DE102008059446A1 - New proteases and agents containing these proteases - Google Patents

Abstract

The present application relates to novel proteases, sufficiently related proteases and their derivatives and their preparation and their use. It also relates to agents, in particular detergents and cleaners containing these proteases, sufficiently related proteases and their derivatives, corresponding washing and cleaning processes and uses of the proteases, in particular for washing and cleaning purposes.

Description

The present application relates to novel proteases, sufficiently related Proteases and their derivatives as well as their preparation and their use. It also relates to agents, especially detergents and cleaners containing these proteases, sufficiently related proteases and their derivatives, appropriate washing and cleaning processes and Uses of the proteases, especially for washing and cleaning purposes.

Proteases are among the most technically important enzymes of all. For detergents and cleaners, they are the longest established and contained in virtually all modern, powerful detergents and cleaners enzymes. They cause the degradation of protein-containing stains on the items to be cleaned. Of these, in turn, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important, which are attributed to the serine proteases due to the catalytically active amino acids. They act as nonspecific endopeptidases, that is, they hydrolyze any acid amide linkages that are internal to peptides or proteins. Their pH optimum is usually in the clearly alkaline range. For an overview of this family, for example, the article "Subtilases: Subtilisinlike Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 , Subtilases are naturally produced by microorganisms; Of these, in particular, the subtilisins formed and secreted by Bacillus species are to be mentioned as the most important group within the subtilases.

Examples of the subtilisin-type proteases preferably used in detergents and cleaners are the subtilisins BPN 'and Carlsberg, the protease P692, the subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the the subtilases, but not the subtilisins in the narrower sense attributable enzyme thermitase, proteinase K and the proteases TW3 and TW7. Other usable proteases are, for example, under the trade names Durazym ^®, relase ^®, Everlase® ^®, Nafizym, Natalase ^®, Kannase® ^® and Ovozyme ^® from Novozymes, the ^® under the trade names Purafect ^®, Purafect ^® OXP, Purafect Prime and Properase.RTM ^® from Genencor, that under the trade name Protosol® ^® from Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi ^® from Wuxi Snyder Bioproducts Ltd., China, the P under the trade names Proleather® ^® and protease ^® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzymes available under the name proteinase K-16 from Kao Corp., Tokyo, Japan.

Regarding the use of proteases in detergents and cleaners known that proteases improve the washing or Cleaning performance together with other enzymes, for example Amylases, cellulases, hemicellulases, mannanases, β-glucosidases, Oxidases, oxidoreductases or lipases can be used. Likewise, the use of proteases in detergents in combination with other active ingredients such as bleaches or soil release agents known to the skilled person.

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

The exemplified here, different technical Applications require proteases with different properties, such as the reaction conditions, the stability or the substrate specificity. Conversely, hang the technical applications of proteases, for example in the context of a detergent or cleaning agent formulation, of others Factors like the stability of the enzyme high temperatures, over oxidizing agents or towards its denaturation by surfactants, from folding effects or of desired synergies with other ingredients from.

Consequently There is still a high demand for technically usable Proteases, due to the variety of their uses in their Entire a wide range of properties to very much cover subtle differences in performance.

The basis for this are new proteases, which in turn can be specifically developed for specific applications. The classical procedure for obtaining new enzymes is to take samples of microorganisms from natural habitats and to cultivate them under the conditions considered suitable, for example in an alkaline medium. In this way, enrichment cultures of microorganisms are obtained, which with a certain probability also contain enzymes, including proteases, which are active under the respective conditions. From this example then wise on plating on proteinaceous agar plates and measuring the lysis farms formed the microorganisms selected and purified with the most powerful enzymes or cloned the respective genes. Such a procedure is for example in the textbook "Alkalophilic Microorganisms. A new microbial world "by K. Horikoshi and T. Akiba (1982), Japan Scientific Societies Press, Springer-Verlag, New York, Heidelberg, Berlin, ISBN 0-387-10924-2, Chapter 2, pages 9-26 described.

task The present invention is further, hitherto unknown Provide proteases that have a proteolytic activity exhibit. Advantageously, the protease should be characterized that their contribution to the performance of an agent containing the protease in particular a washing or cleaning agent, the contribution of one proteolytic enzyme established for this purpose Performance of the agent comes at least close and ideally exceeds. As a contribution to the performance in this regard is particular the contribution to the cleaning performance of a washing or cleaning agent significant.

A Further objects of the invention are proteases, in particular of Subtilisin type, provide that opposite the state The technique provides improved stability Temperature effects, pH fluctuations, denaturing or oxidizing agents, proteolytic degradation, high temperatures, acid or alkaline conditions or against one Have change in the redox ratios. Other tasks may result in decreased immunogenicity or reduced allergenic effects.

A Another object of the present invention is to provide proteases, at lower temperatures, especially between 10 ° C and 50 ° C, and preferably between 10 ° C and 40 ° C or between 20 ° C and 40 ° C a good cleaning performance, preferably an improved cleaning performance compared to the proteases disclosed in the prior art, in particular those of the subtilisin type.

A Another object of the present invention is to provide proteases, the proteases known in the art improved cleaning performance with respect to at least one soiling, preferably with respect to multiple soils.

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

• a) Aminosäuresequenz, die zu der in SEQ ID NO. 3 angegebenen Aminosäuresequenz zu mindestens 98,3% identisch ist
• b) Aminosäuresequenz, die zu der in SEQ ID NO. 2 angegebenen Aminosäuresequenz zu mindestens 98,5% identisch ist,

insbesondere eine Aminosäuresequenz, die ausgewählt ist aus der Gruppe bestehend aus

• a) Aminosäuresequenz, die zu der in SEQ ID NO. 3 angegebenen Aminosäuresequenz zunehmend bevorzugt zu mindestens 98,4%, 98,5%, 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist
• b) Aminosäuresequenz, die zu der in SEQ ID NO. 2 angegebenen Aminosäuresequenz zu mindestens 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist.

An object of the invention is a protease comprising an amino acid sequence selected from the group consisting of

• a) Amino acid sequence corresponding to that shown in SEQ ID NO. 3 amino acid sequence is at least 98.3% identical
• b) Amino acid sequence corresponding to that shown in SEQ ID NO. 2 amino acid sequence is at least 98.5% identical,

in particular an amino acid sequence selected from the group consisting of

• a) Amino acid sequence corresponding to that shown in SEQ ID NO. 3, more preferably at least 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical
• b) Amino acid sequence corresponding to that shown in SEQ ID NO. 2, at least 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99, 5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical.

Herewith are as further subjects of the invention the associated, coding for proteases of the invention Nucleic acids, proteases according to the invention or non-human host cells containing nucleic acids, suitable methods for their preparation, in particular on the nucleic acids or host cell-based molecular biological processes and process elements and agents, in particular detergents and cleaners, detergents and cleaners Purification process and the proteases concerned marked uses connected.

The naturally formed protease on which the present invention is based can be obtained from the culture supernatant of a Bacillus strain which can be obtained from the examples. DSMZ (DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, Inhoffenstr e 7 B, 38124 Braunschweig, Germany) is identified as a Bacillus pumilus strain. For the purpose of reworkability, according to the Budapest Treaty, a plasmid containing the nucleic acid sequence of the protease according to the invention was deposited with DSMZ under the accession number DSM 21891.

A Protease according to the invention has a proteolytic Activity, that is, it is for hydrolysis capable of peptide binding of a polypeptide or protein, especially in a washing or cleaning agent. An inventive Protease is therefore an enzyme which inhibits the hydrolysis of peptide bonds catalyzed and thereby able to add peptides or proteins columns. A protease according to the invention is in particular a subtilisin. For the purposes of the present invention, enzymes, Proteins, fragments and derivatives, unless explicitly stated such need to be addressed, under the generic term proteins or polypeptides, since a protein is a polypeptide is.

A Protease of the invention is due to their proteolytic activity and its further properties, especially regarding their stability Surfactants and / or bleaching agents and / or their temperature profile and / or their pH profile, for use in detergents and cleaners suitable. Surprisingly, she already performs in her Such a good contribution to the cleaning performance of a washing or detergent containing the protease, the the contribution of a proteolytic agent established for this purpose Enzyme comes close to the cleaning performance of the compound and it gets to different soiling even surpasses. It therefore allows an improved Removal of at least one, preferably several protease-sensitive Soiling on textiles and / or hard surfaces, for example, dishes. Next surprisingly it was found that such a protease a washing or cleaning agents a gives advantageous cleaning performance, especially at low Temperatures, for example between 10 ° C and 50 ° C, in particular between 10 ° C and 40 ° C or between 20 ° C and 40 ° C.

Under Cleaning performance according to the invention, the whitening performance a washing or cleaning agent to stains, in particular on protease-sensitive stains and hereunder in particular on protease-sensitive Wäscheanschmutzungen understood. The cleaning performance is preferably determined as below specified.

by virtue of the provided invention Nucleic acids is an additional optimization this protease, for example by amino acid substitutions or other mutations, possible. Furthermore, can Nucleic acids according to the invention in a wide variety Recombination approaches introduced and thus for the production completely new proteases or other polypeptides used become.

numerous Proteases and especially subtilisins are called pre-proteins, so formed together with a propeptide and a signal peptide, the function of the signal peptide is usually therein the release of the protease from the one producing it Cell into the periplasm or to ensure the medium surrounding the cell and the propeptide usually for the correct one Conclusion of the protease is necessary. The signal peptide and the Propeptide are usually the N-terminal part of the preprotein. The Signal peptide is transformed by natural conditions Signal peptidase cleaved from the remaining protease. Subsequently the correct final supported by the propeptide takes place Folding of the protease. The protease is then in its active form and cleaves off the propeptide itself. After cleavage of the propeptide exercises the then mature protease, especially subtilisin, their catalytic activity without the original ones N-terminal amino acids.

For technical applications are due to their enzymatic activity the mature proteases, d. H. the after their production processed enzymes, compared to the preproteins prefers. The proteases can be produced by those who produce them Cells are modified after preparation of the polypeptide chain, for example, by attachment of sugar molecules, Formylations, aminations, etc. Such modifications will become referred to as post-translational modifications. This post translational Modifications can, but do not have to be Exert influence on the function of the protease.

A Nucleic acid sequence according to the invention is under SEQ ID NO. 1 indicated. This nucleic acid encodes for a protease, which is one for subtilisins typical classification into signal peptide, propeptide and mature Protease has. The full-length protein is under SEQ ID NO. 2 and the mature protease under SEQ ID NO. 3 indicated. By this is meant the actually active mature protein, because this exercises the technically relevant function. Particularly according to the invention therefore, the mature, active proteases are preferred. These wise a molecular weight between 25 and 30 kD (kilodaltons), in particular 27 kD, determined by SDS-polyacrylamide gel electrophoresis.

For the protease according to SEQ ID NO. 3 or SEQ ID NO. 2 was a sequence analysis and a sequence comparison with known protein sequences from the publicly available databases UniProtKB and / or Swiss-Prot (see UniProtKB, EMBL Outstation - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom; //www.uniprot.org) and / or GenBank (National Biotechnology Center NCBI, Bethesda, MD, USA), to identify the proteins most closely related. This sequence comparison was carried out using the BLAST algorithm established in the prior art and commonly used (cf., for example, US Pat Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410 , and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs "; Nucleic Acids Res., 25, pp. 3389-3402 ). Another algorithm available in the prior art is the FASTA algorithm.

On the level of amino acids for the entire protease according to SEQ ID NO. 2 was next Protein identified: Sequence Q5XPN0 from Bacillus pumilus (UniProtKB / TrEMBL Q5XPN0 (Q5XPN0_BACPU); "Organic Solvent Tolerant Protease") with 98.4% identity or differences in 6 amino acid positions.

On the level of amino acids for the mature Protease according to SEQ ID NO. 3 was next Protein identified: Sequence Q5XPN0 from Bacillus pumilus UniProtKB / TrEMBL Q5XPN0 (Q5XPN0_BACPU); "Organic Solvent Tolerant Protease") with 98.2% identity or deviations in 5 amino acid positions.

Thus, the protease of the invention differs from this protease in 6 amino acids based on the total length protein according to SEQ ID NO. 2 and in 5 amino acids based on the mature protease according to SEQ ID NO. 3. Thus, the protease found is a new enzyme. Thus, all proteases that are at least 98.5% identical to SEQ ID NO. 2 and / or which differ from the sequence Q5XPN0 (indicated as SEQ ID NO: 5) in at least one, preferably 2, 3, 4, 5 or 6 amino acids or which are at least 98.3% identical to SEQ ID NO , 3 and / or which differ from the sequence of the mature protease according to Q5XPN0 (indicated as SEQ ID NO: 6) in at least one, preferably 2, 3, 4 or 5 amino acids. To the well-established in the art protease from Bacillus lentus (SEQ ID NO 4, see also WO 97/21760 A1 ) gives an identity of 58.2% based on the mature enzyme.

by virtue of the correspondence to be recognized and the relationship to the other specified subtilisins is an inventive Protease as a subtilisin.

The Determination of the identity of nucleic acid or Amino acid sequences are made by a sequence comparison. By such a comparison with known enzymes, for example are stored in generally accessible databases, Eats from the amino acid or nucleotide sequence as well conclude the enzymatic activity of a considered enzyme. This may be due to other areas of the protein that are not attached to the actual reaction involved, qualitatively or quantitatively be modified. This could be, for example, the enzyme stability, the activity, the reaction conditions or the substrate specificity affect.

Such a comparison is made by assigning similar sequences in the nucleotide sequences (or amino acid sequences) to each other. A tabular assignment of the respective positions is referred to as alignment. In the analysis of nucleotide sequences, in turn, both complementary strands and all three possible reading frames are to be taken into account; as well as the degeneracy of the genetic code and the organism-specific use of codons (codon usage). Usually, sequence comparisons and alignments are created by computer programs. For example, Clustal (cf., for example, Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500 ) or T-Coffee (cf., for example Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217 ) as well as BLAST or FASIA for the database search, or programs based on these programs or algorithms. In the present application sequence comparisons and alignments were performed with the computer program Vector NTI ^® Suite 7.0 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, United States) created with the preset default parameters.

A Compilation of all matching in the compared sequences Positions is called a consensus sequence.

Such a comparison also allows a statement about the similarity the compared sequences to each other. It usually becomes in percent identity, that is the proportion of identical nucleotides or amino acid residues on the same or in an alignment corresponding positions indicated. The broader term of homology refers to amino acid sequences Conserved amino acid substitutions into consideration one, so amino acids with similar chemical Activity as these are mostly similar within the protein exercise chemical activities. Therefore, the similarity the sequences compared also percent homology or percent similarity be specified. Identity and / or homology information can be transmitted via whole polypeptides or genes or only over individual areas to be hit.

homologous or identical regions of different nucleic acid or amino acid sequences are by matches defined in the sequences. Such areas can also be characterized by identical functions. You can be small and comprise only a few nucleotides or amino acids. Often, such small areas practice for overall activity of the protein essential functions. It may therefore make sense be, sequence matches only to individual, if necessary to relate to small areas. Examples of such functions may interact with nucleic acids be special molecules, for example in promoter sequences the interaction with certain transcription factors. At amino acid sequences Such functions can also be the smallest subfunctions of the be performed by the entire polypeptide function such as the formation of individual hydrogen bonds to Complexation of a substrate or a transition complex.

• a) Aminosäuresequenz, die zu der in SEQ ID NO. 3 angegebenen Aminosäuresequenz zunehmend bevorzugt zu mindestens 98,4%, 98,5%, 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist
• b) Aminosäuresequenz, die zu der in SEQ ID NO. 2 angegebenen Aminosäuresequenz zu mindestens 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist.

In a preferred embodiment of the invention, the protease is characterized by comprising an amino acid sequence selected from the group consisting of

• a) Amino acid sequence corresponding to that shown in SEQ ID NO. 3, more preferably at least 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical
• b) Amino acid sequence corresponding to that shown in SEQ ID NO. 2, at least 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99, 5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical.

All The above facts and explanations apply to these preferred embodiments accordingly.

• – Blut-Milch/Tusche auf Baumwolle: Produkt Nr. C-05 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande
• – Voll-Ei/Pigment (Ganzei/Ruß) auf Baumwolle: Produkt Nr. 10 N erhältlich von der Firma wfk Testgewebe GmbH; Brüggen-Bracht, Deutschland, oder Produkt C-S-37 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande
• – Schokoladenmilch/Ruß auf Baumwolle: Produkt Nr. C-03 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande
• – Erdnuss Öl-Pigment/Tusche auf Polyester/Baumwolle: Produkt Nr. PC-10 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande
• – Gras auf Baumwolle: Produkt Nr. 164 erhältlich von der Firma Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Schweiz
• – Kakao auf Baumwolle: Produkt Nr. 112 erhältlich von der Firma Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Schweiz,

bestimmt wird durch Messung des Weißheitsgrades der gewaschenen Textilien, der Waschvorgang für mindestens 30 Minuten, optional 60 Minuten, bei einer Temperatur von 40°C erfolgt und das Wasser eine Wasserhärte zwischen 15,5 und 16,5° (deutsche Härte) aufweist.In a further preferred embodiment of the invention, the protease is characterized in that its purification performance corresponds at least to that of a protease which comprises an amino acid sequence which is as described in SEQ ID NO. 4 corresponds to the amino acid sequence indicated, and / or at least corresponds to that of a protease which comprises an amino acid sequence which corresponds to that shown in SEQ ID NO. 6 corresponds to the amino acid sequence indicated, and / or at least that of the protease from Bacillus pumilus according to WO2007 / 131656 , wherein the cleaning performance is determined in a washing system containing a detergent in a dosage between 4.5 and 7.0 grams per liter of wash liquor and the protease, wherein the proteases to be compared are used in the same activity and the cleaning performance against one or more of Soiling blood-milk / ink on cotton, whole egg / pigment (whites / soot) on cotton, chocolate milk / soot on cotton, peanut oil pigment / ink on polyester / cotton, grass on cotton and cocoa on cotton, especially against one or more of the stains

• - Blood milk / cotton ink: Product No. C-05 available from CFT (Center For Test Materials) BV Vlaardingen, the Netherlands
• - Whole egg / pigment (whole egg / carbon black) on cotton: Product No. 10 N available from the company wfk Testgewebe GmbH; Brüggen-Bracht, Germany, or product CS-37 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands
• - Chocolate milk / soot on cotton: Product No. C-03 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands
• - Peanut oil pigment / ink on polyester / cotton: Product No. PC-10 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands
• - Grass on cotton: Product No. 164 available from the company Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Switzerland
• - Cocoa on cotton: Product No. 112 available from the company Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Switzerland,

is determined by measuring the whiteness of the washed fabrics, the washing process for at least 30 minutes, optionally 60 minutes, carried out at a temperature of 40 ° C and the water is a water Hardness between 15.5 and 16.5 ° (German hardness).

According to the invention the terms whole egg / carbon black or whole egg / pigment with regard to of soiling as equivalent and mutually consider.

One preferred liquid detergent for such Washing system is composed as follows (all figures in weight percent): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3-0.5% ethanol, 4-7% FASOS (fatty alcohol ether sulfate), 24-28% nonionic surfactants, 1% Boric acid, 1-2% sodium citrate (dihydrate), 2-4% Soda, 14-16% coconut fatty acids, 0.5% HEDP (1-hydroxyethane- (1,1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.05% optical Brightener, 0-0.001% dye, remainder demineralized water. Preferably, the dosage of the liquid Detergent between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter wash liquor. Preference is given to washing in a pH range between pH 8 and pH 10.5, preferably between pH 8 and pH 9.

One preferred powdered detergent for a such washing system is composed as follows (all information in weight percent): 10% linear alkyl benzene sulfonate (sodium salt), 1.5% C12-C18 fatty alcohol sulfate (sodium salt), 2.0% C12-C18 fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium bicarbonate, 4.0% amorphous sodium disilicate, 17% sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1.0% carboxymethylcellulose, 1.0% phosphonate, 25% sodium sulfate, balance: optional foam inhibitors, optical brightener, Fragrances and, if necessary, water ad 100%. Preferred is the dosage of the powdered detergent between 5.5 and 7.0 grams per liter of wash liquor, for example 5.6, 5.9 or 6.7 grams per liter of wash liquor. Preference is given to washing in one pH range between pH 9 and pH 11.

Of the Whiteness, d. H. the brightening of soiling, as a measure of the cleaning performance is preferred determined by optical measuring methods, preferably photometrically. One this suitable device is for example the Spectrometer Minolta CM508d. Usually, the for previously measuring with a white standard, preferably a supplied white standard, calibrated.

The activity-equivalent use of the respective protease ensures that even if the ratio of active substance to total protein (the values of the specific activity) diverge, the respective enzymatic properties, for example the cleaning performance of certain soils, are compared. In general, a low specific activity can be compensated by adding a larger amount of protein. Methods for the determination of the protease activities are familiar to the expert in the field of enzyme technology and are routinely used by him. For example, such methods are disclosed in Surfactants, Vol. 7 (1970), pp. 125-132 , The protease activity is preferably indicated in PE (protease units). For example, suitable protease activities are 2.25, 5 or 10 PE (protease units) per ml wash liquor. However, the protease activity is not equal to zero.

proteins can also talk about the reaction with an antiserum or a particular antibody to groups immunologically related proteins are summarized. The members A group is characterized by being the same, by one Antibodies recognized to have antigenic determinant. she are therefore structurally so similar to each other that they are of an antiserum or specific antibodies. A further subject of the invention therefore further proteases, which are characterized by having at least one and increasingly preferably two, three or four consensus antigenic determinants having a protease according to the invention. Such proteases are due to their immunological similarities structurally similar to the proteases of the invention, that also a similar function is assumed.

In Another embodiment of the invention is an inventive Protease naturally present in an organism, which can be isolated from a natural habitat.

These Embodiment is therefore particularly advantageous because then the associated organism itself taken into culture can be. Advantageously, can then be from its cell extracts or culture supernatants according to the invention Isolate and produce proteases.

Preferably, the protease is present in a microorganism, more preferably in a fungus, in a Gram-negative or in a Gram-positive bacterium, and of these particularly preferably in one Bacterium of the genus Bacillus. Because especially for these organisms cultivation methods are known and established in the prior art. This is especially true for Bacilli, which play a prominent role in the technical production of enzymes. Most preferably, the protease is present in Bacillus pumilus and especially in Bacillus pumilus DSM ID 08-100.

Further became one for a preferred invention Protease-encoding nucleic acid within the scope of the present invention Invention recombinantly introduced into a host cell and as above described under the number DSM 21891 deposited with the DSMZ. In a further preferred embodiment of the invention the protease is thus characterized by being derived from a Nucleic acid is coded in a host cell with the deposit number DSM 21891 is present.

proteases or enzymes in general can be prepared by various methods, z. B. targeted genetic modification by mutagenesis, evolved and for specific purposes or in terms of specific properties, such as catalytic activity, Stability, etc., to be optimized. amendments the nucleotide or amino acid sequence, as for example brought about by molecular biology methods known per se are therefore called mutations. Depending on the nature of the change, for example, deletion, Insertion or substitution mutations or those in which different genes or parts of genes are fused together. The associated organisms are called mutants. Proteins encoded by mutant nucleic acids are known as Variants called. For example, deletion, Insertion, substitution mutations or fusions to deletion, insertions, substitution mutated genes or fusion genes and at the protein level to corresponding deletion, insertion or Substitution variants or fusion proteins. The goal it is, in the known molecules targeted mutations, for example substitutions, insertions or deletions, to introduce for example, the cleaning performance of inventive To improve enzymes. For this purpose, in particular the surface charges and / or the isoelectric point of the molecules and thereby, for example their interactions with the substrate are changed. Thus, for example, via mutations, the net charge the enzymes are altered to reveal the substrate binding especially for use in detergents and cleaners to influence. Another, especially supplementary Strategy is to provide stability through appropriate mutations of the proteases in question, thereby increasing their To increase cleaning performance.

A modern direction of enzyme development consists of elements known, related proteins via statistical Procedure for new enzymes with previously unattainable properties to combine. Such procedures are also under the generic term Summarized Directed Evolution. This includes For example, the so-called "StEP" method, "Random priming recombination, "DNA shuffling", "RACHITT" or "recombining ligation reaction "(RLR).

For the description of substitutions that exactly one amino acid position affect (amino acid exchanges), the following convention applied: first, the natural existing amino acid in the form of the internationally used Denotes one-letter codes, then follows the associated sequence position and finally the inserted amino acid. Several exchanges within the same polypeptide chain are performed Slashes separated. For insertions are after the sequence position additional amino acids named. For deletions, the missing amino acid is through replaced a symbol, such as a star or a dash. For example, A95G describes the substitution of alanine at position 95 by glycine, A95AG the insertion of glycine after the amino acid Alanine at position 95 and A95 * the deletion of alanine at position 95. This nomenclature is familiar to those skilled in the enzyme technology field known.

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

Fragments are understood as meaning all proteins or peptides which are smaller than natural proteins and, for example, can be obtained synthetically. Due to their amino acid sequences, they can be assigned to the respective complete proteins. For example, they may adopt the same structures or perform enzymatic activities or partial activities, such as the complexation of a particular substrate. Fragments and deletion variants of parent proteins are in principle similar in that one or more amino acids are absent compared to an initial sequence; while fragments usually comprise small pieces of an initial sequence, deletion mice are lacking usually only short areas and thus possibly only individual sub-activities.

insertions like deletions are not limited to single amino acids. Rather, several amino acids or whole fragments or even whole proteins in an initial sequence be inserted or fused with an initial sequence be. In the latter case, it is then a chimeric protein. This includes new combinations of larger ones Enzyme sections, ie fragments, with other enzymes or proteins other function. So it is possible, for example, a enzyme of the invention or parts thereof peptidic linkers or directly as a fusion protein with binding domains from other proteins, such as the cellulose-binding domain, to provide and thereby the hydrolysis of the substrate more effectively shape. Likewise, inventive Enzymes, for example, with amylases or cellulases or fragments linked to perform a dual function.

a Another object of the invention thus represents a protease, which is characterized in that it consists of a Protease is obtainable as a parent molecule by fragmentation, deletion, Insertion or substitution mutagenesis and an amino acid sequence which covers a length of at least 50 and increasingly preferably at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, 266, 267 or 268 contiguous amino acid positions matches the parent molecule.

So For example, it is possible at the terms or in the Loops of the enzyme to delete single amino acids, without that thereby the proteolytic activity is lost. By such deletions, for example, often the Allergenicity of relevant enzymes lowered and thus total their usability can be improved. Regarding the intended Use of these enzymes, it is particularly preferred if they too after fragmentation or deletion mutagenesis a proteolytic Own activity.

Also Substitutes can give rise to advantageous effects. In principle, this also includes individual substitutions of amino acids, but it can also be several contiguous amino acids be exchanged for others.

A further subject of the invention is a protease, which is characterized in that it is obtainable from a protease according to the invention as the starting molecule and has one or more amino acid substitutions in positions which correspond to the positions 3, 4, 36, 42, 47, 56, 61 , 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237 , 242, 243, 255 and 268 of the protease from Bacillus lentus according to SEQ ID NO. 4 are assigned in an alignment. The amino acid positions are determined by an alignment of the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as described in SEQ ID NO. 4 is defined. Such an alignment is in 1 specified. Since the protease from Bacillus lentus in the prior art represents an important reference molecule for the description of new proteases and amino acid changes and the novel proteases described here and thus also their sequence are unknown, it is advantageous in the assignment of the amino acid positions on the count of the protease from Bacillus lentus (SEQ ID NO: 4). Furthermore, the count depends on the mature (mature) protein. This assignment is also to be used in particular if the amino acid sequence of the protease according to the invention comprises a higher number of amino acid residues than the protease from Bacillus lentus according to SEQ ID NO. 4. Starting from said positions in the amino acid sequence of the protease from Bacillus lentus, the alteration positions in a protease according to the invention are those which correspond precisely to these positions in accordance with 1 assigned.

advantageous Positions for sequence changes, in particular Substitutions, the protease from Bacillus lentus, that transmit to homologous positions of the invention Proteases are of importance and the protease advantageous give them functional properties are therefore 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, to be assigned in an alignment with SEQ ID NO. 4 and thus in the count according to SEQ ID NO. 4. In the above positions lie in the wild-type molecule of Protease from Bacillus lentus following amino acid residues: S3, V4, S36, N42, A47, T56, G61, T69, E87, A96, R99, A101, I102, S104, N114, H118, A120, S130, S139, T141, S142, S154, S157, A188, V193, V199, G205, L211, A224, K229, S236, N237, N242, H243, N255 or T268.

Particularly advantageous examples are substitutions 3T, 4I, 61A, 99G, 99A, 99S, 154D, 154E, 211D, 211G and 211E, provided that the correspondingly homologous positions in a protease according to the invention are not naturally taken up by one of these preferred amino acids. The Austau presumably have a stabilizing effect on the molecule to improve the cleaning performance of the protease and thus to an improved cleaning performance of a washing or cleaning agent containing the protease.

A final confirmation in the assignment of homologous positions, ie in particular their functional correspondence, can ultimately provide only comparative experiments, according to which the two positions assigned to each other on the basis of an alignment in both compared proteases are changed in the same way and observed whether in both the enzymatic activity is changed in the same way. If, for example, an amino acid exchange in a certain position of the protease from Bacillus lentus (BLAP) is accompanied by an increase in K _M or another enzymatic parameter, the same or a similar shift of the K _M value is observed in a protease variant according to the invention , whose amino acid exchange has been achieved by the same introduced amino acid, the confirmation of this aspect of the invention is to be seen herein.

One Another object of the invention is therefore a previously described Protease, which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer.

Because an increase in stability during storage and / or during use, for example during the washing process, causes the enzymatic activity to last longer stops and thus the cleaning performance is improved. In addition to the above-mentioned stabilization options all described in the prior art and / or appropriate Stabilization options into consideration.

Prefers These are stabilizations that are due to mutations of the enzyme even possible. Because these require stabilization following the recovery of the enzyme no further steps. Some For this purpose suitable sequence changes are on known from the prior art. So can proteases For example, it can also be stabilized by being certain Replaces tyrosine residues with others.

• – Veränderung der Bindung von Metallionen, insbesondere der Calcium-Bindungsstellen, beispielsweise durch Austauschen von einer oder mehreren der an der Calcium-Bindung beteiligten Aminosäure gegen eine oder mehrere negativ geladene Aminosäuren und/oder durch Einführen von Sequenzveränderungen in mindestens einer der Folgen der beiden Aminosäuren Arginin/Glycin;
• – Schutz gegen den Einfluss von denaturierenden Agentien wie Tensiden durch Mutationen, die eine Veränderung der Aminosäuresequenz auf bzw. an der Oberfläche des Proteins bewirken;
• – Austausch von Aminosäuren, die nahe dem N-Terminus liegen, gegen solche, die vermutlich über nicht-kovalente Wechselwirkungen mit dem Rest des Moleküls in Kontakt treten und somit einen Beitrag zur Aufrechterhaltung der globulären Struktur leisten.

Other options include:

• Changing the binding of metal ions, in particular the calcium binding sites, for example by exchanging one or more of the amino acids participating in the calcium binding for one or more negatively charged amino acids and / or introducing sequence changes in at least one of the sequences of the two amino acids arginine / glycine;
• Protection against the effect of denaturing agents, such as surfactants, on mutations causing an alteration of the amino acid sequence on or on the surface of the protein;
• Replacement of amino acids located near the N-terminus against those likely to contact non-covalent interactions with the rest of the molecule, thus contributing to the maintenance of the globular structure.

preferred Embodiments are those in which the enzyme is on several species is stabilized. Because it can be assumed be that multiple stabilizing mutations additive or synergistic Act.

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

For the purposes of the present application, derivatives are understood as meaning those proteins whose pure amino acid chain has been chemically modified. Such derivatizations can be carried out, for example, biologically in connection with protein biosynthesis by the host cell. In this regard, couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy. For this molecular biological methods can be used. However, derivatizations can also be carried out chemically, for example by the chemical transformation of a side chain of an amino acid or by covalent binding of another compound to the protein. For example, the coupling of amines to carboxyl groups of an enzyme to alter the isoelectric point is possible. However, such a compound can also be other proteins which are bound, for example via bifunctional chemical compounds, to proteins according to the invention. Such modifications may, for example, affect the substrate specificity or binding strength to the substrate or cause a temporary blockage of the enzymatic activity when the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Also, derivatization is understood to mean covalent attachment to a macromolecular carrier, as well as noncovalent inclusion in suitable macromolecular cage structures. Such modifications may further affect 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 enzyme and thus increase its skin tolerance, for example. For example, couplings with macromolecular compounds, for example, polyethylene glycol, can improve the protein in terms of stability and / or skin tolerance.

Under Derivatives of a protein of the invention can understood in the broadest sense also preparations of these proteins become. Depending on extraction, processing or preparation can a protein be associated with various other substances be, for example, from the culture of producing microorganisms. A protein can also, for example, increase its Storage stability, targeted with certain other substances have been added. Therefore, according to the invention also all preparations of an inventive Protein. That is also independent of whether it is in one certain preparation actually this enzymatic Activity unfolded or not. Because it can be desired be that there is little or no activity during storage owns, and only at the time of use its enzymatic Function unfolds. This can, for example, via appropriate Accompanying substances are controlled. In particular, the joint preparation proteases with protease inhibitors is advantageous.

One Another subject of the invention is a nucleic acid, which codes for a protease according to the invention, and a vector containing such a nucleic acid.

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

at DNA is the sequence of both complementary strands to be considered in all three possible reading frames.

Further is to take into account that different codons, ie Base triplets encoding the same amino acids can, allowing a specific amino acid sequence be encoded by several different nucleic acids can, what is called degeneracy of the genetic code. In addition, different organisms have differences in the Use of these codons. For these reasons need both amino acid sequences and nucleotide sequences be included in the consideration of the scope. Therefore are all nucleotide sequences and thus nucleic acids included in this subject of the invention, one of the encoding proteases described above. Of the One skilled in the art is capable of doing these nucleotide sequences beyond doubt because despite the degeneracy of the genetic code individual codons defined amino acids are assigned. Therefore, the person skilled in the art can start from an amino acid sequence for this amino acid sequence encoding nucleic acids determine easily.

A person skilled in the art is now able to use well-known methods such as, for example, chemical synthesis or polymerase chain reaction (PCR) in conjunction with standard molecular biological and / or proteinchemical methods, using known DNA and / or amino acid sequences, the appropriate nucleic acids up to to produce complete genes. Such methods are for example Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press. known.

• a) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz in einem Teilstück von mindestens 300 zusammenhängenden Nukleotiden mindestens zu 97,1% und zunehmend bevorzugt zu mindestens 97,2%, 97,4%, 97,6%, 97,8%, 98%, 98,2%, 98,3%, 98,4%, 98,5%, 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist
• b) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz in den Positionen 325 bis 1149 mindestens zu 97,1% und zunehmend bevorzugt zu mindestens 97,2%, 97,4%, 97,6%, 97,8%, 98%, 98,2%, 98,3%, 98,4%, 98,5%, 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist
• c) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz mindestens zu 97,1% und zunehmend bevorzugt zu mindestens 97,2%, 97,4%, 97,6%, 97,8%, 98%, 98,2%, 98,3%, 98,4%, 98,5%, 98,6%, 98,7%, 98,8%, 98,9%, 99%, 99,1%, 99,2%, 99,3%, 99,4%, 99,5%, 99,6%, 99,7%, 99,8%, 99,9% und ganz besonders bevorzugt zu 100% identisch ist.

In a preferred embodiment of the invention, the nucleic acid coding for a protease according to the invention is characterized in that it comprises a nucleic acid sequence which is selected from the group consisting of

• a) Nucleic acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 nucleic acid sequence in a portion of at least 300 contiguous nucleotides at least 97.1% and increasingly preferably at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99, 8%, 99.9%, and most preferably 100% identical
• b) Nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO. 1 to at least 97.1%, and more preferably at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98, of Nucleic Acid Sequence in positions 325 to 1149. 3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3% , 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical
• c) Nucleic acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 to at least 97.1%, and more preferably at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4 %, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical.

In Another embodiment of the invention is the Nucleic acid characterized in that it has a nucleic acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 indicated nucleic acid sequence in a portion of increasingly preferred 400, 500, 600, 700, 800, 900, 1000, 1100 contiguous nucleotides in each case at least 97.1% and increasingly preferably in each case at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical is.

The p. above) nucleic acid sequence the region coding for the mature protease. The stop codon is included in this consideration because of its Existence ensures that not a bigger, possibly no longer functional, unwanted protein is formed. Thus, when cloning is to pay attention that at this point there is also a stop codon, if not specifically induced via the C-terminus, a protein fusion shall be. Should later turn out that the Mature protein is formed only from part of this sequence is, the scope of protection applies accordingly for this Part. Preference is given to those nucleic acids which are suitable for encode mature proteins, and are increasingly preferred those which are increasingly active variants of the invention Encode proteins.

Farther can in nucleic acids of the invention one or more codons have been replaced by synonymous codons. This aspect relates to the heterologous expression of the invention Enzymes. So every organism, especially every production strain, a certain codon usage. Under codon usage is the translation understood the genetic code in amino acids, d. H. which nucleotide sequence (triplet or base triplet) for which amino acid or for which function, for example onset and end of the region to be translated, binding sites for different proteins, etc., coded. So every organism, in particular, each host cell, a particular codon Usage. It can bottlenecks in protein biosynthesis occur when the on the transgenic nucleic acid codons in the Host cell of a comparatively small number of loaded tRNAs face. Synonymous codons encode for the same amino acids and may be in the respective Host cell be translated better. This possibly necessary Rewriting thus depends on the choice of the expression system. Especially for samples from unknown, possibly not cultivable Organisms may need to be adjusted accordingly.

With All of the above subjects of the invention and embodiments are as further subjects of the invention corresponding vectors, Host cells, production methods in which appropriate vectors or Host cells are used as well as uses accordingly Linked to nucleic acids, vectors and host cells. Therefore The above statements relate to these subjects of the invention corresponding.

One Another object of the invention is thus a vector, the one contains nucleic acid according to the invention, in particular a cloning vector or an expression vector.

Under For the purposes of the present invention, vectors are made from nucleic acids existing elements understood as the characteristic nucleic acid region contain a nucleic acid according to the invention. she they can translocate in a species or cell line several generations or cell divisions away as a stable genetic Establish element. Vectors are especially in use in bacteria special plasmids, so circular genetic elements. It is distinguished between such vectors, the storage and thus to a certain extent also the genetic engineering work serve, so-called cloning vectors, and those that have the function meet, the nucleic acid or on this to realize existing gene (often transgene) in the host cell, that is, the expression of the subject polypeptide to enable. These vectors are called expression vectors designated.

in the The scope of the present invention is an inventive Nucleic acid suitably cloned into a vector. These may include, for example, those whose Origin of bacterial plasmids, viruses or bacteriophages are, or predominantly synthetic vectors or plasmids with elements of various kinds Origin. With the other existing genetic elements Vectors can translocate in the respective host cells several generations as stable units. It is irrelevant in the context of the invention, whether they are extrachromosomal exist as separate units or in a chromosome or chromosomal Integrate DNA. Which of the numerous from the state of the art well-known systems depends on the individual case from. Decisive, for example, the achievable Copy number, the available selection systems, especially antibiotic resistance, or cultivability be the host cells capable of receiving the vectors.

expression vectors include subsequences that enable them to enter the host cells containing them, preferably microorganisms, especially prefers bacteria to replicate and there a contained nucleic acid to bring to expression. The expression is particularly of the promoter (s) that regulate transcription. In principle, expression can be influenced by the natural, originally in front of the nucleic acid to be expressed localized promoter, but also by one on the expression vector provided promoter of the host cell or by a modified or a completely different promoter of another organism or another host cell. In the present case, at least a promoter for the expression of a Nucleic acid provided and for used their expression.

expression vectors can be regulated, for example by changing the cultivation conditions or when a certain Cell density of the host cells they contain or by adding certain Substances, in particular activators of gene expression. An example for such a substance, the galactose derivative is isopropyl-β-dithiogalactopyranoside (IPTG), which activates the bacterial lactose operon (lac operon) is used.

expression vectors allow an inventive Protein heterologous, so in another cell or host cell as the one from which it is naturally derived can, is produced. The cells can be different Belonging to organisms or derived from different organisms. Also, a homologous recovery of an inventive Protein from a host cell containing this protein naturally (ie already in its wild-type form) is expressed with an inventive Vector possible. This may have the advantage that natural, Translational related modifications be performed on the resulting protein as well as they would naturally occur.

To An insertable expression system may further include additional Counting nucleic acids (genes) or proteins encoded by them, for example, those available on other vectors be placed, and the production according to the invention Influence proteins. These may be modifying proteins act or those with the invention Protein should be purified together, about its enzymatic To influence function. These may be, for example, other proteins or enzymes to inhibitors or to act on such elements affect the interaction with different substrates. In usually have to come from the abundance of different ones in the prior art available systems the optimal expression systems for the individual case experimentally be determined.

A further subject of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. A nucleic acid according to the invention or a vector according to the invention is preferably introduced into the host cell by its transformation. Preferably, a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, it is also possible for individual components, ie nucleic acid fragments or fragments of a nucleic acid according to the invention, to be introduced into a host cell in such a way that the resulting host cell contains a nucleic acid or a vector according to the invention. This procedure is particularly suitable when the host cell already contains one or more constituents of a nucleic acid according to the invention or a vector according to the invention and the further constituents are then supplemented accordingly. Methods of transforming cells are well established in the art and well known to those skilled in the art. In principle, all cells, that is to say prokaryotic or eukaryotic cells, are suitable as host cells. Preference is given to those host cells which can be handled genetically advantageous, what in For example, the transformation with the nucleic acid or the vector and its stable establishment, for example, unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenially) expressed protein into the medium surrounding the host cells.

Further preferred embodiments provide such host cells due to genetic regulatory elements, for example be provided on the vector, as well can be present in these cells from the outset, are regulatable in their activity. For example, by controlled addition of chemical compounds acting as activators serve by changing the cultivation conditions or when they reach a certain cell density, they can be excited for expression. This allows an economic Production of the proteins according to the invention. One An example of such a compound is IPTG as above described.

preferred Host cells are prokaryotic or bacterial cells. bacteria are characterized by short generation times and low demands to the cultivation conditions. This can be cost effective Cultivation method or manufacturing process can be established. In addition, the expert has bacteria in the fermentation over a wealth of experience. For a special production can be different, in individual cases experimentally reasons to be determined, such as nutrient sources, Product formation rate, time required, etc., Gram-negative or Gram-positive Bacteria be suitable.

at gram-negative bacteria such as Escherichia coli secretes a variety of proteins in the periplasmic space, that is, into the compartment between the two enclosing the cells Membranes. This can be advantageous for special applications be. Furthermore, Gram-negative bacteria can also be designed in this way be that they express the proteins not only in the periplasmic Space, but in the medium surrounding the bacterium eject. Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of Actinomycetales possess in contrast no outer membrane, leaving secreted proteins immediately into the medium surrounding the bacteria, usually the nutrient medium, are released, from which the expressed proteins purify to let. They can be isolated directly from the medium or be processed further. In addition, gram-positive bacteria with most of the organisms of origin for technically important ones Enzymes are related or identical and mostly self-comparable Enzymes, so they have a similar codon usage and their protein synthesizer of course accordingly is aligned.

invention Host cells can meet their requirements the cultural conditions have changed, others or additional Have selection markers or other or additional Express proteins. In particular, it may also be such host cells which express several proteins or enzymes transgene.

The the present invention is in principle applicable to all microorganisms, in particular to all fermentable microorganisms, especially preferably applicable to those of the genus Bacillus, and leads to that by the use of such microorganisms according to the invention Make proteins. Such microorganisms then put Host cells in the context of the invention.

In Another embodiment of the invention is the host cell characterized in that it is a bacterium, preferably one, that is selected from the group of genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferred one that is selected from the group of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

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

In contrast to prokaryotic cells, eukaryotic cells are capable of post-translationally modifying the protein formed. Examples are fungi such as actinomycetes or yeasts such as sucrose myces or Kluyveromyces. This may be particularly advantageous, for example, if the proteins are to undergo specific modifications in the context of their synthesis that enable such systems. 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 lower the allergenicity of an expressed protein. Also, coexpression with the enzymes naturally produced by such cells, such as cellulases, may be advantageous. Furthermore, for example, thermophilic fungal expression systems may be particularly suitable for the expression of temperature-resistant proteins or variants.

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

• a) Kultivieren einer erfindungsgemäßen Wirtszelle
• b) Isolieren der Protease aus dem Kulturmedium oder aus der Wirtszelle.

Host cells according to the invention are preferably used to produce proteases according to the invention. Another object of the invention is therefore a method for preparing a protease comprising

• a) culturing a host cell according to the invention
• b) isolating the protease from the culture medium or from the host cell.

This Subject of the invention preferably comprises fermentation process. Fermentation are known per se from the prior art and represent the actual large-scale production step, usually followed from a suitable purification method of the product produced, for example, the protease according to the invention. All fermentation processes based on a corresponding procedure for the preparation of a protease according to the invention are based, embodiments of this subject invention represents.

in this connection need each of the optimal conditions for the Production process, in particular the optimal culture conditions for the host cells used, according to the knowledge of the expert be determined experimentally, for example in terms of fermentation volume and / or media composition and / or oxygenation and / or Stirrer.

Fermentation process, which are characterized in that the fermentation over An inflow strategy is also implemented into consideration. Here are the media components, by the continuous cultivation are consumed, fed. This can increase considerably both in the cell density as well as in the cell mass or dry mass and / or especially the activity of the protease of interest become.

Further The fermentation can also be designed to be unwanted Metabolites filtered out or by adding buffer or each appropriate counterions are neutralized.

The Protease prepared can be harvested from the fermentation medium become. Such a fermentation process is opposite isolation of the protease from the host cell, d. H. a product preparation the cell mass (dry matter) preferred, but requires the provision of suitable host cells or of one or more suitable ones Secretion markers or mechanisms and / or transport systems, so that the host cells secrete the protease into the fermentation medium. Without secretion, alternatively, the isolation of the protease from the Host cell, d. H. a purification of the same from the cell mass, respectively. Again, various methods are known as precipitation z. As by ammonium sulfate or ethanol, or the chromatographic purification.

All The circumstances outlined above may apply Combined method to inventive Produce proteases. There are many in this regard possible combinations of process steps conceivable. The optimal procedure must be for each specific case be determined.

One Another object of the invention is an agent which is characterized is that it is a protease according to the invention contains described above. Preferred is the agent a washing or cleaning agent.

Prefers an agent according to the invention is further characterized characterized in that it is an inventive Protease in an amount of 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, more preferably 20 μg to 15 mg and most preferably from 50 μg to 10 contains mg per g of the agent.

To This subject matter includes all conceivable washing or detergent types, both concentrates and undiluted means to be used, for commercial scale use, in the washing machine or in hand washing respectively -cleaning. These include, for example, detergents for Textiles, carpets, or natural fibers, for which the name Detergent is used. These include, for example also dishwashing detergents for dishwashers or manual dishwashing detergent or cleaner for hard surfaces such as metal, glass, porcelain, ceramics, Tiles, stone, painted surfaces, plastics, wood or leather for which the term detergent is used becomes.

One Means according to the invention can be both an agent for bulk consumers or technical users as also be a product for the private consumer, whereby all types of detergents and cleaning agents established in the prior art also embodiments of the present invention represent.

The detergents or cleaners according to the invention, as in particular powdered solids, in nachverdichteter Particle form, as homogeneous solutions or suspensions can, except the inventively used Proteases in principle all known and customary in such agents Contain ingredients, preferably at least one other Ingredient is present in the agent. The invention In particular, builders, surface-active agents can be used Surfactants, bleaching agents based on organic and / or inorganic Peroxygen compounds, bleach activators, water-miscible organic Solvents, enzymes, sequestrants, electrolytes, pH regulators and other auxiliaries such as optical brighteners, grayness inhibitors, Foam regulators, as well as dyes and fragrances, and combinations included hereof.

Especially another combination of the invention Active substances with one or more further ingredients of the Means proves to be advantageous, since such a means a improved cleaning performance through resulting synergisms can have. In particular, by the combination of an inventive Protease with one of the surfactants described below and / or one of the builders described below and / or a The bleaching agent described below becomes such a synergism reached.

The Agents according to the invention can be a surfactant or more surfactants, especially anionic surfactants, nonionic surfactants and their mixtures, but also cationic, zwitterionic and amphoteric surfactants come into question.

suitable Nonionic surfactants are, in particular, alkyl glycosides and ethoxylation and / or propoxylation products of alkyl glycosides or linear or branched alcohols each having 12 to 18 carbon atoms in the alkyl moiety and 3 to 20, preferably 4 to 10 alkyl ether groups. Farther are corresponding ethoxylation and / or propoxylation products of N-alkylamines, vicinal diols, fatty acid esters and fatty acid amides, with respect to the alkyl part said long-chain alcohol derivatives correspond, and of alkylphenols having 5 to 12 carbon atoms in the alkyl radical useful.

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. From coconut, palm, tallow or oleyl alcohol, and on average from 2 to 8 EO per mole of alcohol. The preferred ethoxylated alcohols include, for example, C ₁₂ -C ₁₄ -alcohols with 3 EO or 4 EO, C ₉ -C ₁₁ -alcohols with 7 EO, C ₁₃ -C ₁₅ -alcohols with 3 EO, 5 EO, 7 EO or 8 EO, C ₁₂ -C ₁₈ -alcohols with 3 EO, 5 EO or 7 EO and mixtures of these, such as mixtures of C ₁₂ -C ₁₄ -alcohol with 3 EO and C ₁₂ -C ₁₈ -alcohol with 7 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 range ethoxylates, NRE). In addition to these nonionic surfactants, fatty alcohols with more than 12 EO can also be used. Examples include (tallow) fatty alcohols with 14 EO, 16 EO, 20 EO, 25 EO, 30 EO or 40 EO. In particular, agents for use in mechanical processes usually extremely low-foam compounds are used. These include preferably C ₁₂ -C ₁₈ -Alkylpolyethylenglykolpolypropylenglykolether each with up to 8 moles of ethylene oxide and propylene oxide units in the molecule. One can also use other known low-foaming nonionic surfactants such as C ₁₂ -C _{1 8} alkyl polyethylene glycol polybutylene each having up to 8 moles of ethylene oxide and butylene oxide units in the molecule and end-capped Alkylpolyalkylenglykolmischether. Also particularly preferred are the hydroxyl-containing alkoxylated alcohols, as described in the European patent application EP 0 300 305 are described, so-called Hydroxymischether. The nonionic surfactants also include alkyl glycosides of the general formula RO (G) _x in which R is a primary straight-chain or methyl-branched, in particular 2-methyl-branched aliphatic radical having 8 to 22, preferably 12 to 18 carbon atoms and G represents a glycose unit having 5 or 6 C atoms, preferably glucose. The degree of oligomerization x, which indicates the distribution of monoglycosides and oligoglycosides, is an arbitrary number - which, as a variable to be determined analytically, may also assume fractional values - between 1 and 10; preferably x is 1.2 to 1.4. Also suitable are polyhydroxy fatty acid amides of the formula (III) in which R ¹ CO is an aliphatic acyl radical having 6 to 22 carbon atoms, R ^{2 is} hydrogen, an alkyl or hydroxyalkyl radical having 1 to 4 carbon atoms and [Z] is a linear or branched polyhydroxyalkyl radical having 3 to 10 carbon atoms and 3 to 10 hydroxyl groups:

in der R³ für einen linearen oder verzweigten Alkyl- oder Alkenylrest mit 7 bis 12 Kohlenstoffatomen, R⁴ für einen linearen, verzweigten oder cyclischen Alkylenrest oder einen Arylenrest mit 2 bis 8 Kohlenstoffatomen und R⁵ für einen linearen, verzweigten oder cyclischen Alkylrest oder einen Arylrest oder einen Oxy-Alkylrest mit 1 bis 8 Kohlenstoffatomen steht, wobei C₁-C₄-Alkyl- oder Phenylreste bevorzugt sind, und [Z] für einen linearen Polyhydroxyalkylrest, dessen Alkylkette mit mindestens zwei Hydroxylgruppen substituiert ist, oder alkoxylierte, vorzugsweise ethoxylierte oder propoxylierte Derivate dieses Restes steht. [Z] wird auch hier vorzugsweise durch reduktive Aminierung eines Zuckers wie Glucose, Fructose, Maltose, Lactose, Galactose, Mannose oder Xylose erhalten. Die N-Alkoxy- oder N-Aryloxy-substituierten Verbindungen können dann beispielsweise durch Umsetzung mit Fettsäuremethylestern in Gegenwart eines Alkoxids als Katalysator in die gewünschten Polyhydroxyfettsäureamide überführt werden. Eine weitere Klasse bevorzugt eingesetzter nichtionischer Tenside, die entweder als alleiniges nichtionisches Tensid oder in Kombination mit anderen nichtionischen Tensiden, insbesondere zusammen mit alkoxylierten Fettalkoholen und/oder Alkylglykosiden, eingesetzt werden, sind alkoxylierte, vorzugsweise ethoxylierte oder ethoxylierte und propoxylierte Fettsäurealkylester, vorzugsweise mit 1 bis 4 Kohlenstoffatomen in der Alkylkette, insbesondere Fettsäuremethylester. Auch nichtionische Tenside vom Typ der Aminoxide, beispielsweise N-Kokosalkyl-N,N-dimethylaminoxid und N-Talgalkyl-N,N-dihydroxyethylaminoxid, und der Fettsäurealkanolamide können geeignet sein. Die Menge dieser nichtionischen Tenside beträgt vorzugsweise nicht mehr als die der ethoxylierten Fettalkohole, insbesondere nicht mehr als die Hälfte davon. Als weitere Tenside kommen sogenannte Gemini-Tenside in Betracht. Hierunter werden im Allgemeinen solche Verbindungen verstanden, die zwei hydrophile Gruppen pro Molekül besitzen. Diese Gruppen sind in der Regel durch einen sogenannten ”Spacer” voneinander getrennt. Dieser Spacer ist in der Regel eine Kohlenstoffkette, die lang genug sein sollte, dass die hydrophilen Gruppen einen ausreichenden Abstand haben, damit sie unabhängig voneinander agieren können. Derartige Tenside zeichnen sich im Allgemeinen durch eine ungewöhnlich geringe kritische Micellkonzentration und die Fähigkeit, die Oberflächenspannung des Wassers stark zu reduzieren, aus. In Ausnahmefällen werden unter dem Ausdruck Gemini-Tenside nicht nur derartig ”dimere”, sondern auch entsprechend ”trimere” Tenside verstanden. Geeignete Gemini-Tenside sind beispielsweise sulfatierte Hydroxymischether oder Dimeralkohol-bis- und Trimeralkohol-tris-sulfate und -ethersulfate. Endgruppenverschlossene dimere und trimere Mischether zeichnen sich insbesondere durch ihre Bi- und Multifunktionalität aus. So besitzen die genannten endgruppenverschlossenen Tenside gute Netzeigenschaften und sind dabei schaumarm, so dass sie sich insbesondere für den Einsatz in maschinellen Wasch- oder Reinigungsverfahren eignen. Eingesetzt werden können aber auch Gemini-Polyhydroxyfettsäureamide oder Poly-Polyhydroxyfettsäureamide. Geeignet sind auch die Schwefelsäuremonoester der mit 1 bis 6 Mol Ethylenoxid ethoxylierten geradkettigen oder verzweigten C₇-C₂₁-Alkohole, wie 2-Methylverzweigte C₉-C₁₁-Alkohole mit im Durchschnitt 3,5 Mol Ethylenoxid (EO) oder C₁₂-C₁₈-Fettalkohole mit 1 bis 4 EO. Zu den bevorzugten Aniontensiden gehören auch die Salze der Alkylsulfobernsteinsäure, die auch als Sulfosuccinate oder als Sulfobernsteinsäureester bezeichnet werden, und die Monoester und/oder Diester der Sulfobernsteinsäure mit Alkoholen, vorzugsweise Fettalkoholen und insbesondere ethoxylierten Fettalkoholen darstellen. Bevorzugte Sulfosuccinate enthalten C₈- bis C₁₈-Fettalkoholreste oder Mischungen aus diesen. Insbesondere bevorzugte Sulfosuccinate enthalten einen Fettalkoholrest, der sich von ethoxylierten Fettalkoholen ableitet, die für sich betrachtet nichtionische Tenside darstellen. Dabei sind wiederum Sulfosuccinate, deren Fettalkohol-Reste sich von ethoxylierten Fettalkoholen mit eingeengter Homologenverteilung ableiten, besonders bevorzugt. Ebenso ist es auch möglich, Alk(en)ylbernsteinsäure mit vorzugsweise 8 bis 18 Kohlenstoffatomen in der Alk(en)ylkette oder deren Salze einzusetzen. Als weitere anionische Tenside kommen Fettsäure-Derivate von Aminosäuren, beispielsweise von N-Methyltaurin (Tauride) und/oder von N-Methylglycin (Sarkoside) in Betracht. Insbesondere bevorzugt sind dabei die Sarkoside beziehungsweise die Sarkosinate und hier vor allem Sarkosinate von höheren und gegebenenfalls einfach oder mehrfach ungesättigten Fettsäuren wie Oleylsarkosinat. Als weitere anionische Tenside kommen insbesondere Seifen in Betracht. Geeignet sind insbesondere gesättigte Fettsäureseifen, wie die Salze der Laurinsäure, Myristinsäure, Palmitinsäure, Stearinsäure, hydrierten Erucasäure und Behensäure sowie insbesondere aus natürlichen Fettsäuren, zum Beispiel Kokos-, Palmkern- oder Talgfettsäuren, abgeleitete Seifengemische. Zusammen mit diesen Seifen oder als Ersatzmittel für Seifen können auch die bekannten Alkenylbernsteinsäuresalze eingesetzt werden.The polyhydroxy fatty acid amides are preferably derived from reducing sugars having 5 or 6 carbon atoms, in particular from glucose. The group of polyhydroxy fatty acid amides also includes compounds of the formula (IV)

in the R ^{3 is} a linear or branched alkyl or alkenyl radical having 7 to 12 carbon atoms, R ^{4 is} a linear, branched or cyclic alkylene radical or an arylene radical having 2 to 8 carbon atoms and R ^{5 is} a linear, branched or cyclic alkyl radical or a Aryl radical or an oxy-alkyl radical having 1 to 8 carbon atoms, wherein C ₁ -C 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 of this group. [Z] is also obtained here preferably by reductive amination of a sugar such as glucose, fructose, maltose, lactose, galactose, mannose or xylose. The N-alkoxy- or N-aryloxy-substituted compounds can then 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. Another class of preferred nonionic surfactants used either as the sole nonionic surfactant or in combination with other nonionic surfactants, in particular together with alkoxylated fatty alcohols and / or alkyl glycosides, are alkoxylated, preferably ethoxylated or ethoxylated and propoxylated fatty acid alkyl esters, preferably from 1 to 4 carbon atoms in the alkyl chain, especially fatty acid methyl ester. Nonionic surfactants of the amine oxide type, for example N-cocoalkyl-N, N-dimethylamine oxide and N-tallowalkyl-N, N-dihydroxyethylamine oxide, and the fatty acid alkanolamides may also be suitable. The amount of these nonionic surfactants is preferably not more than that of the ethoxylated fatty alcohols, especially not more than half thereof. Other suitable surfactants are so-called gemini surfactants. These are generally understood as meaning those compounds which have two hydrophilic groups per molecule. These groups are usually separated by a so-called "spacer". This spacer is typically a carbon chain that should be long enough for the hydrophilic groups to be spaced sufficiently apart for them to act independently of each other. Such surfactants are generally characterized by an unusually low critical micelle concentration and the ability to greatly reduce the surface tension of the water. In exceptional cases, the term gemini surfactants not only such "dimer", but also corresponding to "trimeric" surfactants understood. Suitable gemini surfactants are, for example, sulfated hydroxy mixed ethers or dimer alcohol bis and trimer alcohol tris sulfates and ether sulfates. End-capped dimeric and trimeric mixed ethers are characterized in particular by their bi- and multi-functionality. Thus, the end-capped surfactants mentioned have good wetting properties and are low foaming, so that they are particularly suitable for use in machine washing or cleaning processes. However, it is also possible to use gemini-polyhydroxy fatty acid amides or poly-polyhydroxy fatty acid amides. Also suitable are the sulfuric acid monoesters of straight-chain or branched C ₇ -C ₂₁ -alcohols ethoxylated with from 1 to 6 mol of ethylene oxide, such as 2-methyl-branched C ₉ -C ₁₁ -alcohols having on average 3.5 mol of ethylene oxide (EO) or C ₁₂ - C ₁₈ -fatty alcohols with 1 to 4 EO. To the preferred anionic surfactants also include 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 C ₈ - C ₁₈ fatty alcohol radicals or mixtures thereof. Particularly preferred sulfosuccinates contain a fatty alcohol residue derived from ethoxylated fatty alcohols, which by themselves are nonionic surfactants. Sulfosuccinates, whose fatty alcohol residues are derived from ethoxylated fatty alcohols with a narrow homolog distribution, are again particularly preferred. Likewise, it is also possible to use alk (en) ylsuccinic acid having preferably 8 to 18 carbon atoms in the alk (en) yl chain or salts thereof. Suitable further anionic surfactants are fatty acid derivatives of amino acids, for example N-methyltaurine (Tauride) and / or N-methylglycine (sarcosides). Particularly preferred are the sarcosides or the sarcosinates and here especially sarcosinates of higher and optionally monounsaturated or polyunsaturated fatty acids such as oleyl sarcosinate. As further anionic surfactants are particularly soaps into consideration. Particularly 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. Together with these soaps or as a substitute for soaps, it is also possible to use the known alkenylsuccinic acid salts.

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

surfactants are in proportions in inventive compositions of preferably 5% by weight to 50% by weight, in particular 8% by weight to 30 wt .-%, contained.

An agent according to the invention preferably contains at least one water-soluble and / or water-insoluble, organic and / or inorganic builder. The water-soluble organic builder substances include polycarboxylic acids, in particular citric acid and sugar acids, monomeric and polymeric aminopolycarboxylic acids, in particular methylglycinediacetic acid, nitrilotriacetic acid and ethylenediaminetetraacetic acid and polyaspartic acid, polyphosphonic acids, in particular aminotris (methylenephosphonic acid), ethylenediaminetetrakis (methylenephosphonic acid) and 1-hydroxyethane-1,1-diphosphonic acid, polymeric hydroxy compounds such as dextrin and also polymeric (poly) carboxylic acids, in particular the polycarboxylates obtainable by oxidation of polysaccharides or dextrins, polymeric acrylic acids, methacrylic acids, maleic acids and copolymers thereof, which may also contain polymerized small amounts of polymerizable substances without carboxylic acid functionality. The molecular weight of the homopolymers of unsaturated carboxylic acids is generally between 3,000 and 200,000, of the copolymers between 2,000 and 200,000, preferably 30,000 to 120,000, each based on the free acid. A particularly preferred acrylic acid-maleic acid copolymer has a molecular weight of from 30,000 to 100,000. Commercial products are, for example Sokalan ^® CP 5, CP 10 and PA 30 from BASF. Suitable, although less preferred, compounds of this class are copolymers of acrylic or methacrylic acid with vinyl ethers, such as vinylmethyl ethers, vinyl esters, ethylene, propylene and styrene, in which the acid content is at least 50% by weight. It is also possible to use terpolymers which contain two unsaturated acids and / or salts thereof as monomers and also vinyl alcohol and / or an esterified vinyl alcohol or a carbohydrate as the third monomer as water-soluble organic builder substances. The first acidic monomer or its salt is derived from a monoethylenically unsaturated C ₃ -C ₈ -carboxylic acid and preferably from a C ₃ -C ₄ -monocarboxylic acid, in particular from (meth) -acrylic acid. The second acidic monomer or its salt may be a derivative of a C ₄ -C ₈ -dicarboxylic acid, with maleic acid being particularly preferred, and / or a derivative of an allylsulfonic acid which is substituted in the 2-position by an alkyl or aryl radical. Such polymers generally have a molecular weight between 1,000 and 200,000. Further preferred copolymers are those which preferably have as monomers acrolein and acrylic acid / acrylic acid salts or vinyl acetate. The organic builder substances can be used, in particular for the preparation of liquid agents, in the form of aqueous solutions, preferably in the form of 30 to 50 percent by weight aqueous solutions. All of the acids mentioned are generally used in the form of their water-soluble salts, in particular their alkali metal salts.

such If desired, organic builders may be used in amounts up to 40 wt .-%, in particular up to 25 wt .-% and preferably from 1% by weight to 8% by weight. Quantities near the mentioned Upper limit are preferably in pasty or liquid, in particular hydrous, inventive Funds used.

Suitable water-soluble inorganic builder materials are, in particular, alkali metal silicates, alkali metal carbonates and alkali metal phosphates, which may be in the form of their alkaline, neutral or acidic sodium or potassium salts. Examples of these are trisodium phosphate, tetrasodium diphosphate, disodium dihydrogen diphosphate, pentasodium triphosphate, so-called sodium hexametaphosphate, oligomeric trisodium phosphate with degrees of oligomerization of from 5 to 1000, in particular from 5 to 50, and the corresponding potassium salts or mixtures of sodium and potassium salts. In particular, crystalline or amorphous alkali metal aluminosilicates, in amounts of up to 50% by weight, preferably not more than 40% by weight, and in liquid agents, in particular from 1% by weight to 5% by weight, are used as water-insoluble, water-dispersible inorganic builder materials. used. Among these, the crystalline sodium aluminosilicates in detergent quality, more particularly zeolite A, P and optionally X, alone or in mixtures, for example in the form of a co-crystals of zeolites A and X (VEGOBOND ^® AX, a commercial product of Condea August S. p. A.), preferred. Amounts near the above upper limit are preferably used in solid, particulate agents. In particular, suitable aluminosilicates have no particles with a particle size greater than 30 .mu.m and preferably consist of at least 80% by weight of particles having a size of less than 10 .mu.m. Their calcium binding capacity, according to the information of the German Patent DE 24 12 837 can be determined, is usually in the range of 100 to 200 mg CaO per gram.

Suitable substitutes or partial substitutes for the said aluminosilicate are crystalline alkali silicates which may be present alone or in a mixture with amorphous silicates. The alkali metal silicates useful as builders in the compositions according to the invention preferably have a molar ratio of alkali metal oxide to SiO ₂ below 0.95, in particular from 1: 1.1 to 1:12, and may be present in amorphous or crystalline form. Preferred alkali metal silicates are the sodium silicates, in particular the amorphous sodium silicates, with a molar ratio of Na ₂ O: SiO ₂ of 1: 2 to 1: 2.8. The crystalline silicates which may be present alone or in admixture with amorphous silicates, are crystalline layer silicates with the general formula Na ₂ Si _x O _{2x + 1} · are used yH ₂ O, in which x, the so-called module, a number from 1.9 to 22, in particular 1.9 to 4 and y is a number from 0 to 33 and preferred values for x are 2, 3 or 4. Preferred crystalline phyllosilicates are those in which x in the abovementioned general formula assumes the values 2 or 3. In particular, both β- and δ-sodium disilicates (Na ₂ Si ₂ O ₅ .yH ₂ O) are preferred. Also prepared from amorphous alkali silicates, practically anhydrous crystalline alkali metal silicates of the abovementioned general formula in which x is a number from 1.9 to 2.1, can be used in inventive compositions. In a further preferred embodiment of the composition according to the invention, a crystalline sodium layer silicate with a modulus of 2 to 3 is used, as can be prepared from sand and soda. Crystalline sodium silicates with a modulus in the range from 1.9 to 3.5 are used in a further preferred embodiment of compositions according to the invention. Crystalline layer-form silicates of formula (I) given above are sold by Clariant GmbH under the trade name Na-SKS, eg. Na-SKS-1 (Na ₂ Si ₂₂ O ₄₅ .xH ₂ O, Kenyaite), Na-SKS-2 (Na ₂ Si ₁₄ O ₂₉ .xH ₂ O, magadiite), Na-SKS-3 (Na ₂ Si ₈ O ₁₇ .xH ₂ O) or Na-SKS-4 (Na ₂ Si ₄ O ₉ .xH ₂ O, Makatite). Of these, especially Na-SKS-5 (α-Na ₂ Si ₂ O ₅ ), Na-SKS-7 (β-Na ₂ Si ₂ O ₅ , natrosilite), Na-SKS-9 (NaHSi ₂ O ₅ · 3H ₂ O), Na-SKS-10 (NaHSi ₂ O ₅ · 3H ₂ O, kanemite), Na-SKS-11 (t-Na ₂ Si ₂ O ₅₎ and Na-SKS-13 (NaHSi ₂ O ₅ ), but especially Na-SKS-6 (δ-Na ₂ Si ₂ O ₅ ). In a preferred embodiment of inventive compositions is a granular compound of crystalline phyllosilicate and citrate, of crystalline layered silicate and aforementioned (co) polymeric polycarboxylic acid or of alkali silicate and alkali metal as it is available, for example under the name Nabion ^® 15 commercially ,

builders are preferred in the compositions according to the invention in amounts up to 75 wt .-%, in particular 5 wt .-% to 50 included.

When for use in the invention Means of suitable peroxygen compounds come in particular organic peracids or peracid salts organic Acids, such as phthalimidopercaproic acid, perbenzoic acid or salts of diperdodecanedioic acid, hydrogen peroxide and under the washing conditions hydrogen peroxide-releasing inorganic Salts, which include perborate, percarbonate, persilicate and / or persulfate like Caroat belong. If solid peroxygen compounds can be used, they can be in the form of powders or granules are used, which are also known in principle Wrapped up. If an inventive Agent contains peroxygen compounds, these are in Amounts of preferably up to 50% by weight, in particular of 5% by weight to 30 wt .-%, present. The addition of small amounts of known bleach stabilizers such as phosphonates, bristles or metaborates and metasilicates and magnesium salts such as magnesium sulfate be expedient.

Suitable bleach activators are compounds which, under perhydrolysis conditions, have aliphatic peroxycarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or, where appropriate substituted perbenzoic acid, can be used. 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 tetraacetylglycoluril (TAGU), N- Acylimides, in particular N-nonanoylsuccinimide (NOSI), acylated phenolsulfonates, in particular n-nonanoyl or isononanoyloxybenzenesulfonate (n- or iso-NOBS), carboxylic anhydrides, in particular phthalic anhydride, acylated polyhydric alcohols, in particular triacetin, ethylene glycol diacetate, 2,5-diacetoxy- 2,5-dihydrofuran and enol esters and also acetylated sorbitol and mannitol or mixtures thereof (SORMAN), acylated sugar derivatives, in particular pentaacetylglucose (PAG), pentaacetylfruktose, tetraacetylxylose and octaacetyllactose as well as acetylated, optionally N-alkylated glucamine and gluconolactone, and / or N- acylated lactams, for example N-benzoyl-caprolactam. The hydrophilic substituted acyl acetals and the acyl lactams are also preferably used. Combinations of conventional bleach activators can also be used. Such bleach activators can, in particular in the presence of the abovementioned hydrogen peroxide-producing bleach, in the usual amount range, preferably in amounts of from 0.5 wt .-% to 10 wt .-%, in particular 1 wt .-% to 8 wt .-%, based on However, total agent, be included, missing when using percarboxylic acid as the sole bleach, preferably completely.

additionally to the conventional bleach activators or in their place also sulfonimines and / or bleach-enhancing transition metal salts or transition metal complexes as so-called Be included bleach catalysts.

To in the inventive compositions, in particular if they are in liquid or pasty form, include water-usable organic solvents Alcohols having 1 to 4 carbon atoms, in particular methanol, ethanol, isopropanol and tert-butanol, diols having 2 to 4 C atoms, in particular ethylene glycol and propylene glycol, as well as mixtures thereof and those mentioned Classes of derivable ether. Such water-miscible Solvents are in the inventive Preferably in amounts not exceeding 30% by weight, in particular from 6% to 20% by weight.

to Setting a desired, through the mixture the remaining components are not self-generating pH the agents according to the invention can be and environmentally friendly acids, in particular citric acid, Acetic acid, tartaric acid, malic acid, lactic acid, Glycolic acid, succinic acid, glutaric acid and / or adipic acid, but also mineral acids, especially sulfuric acid, or bases, in particular ammonium or alkali hydroxides. Such pH regulators are in the inventive compositions in amounts of preferably not more than 20% by weight, in particular from 1.2% by weight to 17% by weight, contain.

graying have the task of removing the dirt from the textile fiber to keep suspended in the fleet. These are water-soluble Colloids mostly organic nature suitable, such as starch, Glue, gelatin, salts of ether carboxylic acids or ether sulfonic acids starch or cellulose or salts of acidic sulfuric acid esters cellulose or starch. Also water-soluble, Acidic group-containing polyamides are for this purpose suitable. Furthermore, other than the above-mentioned starch derivatives can be used use, for example, aldehyde levels. To be favoured Cellulose ethers, such as carboxymethyl cellulose (Na salt), methyl cellulose, Hydroxyalkylcellulose and mixed ethers, such as methylhydroxyethylcellulose, Methylhydroxypropylcellulose, methylcarboxymethylcellulose and their Mixtures, for example in amounts of 0.1 to 5 wt .-%, based on the means used.

invention Textile detergents can be used as optical brighteners, for example Derivatives of Diaminostilbendisulfonsäure or their alkali metal salts, although they are for the Use as a color detergent preferably free of optical brighteners are. For example, salts of 4,4'-bis (2-anilino-4-morpholino-1,3,5-triazinyl-6-amino) stilbene-2,2'-disulfonic acid are suitable or similar compounds, instead of the morpholino group a diethanolamino group, a methylamino group, an anilino group or carry a 2-methoxyethylamino group. Furthermore you can Brighteners of the type of substituted diphenylstyrene may be present for example, the alkali salts of 4,4'-bis (2-sulfostyryl) -diphenyl, 4,4'-bis (4-chloro-3-sulfostyryl) -diphenyls, or 4- (4-chlorostyryl) -4 '- (2-sulfostyryl) -diphenyls. Mixtures of the aforementioned optical brightener can be used.

In particular, when used in mechanical processes, it may be advantageous to add conventional foam inhibitors to the compositions. As foam inhibitors are, for example, soaps of natural or synthetic origin, which have a high proportion of C ₁₈ -C ₂₄ fatty acids. Suitable non-surfactant Foam inhibitors are, for example, organopolysiloxanes and mixtures thereof with microfine, optionally silanized silica and paraffins, waxes, microcrystalline waxes and mixtures thereof with silanated silicic acid or bis-fatty acid alkylenediamides. It is also advantageous to use mixtures of various foam inhibitors, for example those of silicones, paraffins or waxes. The foam inhibitors, in particular silicone- and / or paraffin-containing foam inhibitors, are preferably bound to a granular, water-soluble or dispersible carrier substance. In particular, mixtures of paraffins and bistearylethylenediamide are preferred.

The to select ingredients as well as the conditions under which the agent is used, such as temperature, pH, ionic strength, redox ratios or mechanical influences, should be for the respective Cleaning problem to be optimized. So are usual temperatures for detergents and cleaners in ranges of 10 ° C over 40 ° C and 60 ° C up to 95 ° for mechanical Means or in technical applications. Because with modern washing and dishwashers the temperature usually infinitely adjustable Also, all intermediate temperatures are included. Preferably, the ingredients of the respective agents become one upon another Voted. Synergies in terms of cleaning performance are preferred. Particularly preferred are synergies in a temperature range between 10 ° C and 60 ° C are present, in particular in a temperature range from 10 ° C to 60 ° C, from 10 ° C to 50 ° C, from 10 ° C to 40 ° C, from 10 ° C to 30 ° C, from 15 ° C to 30 ° C from 10 ° C to 25 ° C and from 15 ° C to 25 ° C.

The Preparation of inventive solid means provides no difficulties and can be done in a known way, for example by spray-drying or granulation, enzymes being used and any other thermally sensitive ingredients like for example bleach, if appropriate later separately be added. For the production according to the invention Agents with increased bulk density, in particular in the range of 650 g / l to 950 g / l, is an extrusion step preferred method.

to Preparation of agents according to the invention in tablet form, the one-phase or multi-phase, monochrome or multicolor and in particular from one or more layers, in particular two layers can exist, it is preferable to proceed in such a way that all components - if necessary one layer each - in a mixer mixed together and the mixture by means of conventional Tablet presses, for example eccentric presses or rotary presses, with compressive forces in the range of about 50 to 100 kN, preferably compressed at 60 to 70 kN. Especially for multi-layered tablets, it may be advantageous if at least a layer is pre-pressed. This is preferably included Pressing forces between 5 and 20 kN, in particular carried out at 10 to 15 kN. You get so easily unbreakable and yet sufficiently fast under conditions of use soluble tablets with breaking and bending strengths of normally 100 to 200 N, but preferably over 150 N. Preferably, a tablet produced in this way has a weight from 10 g to 50 g, especially from 15 g to 40 g. The spatial form the tablets are arbitrary and can be round, oval or angular, although intermediate forms are possible. Corners and edges are advantageously rounded. Round tablets preferably have a diameter of 30 mm to 40 mm. Especially the big one of square or cuboid tablets, which predominantly over the metering device, for example, the dishwasher is introduced depends on the geometry and the volume of this metering device. Exemplary preferred embodiments have a footprint of (20 to 30 mm) × (34 to 40 mm), in particular 26 × 36 mm or 24 × 38 mm up.

liquid or pasty invention Agent in the form of common solvents Solutions are usually made by simply mixing the Ingredients that are in substance or as a solution in one automatic mixer can be given. Process for the production of laundry detergents according to the invention or cleaning agents accordingly constitute a separate subject of the invention All processes by which a washing or cleaning as described in the present application can be obtained can be in the scope of this subject invention locked in.

embodiments The present invention thus encompasses all solid, pulverulent, liquid, gel or pasty Dosage forms of the funds, which may also consist of several Phases can exist as well as in compressed or not compressed form may be present. Another embodiment The invention therefore provides means characterized are that they are present as a one-component system. Such means preferably consist of one phase. On the other hand, funds are out consist of several phases, characterized in that it is in several components is split. To the invention solid dosage forms also include extrudates, granules, Tablets or pouches, both in bulk containers as may also be packaged in portions.

An agent according to the invention can be present as a free-flowing powder, in particular with a bulk weight of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. Alternatively, agents according to the invention may also be liquid, gelatinous or pasty. A further embodiment of the invention is therefore characterized in that the washing or cleaning agent is in liquid, gel or pasty form, in particular in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste.

The Detergents or cleaning agents according to the invention can in a container, preferably an air-permeable Container, be packaged, out of it just before use or released during the washing process. Especially may be the protease contained in the agent and / or other ingredients of the agent at room temperature or in the absence of Water for the enzyme impermeable substance which under application conditions of the agent becomes permeable to the enzyme. Such an embodiment The invention is thus characterized in that the protease with one at room temperature and / or in the absence of water is enveloped for the protease impermeable substance.

invention Detergents or cleaners can only contain a protease. Alternatively, they can also have more hydrolytic enzymes or other enzymes in one for the Efficacy of the agent appropriate concentration contain. Another object of the invention thus provide Means further comprising one or more further enzymes, being in principle all of the prior art for these purposes established enzymes can be used. Preferred as further enzymes all enzymes which can be used in the invention Means can develop a catalytic activity, especially one in particular a protease, amylase, cellulase, Hemicellulase, mannanase, tannase, xylanase, xanthanase, β-glucosidase, Carrageenase, perhydrolase, oxidase, oxidoreductase or a lipase, and preferably mixtures thereof. These enzymes are in principle of natural origin; starting from the natural ones Molecules are available for use in washing and washing Cleaning agents improved variants available which are preferably used accordingly.

Agents of the invention preferably contain enzymes in total amounts of 1 × 10 -8 to 5 weight percent based on active protein. Preferably, the enzymes are from 0.001 to 5% by weight, more preferably from 0.01 to 5% by weight, even more preferably from 0.05 to 4% by weight and most preferably from 0.075 to 3.5% by weight. % contained in agents according to the invention, wherein each enzyme contained can be present in the stated proportions. The protein concentration can be determined by known methods, for example the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall AG, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766 ).

The protease activity in such agents may be as described in U.S. Pat Surfactants, Vol. 7 (1970), pp. 125-132 method described. It is given in PE (protease units).

at The comparison of the performance of two detergent enzymes must be between distinguished from the same and the same activity become. Especially when genetically engineered, largely free of side activity Preparations the same protein use is appropriate. Because that is a statement about whether it is possible the same amount of protein - as a measure of the yield of fermentative production - comparable Results. Gap the respective conditions from active substance to total protein (the values of the specific activity) apart, so is an activity-same comparison too recommend because this is the respective enzymatic properties be compared. Generally, that has a low specific activity balanced by adding a larger amount of protein can be.

Especially Preferably, the other enzymes support the effect agent, for example the cleaning performance of a washing or cleaning agent, with regard to certain stains or Stains. Most preferably, the enzymes show synergistic effects in terms of their effect on certain soils or stains, d. H. those contained in the middle composition Enzymes support each other in their cleaning performance. Synergistic effects can not only be between different Enzymes, but also between one or more enzymes and more Ingredients of the inventive agent occur.

a own subject of the invention provides the use of an inventive Washing or cleaning agent to remove soiling, in particular of protease-sensitive soiling, on textiles or hard surfaces, d. H. for cleaning textiles or hard surfaces.

For means according to the invention can, in particular due to the egg described above properties of the contained protease, can be advantageously used to remove contaminants from textiles or from hard surfaces. Embodiments of this subject invention include, for example, hand washing, manual removal of stains from fabrics or hard surfaces, or use in conjunction with a machine process.

All Facts, objects and embodiments, for washing or Cleaning agents are described are also on this subject of the invention applicable. Therefore, it is explicitly stated here the revelation in the appropriate place with the reference, that this disclosure also for the above inventive Use applies. In preferred embodiments of this Use are the relevant detergents or cleaning agents Therefore, according to one of the embodiments described above provided.

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

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

method for the cleaning of textiles are generally characterized from that in several process steps different cleaning active Substances applied to the items to be cleaned and after the exposure time be washed off, or that the items to be cleaned in any other way with a detergent or a solution or dilution this agent is treated. The same applies to Process for cleaning all materials other than textiles, especially hard surfaces. All conceivable washing or purification processes may be used in at least one of Process steps to the application of an inventive Detergent or cleaning agent are enriched and then put Embodiments of the present invention. All Facts, objects and embodiments, for washing or Detergents are described are also applicable to this subject invention. Therefore, explicit reference is made to the disclosure at this point Corresponding body with the reference that this disclosure also for the above inventive Procedure applies.

a Another object of the invention provide methods for cleaning of textiles or hard surfaces that result are characterized in that in at least one method step a protease of the invention catalytically active is, in particular such that the protease in an amount of 40 micrograms 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g, and most preferably from 200 μg to 1 g per application is used. All facts, objects and embodiments suitable for the invention Proteases are described are also on this subject invention applicable. Therefore, it is explicitly stated here the revelation in the appropriate place with the reference, that this disclosure also for the above inventive Procedure applies.

There Proteases of the invention naturally already have a hydrolytic activity and this also unfold in media that otherwise have no cleaning power as in mere buffer, a single and / or the only step in such a process is if desired, as the only detergent-active component such a protease is brought into contact with the soil, preferably in a buffer solution or in water. This poses a further embodiment of this subject invention represents.

alternative Embodiments of this subject invention provide also processes for the treatment of textile raw materials or for textile care in which in at least one method step, an inventive Protease becomes active. These include processes for textile raw materials, Prefers fibers or textiles with natural ingredients, and especially for those with wool or silk.

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

invention Enzymes are as described above advantageous in agents according to the invention, in particular Detergents and cleaners, and processes, in particular washing and cleaning Cleaning method, can be used. So you can use that be proteinaceous of textiles or hard surfaces Eliminate impurities.

a Another object of the invention is therefore the use of a protease according to the invention, as described above has been described for cleaning textiles or hard surfaces. Preferably, the protease is added in an amount of 40 μg 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g, and most preferably from 200 μg used up to 1 g per application. All facts, objects and embodiments suitable for the invention Proteases are described are also on this subject invention applicable. Therefore, it is explicitly stated here the revelation in the appropriate place with the reference, that this disclosure also for the above inventive Use applies. In a preferred embodiment of this Use are the relevant invention Enzymes in the context of a composition according to the invention, Preferably, a washing or Detergent provided.

A further embodiment of this subject invention provides the use of a protease according to the invention for activating or deactivating ingredients of laundry detergents or detergents.

Because Protein components of detergents or cleaners can be inactivated by the action of a protease. This otherwise to use more unwanted effect targeted, is also an aspect of the present invention. It is the same as above possible that by proteolysis another component only is activated, for example, if it is a hybrid protein from the actual Enzyme and the appropriate inhibitor. Another example for such regulation is that in which an active component to protect or control their activity in one Material is encapsulated, which is attacked by proteolysis. Proteases according to the invention can thus used for inactivation, activation or release reactions especially in multi-phase means.

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

Further, the following uses are also embodiments of the present invention:

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

The The present invention is also in the form of such an inventive Protease-containing agents, which are Cosmetics are. Below are all kinds of cleansing and caring products for human skin or hair, especially cleansers.

Because Proteases also play in the cell renewal process of the human Skin (desquamation) plays a crucial role. Accordingly, become Proteases also used as bioactive components in skin care products, to the degradation of desmosome structures that are increased in dry skin to support. As explained in detail above proteases of the invention can be further developed be, for example via amino acid substitutions and / or other mutations. Thus, also inventive Proteases, in particular those which are present after mutagenesis or by Add appropriate, interacting with them in their materials Activity are controlled as active components in Skin or hair cleaning or care products. Especially preferred are such preparations of these enzymes as described above, for example, by coupling to macromolecular carrier stabilized and / or by substitutions in highly allergenic positions modified or derivatized, so that they are for the people have a higher skin tolerance.

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

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

In addition to the use in detergents and cleaners and cosmetics numerous applications of proteases, in particular subtilases are established in the prior art. For example, the manual provides an overview of this "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 , All of these techniques can be enriched by proteases of the invention. These include in particular the following fields of application:

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

in principle is the use of proteases according to the invention in all other technical fields within the scope of the present Registration included, for which it is suitable it turns.

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

Examples

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

example 1

Isolation and identification of the proteolytic active bacterial strain

0.1 g of a soil sample was suspended in 1 ml of sterile 0.9% NaCl solution and on agar plates containing milk powder (1.5% agar, 0.1% K ₂ HPO ₄ , 0.5% yeast extract, 1% peptone, 1% milk powder, 0.02% MgSO ₄ .7H ₂ O, 0.4% Na ₂ CO ₃ , pH 10) and incubated at 30 ° C. A proteolytically active bacterium was isolated on the basis of a clarification laboratory, which was identified by the German Collection of Microorganisms and Cell Cultures (DSMZ) as Bacillus pumilus (ID 08-100).

Example 2

Isolation and cloning of the new serine protease from the soil

The proteolytically active bacterium was cultured in TBY medium (0.5% NaCl, 0.5% yeast extract, 1% tryptone, pH 7.4) for 16 h at 30 ° C. The total DNA of this bacterium was prepared by standard methods, treated with the restriction enzyme Sau 3A and transferred to a Bacillus vector (derivative of pBC16; Bernhard et al. (1978), J. Bacteriol., Vol. 133 (2), p. 897 et seq. ) cloned. This vector was inserted into the protease-negative host strain Bacillus subtilis DB 104 ( Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444 ).

The Transformants were first regenerated on DM3 medium and then on milk powder agar plates (TBY Skimmilk plates; see Example 1). Proteolytically active clones were identified by their lysis farms. From the preserved proteolytically active clones were selected one whose plasmid (Vector) isolated and the gene fragment contained in this vector (Insert) sequenced by standard methods.

The insert contains an open reading frame of about 1.2 kb, its DNA sequence for a protease of subtilisin type. The sequence was amplified by PCR, cloned into the E. coli vector pUC19 and deposited under the Budapest Treaty with the DSMZ under the number DSM 21891.

Example 3

Determination of the cleaning performance at Use in a commercially available powdery laundry detergent

• A: Gras auf Baumwolle: Produkt Nr. 164 von der Firma Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Schweiz;
• B: Erdnuss Öl-Pigment/Tusche auf Polyester/Baumwolle: Produkt Nr. PC-10 von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande;
• C: Schokoladenmilch/Ruß auf Baumwolle: Produkt Nr. C-03 von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande.

For this example, standardized contaminated textiles were used which had been purchased from EMPA Test Materials AG (St. Gallen, Switzerland), wfk Testgewebe GmbH (Brüggen-Bracht, Germany) or the Center For Testmaterials (CFT, Vlaardingen, The Netherlands). The following stains and textiles were used:

• A: Grass on cotton: product no. 164 from the company Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG, St. Gallen, Switzerland;
• B: Peanut oil pigment / ink on polyester / cotton: Product No. PC-10 from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands;
• C: Chocolate milk / soot on cotton: Product No. C-03 from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands.

With This test material was based on various detergent formulations investigated their cleaning performance. For that were the Bake for 60 minutes at a temperature of 40 ° C washed. The dosage was 5.9 g of the detergent per liter of wash liquor. It was made with city water with a water hardness of washed about 16 ° German hardness.

The control detergent used was a detergent base formulation of the following composition (all figures in percent by weight): 10% linear alkylbenzenesulfonate (sodium salt), 1.5% C12-C18 fatty alcohol sulfate (sodium salt), 2.0 % C12-C18 fatty alcohol with 7 EO, 20% sodium carbonate, 6.5% sodium bicarbonate, 4.0% amorphous sodium disilicate, 17% sodium carbonate peroxohydrate, 4.0% TAED, 3.0% polyacrylate, 1.0% carboxymethylcellulose, 1 , 0% phosphonate, 25% sodium sulfate, balance: foam inhibitors, optical brightener, fragrances. The detergent base formulation was treated with the same activity (5 PE / ml final concentration) with the following proteases for the various test series: Protease according to the invention comprising an amino acid sequence according to SEQ ID NO. 3 (batch 1), protease from Bacillus pumilus according to WO2007 / 131656 (Approach 2) and performance-improved variant F49 of the protease from Bacillus lentus according to WO 95/23221 (Approach 3).

After washing, the whiteness of the washed fabrics was measured. The measurement was carried out on a Minolta CM508d spectrometer (illuminant D65, 10 °). The device was previously calibrated with a supplied white standard. The results obtained are the differential remissions between a wash with a detergent containing a protease and a parallel control wash with a detergent without protease. The results are summarized in the following Table 1 and allow a direct conclusion on the contribution of each enzyme contained to the cleaning performance of the agent used. Table 1: Washing results with a powdered detergent at 40 ° C. soiling Approach 1 Approach 2 Approach 3 A 2.4 0.8 2.4 B 8.0 7.4 7.3 C 6.3 3.0 4.3

It it becomes clear that a protease according to the invention already shows a better cleaning performance in its wild-type form in comparison to a known protease from Bacillus pumilus, the has a similar amino acid sequence, as well even in comparison with the performance-enhanced protease variant according to approach 3, which is not a wild-type molecule is.

Description of the figures

1 Alignment of the mature protease of the invention (SEQ ID NO. 3) with proteases from the prior art, created with the program Vector NTI ^® Suite 7.0 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) under standard parameters.

• 1: Protease aus Bacillus lentus DSM 5483 ( WO 92/21760 A1 )
• 2: Erfindungsgemäße Protease gemäß SEQ ID NO. 3 (reifes Enzym)
• 3: reife (mature) Protease gemäß Datenbankeintrag Q5XPN0 aus Bacillus pumilus (Q5XPN0_BACPU; „Organic solvent tolerant protease”; Bacillus pumilus (Bacillus mesentericus); vgl. SEQ ID NO. 6)

In this mean:

• 1: Protease from Bacillus lentus DSM 5483 ( WO 92/21760 A1 )
• 2: Protease according to the invention according to SEQ ID NO. 3 (mature enzyme)
• 3: Mature protease according to database entry Q5XPN0 from Bacillus pumilus (Q5XPN0_BACPU; "Organic solvent tolerant protease"; Bacillus pumilus (Bacillus mesentericus), see SEQ ID NO.

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

QUOTES INCLUDE IN THE DESCRIPTION

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

Cited patent literature

• WO 97/21760 A1 [0027]
• - WO 2007/131656 [0036, 0167]
• EP 0300305 [0110]
• - DE 2412837 [0116]
• WO 95/23221 [0167]
• WO 92/21760 A1 [0171]

Cited non-patent literature

• - "Subtilases: Subtilisinlike Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 [0002]
• - "Alkalophilic microorganisms. A new microbial world "by K. Horikoshi and T. Akiba (1982), Japan Scientific Societies Press, Springer-Verlag, New York, Heidelberg, Berlin, ISBN 0-387-10924-2, Chapter 2, pages 9-26 [ 0008]
• Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410 [0024]
• Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs "; Nucleic Acids Res., 25, pp. 3389-3402 [0024]
• - Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500 [0030]
• - Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. Biol. 302, 205-217 [0030]
• - Surfactants, Vol. 7 (1970), pp. 125-132 [0041]
• - Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press. [0072]
• Gornall AG, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766 [0135]
• Surfactants, Vol. 7 (1970), pp. 125-132 [0136]
• - "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 [0157]
• Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 [0160]
• Bernhard et al. (1978), J. Bacteriol., Vol. 133 (2), p. 897 et seq. [0162]
• Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444 [0162]

Claims (17)

Protease comprising an amino acid sequence, which is selected from the group consisting of a) Amino acid sequence corresponding to that shown in SEQ ID NO. 3 specified Amino acid sequence is at least 98.3% identical b) Amino acid sequence corresponding to that shown in SEQ ID NO. 2 specified Amino acid sequence is at least 98.5% identical, especially an amino acid sequence selected from consisting of the group a) Amino acid sequence, the to SEQ ID NO. 3 indicated amino acid sequence increasingly preferably at least 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical b) amino acid sequence, which corresponds to that shown in SEQ ID NO. 2 indicated amino acid sequence at least 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and most preferably is 100% identical. Protease according to claim 1, characterized in that that they naturally exist in an organism which is isolable from a natural habitat, especially in a microorganism, preferably in a fungus, in a Gram-negative or in a Gram-positive bacterium, below particularly preferred in a bacterium of the genus Bacillus, in particular in Bacillus pumilus and especially in Bacillus pumilus DSM ID 08-100, or that it is encoded by a nucleic acid, which is present in a host cell with the accession number DSM 21891 is. Protease, characterized in that it consists of a Protease according to one of claims 1 or 2 as starting molecule is obtainable by fragmentation, deletion, insertion or Includes substitution mutagenesis and an amino acid sequence, those over a length of at least 50 and increasing preferably at least 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, 266, 267 or 268 contiguous amino acid positions matches the parent molecule, and / or that it consists of a protease according to one of claims 1 or 2 as the starting molecule is obtainable by one or several amino acid exchanges in positions corresponding to the positions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268 of the protease from Bacillus lentus according to SEQ ID NO. 4 assigned in an alignment are. Nucleic acid coding for a Protease according to one of claims 1 to 3, in particular one comprising a nucleic acid sequence selected is made up of the group a) nucleic acid sequence, which corresponds to that shown in SEQ ID NO. 1 indicated nucleic acid sequence in a section of at least 300 contiguous Nucleotides at least 97.1% and increasingly preferably at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical is b) Nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO. 1 indicated nucleic acid sequence in positions 325 to 1149 at least 97.1% and increasingly preferred to at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and most preferably 100% identical is c) Nucleic acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 specified nucleic acid sequence at least 97.1% and increasingly preferably at least 97.2%, 97.4%, 97.6%, 97.8%, 98%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% and all more preferably 100% identical. Vector containing a nucleic acid according to Claim 4, in particular a cloning vector or an expression vector. Non-human host cell containing a nucleic acid according to claim 4 or a vector according to claim 5, or which comprises a protease according to any one of claims 1 to 3, especially one that surrounds the protease into the host cell Medium secreted. Host cell according to claim 6, characterized in that it has a cell nucleus or that it is a bacterium, preferably one selected from the genera Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas , more preferably one selected from the group of Escherichia coli, Klebsiella planticola, Ba cillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum, Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia. A method for producing a protease comprising a) Culturing a host cell according to any of the claims 6 or 7 b) isolating the protease from the culture medium or from the host cell. Means, in particular a washing or cleaning agent, characterized in that it comprises at least one protease after a of claims 1 to 3, in particular in in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, more preferably from 20 μg to 15 mg and most preferably from 50 μg to 10 mg per g of the agent Agent according to claim 9, characterized that it is present as a one-component system or that it is in several Components is divided. Agent according to one of claims 9 or 10, characterized in that it is in solid form, in particular as free-flowing powder with a bulk density from 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or that it is in liquid, gel or pasty pasty form is present. Agent according to one of claims 9 to 11, characterized in that the protease is at room temperature and / or impermeable to the protease in the absence of water Substance is sheathed. Agent according to one of claims 9 to 12, characterized in that it contains one or more further enzymes in particular a protease, amylase, cellulase, hemicellulase, Mannanase, taunase, xylanase, xanthanase, β-glucosidase, Carrageenase, perhydrolase, oxidase, oxidoreductase or a lipase, and preferably mixtures thereof. Use of an agent according to one of the claims 9 to 13 to remove protease-sensitive stains Textiles or hard surfaces. Process for cleaning textiles or hard Surfaces, characterized in that in at least a method step, an agent according to one of the claims 9 to 13 is applied. Process for cleaning textiles or hard Surfaces, characterized in that in at least a method step, a protease according to one of the claims 1 to 3 is catalytically active, in particular such that the protease in an amount of 40 μg to 4 g, preferably 50 μg to 3 g, more preferably from 100 μg to 2 g and whole more preferably used from 200 μg to 1 g per application is. Use of a protease according to any one of the claims 1 to 3 for cleaning textiles or hard surfaces, in particular such that the protease is in an amount of 40 μg to 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g, and most preferably from 200 μg to 1 g per application is used.