularly preferably endo-peptidases, in particular proteases of the subtilisin type, or parts thereof. The polypeptides according to the invention are therefore preferably able to hydrolyze acid amide bonds of proteins, in particular those which are located inside the proteins. The parts of the polypeptides may, in particular, be protein domains which may be suitable, for example, for the formation of functional chimeric enzymes.

For the development of technical proteases, which can be used in particular in detergents, it can serve as a naturally occurring, microbially formed enzyme as a starting point for mutagenesis methods known per se, for example point mutagenesis, fragmentation, deletion, insertion or fusion with other proteins or protein parts or other modifications for the desired use to be optimized. Such optimizations can be, for example, adjustments to temperature influences, pH fluctuations, redox ratios and / or other influences that are relevant for the technical fields of application. For example, an improvement in oxidation resistance, stability to denaturing agents or proteolytic degradation, high temperatures, acidic or strongly alkaline conditions, a change in sensitivity to calcium ions or other cofactors, a reduction in immunogenicity or allergenic activity are desired.

For this purpose, for example, by targeted point mutations, the surface charges or the loops involved in the catalysis or substrate binding can be changed. One starting point for this is an alignment with known proteases. This makes it possible to locate positions which, if necessary, could lead to an improvement in the properties of the protein.

The mutagenesis methods are based on the associated nucleotide sequence shown in SEQ ID NO. 1, or the nucleotide sequences which are sufficiently similar for this purpose, which are shown below as a separate subject of the invention. Corresponding molecular biology methods are described in the prior art, for example in manuals such as those of Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", CoId Spring Harbor Laboratory Press, New York, 1989.

Further embodiments of the present invention are therefore, in addition to the protein variants based on point or substitution mutation already mentioned above according to the invention, also all of the abovementioned polypeptides according to the invention, in particular polypeptides having an amino acid sequence according to SEQ ID NO: 2 or from position 109 to position 383 according to SEQ ID NO: 2, by insertion mutagenesis and / or substitution mutagenesis and / or inversion mutagenesis and / or by fusion with at least one other protein or protein fragment derived polypeptides, in particular those polypeptides Insertions and / or deletions and / or inversions of up to 50 amino acids, more preferably of up to 40, 30 or 20, in particular of up to 15, 10 or 5, especially of up to 4, 3 or 2 amino acids, especially with deletions and / or insertions of exactly one amino acid.

Thus, for example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without thereby losing the proteolytic activity. Such mutations are described, for example, in WO 99/49057. WO 01/07575 teaches that deletions of this kind lower the allergenicity of the proteases involved and, as a result, their overall substitutability can be improved. Fragmentation benefits the later-described aspect of insertion or substitution mutagenesis and / or fusion with other enzymes. With regard to the intended use of these enzymes, it is particularly preferred if they also have a proteolytic activity after fragmentation or deletion mutagenesis.

Numerous prior art documents also disclose beneficial effects of insertions and substitutions in subtilases. In principle, in addition to the substitution of individual amino acids, this also includes the substitution of several contiguous amino acids. This also includes recombinations of larger enzyme sections, such as the above-mentioned fragments, with other proteases or proteins of a different function. For example, it is possible, based on WO 99/57254, to provide a protein of the invention or parts thereof via peptidic linkers or directly as a fusion protein with binding domains of other proteins, such as the cellulose-binding domain, thereby making the hydrolysis of the substrate more effective. Likewise, proteins according to the invention can also be linked with amylases or cellulases, for example, in order to perform a dual function.

Preferred among the polypeptides according to the invention are those protein variants which have one or more amino acid exchanges in 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, 211, 224, 229, 236, 237, 242, 243, 255 and 268 in the enumeration of Bacillus lentus alkaline protease.

Chimeric proteins according to the invention have in the broadest sense a proteolytic activity. This may be exercised or modified by a moiety derived from a polypeptide of the invention. The chimeric proteins can therefore also lie outside the range claimed above over their entire length. For example, the purpose of such a fusion is to introduce or modify a particular function or partial function using the fused protein portion of the invention. For the purposes of the present invention, it is irrelevant whether such a chimeric protein consists of a single polypeptide chain or several subunits. In order to realize the last-mentioned alternative, it is possible, for example, to break down a single chimeric polypeptide chain into several or more by post-translational or only after a purification step by means of a targeted proteolytic cleavage.

For example, it is possible, based on WO 99/57254, to provide a polypeptide according to the invention or parts thereof via peptidic linkers or directly as a fusion protein with binding domains from other proteins, for example the cellulose-binding domain, thereby making the hydrolysis of the substrate more effective. Such a binding domain could also be derived from a protease, such as to enhance the binding of the protein of the invention to a protease substrate. This increases the local protease concentration, which may be advantageous in individual applications, for example in the treatment of raw materials. Likewise, proteins according to the invention can also be linked with amylases or cellulases, for example, in order to perform a dual function.

The polypeptides according to the invention obtainable by insertion mutation are to be assigned to their basic similarity because of the chimeric proteins according to the invention. This subheading also includes substitution variants, ie those in which individual regions of the molecule have been replaced by elements from other proteins. The purpose of insertion and substitution mutagenesis, as in hybrid formation, is to combine individual properties, functions or partial functions of proteins according to the invention with those of other proteins. This also includes, for example, variants to be obtained via shuffling or recombination of partial sequences from different proteases. As a result, proteins can be obtained which have not previously been described. Such techniques allow drastic effects to very subtle activity modulations.

Preferably, such mutations are random priming recombinations according to a statistical method attributable to the Directed Evolution field, such as StEP (Zhao et al., 1998, Nat. Biotechnol., Vol. 16, pp. 258-261) (Shao et al., (1998), Nucleic Acids Res., Vol. 26, pp. 681-683), DNA shuffling (Stemmer, WPC (1994), Nature, Vol. 370, pp. 389-391) or recursive Sequence recombination (RSR, WO 98/27230, WO 97/20078, WO 95/22625) or the method RACHITT (Coco, WM et al. (2001), Nat. Biotechnol., Volume 19, pp 354-359) performed , Conveniently, such methods are coupled with a selection or screening procedure following mutagenesis and expression to detect variants having the desired properties. Since these techniques are based on the DNA level, the starting point for biotechnological production is available with the respective newly generated genes.

Inversion mutagenesis, ie a partial sequence reversal, can be regarded as a special form of both the deletion and the insertion. Such variants can also be generated purposefully or randomly.

Preference is given to all such polypeptides according to the invention which have hitherto been carried out and which are characterized in that they are capable of hydrolysing protein per se.

Such are summarized under the official enzymes Nomenclature 1992 IUBMB under 3.4 (peptidases). Among them, endopeptidases, especially of the groups 3.4.21 serine proteinases, 3.4.22 cysteine proteinases, 3.4.23 aspartate proteinases and 3.4.24 metalloproteinases, are preferred. Among these are Serine proteinases (3.4.21) are particularly preferred, including subtilases and especially subtilisins (see "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C Betzel, New York, 1996). Among these, in turn, the subtilisins of group IS-2, the highly alkaline subtilisins, are preferred.

In this case, active molecules are preferred to inactive ones, since, for example, the proteolysis carried out is of importance, for example, in the fields of application set out below.

The above-mentioned fragments also have a proteolytic activity in the broadest sense, for example for complexing a substrate or for forming a structural element required for the hydrolysis. They are preferred if, taken alone, they can already be used for the hydrolysis of another protein without the need for further protease components to be present. This refers to the activity that can be exerted by a protease per se; the possibly simultaneously necessary presence of buffer substances, cofactors etc. remains unaffected.

The interaction of different parts of the molecule for the hydrolysis of proteins naturally occurs in deletion mutants rather than in fragments, and arises in particular in fusion proteins, especially those which have resulted from a shuffling of related proteins. Insofar as a proteolytic function in the broadest sense is maintained, modified, specified, or even achieved, the deletion variants are proteins according to the invention, as in the case of the fusion proteins. Preferred representatives of this subject invention include those which per se are capable of hydrolyzing a protein substrate without the need for further protease components to be present.

A preferred embodiment are all such polypeptides according to the invention carried out so far, which are characterized in that they are additionally stabilized. This increases their stability during storage and / or during their use, for example in the washing process, so that their activity lasts longer and is thus reinforced. The stability of proteases according to the invention can be increased, for example, by coupling to polymers. It requires that the proteins be linked to such polymers via a chemical coupling step prior to their use in appropriate agents.

Preference is given to stabilizations which are possible via point mutagenesis of the molecule itself. Because these require no further work steps following the protein recovery. Some point mutations suitable for this purpose are known per se from the prior art. For example, proteases can be stabilized by replacing certain tyrosine residues with others.

Other options include:

- The replacement of certain amino acid residues with proline;

the introduction of polar or charged groups on the surface of the molecule;

- Alteration of the binding of metal ions, in particular the calcium binding sites.

According to the patent US 5453372 proteins can be protected by certain mutations on the surface against the influence of denaturing agents such as surfactants.

Another way to stabilize against elevated temperature and the action of surfactants would be the stabilization via the exchange of amino acids that are close to the N-terminus, against those that come into contact with the rest of the molecule via non-covalent interactions and thus a Contribute to the maintenance of the globular structure.

A preferred embodiment are all such polypeptides according to the invention carried out so far, which are characterized in that they are additionally derivatized. Derivatives are understood as meaning proteins which are derived from the exported proteins by an additional modification. Such modifications may affect, for example, stability, substrate specificity, or binding strength to the substrate or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, increase its skin compatibility.

Such derivatizations can be carried out, for example, biologically, for example in connection with the protein biosynthesis by the producing host organism. Here, couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy.

However, derivatizations can also be carried out chemically, for example by the chemical transformation of a side chain or by covalent bonding of another, for example macromolecular, compound to the protein. For example, such a coupling of amines to carboxyl groups of the enzyme to change the isoelectric point done. Furthermore, macromolecules such as proteins, for example via bifunctional chemical compounds, can be bound to proteins according to the invention. Such a macromolecule may be a binding domain, for example. Such derivatives are particularly suitable for use in detergents or cleaners. Analogously, protease inhibitors can also be linked to the proteins according to the invention via linkers, in particular amino acid linkers. Couplings with other macromolecular compounds such as polyethylene glycol improve the molecule for other properties such as stability or skin compatibility.

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, all preparations of a protein according to the invention are also according to the invention. That is also independent of whether it actually unfolds this enzymatic activity in a particular preparation or not. Because it may be desired that it has no or only low activity during storage, and unfolds its proteolytic function only at the time of use. This can be controlled, for example, via appropriate accompanying substances such as protease inhibitors.

A preferred embodiment is any of those proteins, protein fragments, fusion proteins or derivatives characterized by having at least one antigenic determinant in common with one of the polypeptides of the invention described above.

Because crucial for the enzymatic activities are the secondary structural elements of a protein and its three-dimensional folding. Thus, in their primary structure distinctly different domains can form spatially largely identical structures and thus enable the same enzymatic behavior. Such commonalities in the secondary structure are commonly recognized as consonant antigenic determinants of antisera or pure or monoclonal antibodies. By means of immunochemical cross-reactions, similar proteins or derivatives can thus be detected and assigned. For this reason, the scope of the present invention also includes those proteins which may not be able to be assigned to the above-defined proteins, protein fragments, fusion proteins or derivatives according to the invention via their homology values in the primary structure, but via their immunochemical relationship.

A preferred embodiment are all such polypeptides according to the invention which have hitherto been carried out and which are characterized in that they are obtainable from a natural source, in particular from a microorganism.

These may be, for example, unicellular fungi or bacteria. Because they are usually easier to win and handle as multicellular organisms or cell cultures derived from multicellular organisms; although these are for specific embodiments can represent meaningful options and thus are not excluded in principle from the subject invention.

Although it is possible that naturally occurring producers can produce an enzyme according to the invention, they only express this to a slight extent under the initially determined conditions and / or release it into the surrounding medium. However, this does not exclude that suitable environmental conditions or other factors can be determined experimentally, under the influence of which they can be stimulated to economically meaningful production of the protein according to the invention. Such a regulatory mechanism can be used specifically for biotechnological production. If this too is not possible, they can still serve to isolate the associated gene.

Particularly preferred among these are those from gram-positive bacteria.

Because they have no outer membrane and thus release secreted proteins directly into the surrounding medium.

Very particularly preferred are those from gram-positive bacteria of the genus Bacillus.

Bacillus proteases have from the outset favorable properties for various technical applications. These include some stability to elevated temperature, oxidizing or denaturing agents. In addition, microbial proteases have the greatest experience in terms of their biotechnological production, for example the construction of favorable cloning vectors, the selection of host cells and growth conditions or the estimation of risks, such as allergenicity. Bacilli are also established as production organisms with a particularly high production output in technical processes. The wealth of experience that has been acquired for the production and use of these proteases also benefits further developments of these enzymes according to the invention. This applies, for example, to her Compatibility with other chemical compounds, such as the ingredients of detergents or cleaners.

Among those from the Bacillus species, in turn, those from the species Bacillus pumilus, in particular from the strain of Bacillus pumilus used according to the invention, are preferred.

Because of this, the embodiment of the enzyme according to the invention was originally obtained. Its associated sequences are given in the Sequence Listing. From this or related strains, the variants described above can be prepared in particular using standard molecular biological methods, such as PCR and / or per se known point mutagenesis method.

A further solution of the problem and thus a separate subject of the invention are the nucleic acids which serve to realize the invention.

A person skilled in the art will be able to prepare complete genes by known DNA and / or amino acid sequences via methods that are generally known today, such as, for example, chemical synthesis or the polymerase chain reaction (PCR) in conjunction with molecular biological and / or proteinchemical standard methods. Such methods are known for example from the "Encyclopaedia of Biochemistry", Spektrum Akademischer Verlag, Berlin, 1999, Volume 1, pp. 267-271 and Volume 2, pp. 227-229. This is possible in particular if it is possible to fall back on a strain deposited in a master collection. For example, with PCR primers which can be synthesized by means of a known sequence and / or isolated mRNA molecules, the relevant genes can be synthesized from such strains, cloned and, if desired, further processed, for example mutagenized.

Nucleic acids form the starting point of almost all molecular biological investigations and further developments as well as the production of proteins. These include in particular the sequencing of the genes and the derivation of the associated Amino acid sequence, any kind of mutagenesis (see above) and the expression of the proteins.

Mutagenesis for the development of proteins with specific properties is also referred to as "protein engineering". Properties to be optimized have already been exemplified above. Such a mutagenesis may be targeted or by random methods, for example, with subsequent activity-directed recognition and / or selection (screening and selection) on the cloned genes, such as hybridization with nucleic acid probes, or on the gene products, the proteins, be done about their activity. The further development of the proteases according to the invention can also be carried out in particular in the "Protein engineering" publication by P.N.Bryan (2000) in Biochim. Biophys. Acta, vol. 1543. pp. 203-222, are presented.

Another object of the present invention are therefore also polynucleotides encoding polypeptides according to the invention, in particular hydrolases, especially subtilisin-type alkaline proteases. The present invention therefore relates in particular to polynucleotides selected from the group consisting of: a) polynucleotide having a nucleic acid sequence according to SEQ ID NO: 1, b) polynucleotide having a nucleic acid sequence from position 1 to 153 according to SEQ ID NO: 1, c) polynucleotide with a nucleic acid sequence from position 1 to 324 according to SEQ ID NO: 1, d) polynucleotide having a nucleic acid sequence from position 325 to 1152 according to SEQ ID NO: 1, e) polynucleotide coding for a polypeptide having an amino acid sequence according to SEQ ID NO: 2, f Polynucleotide encoding a polypeptide having an amino acid sequence from position 1 to 51 according to SEQ ID NO: 2, g) polynucleotide coding for a polypeptide having an amino acid sequence from position 1 to 108 according to SEQ ID NO: 2, h) polynucleotide coding for a polypeptide having an amino acid sequence from position 109 to 383 according to SEQ ID NO: 2, i) polynucleotide coding for a polypeptide according to the invention, j) naturally occurring or artificially produced mutants or polymorphic forms or alleles of a polynucleotide according to (a) with up to 55, preferably up to 50, 45, 40 or 30, more preferably up to 25, 20, 15 or 10, especially up to 9, 8, 7, 6, 5, 4, 3 or 2 mutations, especially with exactly one mutation, k) naturally occurring or artificially produced mutants or polymorphic forms or alleles of a polynucleotide according to (b) or (c) with up to 8, preferably up to 7, 6 or 5, more preferably up to 4, 3 or 2 mutations, especially with exactly one mutation,

I) naturally occurring or artificially produced mutants or polymorphic forms or alleles of a polynucleotide according to (d) having up to 40 mutations, preferably up to 35, 30 or 25, more preferably up to 20, 15 or 10, in particular up to 9, 8 , 7, 6, 5, 4, 3, or 2 mutations, especially with exactly one mutation, m) polynucleotides having a sequence homology or identity of at least 95%, preferably at least 96 or 97%, more preferably at least 98% at least 99% with respect to a polynucleotide according to (a), n) polynucleotides having a sequence homology or identity of at least 95% with respect to a polynucleotide according to (b), o) polynucleotides having a sequence homology or identity of at least 98% with respect to a polynucleotide according to (c), p) polynucleotides having a sequence homology or identity of at least 95.5%, preferably at least 96 or 97%, more preferably at least 98%, especially at least 99% in B a polynucleotide according to (d), q) under stringent conditions with a polynucleotide according to (a), (b), (c) or (d) hybridizing polynucleotides, under stringent conditions is preferably to be understood incubation at 60 ⁰ C in a solution containing O ₁ lxSSC and 0.1% sodium dodecylsulfite (SDS), where 2OxSSC denotes a solution containing 3 M sodium chloride and 0.3 M sodium citrate (pH 7.0), r) polynucleotides consisting of at least 200, in particular at least 250, 300, 350 or 400, more preferably at least 450, 500, 550 or 600, especially at least 650, 700, 750 or 800 consecutive nucleic acids of a polynucleotide according to (a), ( c), (d), (g), (h), (m) or (p), s) polynucleotides having deletions and / or insertions and / or inversions of up to 50, preferably up to 40, 30 or 20 , particularly preferably up to 15, 10 or 5, in particular up to 4, 3 or 2 nucleotides, in particular insertions and / or deletions of exactly one nucleotide with respect to a polynucleotide according to (a) to (r), in particular Reference to a polynucleotide according to (a) or (d), t) polynucleotides comprising at least one of the polynucleotides mentioned under (a) to (s), u) to polynucleotides according to (a) to (t) complementary polynucleotides.

The polynucleotides may be present as a single strand or as a double strand. Subject matter of the invention are, besides the deoxyribonucleic acids, also the homologous and complementary ribonucleic acids.

The present invention also relates, in particular, to those polynucleotides in which certain regions have been replaced by other regions, taking into account the differing codon usage of a host organism used for expression, in order to enable the expression of the polypeptide according to the invention.

According to the statements made above, the following are increasingly preferred among the nucleic acids according to the invention described above:

- Those which are characterized by being obtainable from a natural source, in particular from a microorganism;

- including those characterized in that the microorganism is a Gram-positive bacterium;

- including those characterized in that the Gram-positive bacterium is one of the genus Bacillus; and - Among these, those which are characterized in that it is the Bacillus species Bacillus pumilus, in particular the strain used in the invention.

A separate subject of the invention are vectors which contain one of the previously described nucleic acid regions according to the invention, in particular one which codes for one of the polypeptides according to the invention designated above.

Because in order to deal with the invention relevant nucleic acids, and thus in particular to prepare the production of polypeptides according to the invention, they are suitably ligated into vectors. Such vectors as well as the associated working methods are described in detail in the prior art. Vectors are commercially available in large numbers and with a wide variety of variations, both for cloning and for expression. These include, for example, vectors derived from bacterial plasmids, bacteriophages or viruses, or predominantly synthetic vectors. Furthermore, they are differentiated according to the type of cell types in which they are able to establish themselves, for example vectors for gram-negative, gram-positive bacteria, yeast or higher eukaryotes. They form suitable starting points for example for molecular biological and biochemical investigations and for the expression of the relevant gene or associated protein.

In one embodiment, vectors of the invention are cloning vectors.

Because cloning vectors are suitable in addition to the storage, the biological amplification or the selection of the gene of interest for its molecular biological characterization. At the same time they are transportable and storable forms of the claimed nucleic acids and are also starting points for molecular biological techniques that are not bound to cells, such as the PCR or / n-we-mutagenesis method. Preferably, vectors according to the invention are expression vectors.

Because such expression vectors are the basis for realizing the corresponding nucleic acids in biological production systems and thus to produce the associated proteins. Preferred embodiments of this subject matter of the invention are expression vectors carrying genetic elements necessary for expression, for example the natural promoter originally located in front of this gene or a promoter from another organism. These elements can be arranged for example in the form of a so-called expression cassette. Alternatively, individual or all regulatory elements may also be provided by the respective host cell. With particular preference, the expression vectors are matched to the selected expression system, in particular the host cell (see below), with regard to further properties, for example the optimal copy number.

It is furthermore advantageous for a high expression rate if the expression vector contains as far as possible only the relevant gene as an insert and no larger 5 ¹ or 3 'noncoding regions. Such inserts are obtained, for example, when the fragment obtained after statistical treatment of the chromosomal DNA of the starting strain with a restriction enzyme has been cut again after sequencing before integration into the expression vector.

An example of an expression vector is the vector pAWA22. Other vectors are available to those skilled in the art and are commercially available in large numbers.

A separate subject of the invention are cells which contain a polynucleotide according to the invention after genetic engineering modification.

Because these cells contain the genetic information for the synthesis of a protein according to the invention. Among these, in contrast to the natural producers described above, are those cells meant, which have been provided according to known methods with the nucleic acids of the invention, or derived from such cells. For this purpose, suitable suitable host cells are those which can be cultivated relatively easily and / or yield high product yields.

They allow, for example, the amplification of the corresponding genes, but also their mutagenesis or transcription and translation and ultimately the biotechnological production of the proteins in question. This genetic information can either be extrachromosomally as a separate genetic element, ie be present in bacteria in plasmidaler localization or integrated into a chromosome. The choice of a suitable system depends on issues such as the nature and duration of storage of the gene, or the organism or the type of mutagenesis or selection. For example, based on bacteriophages - and their specific host cells - the prior art described mutagenesis and selection processes for the development of detergent enzymes.

The polynucleotide according to the invention is preferably part of one of the above-described vectors according to the invention, in particular of a cloning or expression vector.

Because this makes them relevant to the realization of the present invention.

Furthermore, those cells are preferred which express a polypeptide of the invention and preferably secrete.

Only the host cells that make up the proteins enable their biotechnological production. In principle, all organisms, that is prokaryotes, eukaryotes or cyanophyta, are suitable as host cells for protein expression. Preference is given to those host cells which can be genetically well handled, for example, the transformation with the expression vector, its stable establishment and the regulation of expression, for example, unicellular fungi or bacteria. In addition, preferred host cells are characterized by good microbiological and biotechnological handling. This concerns, for example, light Cultivability, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferably, laboratory strains are selected which are aligned to the expression. Such are available commercially or via publicly available strain collections. Each protein of the invention can thus be theoretically obtained from a variety of host organisms. From the abundance of various systems available in the prior art, the optimal expression systems for the individual case must be determined experimentally.

Particularly advantageous are host cells which are themselves protease-negative and thus do not degrade proteins formed.

Preferred embodiments are those host cells which are regulatable in their activity by virtue of corresponding genetic elements, for example by controlled addition of chemical compounds, by changing the cultivation conditions or depending on the respective cell density. This controllable expression allows very economical production of the proteins of interest; It can be realized, for example, via a corresponding element on the relevant vector. Suitably, the gene, expression vector and host cell are matched to one another, which relates, for example, to the genetic elements required for expression (ribosome binding site, promoters, terminators) or the codon usage.

Of these, preference is given to those expression hosts which secrete the protein formed into the surrounding medium because it can be easily processed by comparison.

Further preferred are host cells which are bacteria.

Because bacteria are characterized by short generation times and low demands on the cultivation conditions. As a result, inexpensive methods can be established. In addition, bacteria have a wealth of experience in fermentation technology. For a special production can out Gram-negative or gram-positive bacteria may be suitable for a wide variety of reasons to be determined experimentally in individual cases, such as nutrient sources, product formation rate, time requirement, etc.

In a preferred embodiment, it is a gram-negative bacterium, in particular one of the genera Escherichia coli or Klebsiella, in particular strains of E. coli K12, E. coli B or Klebsiella planticola, and more particularly derivatives of the strains Escherichia coli BL21 (DE3 ) E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

For Gram-negative bacteria, such as E. coli, a variety of proteins are secreted into the periplasmic space. This can be advantageous for special applications. In the application WO 01/81597 a method is disclosed according to which it is achieved that gram-negative bacteria also eject the expressed proteins. Such a system is also suitable for the production of proteins according to the invention. The Gram-negative bacteria which are mentioned as preferred are generally light, that is to say commercially or accessible via public strain collections and, in conjunction with other components which are likewise available in large numbers, such as vectors, can be optimized for specific production conditions.

In an alternative, no less preferred embodiment, it is a Gram-positive bacterium, in particular one of the genera Bacillus, Staphylococcus or Corynebacteria, more particularly of the species Bacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B. globigii, B gibsonii, B. pumilus or ß. alcalophilus, Staphylococcus carnosus or Corynebacterium glutamicum.

Because gram-positive bacteria have the gram-negative compared to the fundamental difference, secreted proteins readily deliver into the nutrient medium surrounding the cells, from which, if desired, the expressed proteins according to the invention can be purified directly from the nutrient medium. In addition, they are related or identical to most of the organisms of origin for technically important subtilisins and usually form even comparable subtilisins that they have a similar codon usage and their protein synthesizer is naturally aligned accordingly. A further advantage may be that a mixture of proteins according to the invention with the subtilisins formed endogenously by the host strains can be obtained by this process. Such a co-expression is also apparent from the application WO 91/02792. Should it not be desired, the protease genes naturally present in the host cell would have to be permanently or temporarily inactivated.

Further preferred are host cells which are eukaryotic cells, preferably of the genus Saccharomyces.

Examples thereof are fungi such as Actinomycetes or even yeasts such as Saccharomyces or Kluyveromyces. Thermophilic fungal expression systems are presented, for example, in WO 96/02653 A1. Such are particularly suitable for the expression of temperature-resistant variants. Modifications that eukaryotic systems perform, especially in connection with protein synthesis, include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce allergenicity. Also, coexpression with the enzymes naturally produced by such cells, such as cellulases, may be advantageous.

An independent subject of the invention are processes for the preparation of a polypeptide according to the invention.

This includes any method for producing a polypeptide of the invention described above, for example, chemical synthesis methods.

In contrast, however, preference is given to all molecular-biological, microbiological or biotechnological preparation methods which are established in the prior art and are already mentioned above in individual aspects and which are based on the above-described nucleic acids according to the invention. For this purpose, according to the above, for example, in the sequence listing under SEQ ID NO. 1 specified nucleic acids or mutants derived therefrom or partial sequences thereof are used.

These are preferably processes which are carried out using a previously designated vector and more preferably preferably using a previously described, advantageously genetically modified cell. Because this provides the correspondingly preferred genetic information in a microbiologically usable form.

Embodiments of the present invention, based on the associated nucleic acid sequences, may also be cell-free expression systems in which protein biosynthesis is understood in vitro. All of the elements already described above can also be combined to form new methods for producing proteins according to the invention. It is conceivable for each protein according to the invention a variety of possible combinations of process steps, so that optimal procedures must be determined experimentally for each specific case.

In accordance with the above, those methods are preferred in which the nucleotide sequence has been adapted in one, preferably a plurality of codons to the codon usage of the host strain.

A separate subject of the invention are agents which contain the abovementioned polypeptides according to the invention.

This includes all types of agents, in particular mixtures, formulations, solutions, etc., the substitutability of which is improved by addition of a protein according to 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 synergistic with the proteases Ingredients. 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. UhNg ₁ Wiley-Verlag, New York, 1998.

Possible fields of use here are, in particular, the use for obtaining or treating raw materials or intermediates in textile production, in particular for removing protective layers on fabrics, in particular wool or silk, and the use for the care of textiles, the natural fibers, in particular wool or silk, contain.

Because in particular natural fibers, such as wool or silk, are characterized by a characteristic, microscopic surface structure. This can, as the example of wool in the article by R. Breier in Melliand textile reports of 1.4.2000 (p 263) has been carried out in the long term to undesirable effects, such as entanglement lead. To avoid such effects, the natural raw materials are treated with agents according to the invention which, for example, help to smooth the shingled surface structure based on protein structures and thus counteract entanglement.

Accordingly, the invention also relates to processes for the treatment of textile raw materials and for textile care, in which polypeptides according to the invention are used in at least one of the process steps. These include processes for textile raw materials, fibers or textiles with natural constituents, in particular those with wool or silk. These may be, for example, processes in which materials for processing in textiles are prepared, for example for anti-fungal finishing, or, for example, for processes which enrich the cleaning of worn textiles with a nourishing component.

Other possible uses include the use for biochemical analysis or for the synthesis of low molecular weight compounds or of proteins, including preferably the use for end group determination in the context of a peptide sequence analysis;

- the use for the preparation, purification or synthesis of natural substances or biologically valuable substances;

the use for the treatment of natural raw materials, in particular for surface treatment, more particularly in a process for the treatment of leather, in particular for the depilation of leather;

the use for the treatment of photographic films, in particular for the removal of gelatin-containing or similar protective layers; and

the use for the production of food or feed, in particular for the enzymatic treatment of soymilk and / or soymilk products.

In principle, the use of the abovementioned polypeptides according to the invention in all other fields of technology for which it proves to be suitable is included in the scope of protection of the present application.

Another use according to the invention is the use of the polypeptides according to the invention in cosmetic agents. This is understood to mean all types of cleansing and conditioning agents for human skin or hair, in particular cleansing agents. The agent may also be a pharmaceutical agent depending on the purpose of use.

Because proteases also play a crucial role in the cell renewal process of the human skin (desquamation) (T Egelrud et al., Acta Derm. Venerol., Vol. Zl (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. The use of subtilisin proteases with amino acid substitutions in the positions R99G / A / S, S154D / E and / or L211 D / E for cosmetic purposes is described, for example, in WO 97/07770 A1. According to the above, proteases according to the invention can have the corresponding point mutations be further developed. 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. Particular preference is given to those preparations of these enzymes which, as described above, are stabilized, for example by coupling to macromolecular carriers (cf. US Pat. No. 5,230,891) and / or derivatized by point mutations at highly allergenic positions, so that they have a higher skin compatibility for humans.

Examples of cosmetic and / or pharmaceutical compositions according to the invention are shampoos, soaps, washing lotions, creams, peels and mouth, tooth or denture care agents. In particular, these compositions may also contain constituents, as mentioned below for detergents and cleaners.

Accordingly, corresponding cosmetic cleaning and care methods and the use of such proteolytic enzymes for cosmetic purposes are also included in this subject matter, in particular in appropriate agents, such as shampoos, soaps or washing lotions, or in care products that are offered, for example in the form of creams. Also, the use in a peeling drug, or for the use for its production is included in this article.

A particularly preferred subject according to the invention are detergents and cleaners containing polypeptides according to the invention. As is shown in the exemplary embodiments of the present application, washing and cleaning agents with a protease preferred according to the invention surprisingly found an increase in the washing performance compared to agents with conventionally employed proteases.

For the purposes of the present application, the washing performance or the cleaning performance of a washing or cleaning agent is to be understood as the effect which the agent in question has on the soiled articles, for example textiles or fabrics Objects with hard surfaces. Individual components of such agents, in particular the enzymes according to the invention, are assessed with regard to their contribution to the washing or cleaning performance of the entire detergent or cleaning agent. It should be noted in particular that from the enzymatic properties of an enzyme can not be readily deduced to its contribution to the washing performance of an agent. Rather, in addition to the enzymatic activity, factors such as stability, substrate binding, binding to the items to be cleaned or interactions with other ingredients of the detergents or cleaning agents, in particular possible synergy effects in the removal of the contaminants, also play a role here.

As stated above, for the homologous proteases Q5XPN0, Q2HXI3, Q9KWR4 and Q6SIX5 the use in a detergent or cleaning agent is not disclosed.

Another object of the present invention are therefore detergents and cleaning agents, in particular surfactant and / or bleach-containing, containing a polypeptide, in particular a hydrolase, preferably a protease, more preferably a subtilisin-type alkaline protease selected from the group consisting a) polypeptide having an amino acid sequence according to SEQ ID NO: 2, b) polypeptide having an amino acid sequence from position 109 to 383 according to SEQ ID NO: 2, c) naturally occurring or artificially produced mutants, polymorphic forms or alleles of a polypeptide according to ( a) or (b) with up to 50, more preferably with up to 45, 40, 35, 30, 25 or 20, in particular with up to 15, 12, 10, 9, 8, 7, 6 or 5, particularly preferred with up to 4, 3 or 2 mutations, especially with exactly one mutation, d) polypeptides having a sequence homology or identity of at least 80%, preferably at least 81, 82, 83, 84, 85, 86, 87, 88, 89 or 90%, more preferably of at least 91, 92, 93, 94 or 95%, especially of at least 96, 97, 98 or 99% with respect to a polypeptide according to (a) or (b), e) polypeptides consisting of at least 50, more preferably from at least 60, 70, 80, 90 or 100, more preferably from at least 120, 140, 160, 180 or 200, especially from at least 220, 240, 260 or 270 consecutive amino acids the amino acid sequence according to (a) or (b), f) polypeptides having insertions and / or deletions and / or inversions of up to 50, preferably of up to 40, 30 or 20, particularly preferably of up to 15, 10 or 5, in particular up to 4, 3 or 2 amino acids, in particular insertions and / or deletions of exactly one amino acid with respect to a polypeptide according to (a), (b) (c), (d) or (e), g) polypeptides comprising at least one of the polypeptides mentioned under (a) to (f).

The present invention preferably detergents and cleaning agents containing the aforementioned polypeptides according to the invention with higher homology to the inventive polypeptide according to SEQ ID NO: 2 or to the polypeptide of the invention from position 109 to 383 according to SEQ ID NO: 2 ,

The detergents and cleaning agents according to the invention may be any conceivable type of cleaning agent, both concentrates and agents to be used undiluted, for use on a commercial scale, in the washing machine or in hand washing or cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which according to the present invention the term laundry detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather; for such according to the present invention, the term cleaning agent is used. In a broader sense sterilizing and disinfecting agents are to be regarded as detergents and cleaners in the sense of the invention. Embodiments of the present invention include all of the prior art and / or all suitable administration forms of the washing or cleaning agents according to the invention. These include, for example, solid, powdered, liquid, gelatinous or pasty agents, if appropriate also of several phases, compressed or uncompressed; further include, for example: extrudates, granules, tablets or pouches, packed both in large containers and in portions.

In a preferred embodiment, the detergents or cleaners according to the invention contain the above-described polypeptides according to the invention, in particular alkaline subtilisin-type proteases, in an amount of from 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg, most preferably from 50 μg to 10 mg per gram of the agent. Included are all integer and non-integer values lying between these numbers.

The protease activity in such agents can be determined by the method described in Tenside, Vol. 7 (1970), pp. 125-132. It is accordingly stated in PE (protease units).

When comparing the performance of two detergent enzymes, such as in the examples of the present application, a distinction must be made between protein-identical and activity-equivalent use. Particularly in the case of genetically engineered, largely side-activity-free preparations, the use of the same protein is appropriate. Because this is a statement on whether the same amounts of protein - as a measure of the yield of fermentative production - lead to comparable results. If the respective ratios of active substance to total protein (the values of the specific activity) diverge, then an activity-equivalent comparison is recommended because this compares the respective enzymatic properties. In general, a low specific activity can be compensated by adding a larger amount of protein. This is ultimately an economic consideration. In addition to a polypeptide according to the invention, a washing or cleaning agent according to the invention optionally contains further ingredients such as further enzymes, enzyme stabilizers, surfactants, for. As nonionic, anionic and / or amphoteric surfactants, and / or bleaching agents, and / or builder, and optionally other conventional ingredients, which are carried out in the following.

The nonionic surfactants used are preferably alkoxylated, advantageously ethoxylated, in particular primary, alcohols having preferably 8 to 18 carbon atoms and on average 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 may contain linear and methyl-branched radicals in the mixture, 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 example of coconut, palm, tallow or oleyl alcohol, and on average 2 to 8 EO per mole of alcohol are preferred. Preferred ethoxylated alcohols include, for example, Ci ₂ - ₁₄ alcohols with 3 EO or 4 EO, _-11 -alcohol with 7 EO, C _13-15 -alcohols containing 3 EO, 5 EO, 7 EO or 8 EO, C ₁₂ -i ₈ -alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of Ci ₂ -i ₄ -alcohol with 3 EO and C _12-18 -alcohol with 5 EO. The degrees of ethoxylation given represent statistical means which, for a particular product, may be an integer or a fractional number. Preferred alcohol ethoxylates have a narrow homolog distribution (narrow rank ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples of these are tallow fatty alcohol with 14 EO, 25 EO, 30 EO or 40 EO.

Another class of preferred nonionic surfactants 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 from 1 to 4 carbon atoms in the alkyl chain, especially fatty acid methyl esters.

Another class of nonionic surfactants that can be used to advantage are the alkyl polyglycosides (APG). Usable Alkypolyglycosides satisfy the general formula RO (G) _Z in which R is a linear or branched, especially in the 2-position methyl-branched, saturated or unsaturated, 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 glycosylation z is between 1, 0 and 4.0, preferably between 1, 0 and 2.0 and in particular between 1, 1 and 1, 4. Preference is given to using linear alkyl polyglucosides, that is to say alkyl polyglycosides in which the polyglycosyl radical is a glucose radical and the alkyl radical is an n-alkyl radical.

Nonionic surfactants of the amine oxide type, for example N-cocoalkyl N, N-dimethylamine oxide and N-tallow alkyl-N, N-dihydroxyethyl amine oxide, and the fatty acid alkanolamides may also be suitable. The proportion of these nonionic surfactants is preferably not higher than that of the ethoxylated fatty alcohols, especially not more than half of them.

Further suitable surfactants are polyhydroxy fatty acid amides of the formula (II)

I

R-CO-N- [Z] (II)

in which RCO is an aliphatic acyl radical having 6 to 22 carbon atoms, R ^ 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.

The group of polyhydroxy fatty acid amides also includes compounds of the formula R ¹ -OR ²

I

R-CO-N- [Z] (III)

in the 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, wherein d- ₄ alkyl or phenyl radicals are preferred and [Z] is a linear polyhydroxyalkyl radical whose alkyl chain is substituted with at least two hydroxyl groups, or alkoxylated, preferably ethoxylated or propoxylated derivatives this rest.

[Z] is preferably obtained by reductive amination of a reducing sugar, for example glucose, fructose, maltose, lactose, galactose, mannose or xylose. The N-alkoxy- or N-aryloxy-substituted compounds can be converted into the desired polyhydroxy fatty acid amides, for example, by reaction with fatty acid methyl esters in the presence of an alkoxide as catalyst.

As anionic surfactants, for example, those of the sulfonate type and sulfates are used. Suitable surfactants of the sulfonate type are preferably Cg-ia alkyl benzene sulfonates, olefin sulfonates, ie mixtures of alkene and hydroxyalkane sulfonates, and the disulfonates obtained, for example, from C _{_2-18} monoolefins with terminal or internal double bond by sulfonation with gaseous sulfur trioxide and subsequent alkaline or acidic hydrolysis of the sulfonation obtained. Also suitable are alkanesulfonates, from C ₂ - ₁₈ are obtained, for example, alkanes by sulfochlorination or sulfoxidation and subsequent hydrolysis or neutralization. Likewise suitable are the esters of .alpha.-sulfo fatty acids (ester sulfonates), for example the .alpha.-sulfonated methyl esters of hydrogenated coconut, palm kernel or tallow fatty acids. Further suitable anionic surfactants are sulfated fatty acid glycerol esters. Fatty acid glycerol esters are to be understood as meaning the mono-, di- and triesters and mixtures thereof, as obtained in the preparation by esterification of a monoglycerol with 1 to 3 moles of fatty acid or in the transesterification of triglycerides with 0.3 to 2 moles of glycerol. Preferred sulfated fatty acid glycerol esters are the sulfonation products of saturated fatty acids having 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) yl sulfates are the alkali and especially the sodium salts of the Schwefelsäurehalbester C ₂ -C ₈ fatty alcohols, for example coconut fatty alcohol, tallow fatty alcohol, lauryl, myristyl, cetyl or stearyl alcohol, or C ₀ -C ₂ o 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. From the washing are the C ₂ -C ₆ alkyl sulfates and C ₂ -Cis-alkyl sulfates, and C ₄ -C ₅ alkyl sulfates of preferably. 2,3-alkyl sulfates are also suitable anionic surfactants.

Also, the Schwefelsäuremonoester with 1 to 6 moles of ethylene ethoxylated straight-chain or branched C _7-21 alcohols, such as 2-methyl-branched C ₉ n-alcohols containing on average 3.5 mol ethylene oxide (EO) or C _2- ₈ i Fatty alcohols containing 1 to 4 EO are suitable. Due to their high foaming behavior, they are only used in detergents in relatively small amounts, for example in amounts of up to 5% by weight, usually from 1 to 5% by weight.

Further suitable anionic surfactants are also the salts of alkylsulfosuccinic acid, which are also referred to as sulfosuccinates or as sulfosuccinic acid esters and the monoesters and / or diesters of sulfosuccinic acid with alcohols, preferably fatty alcohols and in particular ethoxylated fatty alcohols. Preferred sulfosuccinates contain Cβ-iβ fatty alcohol residues or mixtures of these. In particular, especially preferred sulfosuccinates contain a fatty alcohol radical which is derived from ethoxylated fatty alcohols, which in themselves constitute nonionic surfactants (description see above). 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) yl-succinic acid having preferably 8 to 18 carbon atoms in the alk (en) yl chain or salts thereof.

As further anionic surfactants are particularly 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, soap mixtures derived from natural fatty acids, for example coconut, palm kernel or tallow fatty acids.

The anionic surfactants including the soaps may be in the form of their sodium, potassium or ammonium salts, as well as soluble salts of organic bases such as mono-, di-triethanolamine. The anionic surfactants are preferably present in the form of their sodium or potassium salts, in particular in the form of the sodium salts.

The surfactants may be contained in the detergents or detergents according to the invention overall in an amount of preferably from 5% by weight to 50% by weight, in particular from 8% by weight to 30% by weight, based on the finished composition ,

Detergents or cleaners according to the invention may contain bleaches. Among the compounds which serve as bleaches and provide H ₂ O ₂ in water, sodium percarbonate, sodium perborate tetrahydrate and sodium perborate monohydrate are of particular importance. Other useful bleaching agents are, for example, peroxopyrophosphates, citrate perhydrates and H ₂ O ₂ -producing peracidic salts or peracids, such as persulfates or persulfuric acid. Also useful is the urea peroxohydrate percarbamide, which can be described by the formula H ₂ N-CO-NH ₂ H ₂ O ₂ . In particular, when using the means for cleaning hard surfaces, for example in automatic dishwashing, you can also bleach if desired from the group of organic Bleach contain, although their use is also possible in principle for laundry detergents. Typical organic bleaches are the diacyl peroxides, such as dibenzoyl peroxide. Other typical organic bleaches are the peroxyacids, examples of which include the alkyl peroxyacids and the aryl peroxyacids. Preferred representatives are the peroxybenzoic acid and its ring-substituted derivatives, such as alkylperoxybenzoic acids, but also peroxy-α-naphthoic acid and magnesium monoperphthalate, the aliphatic or substituted aliphatic peroxyacids, such as peroxylauric acid, peroxystearic acid, ε-phthalimidoperoxycaproic acid (Phthalimidoperoxyhexansäure, PAP), o-carboxy benzamidoperoxycaproic acid, N-nonenylamidoperadipic acid and N-nonylamido-persuccinates, and aliphatic and araliphatic peroxydicarboxylic acids, such as 1,12-diperoxycarboxylic acid, 1,9-diperoxyazelaic acid, diperoxysebacic acid, diperoxybrassic acid, the diperoxyphthalic acids, 2-decyldiperoxybutan-1,4-diacid, N, N-Terephthaloyl-di (6-aminopercaproic acid) can be used.

The content of bleach detergent or cleaning agent may be from 1 to 40% by weight and in particular from 10 to 20% by weight, with perborate monohydrate or percarbonate being advantageously used.

In order to achieve an improved bleaching effect during washing at temperatures of 60 ^° C. and below, and in particular during the laundry pretreatment, the agents may also contain bleach activators. As bleach activators, it is possible to use compounds which, under perhydrolysis conditions, give aliphatic peroxycarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Suitable substances are those which carry O- and / or N-acyl groups of the stated C atom number and / or optionally substituted benzoyl groups. 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 1, 3,4,6 Tetraacetylglycoluril (TAGU), N-acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular / 7-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), acylated hydroxycarboxylic acids, such as triethyl-O-acetylcitrate (TEOC) , Carboxylic acid hydrides, in particular phthalic anhydride, isatoic anhydride and / or succinic anhydride, carboxamides, such as N-methyldiacetamide, glycolide, acylated polyhydric alcohols, in particular triacetin, ethylene glycol diacetate, isopropenyl acetate, 2,5-diacetoxy-2,5-dihydrofuran and those from the German patent applications DE 196 16 693 and DE 196 16 767 known enol esters and acetylated sorbitol and mannitol or their mixtures described in European Patent Application EP 0525239 (SORMAN), acylated sugar derivatives, in particular pentaacetylglucose (PAG), pentaacetyl, tetraacetylxylose and Octaacetyllactose and acetylated, optionally N -alkyiated glucamine or gluconolactone, triazole or triazole derivatives and / or particulate caprolactams and / or caprolactam derivatives, preferably N-acylated lactams, for example N-benzoylcaprolactam and N-acetylcaprolactam, which are known from international patent applications WO 94/27970, WO 94/28102, WO 94/28103, WO 95/00626, WO 95/14759 and WO 95/17498 are known. The hydrophilic substituted acyl acetals known from the German patent application DE 196 16 769 and the acyllactams described in the German patent application DE 196 16 770 and the international patent application WO 95/14075 are also preferably used. The combinations of conventional bleach activators known from German patent application DE 4443 177 can also be used. Likewise, nitrile derivatives such as cyanopyridines, nitrile quats, for example N-alkylammonium acetonitriles, and / or cyanamide derivatives can be used. Preferred bleach activators are sodium 4- (octanoyloxy) benzenesulfonate, n-nonanoyl or isononanoyloxybenzenesulfonate (/? - or iso-NOBS), undecenoyloxybenzenesulfonate (UDOBS), sodium dodecanoyloxybenzenesulfonate (DOBS), decanoyloxybenzoic acid (DOBA, OBC 10) and / or dodecanoyloxybenzenesulfonate (OBS 12), as well as N-methylmorpholine-acetonitrile (MMA). Such bleach activators can be used in the customary amount range of from 0.01 to 20% by weight, preferably in amounts of from 0.1 to 15% by weight, in particular from 1% to 10% by weight, based on the total composition, be included.

In addition to or in place of the conventional bleach activators, so-called bleach catalysts may also be included. These substances are Bleach-enhancing transition metal salts or transition metal complexes such as Mn, Fe, Co, Ru or Mo-salene complexes or carbonyl complexes. Mn, Fe, Co, Ru, Mo, Ti, V and Cu complexes with N-containing tripod ligands and also Co, Fe, Cu and Ru ammine complexes are suitable as bleach catalysts, wherein such compounds are preferably used, which are described in DE 19709284 A1.

Detergents or cleaners according to the invention generally comprise one or more builders, in particular zeolites, silicates, carbonates, organic cobuilders and, where there are no ecological reasons against their use, also the phosphates. The latter are particularly preferred builders to be used in automatic dishwashing detergents.

To mention here are crystalline, layered sodium silicates of the general formula

NaMSi _x O _{2x + 1} 7H ₂ O, where M is sodium or hydrogen, x is a number from 1, 6 to 4, preferably 1, 9 to 4.0, and y is a number from 0 to 20 and preferred values for x 2 , 3 or 4 are. Such crystalline layered silicates are described, for example, in European Patent Application EP 164514. Preferred crystalline layered silicates of the formula given are those in which M is sodium and x assumes the values 2 or 3. In particular, both β- and δ-sodium disilicates

Na ₂ Si ₂ O _O yH ₂ O is preferred. Such compounds are commercially available, for example, under the name ^SKS® (Clariant company). That is how it is

SKS-6 ^® mainly to a δ-sodium disilicate with the formula Na ₂ Si ₂ O ₅ yH ₂ O, in SKS-7 ^® mainly to the ß-sodium disilicate. Reaction with acids (for example citric acid or carbonic acid) produces kanemite from the δ-sodium disilicate

NaHSi ₂ O ₅ yH ₂ O, commercially available under the names SKS- ^9® or SKS- ^10® (Clariant). It may also be advantageous to use chemical modifications of these phyllosilicates. For example, the alkalinity of the layered silicates can be suitably influenced. Phyllosilicates doped with phosphate or with carbonate have altered crystal morphologies in comparison with the δ-sodium disilicate, dissolve more rapidly and show an increased calcium binding capacity in comparison with δ-sodium disilicate. So phyllosilicates are the general empirical formula x Na ₂ O • ySiO ₂ • z P ₂ O ₅ , in which the ratio x to y is a number 0.35 to 0.6, the ratio x to z is a number from 1, 75 to 1200 and the ratio y to z correspond to a number from 4 to 2800, described in the patent application DE 196 01 063. The solubility of the layered silicates can also be increased by using particularly finely divided layered silicates. Also compounds from the crystalline layer silicates with other ingredients can be used. In particular, compounds with cellulose derivatives which have advantages in the disintegrating effect and are used in particular in detergent tablets, and compounds with polycarboxylates, for example citric acid, or polymeric polycarboxylates, for example copolymers of acrylic acid, may be mentioned.

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 Delayed 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 also understood to mean "X-ray amorphous". This means 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. However, it may well even lead to particularly good builder properties if the silicate particles provide blurred or even sharp diffraction maxima in electron diffraction experiments. This is to be interpreted as meaning that the products have microcrystalline regions of size 10 to a few hundred nm, values of up to max. 50 nm and in particular up to max. 20 nm are preferred. Particularly preferred are compacted / compacted amorphous silicates, compounded amorphous silicates and overdried X-ray amorphous silicates.

An optionally usable, fine crystalline, synthetic and bound water-containing zeolite is preferably zeolite A and / or P. As zeolite P is 5

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) ), which is sold by the company CONDEA Augusta SpA under the brand name VEGOBOND AX ^® and by the formula

n Na 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. Suitable zeolites have an average particle size of less than 10 μm (volume distribution, measuring method: Coulter Counter) and preferably contain 18 to 22% by weight, in particular 20 to 22% by weight, of bound water.

Of course, a use of the well-known phosphates as builders is possible, unless such use should not be avoided for environmental reasons. Among the large number of commercially available phosphates, the alkali metal phosphates, with particular preference for pentasodium or pentapotassium triphosphate (sodium or potassium tripolyphosphate), are of greatest importance in the washing and cleaning agent industry.

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 ) _n and orthophosphoric H ₃ PO _{4 in} addition to higher 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 also contribute to the cleaning performance.

Sodium dihydrogen phosphate, NaH ₂ PO ₄ , exists as dihydrate (density 1, 91 like ^"3 , melting point 60 °) and as monohydrate (density 2.04 like ^{" 3} ). Both salts are white powders which are very slightly soluble in water and lose the water of crystallization when heated at 200 ^D C into the weak acid diphosphate (disodium hydrogen diphosphate, Na ₂ H ₂ P ₂ O ₇ ), at higher temperature in sodium trimetaphosphate (Na ₃ PaO ₉ ) and Maddrell's salt (see below), go over. NaH ₂ PO _{4 is} acidic; It arises when phosphoric acid is adjusted to a pH of 4.5 with sodium hydroxide solution and the mash is sprayed. Potassium dihydrogen phosphate (potassium phosphate primary or monobasic, potassium biphosphate, KDP), KH ₂ PO ₄ , is a white salt of density 2.33 ^"3 , has a melting point of 253 ⁰ C [decomposition to form potassium polyphosphate (KPO ₃ ) χ] and is easily soluble in water.

Disodium hydrogen phosphate (secondary sodium phosphate), Na ₂ HPO ₄ , is a colorless, very slightly water-soluble crystalline salt. It exists anhydrous and with 2 moles (density 2.066 like ^"3 , loss of water at 95 °), 7 moles (density 1, 68 like ^{" 3} , melting point 48 ° C with loss of 5 H ₂ O) and 12 moles water (Density 1.52 ^"3 , melting point 35 ° C with loss of 5 H ₂ O), becomes anhydrous at 10O ⁰ C and on more intense heating in the diphosphate Na ₄ P ₂ O. ₇ Disodium hydrogen phosphate is by neutralization of phosphoric acid prepared with soda solution using phenolphthalein as an indicator Dipotassium hydrogen phosphate (secondary or dibasic potassium phosphate), K ₂ HPO ₄ , is an amorphous, white salt that is readily soluble in water.

Trisodium phosphate, tertiary sodium phosphate, Na ₃ PO _4, colorless crystals, as the dodecahydrate a density of 1, 62 like ^"3 and a melting point of 73-76 ⁰ C (decomposition), as the decahydrate (corresponding to 19-20% P ₂ O ₅ ) have a melting point of 10O ⁰ C and in anhydrous form (corresponding to 39-40% P ₂ O ₅ ) a density of 2.536 like ^"3 have. Trisodium phosphate is readily soluble in water under alkaline reaction and is prepared by evaporation of a solution of exactly 1 mole of disodium phosphate and 1 mole of NaOH. Tripotassium phosphate (tertiary or tribasic potassium phosphate), K ₃ PO ₄ , is a white, fissile, granular powder of density 2.56 ^'3 , has a melting point of 1340 ° and is readily soluble in water with an alkaline reaction. It is produced, for example, by heating Thomasschlacke with coal and potassium sulfate. Despite the higher price, the more soluble, therefore highly effective, potassium phosphates are often preferred over the corresponding sodium compounds in the detergent industry. Tetrasodium diphosphate (sodium pyrophosphate), Na ₄ P ₂ θ _7l exists in anhydrous form (density 2.534 like ^"3 , melting point 988 ⁰ C, also indicated 88O ⁰ C) and as decahydrate (density 1, 815-1, 836 like ^'3 , melting point 94 ° C with loss of water.) Both substances are colorless crystals which are soluble in water with an alkaline reaction Na ₄ P ₂ O ₇ is formed by heating disodium phosphate to> 200 ° C or by reacting phosphoric acid with soda in a stoichiometric ratio and passing the solution through The decahydrate complexes heavy metal salts and hardness agents and therefore reduces the hardness of the water.Kali diphosphate (potassium pyrophosphate), K ₄ P ₂ O ₇ , exists in the form of the trihydrate and provides a colorless, hygroscopic powder with a density of 2.33 ^'3 , which is soluble in water, wherein the pH of the 1% solution at 25 ° C is 10.4.

Condensation of the NaH ₂ PO ₄ or of the KH ₂ PO ₄ gives rise to relatively high molecular weight sodium and potassium phosphates, in which cyclic representatives, the sodium or potassium metaphosphates and chain-type, the sodium or potassium polyphosphates, can be distinguished. In particular, for the latter are a variety of names in use: hot or cold phosphates, Graham's salt, Kurrolsches and Maddrell's salt. All higher sodium and potassium phosphates are collectively referred to as condensed phosphates.

The industrially important pentasodium triphosphate (Na ₅ P ₃ Oi _O ; sodium tripolyphosphate) is anhydrous or water-soluble salt which crystallizes with 6 H ₂ O and has the general formula NaO- [P (O) (ONa) -O] _n - Na with n = 3. In 100 g of water at room temperature dissolve about 17 g, at 60 ⁰ C about 20 g, at 100 ⁰ C, about 32 g of the salt water-free salt; after two hours of heating the solution to 100 ° C by hydrolysis about 8% orthophosphate and 15% diphosphate. In the preparation of pentasodium triphosphate, phosphoric acid is reacted with soda solution or sodium hydroxide solution in a stoichiometric ratio and the solution is dehydrated by spraying. Similar to Graham's salt and sodium diphosphate, pentasodium triphosphate dissolves many insoluble metal compounds (including lime soaps, etc.). Pentakaliumtriphosphat, K ₅ P ₃ Oi ₀ (potassium tripolyphosphate) comes for example in the form of a 50 wt .-% solution (> 23% P ₂ O ₅ , 25% K ₂ O) in the trade. The potassium polyphosphates are widely used in the washing and cleaning industry. There are also sodium potassium tripolyphosphates which can also be used in the context of the present invention. These arise, for example, when hydrolyzed sodium trimetaphosphate with KOH:

(NaPOa) ₃ + 2 KOH -> Na ₃ K ₂ P ₃ Oi ₀ + H ₂ O

These are used according to the invention exactly as sodium tripolyphosphate, potassium tripolyphosphate or mixtures of these two; Mixtures of sodium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of potassium tripolyphosphate and sodium potassium tripolyphosphate or mixtures of sodium tripolyphosphate and potassium tripolyphosphate and sodium potassium tripolyphosphate can also be used according to the invention.

As organic cobuilders, it is possible in particular to use in the detergents and cleaners according to the invention polycarboxylates or polycarboxylic acids, polymeric polycarboxylates, polyaspartic acid, polyacetals, optionally oxidized dextrins, further organic cobuilders (see below) and phosphonates. These classes of substances are described below.

Useful organic builder substances are, for example, the polycarboxylic acids which can be used in the form of their sodium salts, polycarboxylic acids meaning those carboxylic acids which carry more than one acid function. These are, for example, citric acid, adipic acid, succinic acid, glutaric acid, malic acid, tartaric acid, maleic acid, fumaric acid, sugar acids, aminocarboxylic acids, nitrilotriacetic acid (NTA), if such use can not be avoided for ecological reasons, and mixtures of these. Preferred salts are the salts of polycarboxylic acids such as citric acid, adipic acid, succinic acid, glutaric acid, tartaric acid, sugar acids and mixtures thereof. The acids themselves can also be used. In addition to their builder effect, they also typically have the property of an acidifying component and thus also serve to set a lower and milder pH of detergents or cleaners, unless the pH resulting from the mixture of the other components is desired. In particular, systemic and environmentally compatible acids such as citric acid, acetic acid, tartaric acid, malic acid, lactic acid, glycolic acid, succinic acid, glutaric acid, adipic acid, gluconic acid and any desired mixtures of these can be mentioned in particular. But also mineral acids, in particular sulfuric acid or bases, in particular ammonium or alkali hydroxides can serve as pH regulators. Such regulators are contained in the agents according to the invention in amounts of preferably not more than 20% by weight, in particular from 1.2% by weight to 17% by weight.

Other suitable builders are polymeric polycarboxylates, for example the alkali metal salts of polyacrylic acid or of polymethacrylic acid, for example those having a relative molecular weight of 500 to 70,000 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 from 2,000 to 20,000 g / mol. Because of their superior solubility, the short-chain polyacrylates, which have molecular weights of from 2,000 to 10,000 g / mol, and particularly preferably from 3,000 to 5,000 g / mol, may again be preferred from this group. Also suitable are copolymeric polycarboxylates, in particular those of acrylic acid with methacrylic acid and of acrylic acid or methacrylic acid with maleic acid. Copolymers of acrylic acid with maleic acid which contain 50 to 90% by weight of acrylic acid and 50 to 10% by weight of maleic acid have proven to be particularly suitable. Their molecular weight relative to free acids is generally from 2000 to 70,000 g / mol, preferably from 20,000 to 50,000 g / mol and in particular from 30,000 to 40,000 g / mol. The (co) polymeric polycarboxylates can be used either as a powder or as an aqueous solution. The content of the (co) polymeric polycarboxylates may be from 0.5 to 20% by weight, in particular from 1 to 10% by weight.

To improve the water solubility, the polymers may also contain allylsulfonic acids such as allyloxybenzenesulfonic acid and methallylsulfonic acid as a monomer.

Also particularly preferred are biodegradable polymers of more than two different monomer units, for example those which contain as monomers salts of acrylic acid and maleic acid and vinyl alcohol or vinyl alcohol derivatives or as monomers salts of acrylic acid and 2-alkylallylsulfonic acid and sugar derivatives contain.

Further preferred copolymers are those which have as monomers preferably acrolein and acrylic acid / acrylic acid salts or acrolein and vinyl acetate.

Also to be mentioned as further preferred builders polymeric aminodicarboxylic acids, their salts or their precursors. Particular preference is given to polyaspartic acids or their salts and derivatives.

Further suitable builder substances are polyacetals which can be obtained by reacting dialdehydes with polyolcarboxylic acids which have 5 to 7 C atoms and at least 3 hydroxyl groups. Preferred polyacetals are 5

obtained from dialdehydes such as glyoxal, glutaraldehyde, terephthalaldehyde and mixtures thereof and from polyol carboxylic acids such as gluconic acid and / or glucoheptonic acid.

Further suitable organic builder substances are dextrins, for example oligomers or polymers of carbohydrates, which can be obtained by partial hydrolysis of starches. The hydrolysis can be carried out by customary, for example acid or enzyme catalyzed processes. Preferably, it is hydrolysis products having average molecular weights in the range of 400 to 500 000 g / mol. In this case, a polysaccharide with a dextrose equivalent (DE) in the range from 0.5 to 40, in particular from 2 to 30 is preferred, DE being a customary measure of the reducing action of a polysaccharide in comparison to dextrose, which is a DE of 100 has. Both maltodextrins with a DE of between 3 and 20 and dry glucose syrups with a DE of between 20 and 37 and also yellow dextrins and white dextrins with relatively high molecular weights in the range from 2 000 to 30 000 g / mol are useful.

The oxidized derivatives of such dextrins are their reaction products with oxidizing agents which are capable of oxidizing at least one alcohol function of the saccharide ring to the carboxylic acid function. Particularly preferred organic builders for agents according to the invention are oxidized starches or their derivatives from the applications EP 472042, WO 97/25399, and EP 755944.

Oxydisuccinates and other derivatives of disuccinates, preferably ethylenediamine disuccinate, are other suitable co-builders. Ethylenediamine-N, ^{N'-disuccinate} (EDDS) is preferably in the form of its sodium or magnesium salts. Also preferred in this context are glycerol disuccinates and glycerol trisuccinates. Suitable amounts are in zeolith-, carbonate and / or silicate-containing formulations between 3 and 15 wt .-%.

Other useful organic cobuilders are, for example, acetylated hydroxycarboxylic acids or their salts, which may also be present in Lactone form can be present and which contain at least 4 carbon atoms and at least one hydroxy group and a maximum of two acid groups.

Another substance class with cobuilder properties are the phosphonates. These are, in particular, hydroxyalkane or aminoalkanephosphonates. Among the hydroxyalkane phosphonates, 1-hydroxyethane-1,1-diphosphonate (HEDP) is of particular importance as a co-builder. It is preferably used as the sodium salt, the disodium salt neutral and the tetrasodium salt alkaline (pH 9). Preferred aminoalkanephosphonates are ethylenediamine tetramethylenephosphonate (EDTMP), diethylenetriaminepentamethylenephosphonate (DTPMP) and their higher homologs. They are preferably in the form of neutral sodium salts, eg. B. as hexasodium salt of EDTMP or as hepta- and octa-sodium salt of DTPMP ₁ used. The builder used here is preferably HEDP from the class of phosphonates. The aminoalkanephosphonates also have a pronounced heavy metal binding capacity. Accordingly, in particular if the agents also contain bleach, it may be preferable to use aminoalkanephosphonates, in particular DTPMP, or to use mixtures of the phosphonates mentioned.

Moreover, all compounds capable of forming complexes with alkaline earth ions can be used as co-builders.

Builder substances may optionally be present in the detergents or cleaners according to the invention in amounts of up to 90% by weight. They are preferably contained in amounts of up to 75% by weight. Detergents according to the invention have builder contents of, in particular, from 5% by weight to 50% by weight. In agents according to the invention for the cleaning of hard surfaces, in particular for the automated cleaning of dishes, the content of builder substances is in particular from 5% by weight to 88% by weight, wherein preferably no water-insoluble builder materials are used in such agents. In a preferred embodiment of inventive means for the particular machine cleaning of dishes are 20 wt .-% to 40 wt .-% of water-soluble organic builder, in particular Alkali citrate, 5 wt .-% to 15 wt .-% alkali carbonate and 20 wt .-% to 40 wt .-% Alkalidisilikat included.

Solvents that can be used in the liquid to gelatinous compositions of detergents and cleaners, for example, from the group of monohydric or polyhydric alcohols, alkanolamines or glycol ethers, provided that they are miscible in the specified concentration range with water. The solvents are preferably selected from ethanol, n- or i-propanol, butanols, ethylene glycol methyl ether, ethylene glycol ethyl ether, ethylene glycol propyl ether, ethylene glycol mono-n-butyl ether, diethylene glycol methyl ether, diethylene glycol ethyl ether, propylene glycol methyl, -ethyl or -propyl ether, dipropylene glycol monomethyl -, or -ethyl ether, di-isopropylene glycol monomethyl, or -ethyl ether, methoxy, ethoxy or Butoxytriglykol, i-butoxyethoxy-2-propanol, 3-methyl-3-methoxybutanol, propylene glycol t-butyl ether and mixtures of these solvents ,

Solvents may be used in the liquid to gelled detergents and cleaners according to the invention in amounts of between 0.1 and 20% by weight, but preferably below 15% by weight and in particular below 10% by weight.

To adjust the viscosity, one or more thickeners or thickening systems can be added to the composition according to the invention. These high-molecular substances, which are also called swelling agents, usually absorb the liquids and swell up to finally pass into viscous true or colloidal solutions.

Suitable thickeners are inorganic or polymeric organic compounds. The inorganic thickeners include, for example, polysilicic acids, clay minerals such as montmorillonites, zeolites, silicas and bentonites. The organic thickeners are derived from the groups of natural polymers, modified natural polymers and fully synthetic polymers. Such naturally derived polymers include, for example, agar-agar, carrageenan, tragacanth, gum arabic, alginates, pectins, polyoses, guar gum, locust bean gum, starch, dextrins, gelatin and casein. Modified natural substances that are used as thickeners, come mainly from the group of modified starches and celluloses. Examples include carboxymethylcellulose and other cellulose ethers, hydroxyethyl and propyl cellulose and Kemmehlether called. Fully synthetic thickeners are polymers such as polyacrylic and polymethacrylic compounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines, polyamides and polyurethanes.

The thickeners may be present in an amount of up to 5% by weight, preferably from 0.05 to 2% by weight, and more preferably from 0.1 to 1.5% by weight, based on the finished composition ,

The washing and cleaning agent according to the invention may optionally contain, as further customary ingredients, sequestrants, electrolytes and other auxiliaries, such as optical brighteners, grayness inhibitors, silver corrosion inhibitors, color transfer inhibitors, foam inhibitors, abrasives, dyes and / or fragrances, as well as microbial active ingredients, UV absorbers and or contain enzyme stabilizers.

Detergents according to the invention may contain, as optical brighteners, derivatives of diaminostilbenedisulfonic acid or their alkali metal salts. Suitable are, for example, salts of

4,4'-bis (2-anilino-4-morpholino-1,3,5-triazinyl-6-amino) stilbene-2,2'-disulphonic acid or similarly structured compounds which, instead of the morpholino group, a diethanolamino group, a Methylamino group, an anilino group or a 2-Methoxyethylaminogruppe carry. Further, brighteners of the substituted diphenylstyrene type may be present, for example, the alkali salts of 4,4'-bis (2-sulfostyryl) -diphenyl, 4,4'-bis (4-chloro-3-sulfostyryl) -diphenyl, or 4 - (4-chlorostyryl) -4 '- (2-sulfostyryl). Mixtures of the aforementioned optical brightener can be used.

Graying inhibitors have the task of keeping suspended from the textile fiber dirt suspended in the fleet. Water-soluble colloids of mostly organic nature are suitable for this purpose, for example starch, glue, gelatin, salts of ether carboxylic acids or ether sulfonic acids of starch or of cellulose or salts of acidic sulfuric acid esters of cellulose or starch. Also water-soluble, Acidic group-containing polyamides are suitable for this purpose. Furthermore, other than the above-mentioned starch derivatives can be used, for example aldehyde starches. Preference is given to using cellulose ethers, such as carboxymethylcellulose (Na salt), methylcellulose, hydroxyalkylcellulose and mixed ethers, such as methylhydroxyethylcellulose, methylhydroxypropylcellulose, methylcarboxymethylcellulose and mixtures thereof, for example in amounts of from 0.1 to 5% by weight, based on the compositions ,

To effect silver corrosion protection, silver corrosion inhibitors can be used in dishwashing detergents according to the invention. Such are known in the art, for example benzotriazoles, iron (III) chloride or CoSO ₄ . For example, as known from European Patent EP 0 736 084 B1, particularly suitable silver corrosion inhibitors for use in conjunction with enzymes are manganese, titanium, zirconium, hafnium, vanadium, cobalt or cerium salts and / or complexes where the said metals are present in one of the oxidation states II, III, IV, V or VI. Examples of such compounds are MnSO ₄ , V ₂ O ₅ , V ₂ O ₄ , VO ₂ , TiOSO ₄ , K ₂ TiF ₆ , K ₂ ZrF ₆ , Co (NO ₃ ) ₂ , Co (NO ₃ ) ₃ , and the like mixtures.

"Soil-release" or "soil repellents" are mostly polymers which impart soil repellency when used in a laundry detergent detergent and / or aid in the soil release performance of the other detergent ingredients. A similar effect can also be observed in their use in hard surface cleaners.

Particularly effective and long-known soil release agents are copolyesters with dicarboxylic acid, alkylene glycol and polyalkylene glycol units. Examples of these are copolymers or copolymers of polyethylene terephthalate and polyoxyethylene glycol (DT 16 17 141, or DT 22 00 911). In German Offenlegungsschrift DT 22 53 063 acidic agents are mentioned which contain, inter alia, a copolymer of a dibasic carboxylic acid and an alkylene or cycloalkylene polyglycol. Polymers of ethylene terephthalate and polyethylene terephthalate and their use in detergents are in the German DE 28 57 292 and DE 33 24 258 and European Patent EP 0 253 567. European patent EP 066944 relates to compositions containing a copolyester of ethylene glycol, polyethylene glycol, aromatic dicarboxylic acid and sulfonated aromatic dicarboxylic acid in certain molar ratios. European Patent EP 0 185427 discloses methyl or ethyl group end-capped polyesters having ethylene and / or propylene terephthalate and polyethylene oxide terephthalate units and laundry detergents containing such soil release polymer. European patent EP 0 241 984 relates to a polyester which, besides oxyethylene groups and terephthalic acid units, also contains substituted ethylene units and also glycerine units. European Patent EP 0 241 985 discloses polyesters which, in addition to oxyethylene groups and terephthalic acid units, contain 1, 2-propylene, 1, 2-butylene and / or 3-methoxy-1, 2-propylene groups and also glycerol units and with d - Are capped to C ₄ alkyl groups. European Patent Application EP 0 272 033 discloses, at least in part, end-capped polyesters with poly-propylene terephthalate and polyoxyethylene terephthalate units by means of d-4-alkyl or acyl radicals. European Patent EP 0 274 907 describes sulfoethyl end-capped terephthalate-containing soil release polyesters. According to European Patent Application EP 0 357 280, sulfonation of unsaturated end groups produces soil release polyesters with terephthalate, alkylene glycol and poly-C _2-4 glycol units. International Patent Application WO 95/32232 relates to acidic, aromatic soil release polymers. International Patent Application WO 97/31085 discloses non-polymeric soil repellent active ingredients for multi-functional cotton materials: a first entity, which may be cationic, for example, is capable of adsorption to the cotton surface by electrostatic interaction, and a second Unit that is hydrophobic is responsible for the retention of the drug at the water / cotton interface.

The color transfer inhibitors which are suitable for use in textile detergents according to the invention include, in particular, polyvinylpyrrolidones, polyvinyl imidazoles, polymeric N-oxides such as poly (vinylpyridine-N-oxide) and copolymers of vinylpyrrolidone with vinylimidazole.

When used in automated cleaning processes, it may be advantageous to add foam inhibitors to the agents concerned. As foam inhibitors are, for example, soaps of natural or synthetic origin, which have a high proportion of Ci ₈ -C ₂₄ fatty acids. Suitable non-surfactant foam inhibitors are, for example, organopolysiloxanes and mixtures thereof with microfine, optionally signed silica and paraffins, waxes, microcrystalline waxes and mixtures thereof with signed silica or bistearylethylenediamide. It is also advantageous to use mixtures of various foam inhibitors, for example those of silicones, paraffins or waxes. Preferably, the foam inhibitors, in particular silicone and / or paraffin-containing foam inhibitors, are bound to a granular, water-soluble, or dispersible carrier substance. In particular, mixtures of paraffins and bistearylethylene diamides are preferred.

In addition, a hard surface cleaning agent according to the invention may contain abrasive constituents, in particular from the group comprising quartz flours, wood flours, plastic flours, chalks and glass microspheres and mixtures thereof. Abrasives are preferably present in the detergents according to the invention in an amount of not more than 20% by weight, in particular in an amount of from 5 to 15% by weight.

Dyes and fragrances are added to detergents and cleaners in order to improve the aesthetic appearance of the products and to provide the consumer with a visually and sensory "typical and unmistakable" product in addition to the washing and cleaning performance. As perfume oils or fragrances, individual perfume compounds, for example the synthetic products of the ester type, ethers, aldehydes, ketones, alcohols and hydrocarbons can be used. Fragrance compounds of the ester type are, for example, benzyl acetate, phenoxyethyl isobutyrate, p-tert-butylcyclohexyl acetate, linalyl acetate, dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalyl benzoate, Benzyl formate, ethyl methyl phenyl glycinate, allyl cyclohexyl propionate, styrallyl propionate and benzyl salicylate. The ethers include, for example, benzyl ethyl ether, to the aldehydes, for example, the linear alkanals having 8-18 C atoms, citral, citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde, hydroxycitronellal, lilial and bourgeonal, to the ketones, for example, the ionone, α-lsomethylionon and methyl cedryl ketone, to the alcohols anethole, citronellol, eugenol, geraniol, linalool, phenylethyl alcohol and terpineol, the hydrocarbons include mainly the terpenes such as limonene and pinene. Preferably, however, mixtures of different fragrances are used, which together produce an attractive fragrance. Such perfume oils may also contain natural fragrance mixtures such as are available from vegetable sources, for example, pine, citrus, jasmine, patchouly, rose or ylang-ylang oil. Also suitable are muscatel, sage, chamomile, clove, lemon balm, mint, cinnamon, lime, juniper, vetiver, olibanum, galbanum and labdanum, and orange blossom, neroliol, orange peel and sandalwood. Usually, the content of detergents and cleaners to dyes is less than 0.01 wt .-%, while perfumes can account for up to 2 wt .-% of the total formulation.

The fragrances can be incorporated directly into the detergents or cleaners, but it can also be advantageous to apply the fragrances to carriers, which enhance the adhesion of the perfume to the items to be cleaned and provide a slower fragrance release for long-lasting fragrance, especially of treated textiles. As such carrier materials, for example, cyclodextrins have been proven, the cyclodextrin-perfume complexes can be additionally coated with other excipients. A further preferred carrier for fragrances is the described zeolite X, which can also absorb fragrances instead of or in mixture with surfactants. Preference is therefore given to washing and cleaning agents containing the described zeolite X and fragrances, which are preferably at least partially absorbed on the zeolite.

Preferred dyes, the choice of which presents no difficulty to the skilled person, have a high storage stability and insensitivity to the others Ingredients of the agents and against light and no pronounced substantivity to textile fibers, so as not to stain them.

Detergents or cleaners may contain antimicrobial agents to combat microorganisms. Depending on the antimicrobial spectrum and mechanism of action, a distinction is made between bacteriostatic agents and bactericides, fungistatics and fungicides, etc. Important substances from these groups are, for example, benzalkonium chlorides, alkylarylsulfonates, halophenols and phenolmercuric acetate. Within the scope of the teaching according to the invention, the terms antimicrobial action and antimicrobial active substance have the customary meaning which is described, for example, by KH Wallhäußer ^' m "Practice of Sterilization, Disinfection - Preservation: Germ Identification - Company Hygiene" (5th Edition - Stuttgart; New York: Thieme, Suitable antimicrobial agents are preferably selected from the groups of alcohols, amines, aldehydes, antimicrobial acids or their salts, carboxylic acid esters, acid amides, phenols, phenol derivatives, diphenyls, diphenylalkanes , Urea derivatives, oxygen, nitrogen acetals and formals, benzamidines, isothiazolines, phthalimide derivatives, pyridine derivatives, antimicrobial surface active compounds, guanidines, antimicrobial amphoteric compounds, quinolines, 1, 2-dibromo-2,4-dicyanobutane, iodo-2-propyl butyl-ca rbamate, iodine, iodophores, peroxo compounds, halogen compounds and any mixtures of the above.

The antimicrobial agent may be selected from ethanol, n-propanol, i-propanol, 1,3-butanediol, phenoxyethanol, 1,2-propylene glycol, glycerol, undecylenic acid, benzoic acid, salicylic acid, dihydracetic acid, o-phenylphenol, N-methylmorpholine. acetonitrile (MMA), 2-benzyl-4-chlorophenol, 2,2'-methylenebis (6-bromo-4-chlorophenol), 4,4'-dichloro-2'-hydroxydiphenyl ether (dichlosan), 2,4 , 4'-trichloro-2'-hydroxydiphenyl ether (trichlosan), chlorhexidine, N- (4-chlorophenyl) -N- (3,4-dichlorophenyl) -urea,

N, N '- (1, 10-decanediyldi-1-pyridinyl-4-ylidene) bis (1-octanamine) dihydrochloride, N, N'-bis (4-chlorophenyl) -3,12- diimino-2,4,11,13-tetraaza-tetradecane diimidamide, Glucoprotamines, antimicrobial surface active quaternaries,

Guanidines including the bi- and polyguanidines, such as

1, 6-bis- (2-ethylhexyl-biguanido-hexane) -dihydrochloride,

1, 6-di- (N ₁ , Ni'-phenyldiguanido-N ₅ _l N ₅ ') -hexane-tetrahydrochloride,

1, 6-di- (N ₁ , N _T '-phenyl-N _L -methyldiguanido-Ns.Ns'J-hexane dihydrochloride,

1, 6-di- (N ₁ , N ₁ '-o-chlorophenyl-diguanido- N ₅ , N ₅ ') -hexane-dihydrochloride,

1, 6-di- (N ₁₎ N ₁ '-2,6-dichlorophenyldiguanido-N _{5 I} N _5') hexane dihydrochloride _I

1, 6-di- [Ni, N ₁ '-beta- (p-methoxyphenyl) diguanido-N _5, N _5'] -hexanes dihydrochloride,

1, 6-di- (N ₁ , N ₁ '-alpha-methyl-.beta.-phenyldiguanido-N ₅ , N ₅ ^< ) -hexane dihydrochloride,

1,6-di- (N ₁ , N ₂ -p-nitrophenyldiguanido-N, N, n-hexane dihydrochloride, omega: omega-di- (

Ni.N ^ -phenyldiguanido-Ns.Ns'J-di-n-propyl ether dihydrochloride, omega: omega'-di (N _1l N ₁ '-p-chlorophenyldiguanido-N _5, N _5') -di-n propyl ether tetrahydro chloride, 1,6-di- (N ₁ , N 1 -2,4-dichlorophenyldiguanido N, N, n-hexane tetrahydrochloride,

1, 6-di- (N ₁ , N 1 -p-methylphenyl-diguanido- N ₅ , N ₅ ') hexane-dihydrochloride,

1, 6-di- (N ₁ , N _T ') Aδ-trichlorophenyldiguanido-N, N, N'-hexane-tetrahydrochloride,

1, 6-di- [N ₁ , N 1 -alpha-fp-chlorophenyl) ethyldiguanido-N ₅ , N ₅ '] hexane dihydrochloride, omega: omega-di- (N ₁ , N ₁ ' -p-chlorophenyldiguanido- N ₅ , N ₅ ') m-xylene dihydrochloride,

1, 12-di- (N ₁ , NV-p-chlorophenyldiguanido-Ns.Ns ^ dodecane dihydrochloride,

1, 10-di- (N ₁ , N 1 -phenyl-diguanido-, N, N, N'-decan-tetrahydrochloride,

1, 12-di- (N ₁ , N 1 -phenyl-diguanido- N ₅ , N ₅ ') dodecane-tetrahydrochloride,

1, 6-di- (N ₁ , N ₂ -o-chlorophenyl-diguanido- N ₅ , N ₅ ') hexane-dihydrochloride,

1, 6-di- (N ₁ , N ₂ -o-chlorophenyl-diguanido- N ₅ , N ₅ ') hexane-tetrahydrochloride,

Ethylene bis (1-tolyl biguanide), ethylene bis (p-tolyl biguanide),

Ethylene bis (3,5-dimethylphenylbiguanide), ethylene bis (p-tert-amylphenyl biguanide),

Ethylene bis (nonylphenylbiguanide), ethylene bis (phenylbiguanide),

Ethylene bis (N-butylphenylbiguanide), ethylene bis (2,5-diethoxyphenylbiguanide),

Ethylene bis (2,4-dimethylphenyl biguanide), ethylene bis (o-diphenylbiguanide),

Ethylene bis (mixed amyl naphthyl biguanide), N-butyl ethylene bis (phenylbiguanide),

Trimethylene bis (o-tolylbiguanide), N-butyl-trimethyl-bis (phenyl biguanide) and the corresponding salts such as acetates, gluconates, hydrochlorides, hydrobromides, citrates,

Bisulfites, fluorides, polymaleates, N-cocoalkyl sarcosinates, phosphites, hypophosphites,

Perfluorooctanoates, silicates, sorbates, salicylates, maleates, tartrates, fumarates,

Ethylenediamine tetraacetates, iminodiacetates, cinnamates, thiocyanates, arginates, Pyromellitate, Tetracarboxybutyrate, benzoates, glutarates, monofluorophosphates, perfluoropropionates and any mixtures thereof. Also suitable are halogenated xylene and cresol derivatives, such as p-chlorometacresol or p-chloro-meta-xylene, and natural antimicrobial agents of plant origin (for example, from spices or herbs), animal and microbial origin. Preferably, antimicrobial surface-active quaternary compounds, a natural antimicrobial agent of plant origin and / or a natural antimicrobial agent of animal origin, most preferably at least one natural antimicrobial agent of plant origin from the group comprising caffeine, theobromine and theophylline and essential oils such as eugenol, thymol and geraniol, and / or at least one natural antimicrobial agent of animal origin from the group, comprising enzymes such as protein from milk, lysozyme and lactoperoxidase, and / or at least one antimicrobial surface-active quaternary compound with an ammonium, sulfonium, phosphonium, iodonium - or Arsoniumgruppe, peroxo compounds and chlorine compounds are used. Also substances of microbial origin, so-called bacteriocins, can be used.

The suitable as antimicrobial agents quaternary ammonium compounds (QAV) have the general formula (R ¹ ) (R ² ) (R ³ ) (R ⁴ ) N ⁺ X ^" , in which R ¹ to R ⁴ identical or different Ci-C _22nd Alkyl radicals, C ₇ -C ₂₈ -Aralkylreste or heterocyclic radicals, wherein two or in the case of an aromatic inclusion as in pyridine even three radicals together with the nitrogen atom, the heterocycle, for example a pyridinium or imidazolinium compound, form, and X ^" halide ions , Sulfate ions, hydroxide ions or like anions. For optimum antimicrobial activity, preferably at least one of the radicals has a chain length of 8 to 18, in particular 12 to 16, carbon atoms.

QACs can be prepared by reacting tertiary amines with alkylating agents, such as, for example, methyl chloride, benzyl chloride, dimethyl sulfate, dodecyl bromide, but also ethylene oxide. The alkylation of tertiary amines with a long alkyl radical and two methyl groups succeeds particularly easily, the quaternization of tertiary amines with two long radicals and a methyl group can with the aid of methyl chloride under mild conditions are carried out. Amines which have three long alkyl radicals or hydroxy-substituted alkyl radicals are less reactive and are preferably quaternized with dimethyl sulfate.

Suitable QACs are, for example, benzalkonium chloride

(N-alkyl-N, N-dimethyl-benzyl-ammonium chloride, CAS No. 8001-54-5), benzalkone B (mp-dichlorobenzyl-dimethyl-C 1-4 -alkylammonium chloride, CAS No. 58390-78-6), benzoxonium chloride (benzyl dodecyl bis (2-hydroxyethyl) ammonium chloride), cetrimonium bromide (N-hexadecyl-N, N-trimethyl ammonium bromide, CAS No. 57-09-0), benzetonium chloride (N, N-dimethyl-N- [2- [2- [p- (1,1,3,3-tetramethylbutyl) phenoxy] ethoxy] ethyl] benzylammonium chloride, CAS No. 121-54-0), dialkyldimethylammonium chlorides such as N-decyl-dimethyl-ammonium chloride (CAS No. 7173-51-5-5), didecyldi-methylammonium bromide (CAS No. 2390-68-3), dioctyl-dimethyl-ammonium chloride, 1-cetylpyridinium chloride (CAS No. 123-03 -5) and thiazoline iodide (CAS No. 15764-48-1) and mixtures thereof. Particularly preferred QUATS are the benzalkonium chlorides ₈ alkyl radicals with C ₈ -C, especially C ^ -Cu-Aklyl-benzyl-dimethyl-ammonium chloride.

Benzalkonium halides and / or substituted benzalkonium halides are for example commercially available as Barquat ^® ex Lonza, Marquat® ^® ex Mason, Variquat ^® ex Witco / Sherex and Hyamine ^® ex Lonza and as Bardac ^® ex Lonza. Other commercially obtainable antimicrobial agents are N- (3-chloroallyl) hexaminium chloride such as Dowicide and Dowicil ^® ^® ex Dow, benzethonium chloride such as Hyamine ^® 1622 ex Rohm & Haas, methylbenzethonium as Hyamine ^® 1ox ex Rohm & Haas, cetylpyridinium chloride such as Cepacol ex Merrell Labs ,

The antimicrobial agents are used in amounts of 0.0001 wt .-% to 1 wt .-%, preferably from 0.001 wt .-% to 0.8 wt .-%, particularly preferably from 0.005 wt .-% to 0.3 wt .-% and in particular from 0.01 to 0.2 wt .-% used.

The washing or cleaning agents according to the invention may contain UV absorbers (UV absorbers) which are applied to the treated textiles and the Improve light stability of the fibers and / or the light resistance of other formulation ingredients. Under UV absorber are organic substances (sunscreen) to understand, which are able to absorb ultraviolet rays and the absorbed energy in the form of longer-wave radiation, for example, to give off heat.

Compounds having these desired properties include, for example, the non-radiative deactivating compounds and derivatives of benzophenone having substituents in the 2- and / or 4-position. Also suitable are substituted benzotriazoles, in the 3-position phenyl-substituted acrylates (cinnamic acid derivatives, optionally with cyano groups in the 2-position), salicylates, organic Ni complexes and natural substances such as umbelliferone and the body's own urocanic acid. The biphenyl and, above all, stilbene derivatives described, for example in EP 0728749 A and are available as Tinosorb FD ^® ^® or Tinosorb FR ex Ciba commercially. As UV-B absorbers may be mentioned: 3-Benzylidencampher or 3-Benzylidennorcampher and its derivatives, for example 3- (4-Methylbenzy- liden) camphor, as described in EP 0693471 B1; 4-aminobenzoic acid derivatives, preferably 2-ethylhexyl 4- (dimethylamino) benzoate, 2-octyl 4- (dimethylamino) benzoate and

4- (dimethylamino) benzoesäureamylester; Esters of cinnamic acid, preferably 4-methoxycinnamic acid 2-ethylhexyl ester, 4-methoxycinnamic acid propyl ester, 4-methoxycinnamic acid isoamyl ester, 2-cyano-3,3-phenylcinnamic acid 2-ethylhexyl ester (octocrylene); Esters of salicylic acid, preferably 2-ethylhexyl salicylate, 4-isopropylbenzyl salicylate, homomenthyl salicylate; Derivatives of benzophenone, preferably 2-hydroxy-4-methoxybenzophenone, 2-hydroxy-4-methoxy-4'-methylbenzophenone, 2,2'-dihydroxy-4-methoxybenzophenone; Esters of benzalmalonic acid, preferably di-2-ethylhexyl 4-methoxybenzmalonate; Triazine derivatives, such as

2,4,6-Trianilino- (p-carbo-2'-ethyl-1'-hexyloxy) -1, 3,5-triazine and octyl triazone, as described in EP 0818450 A1 or dioctyl butamido triazone (Uvasorb® HEB ); Propane-1,3-diones such as 1- (4-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1,3-dione; Ketotricyclo (5.2.1.0) decane derivatives, as described in EP 0694521 B1. Also suitable are 2-phenylbenzimidazole-5-sulfonic acid and its alkali metal, alkaline earth metal, ammonium, alkylammonium, alkanolammonium and glucammonium salts; Sulfonic acid derivatives of benzophenones, preferably

2-hydroxy-4-methoxybenzophenone-5-sulfonic acid and its salts; Sulfonic acid derivatives of 3-Benzylidencamphers, such as 4- (2-oxo-3-bornylidenme- thyl) benzenesulfonic acid and 2-methyl-5- (2-oxo-3-bomylidene) sulfonic acid and salts thereof.

As a typical UV-A filter, in particular derivatives of benzoylmethane come into question, such as 1- (4'-tert-butylphenyl) -3- (4'-methoxyphenyl) propane-1, 3-dione, 4-tert-butyl 4'-methoxydibenzoylmethane (Parsol 1789),

1-phenyl-3- (4'-isopropylphenyl) -propane-1, 3-dione and enamine compounds, as described in DE 19712033 A1 (BASF). Of course, the UV-A and UV-B filters can also be used in mixtures. In addition to the soluble substances mentioned, insoluble photoprotective pigments, namely finely dispersed, preferably nano-metal oxides or salts, are also suitable for this purpose. Examples of suitable metal oxides are in particular zinc oxide and titanium dioxide and, in addition, oxides of iron, zirconium, silicon, manganese, aluminum and cerium and mixtures thereof. As salts silicates (talc), barium sulfate or zinc stearate can be used. The oxides and salts are already used in the form of the pigments for skin-care and skin-protecting emulsions and decorative cosmetics. The particles should have an average diameter of less than 100 nm, preferably between 5 and 50 nm and in particular between 15 and 30 nm. They may have a spherical shape, but it is also possible to use those particles which have an ellipsoidal or otherwise deviating shape from the spherical shape. The pigments may also be surface-treated, that is to say hydrophilized or hydrophobicized. Typical examples are coated titanium dioxides, such as, for example, titanium dioxide T 805 (Degussa) or Eusolex® T2000 (Merck; preferred hydrophilic coating agents are silicones and particularly preferably trialkoxyoctylsilanes or simethicones UV light protection filters can be found in the review by P. Finkel in SÖFW-Journal 122 (1996), p. 543.

The UV absorbents are usually used in amounts of from 0.01% by weight to 5% by weight, preferably from 0.03% by weight to 1% by weight.

Compositions according to the invention may comprise further enzymes in addition to the proteins according to the invention for increasing the washing or cleaning performance, it being possible in principle to use all enzymes established for this purpose in the prior art. These include in particular other proteases, amylases, lipases, hemicellulases, cellulases or oxidoreductases, and preferably mixtures thereof. 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. Agents according to the invention preferably contain these further enzymes in total amounts of 1 × 10 ^-6 to 5-weight percent, based on active protein.

Among the other proteases, those of the subtilisin type are preferred. Examples of these are the subtilisins BPN ¹ and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K and the subtilases, but not the subtilisins in the narrower sense Proteases TW3 and TW7. Subtilisin Carlsberg in a developed form under the trade names Alcalase ^® from Novozymes A / S, Bagsvaerd, Denmark. The subtilisins 147 and 309 are sold under the trade names Esperase ^®, or Savinase ^® from Novozymes. Of the protease from Bacillus lentus DSM 5483 (WO 91/02792 A1) the listed under the name BLAP ^® variants, particularly in WO 92/21760 A1, WO 95/23221 A1, WO 02/088340 A2 and WO 03 derive, / 038082 A2. Other useful proteases from various Bacillus sp. And B. g / öson / 7 strains are found in the patent applications WO 03/054185, WO 03/056017, WO 03/055974 and WO 03/054184. Other usable proteases are, for example, under the trade names Durazym ^®, relase ^®, Everlase® ^®, Nafizym, Natalase ^®, Kannase® ^® and Ovozymes ^® from Novozymes, under the trade names Purafect ^®, Purafect ^® OxP and Properase.RTM ^® by the company Genencor, that under the trade name Protosol® ^® from Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® ^® and protease P ^® by the company Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan.

Examples of amylases which can be used according to the invention are the α-amylases from Bacillus licheniformis, B. amyloliquefaciens or S. stearothermophilus and their further developments, which are improved for use in detergents and cleaners. The enzyme from ß. licheniformis is available from Novozymes under the name Termamyl ^® and from Genencor under the name Purastar® ^® ST. Development products of this α-amylase are available from Novozymes under the trade names Duramyl ^® and Termamyl ^® ultra, from Genencor under the name Purastar® ^® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, as Keistase ^®. The α-amylase from B. amyloliquefaciens is marketed by Novozymes under the name BAN ^®, and variants derived from the α-amylase from 8 stearothermophilus under the names BSG ^® and Novamyl ^®, likewise from Novozymes. Further usable commercial products are, for example, the amylase LT® and Stainzyme®, the latter also from Novozymes.

Furthermore, the α-amylase from Bacillus sp. Disclosed in the application WO 02/10356 A2 for this purpose. A 7-7 (DSM 12368) and in the application

WO 02/44350 A2 described cyclodextrin glucanotransferase (CGTase)

B. agaradherens (DSM 9948). Furthermore, the amylolytic enzymes belonging to the sequence space of α-amylases can be used, which in the application

WO 03/002711 A2 is defined, and those described in the application WO 03/054177 A2 to be discribed. Likewise, fusion products of the molecules mentioned can be used, for example those from application DE 10138753 A1.

In addition, the enhancements available under the trade names Fungamyl.RTM ^® by Novozymes of α-amylase from Aspergillus niger and A. oryzae. Another commercial product is, for example, the amylase ^LT® .

Compositions according to the invention may contain lipases or cutinases, in particular because of their triglyceride-cleaving activities, but also in order to generate in situ peracids from suitable precursors. These include, for example, the lipases originally obtainable from Humicola lanuginosa (Thermomyces lanuginosus) or further developed, in particular those with the amino acid exchange D96L. They are for example marketed by Novozymes under the trade names Lipolase ^®, Lipolase Ultra ^®, LipoPrime® ^®, Lipozyme® ^® and Lipex ^®. Furthermore, for example, the cutinases can be used, which were originally isolated from Fusarium solani pisi and Humicola insolens. Likewise useable lipases are available from Amano under the designations Lipase CE ^®, Lipase P ^®, Lipase B ^®, or lipase CES ^®, Lipase AKG ^®, Bacillis sp. ^Lipase® , Lipase ^AP® , Lipase M- ^AP® and Lipase ^{AML® are} available. From the company Genencor, for example, the lipases, or cutinases can be used, the initial enzymes were originally isolated from Pseudomonas mendocina and Fusarium solanii. Other important commercial products, the preparations originally sold by Gist-Brocades M1 Lipase ^® and Lipomax® ^® and the enzymes marketed by Meito Sangyo KK, Japan under the names Lipase MY-30 ^®, Lipase OF ^® and lipase PL ^® to mention also the product Lumafast® ^® from Genencor.

Detergents according to the invention, especially if they are intended for the treatment of textiles, may contain cellulases, depending on the purpose, as pure enzymes, as enzyme preparations or in the form of mixtures in which the individual components advantageously supplement each other in terms of their various performance aspects. These performance aspects include in particular contributions to the Primary washing power, to the secondary washing power of the medium

(Anti-redeposition effect or graying inhibition) and softening (fabric effect), up to the exercise of a "stone washed" effect.

A useful fungal, endoglucanase (EG) -rich cellulase preparation, or its further developments are offered by Novozymes under the trade name Celluzyme ^®. The products Endolase® ^® and Carezyme ^® available also from Novozymes based on the 50 kD EG and 43 kD EG applicable from H. insolens DSM 1800. Further commercial products of this company are Cellusoft® ^® and Renozyme ^®. The latter is based on the application WO 96/29397 A1. Performance-enhanced cellulase variants are disclosed, for example, in the application WO 98/12307 A1. Likewise, the cellulases disclosed in the application WO 97/14804 A1 can be used; For example, it revealed 20 kD EG Melanocarpus, available from AB Enzymes, Finland, under the trade names Ecostone® ^® and Biotouch ^®. Further commercial products from AB Enzymes are Econase® ^® and ECOPULP ^®. Other suitable cellulases from Bacillus sp. CBS 670.93 and CBS 669.93 are disclosed in WO 96/34092 A2, wherein those derived from Bacillus sp. CBS 670.93 from the company Genencor under the trade name Puradax ^{® is} available. Further commercial products of the company Genencor are "Genencor detergent cellulase L" and lndiAge ^® Neutra.

Compositions according to the invention may, in particular for the removal of certain problem soiling, comprise, in addition to the polypeptides according to the invention, further enzymes which are combined under the term hemicellulases. These include, for example, mannanases, xanthan lyases, pectin lyases (= pectinases), pectin esterases, pectate lyases, xyloglucanases (= xylanases), pullulanases and β-glucanases. Suitable mannanases are available, for example under the name Gamanase ^® and Pektinex AR ^® from Novozymes, under the name Rohapec ^® B1 from AB Enzymes and under the name Pyrolase® ^® from Diversa Corp., San Diego, CA, United States. A suitable ß-glucanase from a ß. alcalophilus is for example from the application WO 99/06573 A1. The .beta.-glucanase obtained from B. subtilis is available under the name Cereflo ^® from Novozymes.

To increase the bleaching effect, detergents and cleaners according to the invention may be oxidoreductases, for example oxidases, oxygenases, catalases, peroxidases, such as halo, chloro, bromo, lignin, glucose or manganese peroxidases, dioxygenases or laccases (phenol oxidases, polyphenol oxidases) contain. Suitable commercial products Denilite® ^® 1 and 2 from Novozymes should be mentioned. Advantageously, it is additionally preferred to add organic, particularly preferably aromatic, compounds which interact with the enzymes in order to enhance the activity of the relevant oxidoreductases (enhancers) or to ensure the flow of electrons (mediators) at greatly varying redox potentials between the oxidizing enzymes and the soils.

The enzymes additionally employed in agents according to the invention are either originally derived from microorganisms, such as the genera Bacillus, Streptomyces, Humicola or Pseudomonas, and / or are produced by biotechnological methods known per se by suitable microorganisms, for example by transgenic expression hosts of the genera Bacillus or filamentous fungi.

The purification of the relevant enzymes is conveniently carried out by conventional methods, for example by precipitation, sedimentation, concentration, filtration of the liquid phases, microfiltration, ultrafiltration, exposure to chemicals, deodorization or suitable combinations of these steps.

Agents according to the invention can be added to the polypeptides according to the invention as well as the additionally used enzymes in any form established according to the prior art. These include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, especially in the case of liquid or gel-form detergents, solutions of the enzymes, advantageously as concentrated as possible, sparing in water and / or added with stabilizers. Alternatively, these proteins can be encapsulated for both the solid and liquid dosage forms, for example, by spray-drying or extruding the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are entrapped as in a solidified gel or in those of the core-shell type, in which an enzyme-containing core is coated with a water, air and / or chemical impermeable protective layer. In deposited layers, further active ingredients, for example stabilizers, emulsifiers, pigments, bleaches or dyes, may additionally be applied. Such capsules are applied by methods known per se, for example by shaking or rolling granulation or in fluid-bed processes. Advantageously, such granules, for example by applying polymeric film-forming agent, low in dust and storage stable due to the coating.

Furthermore, it is possible to assemble two or more enzymes together, such as a polypeptide according to the invention and another enzyme, so that a single granulate has a plurality of enzyme activities.

A protein contained in an agent according to the invention, in particular also the polypeptide according to the invention, can be protected against damage, for example inactivation, denaturation or decomposition, for example by physical influences, oxidation or proteolytic cleavage, in particular during storage. In microbial recovery of proteins and / or enzymes, inhibition of proteolysis is particularly preferred, especially if the agents also contain proteases. Preferred agents according to the invention contain stabilizers for this purpose.

One group of stabilizers are reversible protease inhibitors. Benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters are frequently used for this purpose, including, in particular, derivatives with aromatic groups, for example ortho, meta or para-substituted phenylboronic acids, in particular 4-formylphenylboronic acid, or the salts or Esters of the compounds mentioned. Also peptide aldehydes, that is oligopeptides with reduced C-terminus, especially those of 2 to 50 monomers are used for this purpose. Among the peptidic reversible protease inhibitors include ovomucoid and leupeptin. Also, specific, reversible peptide inhibitors for the protease subtilisin and fusion proteins from proteases and specific peptide inhibitors are suitable.

Other enzyme stabilizers are amino alcohols such as mono-, di-, triethanol- and -propanolamine and mixtures thereof, aliphatic carboxylic acids up to C12, such as succinic acid, other dicarboxylic acids or salts of said acids. End-capped fatty acid amide alkoxylates are also suitable for this purpose. Certain organic acids used as builders are capable, as disclosed in WO 97/18287, of additionally stabilizing a contained enzyme.

Lower aliphatic alcohols, but especially polyols such as glycerol, ethylene glycol, propylene glycol or sorbitol are other frequently used enzyme stabilizers. Di-glycerol phosphate also protects against denaturation due to physical influences. Likewise, calcium and / or magnesium salts are used, such as calcium acetate or calcium formate.

Polyamide oligomers or polymeric compounds such as lignin, water-soluble vinyl copolymers or cellulose ethers, acrylic polymers and / or polyamides stabilize the enzyme preparation, inter alia, against physical influences or pH fluctuations. Polyamine N-oxide containing polymers act simultaneously as enzyme stabilizers and as dye transfer inhibitors. Other polymeric stabilizers are linear C ₈ -C ₈ polyoxyalkylenes. Also, alkylpolyglycosides can stabilize the enzymatic components of the agent according to the invention and are able, preferably, to additionally increase their performance. Crosslinked N-containing compounds preferably perform a dual function as soil release agents and as enzyme stabilizers. Hydrophobic, nonionic polymer stabilizes in particular an optionally contained cellulase. Reducing agents and antioxidants increase the stability of the enzymes to oxidative degradation; For example, sulfur-containing reducing agents are familiar. Other examples are sodium sulfite and reducing sugars.

Particular preference is given to using combinations of stabilizers, for example of polyols, boric acid and / or borax, the combination of boric acid or borate, reducing salts and succinic acid or other dicarboxylic acids or the combination of boric acid or borate with polyols or polyamino compounds and with reducing salts. The effect of peptide-aldehyde stabilizers is favorably enhanced by the combination with boric acid and / or boric acid derivatives and polyols, and still further by the additional action of divalent cations, such as calcium ions.

Since agents according to the invention can be offered in all conceivable forms, polypeptides according to the invention in all formulations suitable for addition to the respective compositions represent respective embodiments of the present invention. These include, for example, liquid formulations, solid granules or capsules.

The encapsulated form lends itself to protecting the enzymes or other ingredients from other ingredients, such as bleaches, or to allow for controlled release. Depending on the size of these capsules, a distinction is made to MiIIi-, micro- and nanocapsules, with microcapsules for enzymes being particularly preferred. Such capsules are disclosed, for example, with the patent applications WO 97/24177 and DE 19918267. A possible encapsulation method is that the proteins are encapsulated in this substance, starting from a mixture of the protein solution with a solution or suspension of starch or a starch derivative. Such an encapsulation process is described in the application WO 01/38471.

In the case of solid compositions, the proteins - polypeptides according to the invention as well as optionally contained further enzymes - can be used, for example, in dried, granulated and / or encapsulated form. You can do that separately means added as a separate phase, or with other ingredients together in the same phase, with or without compaction. If microencapsulated enzymes are to be processed in solid form, the water can be removed by methods known from the prior art from the aqueous solutions resulting from the workup, such as spray drying, centrifuging or by solubilization. The particles obtained in this way usually have a particle size between 50 and 200 microns.

The proteins may be added to liquid, gelatinous or pasty agents according to the invention in a concentrated aqueous or non-aqueous solution, suspension or emulsion starting from a protein recovery and preparation carried out in the prior art, but also in gel form or encapsulated or as a dried powder. Such detergents or cleaners according to the invention are generally prepared by simple mixing of the ingredients which can be added in bulk or as a solution in an automatic mixer.

A cleaning agent according to the invention, in particular a hard surface cleaner according to the invention, may also contain one or more propellants (INCI propellants), usually in an amount of 1 to 80% by weight, preferably 1 to 5 to 30% by weight, in particular 2 to 10 wt .-%, particularly preferably 2.5 to 8 wt .-%, most preferably 3 to 6 wt .-%, contained.

Propellants are inventively usually propellants, especially liquefied or compressed gases. The choice depends on the product to be sprayed and the field of application. When using compressed gases such as nitrogen, carbon dioxide or nitrous oxide, which are generally insoluble in the liquid detergent, the operating pressure decreases with each valve actuation. Detergent-soluble or even solvent-acting liquefied gases (liquefied gases) as propellants offer the advantage of constant operating pressure and uniform distribution, because the propellant vaporizes in the air and takes up a volume of several hundred times. According to INCI suitable blowing agents are accordingly: butanes, carbon dioxides, dimethyl carbonates, dimethyl ether, ethanes, hydrochlorofluorocarbon 22, hydrochlorofluorocarbon 142b, hydrofluorocarbon 152a, hydrofluorocarbon 134a, hydrofluorocarbon 227ea, isobutanes, isopentanes, nitrogen, nitrous oxides, pentanes, propanes. On Chlorfluorkohlenstoffe (chlorofluorocarbons, CFCs) as blowing agent, however, because of their harmful effect on the -vor hard UV radiation protective - ozone shield of the atmosphere, the so-called ozone layer, preferably largely and in particular completely omitted.

Preferred blowing agents are liquefied gases. Liquefied gases are gases that can be converted from the gaseous to the liquid state at usually already low pressures and 20 ^° C. In particular, under liquefied gases, however, are the hydrocarbons propane, propene, butane, butene, isobutane (2-methylpropane), isobutene (2-methylpropene), which are obtained in oil refineries as by-products from distillation and cracking of petroleum and in natural gas treatment during gasoline separation. Isobutylene) and mixtures thereof.

The cleaning agent particularly preferably contains propane, butane and / or isobutane, in particular propane and butane, as one or more propellants, more preferably propane, butane and isobutane.

An important task of the enzyme preparation and in particular of the polypeptides according to the invention is, as stated above, the primary washing performance. In addition to the primary washing performance, however, the proteases contained in detergents can also fulfill the function of activating other enzymatic constituents by proteolytic cleavage or inactivating them after a corresponding action time. An embodiment of the present invention are also those agents with protease-sensitive material capsules which are hydrolyzed, for example, by proteins of the invention at an intended time and release their contents. Polypeptides of the invention can thus also be used for inactivation, activation or release reactions, in particular in multiphase agents. A further embodiment of this subject matter accordingly also represents the use of a polypeptide according to the invention for the activation, deactivation or release of ingredients of detergents or cleaners.

In a preferred embodiment, the agent with a polypeptide according to the invention is designed so that it can be used regularly as a care agent, for example by being added to the washing process, applied after washing or applied independently of the washing. The desired effect is to maintain a smooth surface texture of the fabric over a long period of time and / or to prevent and / or reduce damage to the fabric.

A separate subject of the invention are processes for the automated cleaning of textiles or of hard surfaces, in which at least one of the process steps uses a polypeptide according to the invention.

Of these, preference is given to those methods in which the polypeptide according to the invention is used in an amount of 40 μg to 4 g, preferably from 50 μg to 3 g, particularly preferably from 100 μg to 2 g and very particularly preferably from 200 μg to 1 g per application becomes. Included are all integer and non-integer values lying between these numbers.

This includes both manual and mechanical processes, with mechanical processes being preferred on account of their more precise controllability, for example with regard to the quantities and reaction times used.

Methods for cleaning textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned in a plurality of process steps and washed off after the action time, or that the items to be cleaned are otherwise treated with a detergent or a solution of this agent. The same applies to methods of cleaning all materials other than textiles, which are termed hard surfaces be summarized. All conceivable washing or cleaning methods can be enriched in at least one of the method steps by proteins of the invention, and then represent embodiments of the present invention.

Since preferred polypeptides according to the invention naturally already have a protein-dissolving activity and also unfold them in media which otherwise have no cleaning power, for example in bare buffer, a single substep of such a process for the automated cleaning of textiles may consist in optionally adding, in addition to stabilizing compounds, Salts or buffer substances is the only detergent-active component of a polypeptide of the invention is applied. This represents a particularly preferred embodiment of the present invention.

In a further preferred embodiment of such methods, the relevant polypeptides according to the invention are provided in the context of one of the formulations set forth above for agents according to the invention, preferably detergents or cleaners according to the invention.

A separate subject of the invention is the use of an inventive alkaline protease described above for cleaning textiles or hard surfaces.

Accordingly, the above-mentioned concentration ranges apply correspondingly for these uses.

Proteases according to the invention can be used, in particular according to the properties described above and the processes described above, to eliminate proteinaceous impurities from textiles or from hard surfaces. Embodiments include, for example, hand washing, manual removal of stains from fabrics or hard surfaces, or use in conjunction with a machine process. In a preferred embodiment of this use, the alkaline proteases according to the invention are provided in the context of one of the formulations set forth above for compositions according to the invention, preferably detergents or cleaners.

Another object of the present invention is also a product comprising a composition according to the invention or a detergent or cleaning agent according to the invention, in particular a hard surface cleaner according to the invention, and a spray dispenser. The product may be both a single-chamber and a multi-chamber container, in particular a two-chamber container. The spray dispenser is preferably a manually activated spray dispenser, in particular selected from the group consisting of aerosol spray dispensers (also known as spray can), pressure-building spray dispensers, pump spray dispensers and trigger spray dispensers, in particular pump spray dispensers and trigger spray dispensers with a transparent polyethylene or polyethylene terephthalate container. Spray dispensers are described in more detail in WO 96/04940 (Procter & Gamble) and the US patents cited therein about spray dispensers, to which reference is made in this regard and the contents of which are hereby incorporated by reference. Triggersprühspender and pump sprayer have over compressed gas tanks the advantage that no propellant must be used. By means of suitable particle-like attachments, nozzles, etc. (so-called "nozzle valves") on the spray dispenser, the enzyme in this embodiment may optionally also be added to the composition in a form immobilized on particles and thus metered in as a cleaning foam

The following examples further illustrate the invention without being limited thereto.

embodiments

All molecular biological work steps follow standard methods, as described for example in the handbook of Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual ", CoId Spring Harbor Laboratory Press, New York, 1989, etc. Enzymes and kits were used according to the instructions of the respective manufacturers.

example 1

Isolation and identification of a proteolytically active bacterial strain

0.1 g of a soil sample was suspended in 1 ml of sterile NaCl and on agar plates containing milk powder (1, 5% agar, 0.1% K ₂ HPO4, 0.5% yeast extract, 1% peptone, 1% milk powder, 0.02% MgSO ₄ .7H ₂ O, 0.4% Na ₂ CO ₃ , pH 9.6) and incubated at 30 °. A proteolytically active bacterium was isolated on the basis of a clarification laboratory, which was identified as Bacillus pumilus by the German Collection of Microorganisms and Cell Cultures (DSMZ).

Table 1: Microbiological properties of Bacillus pumilus stacnmes (determination of DSMZ)

Example 2

Cloning and sequencing of the mature protease Bacillus pumilus chromosomal DNA was prepared according to standard methods, treated with the restriction enzyme Sau 3A and the fragments obtained were cloned into the vector pAWA22. This is a pBC16-derived expression vector for use in Bacillus species (Bernhard et al., (1978) J. Bacteriol., Vol. 133 (2), pp. 897-903). This vector was transformed into the host strain Bacillus subtilis DB 104 (Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444).

The 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 Kh ^ POφ 20 mM MgCl2, 5 g / l casiaminoacids,

5 g / l yeast extract, 6 g / l glucose, 0.1 g / l BSA) and then on TBY Skimmilk plates (10 g / l peptone, 10 g / l milk powder (see above), 5 g / l Yeast, 5 g / l NaCl, 15 g / l agar). Proteolytically active clones were identified by their lysis sites. One of the resulting proteolytically active clones was selected, the plasmid was isolated and the insert was sequenced according to standard methods.

The resulting insert contained an open reading frame of about 1.2 kb. Whose sequence is in the sequence listing under the name SEQ ID NO. 1 indicated. It covers 1152 bp. The deduced amino acid sequence comprises 383 amino acids followed by a stop codon. It is in the sequence listing under SEQ ID NO. 2 indicated. Of these, the first 108 amino acids are unlikely to be present in the mature protein, so the mature protein is expected to be 275 amino acids in length.

These sequences were synthesized using the Swiss-Prot databases (Geneva Bioinformatics (GeneBio) SA, Geneva, Switzerland, http://www.genebio.com/sprot.html) and GenBank (National Center for Biotechnology Information NCBI, National Institutes of Health, Bethesda, MD, USA). The next similar enzymes were identified in Table 2 below.

Table 2: Homology of the Bacillus pumilus alkaline protease to the nearest similar proteins. In this mean:

ID The registration numbers of the databases Genbank and

Swiss-Prot;

Ident. k. DNA identity at the DNA level for the complete DNA in%;

Ident. m. DNA identity at the DNA level for those coding for the mature protein

DNA in%;

Ident. Proprä. Identity at the amino acid level, based on the proprote protein, in%;

Ident. m. Prot. Identity at the amino acid level, based on the mature protein, in%; n. not specified in the databases.

The amino acid sequences of these proteases are also compared with one another in the alignment of FIG.

Example 3

SDS polyacrylic gel electrophoresis and isoelectric focusing

In denaturing SDS polyacrylamide gel electrophoresis in the PHAST ^® system from Pharmacia-Amersham Biotech, Sweden, has the alkaline protease obtained in Example 2 and 3 from B. pumilus on a molecular weight of 27 kD.

According to isoelectric focusing, also in the PHAST ^® system from Pharmacia-Amersham Biotech, the isoelectric point of the alkaline protease from 8 gibsonii than 8.5.

Example 4 Determination of the washing performance when used in commercial liquid detergent

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 (salad dressing on cotton, CFT CS-6), B (grass on cotton, CFT CS-8), C (blood on cotton, EMPA E-111) and D (milk / cocoa on Cotton, EMPA E-112). In addition, the average was formed over all soils tested (E).

With this test material different detergent formulations were launderometrically examined for their washing performance. For each, a liquor ratio of 1: 12 was set and washed for 30 min at a temperature of 30 ⁰ C or 60 ⁰ C. The dosage was 4.4 g of the respective agent per liter of wash liquor. The water hardness was 16 ° German hardness.

The control detergent used was a detergent base formulation of the following composition (all figures in percent by weight): 0.3- 0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0 , 3-0.5% ethanol, 4-7% FAEOS, 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0 , 5% HEDP, 0-0.4% PVP, 0-0.05% optical brightener, 0-0.001% dye, balance demineralized water. It was treated with the following proteases for the different series of experiments so that in each case a final concentration of 5625 PE of proteolytic activity per liter of wash liquor resulted: β. / en.us- alkaline protease F 49 (WO 95/23221), B. / e / rtus alkaline protease X (WO 92/21760) or the protease according to the invention from 8. pumilus.

After washing, the whiteness of the washed fabrics was measured. The measurement was carried out on a Datacolor SF500-2 spectrometer at 460 nm (UV blocking filter 3), 30 mm aperture, without gloss, illuminant D65, 10 °, d / 8 °. Indicated are the mean values from 4 measurements each. They allow an immediate Conclusion on the contribution of the enzyme contained to the washing performance of the agent used.

Table 3: washing results at 30 ⁰ C

Table 4: Wash results at 60 ⁰ C

It can be seen that the protease of the invention from ß. pumilus on all tested soils and at both temperatures tested the established proteases ß. e / rtus alkaline protease F 49 and B. / enfc / s alkaline protease X exceeds.

Example 5

Enzymatic properties

With the purified alkaline protease from B. pumilus according to the invention, experiments were carried out with casein at various temperatures and pH values.

It turned out that the optimum activity for the cleavage of casein at 60 ⁰ C, while the optimum pH for the cleavage of casein is 10.5.

Description of the figures 9

FIG. 1: Alignment of the amino acid sequences of the invention

Protease from Bacillus pumilus with the next similar known subtilisins, each in the mature, that is processed form.

The following numbers stand for the following proteases:

1 Protease from Bacillus pumilus according to the invention

2 protease Q5XPN0 from Bacillus pumilus (Swiss-Prot)

3 protease Q6SIX5 from Bacillus pumilus (Swiss-Prot)

4 protease Q9KWR4 from Bacillus pumilus (Swiss-Prot)

5 Protease Q2HXI3 from Bacillus pumilus (Swiss-Prot)

FIG. 2: The pBC16-derived expression vector pAWA22, which has a

Promoter from B. licheniformis (PromPLi) and downstream of which has a Bcl restriction site (see Example 2 and Bernhard et al. (1978), J. Bacteriol., 133 (2), pp. 897-903).

INFORMATION ON A MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(Rule I3 ^bls PCT)

A. The following information relates to the deposited micro-organism or other biological material mentioned in the description on page _ line - * ^•

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Agent Plenipotentiary Plenipotentiary

Todorova, Antoaneta

Formal PCT / RO / l34 (July 1998)

Claims

claims

1. Polynucleotide selected from the group consisting of: a) polynucleotide having a nucleic acid sequence according to SEQ ID NO: 1, b) polynucleotide having a nucleic acid sequence from position 1 to 153 according to SEQ ID NO: 1, c) polynucleotide having a nucleic acid sequence from position 1 to 324 according to SEQ ID NO: 1, d) polynucleotide having a nucleic acid sequence from position 325 to 1152 according to SEQ ID NO: 1, e) polynucleotide coding for a polypeptide having an amino acid sequence according to SEQ ID NO: 2, f) polynucleotide encoding a A polypeptide having an amino acid sequence from position 1 to 51 according to SEQ ID NO: 2, g) polynucleotide coding for a polypeptide having an amino acid sequence from position 1 to 108 according to SEQ ID NO: 2, h) polynucleotide coding for a polypeptide having an amino acid sequence of

Position 109 to 383 according to SEQ ID NO: 2, i) polynucleotide encoding a polypeptide according to claim 13, j) naturally occurring or artificially produced mutants or polymorphic

Forms or alleles of a polynucleotide according to (a) with up to 55 mutations, k) naturally occurring or artificially produced mutants or polymorphic

Forms or alleles of a polynucleotide according to (b) or (c) with up to 8 mutations, I) naturally occurring or artificially produced mutants or polymorphic

Forms or alleles of a polynucleotide according to (d) with up to 40 mutations, m) polynucleotides with a sequence homology or identity of at least 95% in

With respect to a polynucleotide according to (a), n) polynucleotides having a sequence homology or identity of at least 95% in

With respect to a polynucleotide according to (b), o) polynucleotides having a sequence homology or identity of at least 98% in

Pertaining to a polynucleotide according to (c), p) polynucleotides having a sequence homology or identity of at least 95.5% with respect to a polynucleotide according to (d), 1

q) under stringent conditions with a polynucleotide according to (a), (b), (c) or (d) hybridizing polynucleotides, r) polynucleotides consisting of at least 200 consecutive

Nucleic acids of a polynucleotide according to (a), (c), (d), (g), (h), (m), (o) or (p), s) polynucleotides with deletions and / or insertions and / or inversions of up to

50 nucleotides to a polynucleotide according to (a) to (r), t) polynucleotides comprising at least one of the mentioned under (a) to (s)

Polynucleotides, u) to polynucleotides according to (a) to (t) complementary polynucleotides.

2. polynucleotide according to claim 1, characterized in that it codes for a hydrolase.

3. polynucleotide according to claim 2, characterized in that it is a protease in the hydrolase.

Polynucleotide according to claim 3, characterized in that the protease is an alkaline protease of the subtilisin type.

5. Polynucleotide according to one of claims 1 to 4, characterized in that the encoded polypeptide is capable of cleaving peptide bonds.

6. Polynucleotide according to one of claims 1 to 5, characterized in that it is obtainable from a natural source, in particular from a microorganism.

7. polynucleotide according to claim 6, characterized in that it is the microorganism is a gram-positive bacterium.

Polynucleotide according to claim 7, characterized in that the gram-positive bacterium is one of the genus Bacillus.

9. polynucleotide according to claim 8, characterized in that it is the Bacillus species Bacillus pumilus.

10. A method for producing and / or identifying a polynucleotide according to any one of claims 1 to 9, characterized in that the polynucleotide is chemically synthesized, isolated from a Genbank by means of a probe or obtained by polymerase chain reaction.

11. A vector comprising a polynucleotide according to one of claims 1 to 9.

12. Vector according to claim 11, characterized in that it is a cloning vector or expression vector.

13. Polypeptide selected from the group consisting of:

a) polypeptide having an amino acid sequence according to SEQ ID NO: 2, b) polypeptide having an amino acid sequence from position 1 to 51 according to SEQ ID NO:

1, c) polypeptide having an amino acid sequence from position 1 to 108 according to SEQ ID NO:

1, d) polypeptide having an amino acid sequence from position 109 to 383 according to SEQ ID NO: 2, e) polypeptide having an amino acid sequence from position 207 to 378 of the sequence given in SEQ ID NO: 2, f) naturally occurring or artificially produced mutants, polymorphic forms or alleles of a polypeptide according to (a) having up to 6 mutations, g) naturally occurring or artificially produced mutants, polymorphic forms or alleles of a polypeptide according to (b) or (c) with one mutation, h) naturally occurring or artificially produced Mutants, polymorphic forms or alleles of a polypeptide according to (d) or (e) with up to 4 mutations, i) polypeptides having a sequence homology or identity of at least 98.5% with respect to a polypeptide according to (a) or (d j) polypeptides encoded by a polynucleotide according to claim 1, k) polypeptides consisting of at least 100 consecutive amino acids of the amino acid sequence according to (d), I) polypeptides consisting of at least 185 consecutive amino acids of the amino acid sequence according to (d), wherein optionally a deviation in a

M) polypeptides consisting of at least 225 consecutive amino acids of the

Amino acid sequence according to (d), wherein optionally deviations in up to two

N) polypeptides consisting of at least 230 consecutive amino acids of the

Amino acid sequence according to (d), wherein optionally deviations in up to three

O) polypeptides with insertions and / or deletions and / or inversions of up to 50

Amino acids with respect to a polypeptide according to (a) to (o), p) polypeptides comprising at least one of those mentioned under (a) to (p)

Polypeptides.

14. Polypeptide according to claim 13, characterized in that it is a hydrolase.

15. Polypeptide according to claim 14, characterized in that it is a protease in the hydrolase.

16. Polypeptide according to claim 15, characterized in that the protease is an alkaline protease of the subtilisin type.

17. Polypeptide according to one of claims 13 to 16, characterized in that the polypeptide is capable of cleaving peptide bonds.

18. Polypeptide according to one of claims 13 to 17, characterized in that the polypeptide is additionally derivatized.

19. Polypeptide according to one of claims 13 to 18, characterized in that it is additionally stabilized.

20. Polypeptide according to one of claims 13 to 19, characterized in that it is obtainable from a microorganism.

21. Polypeptide according to claim 20, characterized in that the microorganism is a gram-positive bacterium.

22. The polypeptide according to claim 21, characterized in that the gram-positive bacterium is one of the genus Bacillus.

23. Polypeptide according to claim 22, characterized in that the Bacillus species is Bacillus pumilus.

24. A cell comprising a nucleic acid according to any one of claims 1 to 9 or a vector according to claim 11 or 12.

25 cell, which expresses one of the polypeptide designated in claims 13 to 23 or can be stimulated to express it, in particular using one of the polynucleotide designated in claims 1 to 9 and / or using a vector according to claim 11 or 12th

26. Cell according to claim 25, characterized in that it is a bacterium, in particular one which secretes the protein formed into the surrounding medium.

27. Cell according to claim 26, which is a gram-negative bacterium, in particular one of the genera Escherichia coli or Klebsiella, in particular strains of E. coli K12, E. coli B or Klebsiella planticola, and more particularly derivatives of the strains Escherichia coli BL21 (DE3),

E. coli RV308, E. coli DH5α, E. coli JM109, E. coli XL-1 or Klebsiella planticola (Rf).

A cell according to claim 26, which is a Gram-positive bacterium, in particular one of the genera Bacillus, Staphylococcus or Corynebacterium, more particularly of the species Bacillus lentus, B. licheniformis, B. amyloliquefaciens, B. subtilis, B. globigii, B gibsonii, B. pumilus or

Alcalophilus, Staphylococcus carnosus or Corynebacterium glutamicum.

29. Cell according to claim 25, wherein it is a eukaryotic cell, preferably one of the genus Saccharomyces.

30. A process for producing a polypeptide according to any one of claims 13 to 23, characterized in that the preparation using a polynucleotide according to any one of claims 1 to 9 and / or a vector according to claim 11 or 12 and / or a cell according to one of Claims 24 to 29, wherein the nucleic acid sequence is optionally adapted to the codon usage of the host cell.

31. Composition, characterized in that it contains at least one polypeptide according to one of claims 13 to 23.

32. The composition according to claim 31, characterized in that it is a washing or cleaning agent.

33. Composition according to claim 31, characterized in that it is a cosmetic or pharmaceutical agent.

34. A composition according to claim 33, characterized in that it is a shampoo, a soap, a washing lotion, a cream, a scrub or an oral, dental or dental prosthesis care agent.

35. Composition selected from the group consisting of detergents and cleaners, characterized in that it contains at least one olypeptide selected from the following group: a) polypeptide having an amino acid sequence according to SEQ ID NO: 2, b) polypeptide having an amino acid sequence from position 109 to 383 according to SEQ ID NO: 2, c) naturally occurring or artificially produced mutants, polymorphic forms or alleles of a polypeptide according to (a) or (b) with up to 50 mutations, d) polypeptides which have a sequence homology or identity of at least 80% with respect to a polypeptide according to (a) or (b), e) polypeptides consisting of at least 50 consecutive amino acids of the amino acid sequence according to (a) or (b), f) polypeptides with insertions and / or deletions and / or inversions of up to 50 amino acids with respect to a polypeptide according to (a), (b), (c), (d) or (e), g) polypeptides comprising at least one of the (a) to (f) mentioned polypeptides.

36. Composition according to one of claims 31 to 35, characterized in that it more preferably the polypeptide in an amount of 2 micrograms to 20 mg, preferably from 5 micrograms to 17.5 mg, more preferably from 20 micrograms to 15 mg from 50 μg to 10 mg per g of the agent.

37. A composition according to any one of claims 31 to 36, characterized in that it contains further enzymes, in particular other proteases, amylases, cellulases, hemicellulases, oxidoreductases and / or lipases.

38. A composition according to any one of claims 31 to 37, 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, abrasives, antistatic agents, pearlescing agents and skin protection agents.

39. A composition according to any one of claims 31 to 38, characterized in that it is a solid, gel-like, pasty or liquid composition.

40. A product comprising a polypeptide according to any one of claims 13 to 23 or a composition according to any one of claims 31 to 39 and a spray dispenser for dosing the enzyme preparation or the cleaning agent as an aerosol and / or as a foam.

41. A method for the automated cleaning of textiles or hard surfaces, characterized in that in at least one of the method steps, a polypeptide according to any one of claims 13 to 23 is active, in particular in an amount of 40 micrograms to 96 g, preferably from 50 micrograms 72 g, more preferably from 100 μg to 48 g and most preferably from 200 μg to 24 g per application.

42. A process for the treatment of textile raw materials or for textile care, characterized in that in at least one of the method steps, a polypeptide according to any one of claims 13 to 23 is active, especially for textile raw materials, fibers or textiles with natural ingredients and especially those with wool or Silk.

43. A process for the hydrolysis of biofilms on surfaces or for the removal of soil residues from surfaces by incubation with a polypeptide according to any one of claims 13 to 23 or a composition according to any one of claims 31 to 39, optionally using a product according to claim 40, characterized in that the surface to be treated is treated with the polypeptide and / or the composition.

44. Use of a polypeptide according to any one of claims 13 to 23, a composition according to any one of claims 31 to 39 or a product according to claim 40 for the destruction and / or removal of Biofilms and / or removal of debris from surfaces and / or for cleaning textiles.

45. Use of a polypeptide according to any one of claims 13 to 23 for the activation or deactivation of ingredients of detergents or cleaning agents.

46. Use of a polypeptide according to any one of claims 13 to 23, a composition according to any one of claims 31 to 39 or a product according to claim 40 for the hydrolytic cleavage of peptide bonds.

47. Use of a polypeptide according to any one of claims 13 to 23 for biochemical analysis or for the synthesis of low molecular weight compounds or of proteins.

48. Use of a polypeptide according to any one of claims 13 to 23 for the preparation, purification or synthesis of natural substances or biologically valuable substances.

49. Use of a polypeptide according to any one of claims 13 to 23 for the treatment of natural raw materials, in particular for surface treatment, especially in a process for the treatment of leather.

50. Use of a polypeptide according to any one of claims 13 to 23 for the recovery or treatment of raw materials or intermediates in textile production, in particular for the removal of protective layers on fabrics.

51. Use of a polypeptide according to any one of claims 13 to 23 for the treatment of textile raw materials or for textile care, in particular for the treatment of wool or silk or wool or silk-containing blended textiles. yy

52. Use of a polypeptide according to any one of claims 13 to 23 for the treatment of photographic films, in particular for the removal of gelatin-containing or similar protective layers.

53. Use of a polypeptide according to any one of claims 13 to 23 for the production of food or animal feed.

54. Product of a composition according to any one of claims 31 to 39 and a multi-chamber bottle.

55. Use of polypeptides according to any one of claims 13 to 23 for the preparation of a cosmetic or pharmaceutical composition.