DE102009027540A1 - New protease comprising a specific amino acid sequence, useful e.g. for biochemical analysis or synthesis of low molecular weight compounds or proteins - Google Patents

Abstract

Protease comprising an amino acid sequence, which is an amino acid sequence specified in SEQ ID NO: 3 (comprising a fully defined 51-319 amino acid sequence given in the specification), or an amino acid sequence specified in SEQ ID NO: 2 (comprising a fully defined 51-319 amino acid sequence given in the specification), is new. Independent claims are included for: (1) a nucleic acid coding for a protease, comprising a nucleic acid sequence, which is identical to a nucleic acid sequence specified in SEQ ID NO: 1 (comprising a fully defined 51-319 amino acid sequence given in the specification) in a part of at least 300 contiguous nucleotides, a nucleic acid sequence, which is identical to the nucleic acid sequence specified in SEQ ID NO: 1 in the position of 277-1083 and a nucleic acid sequence, which is identical to the nucleic acid sequence specified in SEQ ID NO: 1; (2) a vector comprising a nucleic acid, preferably a cloning vector or an expression vector; (3) a non-human host cell comprising a nucleic acid or a vector, or a protease, preferably the protease secreted into the medium surrounding the host cell; (4) the preparation of the protease; (5) an agent, preferably a washing- or cleaning-agent, comprising at least one protease in an amount of 2 mu g to 20 mg, preferably 50 mu g to 10 mg per g of the agent, where the protease in the agent is encased with a substance impermeable at room temperature or in the absence of water for the protease; and (6) cleaning textiles or hard surfaces, comprising applying an agent in at least one step to the textiles or hard surfaces.

Description

The The present application is directed to novel proteases and their Production and use thereof. It also concerns Agents, in particular detergents and cleaners, containing these Proteases, appropriate washing and cleaning processes and uses 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 due to the catalytically active amino acids serine proteases. They act as nonspecific endopeptidases and hydrolyze any acid amide linkages that are internal to peptides or proteins. Their pH optimum is usually in the clearly alkaline range. An overview of this family offers for example the Article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 , Subtilases are naturally produced by microorganisms. Of these, in particular, the subtilisins formed and secreted by Bacillus species are to be mentioned as the most important group within the subtilases.

Examples of the subtilisin-type proteases preferably used in detergents and cleaners are the subtilisins BPN 'and Carlsberg, the protease PB92, 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.

In Detergents are often added to proteases Improvement of the washing or cleaning performance together with further enzymes, in particular amylases, cellulases, hemicellulases, Mannanases, β-glucosidases, oxidases, oxidoreductases or Used lipases. 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 is also known that some for the use in detergents established proteases also for cosmetic purposes or for organic chemical synthesis are suitable.

different Technical applications require proteases with different Properties, such as the reaction conditions, the Stability or substrate specificity. Furthermore, the potential uses of a protease, for example, in a washing or cleaning agent, starting from another Factors like the stability of the enzyme, especially against high or low temperatures, oxidizing agents or surfactants, convolution effects or desired synergies with other ingredients.

There continues to be a high demand for new technically usable proteases. 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, the microorganisms with the most powerful enzymes are then selected and purified, for example by plating on proteinaceous agar plates and measuring the lysis farms formed, or the respective genes are cloned. 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.

The object of the invention is to provide further, hitherto unknown proteases which have a have proteolytic activity. Advantageously, the protease should excel in that its contribution to the performance of an agent containing the protease, in particular a detergent or cleaning agent, is at least close to and ideally better than the contribution of a proteolytic enzyme established for this purpose to the performance of the agent. As a contribution to performance in this regard, in particular the contribution to the cleaning performance of a detergent or cleaning agent is important.

A Another object of the invention is proteases, in particular from Subtilisin type, provide that opposite the state The technique provides improved stability Temperature influences, especially high or low Temperatures, acidic or alkaline conditions or pH changes, denaturing or oxidizing agents, proteolytic degradation, or to a change in the redox ratios exhibit. Further tasks can be reduced in one Immunogenicity and / or decreased allergenic effects be seen.

A Another object of the invention is to provide proteases, at lower temperatures, especially between 10 ° C and 40 ° C, and more preferably between 10 ° C and 30 ° C and between 10 ° C and 25 ° C, especially at 20 ° C, an improved cleaning performance in comparison to the proteases known in the art, in particular those of the subtilisin type, wherein the improved cleaning performance on at least one soiling, preferably on several soils, is present. Advantageously, the cleaning performance in a Wash liquor provided by a non-solid detergent formulation was formed.

• a) der in SEQ ID NO. 3 angegebenen Aminosäuresequenz, und
• b) der in SEQ ID NO. 2 angegebenen Aminosäuresequenz.

The invention relates to a protease comprising an amino acid sequence which is selected from the group consisting of

• a) in SEQ ID NO. 3 indicated amino acid sequence, and
• b) in SEQ ID NO. 2 indicated amino acid sequence.

Herewith are as further subjects of the invention for inventive Proteases encoding nucleic acids, according to the invention Non-human proteases or nucleic acids Host cells, method for producing inventive Proteases and proteases according to the invention Agents, in particular detergents and cleaners, washing and cleaning processes, and proteases of the invention linked to defined uses.

The of the present invention, naturally formed protease is, as will be understood from the examples can, from the culture supernatant of a Bacillus halodurans Tribal available. The soil sample containing this strain comes from the People's Republic of China from the environment of an alkaline Lake in Inner Mongolia.

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.

A protease according to the invention is suitable for use in detergents and cleaners on account of its proteolytic activity and its further properties, in particular with regard to its stability to surfactants and / or bleaches and / or its temperature profile and / or its pH profile. Surprisingly, it already makes in its wild-type form such a good contribution to the cleaning performance of a washing or cleaning agent containing the protease, which comes close to the contribution of a proteolytic enzyme established for this purpose to the cleaning performance of the agent and even surpasses it on different soils , It therefore makes possible an improved removal of at least one, preferably of several protease-sensitive stains on textiles and / or hard surfaces, for example dishes. Particularly advantageous cleaning performances show proteases according to the invention, for example, of stains containing blood and / or grass and / or grass, for example the stains blood milk / ink on cotton: Product No. C-05 available from CFT (Center For Test Materials) BV Vlaardingen, Netherlands, Chocolate milk / soot on cotton: Product No. C-03 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands, and / or grass on cotton: Product No. 164 available from Eidgenössische Material- und Prüfanstalt (EMPA) Testmaterialien AG , St. Gallen, Switzerland. In this regard, the advantageous cleaning performance of a protease according to the invention of blood and / or milk, especially in the context of liquid detergent or cleaning agent formulations, and the advantageous cleaning performance of a protease according to the invention on grass-containing soils in Frame of liquid and / or solid detergent or cleaner formulations. Furthermore, it has surprisingly been found that such advantageous cleaning performances are also achieved at low temperatures between 10 ° C. and 40 ° C., between 10 ° C. and 30 ° C. and between 10 ° C. and 25 ° C., for example at 20 ° 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 substitutions, insertions or deletions of one or more amino acids, or further sequence changes possible. Further may be nucleic acids according to the invention introduced into recombination approaches and thus for 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 then usually 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.

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 exerts the technically relevant proteolytic function. Therefore, according to the invention are particularly preferred the mature, active proteases. These have a molecular weight between 25 and 30 kD (kilodaltons), in particular 27 kD 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 Gutstation - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 ISD, United Kingdom; http://www.uniprot.org ) and / or GenBank (National Center for Biotechnology Information, NCBI, National Institutes of Health, Bethesda, MD, USA) to identify the proteins most closely related. For the nucleic acid sequence according to SEQ ID NO. 1 was performed according to a sequence comparison with known nucleic acid sequences.

This sequence comparison was based on the BLAST algorithm established and commonly used in the prior art (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 ) and in principle takes place in that similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences are assigned to one another. A tabular assignment of the respective positions is referred to as alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created with computer programs. Frequently used, for example, the Clustal series (see, for example Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500 ), T-Coffee (cf., for example Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217 ) or programs based on these programs or algorithms. In the present application, all sequence comparisons (alignments) with the computer program Vector NTI ^® Suite 10.3 were (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, Califomia, USA) created with the preset default parameters whose AlignX module for sequence comparisons ClustalW based ,

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 areas of different Nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such Areas often have 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. Unless otherwise stated identity or homology information in the present Registration but on the total length of each specified Nucleic acid or amino acid sequence.

By such a comparison with known enzymes, for example are stored in generally accessible databases, can be for an amino acid sequence further conclude their enzymatic activity.

For the mature protease according to SEQ ID NO. 3 has been expressed at the amino acid level over the entire length of SEQ ID NO. 3 identified as next-nearest protein: AprM serine protease from Bacillus sp. with a deviation in one amino acid position (UNIPROT database entry Q45521 (Q45521_BACSP); Masui, A. et al. (1994): Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions. Appl. Environ. Microbiol. 60: 3579-3584 ). This sequence is given as SEQ ID NO. 5th

For the entire protease according to SEQ ID NO. 2 became at the amino acid level over the total length of SEQ ID NO. 2 with a deviation in one amino acid position also the mentioned AprM serine protease from Bacillus sp. as next-similar Protein identified.

Thus, the protease found is a new enzyme. 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 64.7% based on each mature enzyme. A mature protease according to the invention is therefore based on SEQ ID NO. 5 and in the count of SEQ ID NO. Figure 5 shows a variant with the amino acid substitution T127A.

A Total length protease according to the invention is therefore starting from SEQ ID NO. 6 and in the count of SEQ ID NO. 6 a variant with the amino acid substitution T127A, where negative values refer to positions in the signal or Procpeptide, d. H. Amino acid position one is in turn the beginning of the mature protease.

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.

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

• – Blut-Milch/Tusche auf Baumwolle: Produkt Nr. C-05 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,

bestimmt wird durch Messung des Weißheitsgrades der gewaschenen Textilien, der Waschvorgang für mindestens 30 Minuten, optional 60 Minuten, bei einer Temperatur von 20°C erfolgt und das Wasser eine Wasserhärte zwischen 15,5 und 16,5° (deutsche Härte) aufweist.In a further 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 corresponds to the amino acid sequence shown 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. 5 amino acid indicated corresponds to the sequence, and / or at least that of a protease according to WO 03/057713 , 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 Stains blood milk / ink on cotton, chocolate milk / soot on cotton, peanut oil pigment / ink on polyester / cotton and grass 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
• - 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,

is determined by measuring the whiteness of the washed textiles, the washing process for at least 30 minutes, optionally 60 minutes, carried out at a temperature of 20 ° C and the water has a water hardness between 15.5 and 16.5 ° (German hardness).

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% FAEOS (fatty alcohol ether sulfate), 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% Soda, 14-16% coconut fatty acids, 0.5% HEDP (1-hydroxyethane- (1,1-di-phosphonic acid)), 0-0.4% PVP (polyvinylpyrrolidone), 0-0.05% optical Brightener, 0-0.001% dye, 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.

in the According to the invention, the determination of the cleaning performance preferably at 20 ° C using a liquid Detergent as indicated above.

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 protease activity 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 ,

Alternatively, the protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-Nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of the pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979 ). The measurement he follows at a temperature of 25 ° C, at pH 8.6, and a wavelength of 410 nm. The measuring time is 5 min and the measuring interval 20 s to 60 s.

The Protease activity is usually in protease units (PE) indicated. Suitable protease activities amount for example, 2.25, 5 or 10 PE per ml wash liquor. The protease activity is not equal to zero.

proteins can be about the reaction with an antiserum or a particular antibody to groups more immunologically related Proteins are summarized. The relatives of one such groups are characterized by the fact that they are the same, from have an antigenic determinant recognized by an antibody. They are therefore structurally so similar to each other that they recognized by an antiserum or specific antibodies become. Another subject of the invention is therefore proteases, which are characterized by having at least one and increasingly preferably two, three or four matching antigens Determinants with a protease according to the invention exhibit. 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.

Prefers if the protease is present in a microorganism, further preferred in a mushroom, gram-negative or gram-positive Bacterium, and among these particularly preferred in a bacterium of the genus Bacillus. Because especially for these organisms In the prior art cultivation methods are known and established. This is especially true for Bacilli, which in the technical Enzyme production play a prominent role. Most notably Preferably, the protease is present in Bacillus halodurans.

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 special properties (for example, in terms of their catalytic Activity, stability, etc.). In particular, changes in the nucleotide or amino acid sequence and as mutations designated. Basically, deletion, insertion or substitution mutations or those in which different genes or parts of genes are fused together. The associated organisms are corresponding mutants, and proteins encoded by mutant nucleic acids Enzyme variants. For example, deletion, insertion, Substitution mutations or fusions to deletions-, insertions-, substitution mutated genes or fusion genes and at the protein level to corresponding deletion, insertion or substitution variants or fusion proteins. The goal is to become familiar Molecules target mutations such as substitutions, insertions or introduce deletions, for example, the cleaning performance to improve the enzymes of the invention. For this In particular, the surface charges and / or the isoelectric point of the molecules and thereby theirs Interactions with the substrate are changed. So For example, the net charge of the enzymes can be changed in order to discuss substrate binding in particular for to influence the use in detergents and cleaners. alternative or in addition, by one or more appropriate Mutations increase the stability of the protease and thereby their cleaning performance can be improved. advantageous Properties of individual mutations, eg. B. individual substitutions, can complement each other. One regarding certain Properties already optimized protease, for example as regards their stability towards surfactants and / or bleaches and / or other components, therefore, in addition to the invention be further developed.

Under Fragments are understood to mean all proteins or peptides that are smaller are as natural proteins and, for example, also synthetic can be obtained. Because of their amino acid sequences They can be the respective complete proteins be assigned. For example, you can use the same structures accept or enzymatic activities or partial activities exercise, such as the complexation of a particular Substrate. Fragments and deletion variants of starting proteins are basically similar in that one or more amino acids lacking compared to an initial sequence; while fragments usually include small pieces of an initial sequence, deletion mutants are usually absent 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.

One Another object of the invention is a protease which is characterized characterized in that it consists of a protease as described above obtainable by single or multiple conservative amino acid substitution is, in particular such that the resulting protease is still at least 85% identical to SEQ ID NO. 3 or SEQ ID NO. 2 is and / or at position 127 in the count of SEQ ID NO. 3 or SEQ ID NO. 2 has the amino acid alanine (Ala, A). advantageously, the protease obtained is still at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 3 or SEQ ID NO. 2 and / or includes at position 127 in the counting of SEQ ID NO. 3 or SEQ ID NO. 2 the Amino acid alanine (Ala, A). The term "conservative Amino acid substitution "means the substitution one amino acid residue against another amino acid residue, this exchange does not change the polarity or charge at the position of the exchanged amino acid leads, z. B. the replacement of a nonpolar amino acid residue against another nonpolar amino acid residue. Conservative amino acid substitutions in the context of the invention, for example, include: G = A = S, I = V = L = M, D = E, N = Q, K = R, Y = F, S = T, G = A = I = V = L = M = Y = F = W = P = S = T.

alternative or additionally, the protease is characterized that they are from a protease according to the invention as the starting molecule is obtainable by fragmentation, Deletion, insertion or substitution mutagenesis and an amino acid sequence which covers a length of at least 50 or 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, the allergenicity lowered enzymes and thus improved their overall applicability become. Advantageously, the enzymes retain even after fragmentation or deletion mutagenesis their proteolytic activity.

Also Substitutions can produce beneficial effects. Either single as well as several contiguous amino acids can be exchanged for other amino acids.

Alternatively or additionally, the protease 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 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, of the protease from Bacillus lentus, which are transferred to homologous positions of the proteases according to the invention preferably from Be Accordingly, the positions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268, in an alignment with SEQ ID NO. 4 and thus in the count according to SEQ ID NO. 4. In said positions, the wild-type molecule of the Bacillus lentus protease has the following amino acid residues: S3, V4, S36, N42, A47, T56, G61, T69, E87, A96, R99, A101, I102, S104, N114, H118, A120, S130, S139, T141, S142, S154, S157, A188, V193, V199, G205, L211, A224, K229, S236, N237, N242, H243, N255 and T268, respectively.

Advantageous In particular, for example, substitutions 3T, 4I, 61A, 99G, 99A, 99S, 154D, 154E, 211D, 211G and 211E, as appropriate homologous positions in an inventive Protease does not already naturally belong to one of these preferred amino acids are taken. The exchanges 3T and 4I have a stabilizing effect on the molecule to improve the cleaning performance the protease and thus to an improved cleaning performance a washing or cleaning agent containing the protease.

Further confirmation of the correct assignment of the amino acids to be changed, ie in particular their functional correspondence, can provide comparative experiments, according to which the two positions assigned to each other on the basis of an alignment are changed in the same way in both compared proteases, and if both are observed the enzymatic activity is changed in the same way. If, for example, an amino acid exchange in a specific position of the protease from Bacillus lentus according to SEQ ID NO. 4 associated with a change in an enzymatic parameter, for example, with the increase of the K _M value, and a corresponding change in the enzymatic parameter, for example, also an increase in the K _M value, observed in a protease variant according to the invention, the amino acid exchange the same introduced amino acid has been achieved, this is an acknowledgment of this aspect of the invention.

One Another object of the invention is a protease described above, which is additionally stabilized, in particular by a or several 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, that causes the enzymatic activity lasts longer and thus the cleaning performance is improved. Basically everyone comes in the state of Technology described and / or appropriate stabilization options into consideration. Preference is given to those stabilizations which are via mutations of the enzyme itself, since such stabilization in the Following the recovery of the enzyme no further steps require. Examples of suitable sequence changes are mentioned above. Further suitable sequence changes are known from the prior art. So can proteases For example, be stabilized by one or exchanged several tyrosine residues for other amino acids become.

• – 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.

Further possibilities of stabilization are, for example:

• 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 of several stabilizing mutations act additive or synergistic.

One Another object of the invention is a protease as above described, which is characterized in that they at least has a chemical modification. A protease with such a change is called a 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 enhancements can be made, for example, in vivo by the host cell expressing the protein. In this regard, couplings of low molecular weight compounds such as lipids or oligosaccharides are particularly noteworthy. derivatizations however, they can also be carried out in vitro, 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. Such another compound may also be another protein that is bound to a protein of the invention via bifunctional chemical compounds, for example. Similarly, derivatization is to be understood as meaning the covalent binding to a macromolecular carrier, or else a noncovalent inclusion in suitable macromolecular cage structures. Derivatizations 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. Such modifications may further affect stability or enzymatic activity. They can also serve to reduce the allergenicity and / or immunogenicity of the protein and thus, for example, increase its skin compatibility. 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 Shelf life, with other substances targeted be. Therefore, all preparations are also according to the invention a protein of the invention. This is also regardless of whether it is in a particular preparation actually unfolds this enzymatic activity or not. Because it may be desired that it at the Storage has little or no activity, and only at the time of use its enzymatic function unfolds. This can be done, for example, using appropriate accompanying substances to be controlled. In particular, the joint preparation of Proteases with protease inhibitors is advantageous.

With regard to all the proteases or protease variants and / or derivatives described above, particular preference is given in the context of the present invention to those whose activity is at least that of the protease according to SEQ ID NO. 3, and / or their cleaning performance of at least that of the protease according to SEQ ID NO. 3, wherein the cleaning performance is determined in a washing system as described above. The determination of the protease activity is a customary measure and takes place in this regard preferably according to the in Surfactants, Vol. 7 (1970), pp. 125-132 described method. Usually, the protease activity is indicated in PE (protease units).

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 Amino acid sequence in proteins or enzymes encode, in particular DNA or RNA molecules. They can be used as a single strand, as a single strand complementary to this single strand or as a double strand. Especially with DNA molecules are the sequences of both complementary strands to be considered in all three possible reading frames.

It should also be noted that different codons, base triplets, can code for the same amino acids so that a particular amino acid sequence can be encoded by several different nucleic acids, which is referred to as the degeneracy of the genetic code. For these reasons, both amino acid sequences and nucleic acid sequences are included in the scope of protection. Therefore, all nucleic acid sequences are included in this subject of the invention which can encode any of the proteases described above. The person skilled in the art is able to determine these nucleic acid sequences unequivocally since, despite the degeneracy of the genetic code, individual codons are assigned defined amino acids. Therefore, the person skilled in the art can easily determine nucleic acids coding for this amino acid sequence on the basis of an amino acid sequence. Furthermore, with nucleic acids according to the invention, one or more codons may have been replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Thus, every organism, for example a host cell of a production strain, has a certain codon usage. Codon usage is the translation of the genetic code into amino acids by the respective organism. Bottlenecks in protein biosynthesis can occur if the codons lying on the nucleic acid in the organism face a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid As a result, a codon in the organism is translated less efficiently than a synonymous codon coding for the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be more efficiently translated in the organism.

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

• a) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz in einem Teilstück von mindestens 300 zusammenhängenden Nukleotiden identisch ist
• b) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz in den Positionen 277 bis 1083 identisch ist
• c) Nukleinsäuresequenz, die zu der in SEQ ID NO. 1 angegebenen Nukleinsäuresequenz 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 identical
• b) Nucleic acid sequence corresponding to the amino acid sequence shown in SEQ ID NO. 1 nucleic acid sequence in positions 277 to 1083 is identical
• c) Nucleic acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 nucleic acid sequence is 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 is identical.

Concerning the overall sequence of SEQ ID NO. 1 has these in the positions 1 to 3 alternatively the codon GTG on. Since this is the Start codon, this also encodes the amino acid methionine (or formyl-methionine (fMet)) and not for valine (Val).

The p. above) nucleic acid sequence the region coding for the mature protease. If it turns out that the mature (mature) protein only from a part of this sequence is formed, the scope of protection applies accordingly for this part. Preferred are such nucleic acids, coding for mature proteins.

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. A distinction is made between cloning vectors and storage and thus to a certain extent also the genetic engineering work serve, and expression vectors that perform the function, the nucleic acid or the gene present on it (often Transgene) in the host cell, that is, to allow expression of the subject polypeptide.

In the context of the present invention, a nucleic acid according to the invention is cloned into a vector. The vectors include, for example, those whose origin are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They can be extrachomosomal as their own units or integrated into a chromosome or chromosomal DNA. Which of the numerous systems known from the prior art is chosen depends on the individual case. Decisive, for example, the achievable copy number, the available Selekti or, in particular, antibiotic resistance, or the cultivability of the host cells capable of accepting the vectors.

expression vectors include nucleic acid sequences that enable them to in the host cells containing them, preferably microorganisms, particularly preferred bacteria, to replicate and there contained one To bring nucleic acid for expression. The expression is particularly influenced by the promoter (s) which regulate transcription. In principle, the expression can be through the natural, originally to be expressed before Nucleic acid localized promoter, but also by a promoter provided on the expression vector the host cell or even by a modified or a completely another promoter of another organism or another host cell. In the present case, at least one promoter for the Expression of a nucleic acid according to the invention provided and for their expression used. Furthermore, expression vectors can be regulated be, for example, by changing the culture conditions or upon reaching a certain cell density of the host cells they contain or by adding certain substances, in particular activators gene expression. An example of such a substance the galactose derivative is isopropyl-β-D-thiogalactopyranoside (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, translation related changes the resulting protein as well as they would naturally expire.

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.

One Another object of the invention is a non-human host cell, the one nucleic acid according to the invention or contains a vector according to the invention, or which contains a protease according to the invention, especially one that surrounds the protease into the host cell Medium secreted. A nucleic acid according to the invention or a vector according to the invention is preferred introduced into the host cell by their transformation. Prefers becomes a nucleic acid according to the invention or a vector according to the invention in a microorganism transformed, which then a inventive Represents host cell. Alternatively, individual components, d. H. Nucleic acid parts or fragments of a novel Nucleic acid are introduced into a host cell in this way, that the resulting host cell is an inventive Nucleic acid or a novel Vector contains. This procedure is particularly suitable if the host cell already has one or more components of a Nucleic acid according to the invention or a contains vector of the invention and the other components are then supplemented accordingly. Methods of transforming cells are known in the art established and well known to the skilled person. As host cells In principle, all cells are suitable, that is prokaryotic or eukaryotic cells. Preferred are such host cells, the be genetically advantageous to handle what, for example the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular Mushrooms or bacteria. Furthermore, preferred host cells are distinguished through a good microbiological and biotechnological handling out. This concerns, for example, easy cultivability, high Growth rates, low requirements for fermentation media and good production and secretion rates for foreign proteins. Secrete preferred host cells according to the invention the (transgene) expressed protein into the surrounding host cells Medium. Furthermore, the proteases can be produced by those producing them Cells are modified after their preparation, for example by attachment of sugar molecules, formylations, Aminations, etc. Such posttranslational modifications can functionally affect the protease.

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 camosus, 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 able to modify the protein formed posttranslationally. Examples for it are mushrooms like Actinomyceten or yeasts like Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example when the proteins are specific in the context of their synthesis To undergo modifications that allow such systems. Among the modifications, the eukaryotic systems particularly related to do with protein synthesis, for example the binding of low molecular weight compounds such as membrane anchors or Oligosaccharides. Such oligosaccharide modifications can for example, to lower the allergenicity of an expressed Protein be desirable. Also a coexpression with the enzymes naturally produced by such cells, such as cellulases or lipases, may be advantageous. Furthermore, for example, thermophilic fungal Expression systems especially for the expression of temperature-resistant Proteins or variants are suitable.

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.

Fermentation process, which are characterized in that the fermentation over An inflow strategy is carried out in particular 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. Furthermore, the fermentation can also be designed so that unwanted metabolic products filtered out or neutralized by adding buffer or appropriate counterions become.

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.

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. As inventive Proteases advantageous cleaning services in particular to blood and / or Milk and / or grass containing soils are the means in particular for the removal of such soiling suitable and advantageous.

This subject matter of the invention includes all conceivable types of detergents or cleaners, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or in hand washing or cleaning. These include detergents for textiles, carpets, or natural fibers, for which the term detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, ie in addition to manual and machine Dishwashing agents, for example, scouring agents, glass cleaners, toilet scenters, etc. The washing and cleaning agents in the invention also include washing aids which are added to the actual detergent in the manual or machine textile laundry to achieve a further effect. In addition, detergents and cleaners in the context of the invention also include textile pre-treatment and post-treatment agents, ie those agents with which the item of laundry is brought into contact before the actual laundry, for example, for stubborn stains, and also those agents which are in one of the actual Textile laundry downstream step the laundry further desires properties such as comfortable grip, crease resistance or low static charge. Amongst others, the fabric softeners are calculated.

The detergents or cleaners according to the invention, as in particular powdered solids, in nachverdichteter Particle form, as homogeneous solutions or suspensions can, in addition to a invention Protease 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 others Excipients such as optical brighteners, grayness inhibitors, foam regulators and colorings and fragrances and combinations thereof.

Especially by a combination of a erfindungegemäßen Protease with one or more other ingredients) of the agent is advantageous because such a means improved cleaning performance having resulting synergisms. In particular, through the combination a protease according to the invention with one of the surfactants described below and / or one of the following builders described and / or one of those described below Bleach will achieve such synergism.

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 at 8 mol ethylene oxide and propylene oxide in the molecule. However, it is also possible to use other known low-foam nonionic surfactants, such as, for example, C ₁₂ -C ₁₈ -alkyl polyethylene glycol-polybutylene glycol ethers having in each case up to 8 mol of ethylene oxide and butylene oxide units in the molecule and end-capped alkylpolyalkylene glycol mixed ethers. 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 is} CO for 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] represents 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 Fettsäuremethylestem 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 Alkenylbemsteinsäuresalze 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. 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, hydrogenation erucic acid and behenic acid and in particular from natural fatty acids, for example coconut, palm kernel or tallow fatty acids, derived soap mixtures. Together with these soaps or as a substitute for soaps, the known Alkenylbemsteinsäuresalze can be used.

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 acid or methacrylic acid with vinyl ethers, such as vinylmethyl ethers, vinyl esters, ethylene, propylene and styrene, in which the proportion of acid 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 μm and preferably consist of at least 80% by weight of particles having a size of less than 10 μm. Your 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. B. Na-SKS-1 (Na ₂ Si ₂₂ O ₄₅ · xH ₂ O, kenyaite), Na-SKS-2 (Na ₂ Si ₁₄ O ₂₉ x H ₂ O, magadiite), Na-SKS-3 (Na ₂ Si ₈ O, 7 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.

As bleach activators, it is possible to use compounds which, under perhydrolysis conditions, give aliphatic peroxycarboxylic acids having preferably 1 to 10 C atoms, in particular 2 to 4 C atoms, and / or optionally substituted perbenzoic acid. Suitable substances are those which carry O- and / or N-acyl groups of the stated C atom number and / or optionally substituted benzoyl groups. Preference is given to polyacylated alkylenediamines, in particular tetraacetylethylenediamine (TAED), acylated triazine derivatives, in particular 1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine (DADHT), acylated glycolurils, in particular 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 bleaching acti vatoren can, in particular in the presence of the above hydrogen peroxide-supplying bleach, in the usual amount range, preferably in amounts of 0.5 wt .-% to 10 wt .-%, in particular 1 wt .-% to 8 wt .-%, based on the total However, agents which are included are preferably completely absent when using percarboxylic acid as the sole bleaching agent.

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 ingredients to be selected as well as the conditions under which the agent is used, such as temperature, pH, ionic strength, redox ratios or mechanical influences, should be optimized for the respective cleaning problem. For example, the usual temperatures for detergents and cleaners range from 10 ° C over 40 ° C and 60 ° C up to 95 ° for mechanical or technical applications. Preferably, the ingredients of the respective funds are coordinated, esp especially such that there are synergies in terms of cleaning performance. Particularly preferred are synergies which are present in a temperature range between 10 ° C and 60 ° C, in particular in a temperature range of 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, from 15 ° C to 25 ° C, and most preferably at 20 ° 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 size 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 agent, as described in the present application can be obtained can be in the scope of this subject invention locked in.

In a further preferred embodiment an inventive agent the protease in one 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. Furthermore, the protease contained in the agent, and / or other ingredients of the agent, with one at room temperature or impermeable in the absence of water for the enzyme Substance be wrapped, which under application conditions the agent becomes permeable to the enzyme. A Such embodiment of the invention is thus characterized that the protease with a at room temperature or in the absence of water for the protease impermeable substance is wrapped. Furthermore, the washing or cleaning agent even in a container, preferably an air-permeable Container, be packaged, out of it just before use or released during the washing process.

• (a) in fester Form vorliegt, insbesondere als rieselfähiges Pulver mit einem Schüttgewicht von 300 g/l bis 1200 g/l, insbesondere 500 g/l bis 900 g/l, oder
• (b) in pastöser oder in flüssiger Form vorliegt, und/oder
• (c) als Einkomponentensystem vorliegt, oder
• (d) in mehrere Komponenten aufgeteilt ist.

In further embodiments of the invention, the agent is characterized in that it

• (A) is in solid form, in particular as a free-flowing powder having a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or
• (b) is in pasty or liquid form, and / or
• (c) is present as a one-component system, or
• (d) is divided into several components.

These embodiments of the present invention include all solid, powdered, liquid, gelatinous or paste-like administration forms of compositions according to the invention, which if appropriate can also consist of several phases and can be present in compressed or uncompressed form. The agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. The solid dosage forms of the composition also include extrudates, granules, tablets or pouches. Alternatively, the agent may also be liquid, gel or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste. Furthermore, the agent may be present as a one-component system. Such means preferably consist of one phase. Alternatively, an agent can also consist of several phases. Such an agent is therefore divided into several components.

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.

^{Compositions according} to the invention preferably contain enzymes in total amounts of 1 × 10 ^-8 to 5 percent by weight based on active protein. Preferably, the enzymes are from 0.001 to 5% by weight, more preferably from 0.01 to 5% by weight, even more preferably from 0.05 to 4% by weight and most preferably from 0.075 to 3.5% by weight. % contained in agents according to the invention, wherein each enzyme contained can be present in the stated proportions. The protein concentration can be determined 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 can in turn according to a in Surfactants, Vol. 7 (1970), pp. 125-132 method described.

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 is 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 stains, on textiles or hard surfaces, d. H. for cleaning textiles or hard surfaces.

Because Agents according to the invention can, in particular due to the above-described properties of the contained Protease, used to advantage from textiles or from hard surfaces corresponding contaminants too remove. Embodiments of this subject invention For example, hand wash, manual removal of patches of textiles or of hard surfaces or the use in connection with a machine process represents.

All facts, objects and embodiments described for washing or cleaning agents according to the invention are also applicable to this subject of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above inventive use. In preferred embodiments of this use, the washing or cleaning agents in question are therefore according to one of the above provided embodiments described.

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 revelation at this point in the appropriate place 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 or agents containing them are also described applicable to this subject invention. Therefore, at this Make explicit reference to the disclosure to the corresponding Pointed by pointing out that this revelation also applies to the above method according to the invention 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.

Another object of the invention is therefore the use of a protease according to the invention, as described above for the cleaning of textiles or hard surfaces. Preferably, the protease is in an amount of 40 ug to 4 g, preferably from 50 micrograms to 3 g, especially before from 100 μg to 2 g and most preferably from 200 μg to 1 g per application. All facts, subjects and embodiments described for proteases according to the invention or agents containing them are also applicable to this subject of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above inventive use. In a preferred embodiment of this use, the relevant enzymes according to the invention are provided within the scope of an agent according to the invention, preferably a washing or cleaning agent according to the invention.

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 the human skin (desquamation) a crucial role. Accordingly Proteases are also called bioactive components in skin care products used to break down the increased in dry skin desmosomal structures 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. An overview over this offers for example the Handbook "Industrial Encyclopedia and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 , All of these techniques can be enriched by the present 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 the context of a peptide Sequenzana 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.

Examples

All molecular biology work steps follow standard methods, such as those in the Handbook 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

Cloning of the new serine protease

The proteolytically active bacterium (Bacillus halodurans) was dissolved in medium (10 g / l glucose, 5 g / l polypeptone, 5 g / l yeast extract, 1 g / l K ₂ HPO ₄ , 0.2 g / l MgSO ₄ .7H ₂ O , 50 g / l NaCl, 8 g / l Na ₂ CO ₃ , pH 10.0). 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 agar plates containing milk powder (TBY Skimmilk plates, 1.5% agar, 0.1% K ₂ HPO ₄ , 0.5% yeast extract, 1% Peilton, 1% milk powder, 0.02% MgSO ₄ .7H ₂ O, 0.4% Na ₂ CO ₃ , pH 10) and incubated at 30 ° C. Proteolytically active clones were identified by their lysis sites. One of the resulting proteolytically active clones was selected, the plasmid (vector) of which was isolated and the gene fragment (insert) contained in this vector was sequenced by standard methods.

The Insert contains an open reading frame of approx. 1.2 kb, its DNA sequence for a subtilisin-type protease coded. The sequence was amplified by PCR and inserted into the E. coli vector pUC19 cloned. The coding for the protease Nucleic acid sequence is shown in SEQ ID NO. 1 indicated.

Example 2: Determination of the cleaning performance when used in a commercially available powdery laundry detergent

A:

Gras auf Baumwolle: Produkt Nr. 164 erhältlich 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 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande

C:

Schokoladenmilch/Ruß auf Baumwolle: Produkt Nr. C-03 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande

Standardized dirty textiles were used for this example. The following soiling and textiles were used:

A:

Grass on cotton: Product No. 164 available 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 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands

C:

Chocolate milk / soot on cotton: Product No. C-03 available 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 Washed at 20 ° C. 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 16 ° German hardness.

The control detergent used was a detergent base formulation of the following composition (all In percentages 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 (approach 1) and a performance-enhanced variant of the alkaline protease from Bacillus lentus, as described in WO 97/21760 is open (approach 2).

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. STABW denotes the standard deviation in parallel experimental approaches. 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 20 ° C. soiling Approach 1 STDEV Approach 2 STDEV A 3.1 0.7 1.9 0.1 B 9.3 0.2 8.4 0.4 C 8.3 0.2 8.1 0.8

It it becomes clear that the protease according to the invention already shows a better cleaning performance in its wild-type form compared with one established for detergents and performance-enhanced protease variant according to approach 2, which is not a wild-type molecule.

Example 3: Determination of the cleaning performance when used in a commercial liquid laundry detergent

A:

Gras auf Baumwolle: Produkt Nr. 164 erhältlich 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 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande

C:

Schokoladenmilch/Ruß auf Baumwolle: Produkt Nr. C-03 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande

D:

Blut-Milch/Tusche auf Baumwolle: Produkt Nr. C-05 erhältlich von CFT (Center For Testmaterials) B. V. Vlaardingen, Niederlande

Standardized dirty textiles were used for this example. The following soiling and textiles were used:

A:

Grass on cotton: Product No. 164 available 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 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands

C:

Chocolate milk / soot on cotton: Product No. C-03 available from CFT (Center For Test Materials) BV Vlaardingen, The Netherlands

D:

Blood Milk / Ink on Cotton: Product No. C-05 available 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 Washed at 20 ° C. 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 16 ° German hardness.

The control detergent used was a detergent base formulation of the following composition (all figures in percentages by weight): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol , 0.3-0.5% ethanol, 4-7% FAEOS (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 (polyvinyl pyrrolidone), 0-0.05% optical brightener, 0-0.001% dye , Rest demineralized water. The detergent base formulation was for the various series of experiments activity (10 PE / ml final concentration) with the following proteases added: Protease according to the invention comprising an amino acid sequence according to SEQ ID NO. 3 (approach 1) and protease according to WO 03/057713 (Approach 2). The further experimental procedure and evaluation was carried out as described in Example 2. The results are summarized in Table 2 below. Table 2: Wash results with a liquid detergent at 20 ° C soiling Approach 1 STDEV Approach 2 STDEV A 4.4 0.0 2.0 0.1 B 8.2 0.2 5.1 0.8 C 6.9 0.4 4.6 0.7 D 15.9 3.3 9.7 1.9

It again becomes clear that an inventive Protease already in its wild-type form, especially in a liquid detergent at 20 ° C, a better cleaning performance shows in comparison with another improved performance for detergents established Protease Varitant according to Approach 2.

Description of the figure:

1:

Erfindungsgemäße Protease gemäß SEQ ID NO. 3 (reifes Enzym)

2:

AprM Serinprotease aus Bacillus sp. (UNIPROT-Datenbankeintrag Q45521; vgl. SEQ ID NO. 5)

3:

Protease aus Bacillus lentus DSM 5483 (reifes Enzym, vgl. auch WO 92/21760 A1 bzw. SEQ ID NO. 4)

1 Sequence comparison (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 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) using standard parameters. Then mean:

1:

Protease according to the invention according to SEQ ID NO. 3 (mature enzyme)

2:

AprM serine protease from Bacillus sp. (UNIPROT database entry Q45521, see SEQ ID NO. 5)

3:

Protease from Bacillus lentus DSM 5483 (mature enzyme, see also WO 92/21760 A1 or SEQ ID NO. 4)

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 [0025]
• WO 03/057713 [0029, 0150]
• - EP 0300305 [0093]
• - DE 2412837 [0099]
• WO 97/21760 [0145]
• WO 92/21760 A1 [0152]

Cited non-patent literature

• - Article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 [0002]
• - 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 [ 0006]
• - http://www.uniprot.org [0019]
• Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410 [0020]
• 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 [0020]
• - Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500 [0020]
• - Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. Biol. 302, 205-217 [0020]
• - Masui, A. et al. (1994): Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions. Appl. Environ. Microbiol. 60: 3579-3584 [0023]
• Surfactants, Vol. 7 (1970), pp. 125-132 [0034]
• Del Mar et al., 1979 [0035]
• Surfactants, Vol. 7 (1970), pp. 125-132 [0057]
• Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press [0061]
• Gornall AG, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766 [0118]
• - Surfactants, Vol. 7 (1970), pp. 125-132 [0118]
• - Handbook "Industrial Enzymes and their Applications" by H. Uhlig, Wiley-Verlag, New York, 1998 [0138]
• - Handbook of Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 [0139]
• Bernhard et al. (1978), J. Bacteriol., Vol. 133 (2), p. 897 et seq. [0140]
• Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444 [0140]

Claims (15)

Protease comprising an amino acid sequence, which is selected from the group consisting of a) the in SEQ ID NO. 3 indicated amino acid sequence, and b) the in SEQ ID NO. 2 indicated amino acid sequence. 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 halodurans. Protease, characterized in that it consists of a Protease according to one of claims 1 or 2 by one or multiple conservative amino acid substitution obtainable is, in particular such that the resulting protease is still at least 85% identical to SEQ ID NO. 3 or SEQ ID NO. 2 is and / or at position 127 in the count of SEQ ID NO. 3 or SEQ ID NO. 2 has the amino acid alanine, and or that from a protease according to one of claims 1 or 2 as the starting molecule is obtainable 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, and or that from a protease according to one of claims 1 or 2 as the starting molecule is obtainable by one or more 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 is identical. b) nucleic acid sequence, which corresponds to that shown in SEQ ID NO. 1 indicated nucleic acid sequence identical in headings 277 to 1083. c) nucleic acid sequence, which corresponds to that shown in SEQ ID NO. 1 indicated nucleic acid sequence is 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 that it has a nucleus, or that it is a bacterium, preferably one selected from the group of Genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably 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 pumilus, Staphylococcus camosus, 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 By means of, and / or that there is at least one protease according to one of Claims 1 to 3 contains and the protease in the agent with one at room temperature or in the absence of Water for the protease impermeable substance is wrapped. Agent according to claim 9, characterized that it (A) is in solid form, especially as a free-flowing Powder having a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l, or (b) in pasty or in liquid form, and / or (c) as One-component system is present, or (d) into several components is divided. Agent according to one of claims 9 or 10, characterized in that it contains one or more further enzymes in particular a protease, amylase, cellulase, hemicellulase, Mannanase, tannase, 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 11 to remove protease-sensitive soils 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 11 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.