DE102021207704A1 - Performance-enhanced protease variants VIII - Google Patents

Abstract

The invention relates to proteases comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO: 1 (a) at the positions which correspond to the 9th , 130, 133, 144, 217, 224, 252 and 271, corresponding to amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E; and (b) at least one, preferably both, of the positions corresponding to positions 6 and 166, have at least one further amino acid substitution, preferably selected from 6W and 166M, and their production and use. Such proteases show a very good cleaning performance.

Description

The invention is in the field of enzyme technology. The invention relates to proteases from Bacillus pumilus, the amino acid sequence of which has been modified, in particular with regard to use in detergents and cleaning agents, in order to give them better cleaning performance, and the nucleic acids coding for them, and their production. The invention also relates to the uses of these proteases and methods in which they are used, and agents containing them, in particular detergents and cleaning agents.

Proteases are among the most technically important enzymes of all. They are the longest-established enzymes for detergents and cleaning agents and are contained in practically all modern, high-performance detergents and cleaning agents. They break down proteinaceous soiling on the items to be cleaned. Among these, proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62) are particularly important, which are serine proteases because of the catalytically active amino acids. They act as non-specific endopeptidases and hydrolyze any acid amide bonds that are inside peptides or proteins. Their pH optimum is usually in the clearly alkaline range, in particular around pH 9. The article “Subtilases: Subtilisin-like Proteases” by R. Siezen, pages 75-95 in “Subtilisin enzymes”, for example, offers an overview of this family by R. Bott and C. Betzel, New York, 1996. Subtilases are naturally produced by microorganisms. Among these, the subtilisins formed and secreted by Bacillus species should be mentioned in particular as the most important group within the subtilases.

Examples of the subtilisin-type proteases preferably used in detergents and cleaning agents are the subtilisins BPN' from Bacillus amyloliquefaciens and Carlsberg from Bacillus licheniformis, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the subtilases, but no longer the subtilisins in the narrower sense to be assigned enzymes thermitase, proteinase K and the proteases TW3 and TW7, as well as variants of the proteases mentioned, which have an altered amino acid sequence compared to the starting protease. Proteases are modified in a targeted or random manner using methods known from the prior art and are thus optimized, for example, for use in detergents and cleaning agents. These include point mutagenesis, deletion or insertion mutagenesis, or fusion with other proteins or protein parts. Correspondingly optimized variants are known for most of the proteases known from the prior art.

In the European patent application EP 2016175 A1 for example, a protease from Bacillus pumilus intended for detergents and cleaning agents is disclosed. In general, only selected proteases are suitable at all for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such preparations. For the use of proteases in detergents, therefore, a high catalytic activity under conditions such as those found during a wash cycle and a high storage stability are particularly desirable.

Consequently, protease- and surfactant-containing liquid formulations from the prior art have the disadvantage that the proteases contained under standard washing conditions (e.g. in a temperature range of 20°C to 40°C) do not have a satisfactory proteolytic activity or are not storage-stable and the formulations therefore do not show optimal cleaning performance on protease-sensitive soiling.

One goal in the development of proteases is therefore to modify them in a targeted or random manner using methods known from the prior art and thus optimize them for use in detergents and cleaning agents. These include, for example, point mutagenesis, deletion or insertion mutagenesis or fusion with other proteins or protein parts.

Surprisingly, it has now been found that a protease from Bacillus pumilus or a protease sufficiently similar thereto (in terms of sequence identity) which, based on the numbering according to SEQ ID NO:1, is located at the positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, and at least one more, preferably both, of the positions corresponding to positions 6 and 166, at least one more Has amino acid substitution, preferably selected from 6W and 166M, is improved in terms of cleaning performance compared to the wild-type form and / or reference mutants and is therefore particularly suitable for use in detergents or cleaning agents. In particular, it has been found that the inven Proteases according to the invention show improved cleaning performance on stubborn soiling, in particular soiling containing egg.

1. (a) an den Positionen, die den Positionen 9, 130, 133, 144, 217, 224, 252 und 271 entsprechen, Aminosäuresubstitutionen, insbesondere die Aminosäuresubstitutionen 9T, 130D, 133A, 144K, 217M, 224A, 252T und 271 E; sowie
2. (b) an mindestens einer, vorzugsweise beiden, der Positionen, die den Positionen 6 und 166 entsprechen, mindestens eine weitere Aminosäuresubstitution aufweist, insbesondere ausgewählt aus 6W und 166M.

The invention therefore relates in a first aspect to a protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO: 1:

1. (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271 amino acid substitutions, in particular amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E; such as
2. (b) at least one, preferably both, of the positions corresponding to positions 6 and 166 has at least one further amino acid substitution, in particular selected from 6W and 166M.

Another object of the invention is a method for producing a protease as defined above, comprising substituting amino acids in a starting protease which has at least 70% sequence identity to the amino acid sequence given in SEQ ID NO: 1 over its entire length (i) at the positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271 in SEQ ID NO:1, such that the protease at positions amino acid substitutions, in particular amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A , 252T and 271E, and (ii) at least one, preferably both, of the positions corresponding to positions 6 and 166 in SEQ ID NO:1 has at least one further amino acid substitution, in particular selected from 6W and 166M. The protease obtainable by means of this method has at least 70% sequence identity to the amino acid sequence given in SEQ ID NO:1 over its entire length.

A protease within the meaning of the present patent application therefore includes both the protease as such and a protease produced using a method according to the invention. All statements on the protease therefore relate both to the protease as such and to the proteases produced by means of corresponding processes.

Further aspects of the invention relate to the nucleic acids coding for these proteases, proteases according to the invention or non-human host cells containing nucleic acids and agents according to the invention comprising proteases, in particular detergents and cleaning agents, washing and cleaning methods, and uses of the proteases according to the invention in detergents or cleaning agents for removing proteinaceous soiling.

A preferred aspect of the invention relates to the use of proteases according to the invention, i.e. proteases which comprise an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO: 1 (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, in particular amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, and ( b) at least one, preferably both, of the positions corresponding to positions 6 and 166 has at least one further amino acid substitution, in particular selected from 6W and 166M, for removing egg-containing stains from textiles and/or hard surfaces, in particular crockery.

These and other aspects, features and advantages of the invention will become apparent to those skilled in the art from a study of the following detailed description and claims. Each feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it should be understood that the examples contained herein are intended to describe and illustrate the invention, but not to limit it, and in particular the invention is not limited to these examples.

All percentages are by weight (wt%) unless otherwise indicated.

Numeric ranges given in the format "from x to y" include the stated values. Where multiple preferred numeric ranges are given in this format, it is understood that any ranges resulting from the combination of the different endpoints are also included.

"At least one" as used herein means one or more, ie 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

The term "detergent and cleaning agent" or "detergent or cleaning agent" as used herein is synonymous with the term "agent" and refers to a composition for cleaning textiles and / or hard surfaces, especially dishes, as in the description explained.

"About", "about" or "approximately" as used herein in relation to a numerical value refers to the corresponding numerical value ±10%, preferably ±5%.

"Liquid" as used herein includes liquids and gels as well as pasty compositions. It is preferred that the liquid compositions are flowable and pourable at room temperature, but it is also possible for them to have a yield point.

According to the definition of the invention, a substance, e.g. a composition or an agent, is solid if it is in the solid state of aggregation at 25° C. and 1,013 mbar.

According to the definition of the invention, a substance, e.g. a composition or an agent, is liquid if it is in the liquid state at 25° C. and 1,013 mbar. Liquid also includes gel.

"Variant" as used herein refers to natural or artificially generated variations of a native protease that have an altered amino acid sequence from the reference form.

The present invention is based on the inventors' surprising finding that amino acid substitutions at the positions described herein bring about an improved cleaning effect of this modified protease in detergents and cleaning agents.

In preferred embodiments of the protease according to the invention, the protease has substitutions selected from amino acid substitutions 6W and 166M at positions corresponding to positions 6 and 166. In various embodiments, the protease has corresponding substitutions at both of these positions. These two substitutions can be at positions 6 and 166. These are preferably 6W and 166M.

1. (a) an den Positionen, die den Positionen 9, 130, 133, 144, 217, 224, 252 und 271 entsprechen, Aminosäuresubstitutionen, insbesondere die Aminosäuresubstitutionen 9T, 130D, 133A, 144K, 217M, 224A, 252T und 271E, sowie
2. (b) an mindestens einer, vorzugsweise beiden, der Positionen, die den Positionen 6 und 166 entsprechen, mindestens eine weitere Aminosäuresubstitution, insbesondere ausgewählt aus 6W und 166M, aus.

In various embodiments, the protease

1. (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, in particular amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, as well as
2. (b) at least one further amino acid substitution, in particular selected from 6W and 166M, at at least one, preferably both, of the positions corresponding to positions 6 and 166.

• (a) an den Positionen, die den Positionen 9, 130, 133, 144, 217, 224, 252 und 271 entsprechen, Aminosäuresubstitutionen, die Aminosäuresubstitutionen 9T, 130D, 133A, 144K, 217M, 224A, 252T und 271E, und
• (b1) an der Position, die der Position 6 entspricht, die Aminosäuresubstitution 6W,
• (b2) an der Position, die der Position 166 entspricht, die Aminosäuresubstitution 166M, oder
• (b3) an den Positionen, die den Positionen 6 und 166 entsprechen, die Aminosäuresubstitutionen 6W und 166M,

auf.In various embodiments, the protease

• (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, and
• (b1) at the position corresponding to position 6, the amino acid substitution 6W,
• (b2) at the position corresponding to position 166, the amino acid substitution 166M, or
• (b3) at positions corresponding to positions 6 and 166, amino acid substitutions 6W and 166M,

on.

In various embodiments, preference is given to proteases which have at least 70% sequence identity with the amino acid sequence given in SEQ ID NO:1 over their entire length and in each case based on the numbering according to SEQ ID NO:1 compared to a protease according to SEQ ID NO:1 one of the following Amino acid substitution combinations having: (i) P9T-N130D-T133A-N144K-G166M-Y217M-S224A-N252T-Q271E, (ii) Y6W-P9T-N130D-T133A-N144K-Y217M-S224A-N252T-Q271E, or (iii) Y6W-P9T-N130D-T133A-N144K-G166M-Y217M-S224A-N252T-Q271E. Very particularly preferably, such a protease has no further amino acid substitutions than those mentioned above ((i) to (iii) compared to a protease according to SEQ ID NO:1.

The remainder of the sequence of the above proteases has sufficient sequence identity to SEQ ID NO:1 that the overall sequence identity of the protease is at least 70%, preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96% or 97%. In various embodiments the remainder of the sequence of the protease, i.e. the sequence excluding the positions mentioned herein which may be mutated, is at least 80%, preferably at least 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the corresponding sequence of SEQ ID NO:1. This means that the sequence of the protease can correspond to the sequence of SEQ ID NO:1 with the exception of the substitutions mentioned.

In one embodiment, the protease according to the invention has more than 70% and less than 100% sequence identity to SEQ ID NO:1. Preferably, the protease of the invention has more than 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97%% and less than 100% sequence identity to SEQ ID NO:1 on. The sequence of the protease may correspond to the sequence of SEQ ID NO:1 with the exception of the substitutions mentioned herein. The protease according to the invention preferably has, based on the numbering according to SEQ ID NO:1, compared to a protease according to SEQ ID NO:1, in addition to one of the following amino acid substitution combinations (i) P9T-N130D-T133A-N144K-G166M-Y217M-S224AN252T-Q271E, ( ii) Y6W-P9T-N130D-T133A-N144K-Y217M-S224A-N252T-Q271E, or (iii) Y6W-P9T-N130D-T133A-N144K-G166M-Y217M-S224A-N252T-Q271E, no further amino acid changes.

The proteases according to the invention have improved cleaning performance. In many different embodiments, the proteases according to the invention can have a proteolytic activity which, based on the wild type (SEQ ID NO:1), is at least 101%, preferably at least 102% or more. Such performance-enhanced proteases enable improved washing results on proteolytically sensitive soiling in different temperature ranges, in particular a temperature range from 20°C to 40°C.

Independently of or in addition to the increased cleaning performance, the proteases according to the invention can have increased storage stability in detergents or cleaning agents. This means that compared to the wild-type enzyme and in particular also compared to the starting variant of the protease (SEQ ID NO:1 with P9T-N130D-T133A-N144K-Y217M-S224AN252T-Q271E), especially when stored for 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 or more days or 28 or more days of comparable (i.e. ±10%) or increased stability in washing or cleaning agents available.

The proteases according to the invention have enzymatic activity, i.e. they are capable of hydrolyzing peptides and proteins, in particular in a washing or cleaning agent. A protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of amide/peptide bonds in protein/peptide substrates and is thereby able to cleave proteins or peptides. Furthermore, a protease according to the invention is preferably a mature protease, i.e. the catalytically active molecule without signal and/or propeptide(s). Unless otherwise stated, the sequences given also relate to mature (processed) enzymes in each case.

In various embodiments of the invention, the protease is a free enzyme. This means that the protease can act directly with all components of an agent and, if the agent is a liquid agent, that the protease is in direct contact with the agent's solvent (e.g. water). In other embodiments, an agent may contain proteases that form an interaction complex with other molecules or that contain an "envelope." A single or several protease molecules can be separated from the other components of the agent by a structure surrounding them. Such a separating structure can arise from, but is not limited to, vesicles, such as a micelle or a liposome. However, the surrounding structure can also be a virus particle, a bacterial cell or a eukaryotic cell. In various embodiments, an agent may contain Bacillus pumilus or Bacillus subtilus cells expressing the proteases of the invention, or cell culture supernatants of such cells.

In various embodiments of the invention, the protease comprises an amino acid sequence that corresponds to the amino acid sequence given in SEQ ID NO:1 over its entire length by at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91, 5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% or 97% is the same (where “at least” refers to each of the values mentioned) and in each case based on the numbering according to SEQ ID NO:1 has the amino acid substitutions indicated above.

In the context of the present invention, the feature that a protease has the specified substitutions means that it contains one (of the specified) substitution(s) at the respective position, i.e. at least the specified positions are not otherwise mutated or, for example, by fragmentation of the protease , are deleted. In various embodiments, the proteases described herein have the sequence of SEQ ID NO:1 with the exception of the substitutions explicitly mentioned, i.e. are 100% identical to the sequence according to SEQ ID NO:1 apart from the substituted positions.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm, which is established and commonly used in the prior art (cf., for example, 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, p.3389-3402) and is done in principle by assigning similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another will. A tabular assignment of the relevant positions is referred to as an 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. For example, the Clustal series (cf., 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. Sequence comparisons (alignments) are also possible using the Vector NTI® Suite 10.3 computer program (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the specified standard parameters, whose AlignX module for sequence comparisons is based on ClustalW. Unless otherwise indicated, sequence identity given herein is determined using the BLAST algorithm.

Such a comparison also allows a statement to be made about the similarity of the compared sequences to one another. It is usually given as a percentage of identity, ie the proportion of identical nucleotides or amino acid residues in the same positions or in positions that correspond to one another in an alignment. In the case of amino acid sequences, the broader concept of homology includes conserved amino acid exchanges, i.e. amino acids with similar chemical activity, since these usually have similar chemical activities within the protein. Therefore, the similarity of the compared sequences can also be indicated as percent homology or percent similarity. Identity and/or homology statements can be made about entire polypeptides or genes or only about individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by similarities in the sequences. Such areas often have identical functions. They can be small, containing only a few nucleotides or amino acids. Such small regions often perform essential functions for the overall activity of the protein. It can therefore make sense to relate sequence matches only to individual, possibly small, areas. However, unless otherwise stated, identity or homology statements in the present application relate to the total length of the particular nucleic acid or amino acid sequence stated.

In the context of the present invention, the indication that an amino acid position corresponds to a numerically indicated position in SEQ ID NO:1 therefore means that the corresponding position is assigned to the numerically indicated position in SEQ ID NO:1 in an alignment as defined above.

In addition to the amino acid changes explained above, proteases according to the invention can have further amino acid changes, in particular amino acid substitutions, -insertions or -deletions. Such proteases are further developed, for example, by targeted genetic modification, ie by mutagenesis methods, and for specific purposes or with regard to special properties Properties (for example, in terms of their catalytic activity, stability, etc.) optimized. Furthermore, nucleic acids according to the invention can be introduced into recombination batches and thus used to generate completely new types of proteases or other polypeptides. The aim is to introduce targeted mutations such as substitutions, insertions or deletions into the known molecules in order, for example, to improve the cleaning performance of enzymes according to the invention. For this purpose, in particular the surface charges and/or the isoelectric point of the molecules and thereby their interactions with the substrate can be changed. For example, the net charge of the enzymes can be changed in order to influence substrate binding, in particular for use in detergents and cleaning agents. Alternatively or additionally, the stability or catalytic activity of the protease can be increased by one or more corresponding mutations, thereby improving its cleaning performance. Advantageous properties of individual mutations, eg individual substitutions, can complement one another. A protease that has already been optimized with regard to certain properties can therefore be further developed within the scope of the invention, for example with regard to its stability during storage or with regard to its stability to surfactants and/or bleaches and/or other components.

The following convention is used here for the description of substitutions that affect exactly one amino acid position (amino acid exchanges): first the naturally occurring amino acid is designated in the form of the internationally customary one-letter code, followed by the associated sequence position and finally the inserted amino acid. Multiple or alternative substitutions within the same polypeptide chain are separated by slashes. “130D/V” thus means that position 130 has mutated to D or V. In the case of insertions, additional amino acids are named after the sequence position. For deletions, the missing amino acid is replaced by a symbol, such as an asterisk or a dash, or a Δ is given in front of the corresponding position. For example, P9T describes the substitution of proline at position 9 by threonine, P9TH the insertion of histidine after the amino acid threonine at position 9 and P9* or ΔP9 the deletion of proline at position 9. This nomenclature is known to those skilled in the field of enzyme technology.

A further object of the invention is therefore a protease which is characterized in that it can be obtained as a starting molecule from a protease as described above by one or more conservative amino acid substitutions, the protease in the count according to SEQ ID NO:1 being the ones described above has amino acid substitutions. The term "conservative amino acid substitution" means the exchange (substitution) of one amino acid residue for another amino acid residue, such exchange not resulting in a change in polarity or charge at the position of the exchanged amino acid, e.g., the exchange of a non-polar amino acid residue for another non-polar amino acid residue. Conservative amino acid substitutions within the scope of the invention include, for example: 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.

Alternatively or additionally, the protease is characterized in that it can be obtained from a protease according to the invention as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which has a length of at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 or 274 contiguous amino acids corresponding to the starting molecule with amino acid substitutions described above, i.e. substitutions 9T, 130D, 133A, 144K, 217M, 252T and 271E at the positions corresponding to positions 9, 130, 133, 144, 217, 252 and 271; and another amino acid substitution at at least one, preferably both, of the positions corresponding to positions 6 and 166 are still present.

For example, it is possible to delete individual amino acids at the termini or in the loops of the enzyme without the proteolytic activity being lost or reduced as a result. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also, for example, also reduce the allergenicity of the enzymes in question and thus improve their usability overall. Advantageously, the enzymes retain their proteolytic activity even after mutagenesis, i.e. their proteolytic activity corresponds at least to that of the starting enzyme, i.e. in a preferred embodiment the proteolytic activity is at least 80%, preferably at least 90%, more preferably at least 100% of the activity of the starting enzyme. Other substitutions can also show beneficial effects. Both individual and several contiguous amino acids can be exchanged for other amino acids.

The other amino acid positions are determined here by aligning the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus pumilus, as shown given in SEQ ID NO:1. Furthermore, the assignment of the positions depends on the mature (mature) protein. This assignment is also to be used in particular when the amino acid sequence of a protease according to the invention comprises a higher number of amino acid residues than the protease from Bacillus pumilus according to SEQ ID NO:1. Starting from the positions mentioned in the amino acid sequence of the protease from Bacillus pumilus, the positions of change in a protease according to the invention are those which are assigned precisely to these positions in an alignment.

Advantageous positions for sequence changes, in particular substitutions, of the protease from Bacillus pumilus, which are preferably of importance when transferred to homologous positions of the proteases according to the invention and give the protease advantageous functional properties, are accordingly the positions which correspond to the positions described herein in an alignment, i.e. in the count according to SEQ ID NO:1. The wild-type molecule of the protease from Bacillus pumilus has the following amino acid residues at the positions mentioned: P9, N130, T133, N144, Y217, S224, N252 and Q271 as well as Y6 and G166.

A further confirmation of the correct assignment of the amino acids to be changed, ie in particular their functional correspondence, can be provided by comparative experiments, according to which the two positions assigned to one another on the basis of an alignment are changed in the two compared proteases in the same way and it is observed whether both the enzymatic activity is altered in the same way. If, for example, an amino acid exchange in a specific position of the protease from Bacillus pumilus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example with an increase in the K _M value, and a corresponding change in the enzymatic parameter, for example also a An increase in the K _M value observed in a protease variant according to the invention, the amino acid replacement of which was achieved by the same introduced amino acid, can be seen as confirmation of the correct assignment.

Another object of the invention is a previously described protease which is additionally stabilized, in particular by one or more mutations, for example substitutions, or by coupling to a polymer. An increase in stability during storage and/or during use, for example during the washing process, means that the enzymatic activity lasts longer and the cleaning performance is therefore improved. In principle, all stabilization options described and/or expedient in the prior art can be considered. Such stabilizations are preferred which are achieved via mutations of the enzyme itself, since such stabilizations do not require any further work steps after the enzyme has been obtained. Examples of sequence changes suitable for this are mentioned above. Other suitable sequence changes are known in the art.

• - Veränderung der Bindung von Metallionen, insbesondere der Calcium-Bindungsstellen, beispielsweise durch Austauschen von einer oder mehreren der an der Calcium-Bindung beteiligten Aminosäure(n) 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 oder an der Oberfläche des Proteins bewirken;
• - Austausch von Aminosäuren, die nahe dem N-Terminus liegen, gegen solche, die vermutlich über nicht-kovalente Wechselwirkungen mit dem Rest des Moleküls in Kontakt treten und somit einen Beitrag zur Aufrechterhaltung der globulären Struktur leisten.

Other stabilization options include:

• - Changing the binding of metal ions, in particular the calcium binding sites, for example by exchanging one or more of the amino acid(s) involved in calcium binding for one or more negatively charged amino acids and/or by introducing sequence changes in at least one of the sequences of the two amino acids arginine/glycine;
• - Protection against the influence of denaturing agents such as surfactants by mutations that cause a change in the amino acid sequence on or at the surface of the protein;
• - Replacement of amino acids that are close to the N-terminus with those that presumably make contact with the rest of the molecule via non-covalent interactions and thus contribute to the maintenance of the globular structure.

Preferred embodiments are those in which the enzyme is stabilized in multiple ways, since multiple stabilizing mutations act additively or synergistically.

Another object of the invention is a protease as described above, which is characterized in that it has at least one chemical modification. A protease with such a change is referred to as a derivative, i.e. the protease is derivatized.

In the context of the present application, derivatives are therefore understood to mean proteins whose pure amino acid chain has been chemically modified. Such derivatizations can, for example, be carried out in vivo by the host cell expressing the protein. In this regard, couplings of low-molecular compounds such as lipids or oligosaccharides are particularly noteworthy. However, derivatizations can also be carried out in vitro, for example by chemical conversion of a side chain of an amino acid or by covalently linking another compound to the protein. For example, it is possible to couple amines to carboxyl groups of an enzyme to change the isoelectric point. Such a different compound can also be a further protein which is bound to a protein according to the invention, for example via bifunctional chemical compounds. Derivatization also means covalent binding to a macromolecular carrier, or non-covalent inclusion in suitable macromolecular cage structures. Derivatizations can, for example, affect the substrate specificity or the binding strength to the substrate or bring about a temporary blocking of the enzymatic activity if the coupled substance is an inhibitor. This can be useful, for example, for the period of storage. Such modifications may also affect stability or enzymatic activity. Furthermore, they can also serve to reduce the allergenicity and/or immunogenicity of the protein and thus, for example, to 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 compatibility.

In the widest sense, derivatives of a protein according to the invention can also be understood to mean preparations of these proteins. Depending on how it is obtained, processed or prepared, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein can also have other substances added to it, for example to increase its storage stability. All preparations of a protein according to the invention are therefore also in accordance with the invention. This is also independent of whether it actually develops this enzymatic activity in a specific preparation or not. Because it can be desirable that it has little or no activity during storage and only develops its enzymatic function at the time of use. This can be controlled, for example, via corresponding accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.

Of 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 storage stability corresponds to at least one of the proteases according to SEQ ID NO:1 or the starting variant or the variants tested in the examples and/or the cleaning performance of which corresponds to at least one of the proteases according to SEQ ID NO:1 or the starting variant or the variants tested in the examples, the cleaning performance being determined in a washing system as described above.

Numerous proteases and in particular subtilisins are formed as so-called preproteins, i.e. together with a propeptide and a signal peptide, the function of the signal peptide usually being to ensure the ejection of the protease from the cell producing it into the periplasm or the medium surrounding the cell, and the propeptide is usually necessary for the correct folding of the protease. The signal peptide and the propeptide are usually the N-terminal part of the preprotein. Under natural conditions, the signal peptide is cleaved off from the rest of the protease by a signal peptidase. This is followed by the correct final folding of the protease, supported by the propeptide. The protease is then in its active form and cleaves off the propeptide itself. After the propeptide has been split off, the then mature protease, in particular subtilisin, exerts its catalytic activity without the N-terminal amino acids originally present. For technical applications in general and in particular within the scope of the invention, the mature (mature) proteases, i.e. the enzymes processed after their production, are preferred over the preproteins. The proteases can also be modified by the cells producing them after the production of the polypeptide chain, e.g. by attachment of sugar molecules, formylation, amination, etc. Such modifications are post-translational modifications and can, but do not have to, have an impact on the function of the protease.

1. a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution in die Protease, wobei die Protease die Substitutionen 9T, 130D, 133A, 144K, 217M, 224A, 252T und 271E an den Positionen, die den Positionen 9, 130, 133, 144, 217, 224, 252 und 271 entsprechen; sowie mindestens eine weitere Aminosäuresubstitution an mindestens einer oder mindestens zwei der Positionen, die den Positionen 6 und 166 entsprechen, umfasst;
2. b) Veränderung der Aminosäuresequenz durch Fragmentierung, Deletions-, Insertions- oder Substitutionsmutagenese derart, dass die Protease eine Aminosäuresequenz umfasst, die über eine Länge von mindestens 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 oder 274 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die Protease die Substitutionen 9T, 130D, 133A, 144K, 217M, 224A, 252T und 271E an den Positionen, die den Positionen 9, 130, 133, 144, 217, 224, 252 und 271 entsprechen; sowie mindestens eine weitere Aminosäuresubstitution an mindestens einer oder mindestens zwei der Positionen, die den Positionen 6 und 166 entsprechen, umfasst.

All the facts mentioned are also applicable to the methods according to the invention for the production of a protease. Accordingly, a method according to the invention also comprises one or more of the following method steps:

1. a) Introducing a single or multiple conservative amino acid substitution into the protease, the protease having the substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 217 , 224, 252 and 271 correspond; and at least one other amino acid substitution at at least one or at least two of the positions corresponding to positions 6 and 166;
2. b) Modification of the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis in such a way that the protease comprises an amino acid sequence which is at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, or 274 contiguous amino acids to the starting molecule match where the protease matches the substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T, and 271E to the positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271; and at least one other amino acid substitution at at least one or at least two of the positions corresponding to positions 6 and 166.

All embodiments also apply to the methods according to the invention.

In further embodiments of the invention, the protease or the protease produced using a method according to the invention is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92 .5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5% or 97% identical to the amino acid sequence given in SEQ ID NO:1 over their entire length. The protease or the protease produced using a method according to the invention has the amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E at positions which correspond to positions 9, 130, 133, 144, 224, 252 and 271 ; and at least one, preferably at least two, further amino acid substitution(s) at at least one of the positions which correspond to positions 6 or 166, in each case based on the numbering according to SEQ ID NO:1. Examples include the following amino acid substitution variants: P9T, N130D, T133A, N144K, Y217M, S224A, N252T and Q271E combined with one of (i) Y6W; (ii) G166M, or (iii) Y6W and G166M, where the numbering in each case relates to the numbering according to SEQ ID NO:1.

A further object of the invention is a nucleic acid which codes for a protease according to the invention, and a vector containing such a nucleic acid, in particular a cloning vector or an expression vector.

These can be DNA or RNA molecules. They can be present as a single strand, as a single strand complementary to this single strand, or as a double strand. Particularly in the case of DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. It should also be taken into account that different codons, ie base triplets, can code for the same amino acids, so that a specific amino acid sequence can be coded by a number of different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences which can encode one of the proteases described above are included in this subject matter of the invention. The person skilled in the art is able to determine these nucleic acid sequences unequivocally since, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Therefore, starting from an amino acid sequence, the person skilled in the art can easily determine nucleic acids coding for this amino acid sequence. Furthermore, in the case of nucleic acids according to the invention, one or more codons can be replaced by synonymous codons. This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Each organism, for example a host cell of a production strain, has a specific codon usage. Codon usage is the translation of the genetic code into amino acids by the organism in question. Bottlenecks in protein biosynthesis can occur if the codons located on the nucleic acid face a comparatively small number of loaded tRNA molecules in the organism. Although coding for the same amino acid, this results in a codon being translated less efficiently in the organism 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 translated more efficiently in the organism.

A person skilled in the art can use methods that are generally known today, such as chemical synthesis or the polymerase chain reaction (PCR) in conjunction with standard molecular biological and/or protein chemical methods, to use known DNA and/or amino acid sequences to produce the corresponding nucleic acids up to complete genes to manufacture. Such methods are known, for example, from Sambrook, J., Fritsch, E.F. and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press. known.

In the context of the present invention, vectors are understood to mean elements consisting of nucleic acids which, as a characterizing nucleic acid region, contain a nucleic acid according to the invention keep. They are able to establish this as a stable genetic element in a species or a cell line over several generations or cell divisions. Vectors are special plasmids, i.e. circular genetic elements, particularly when used in bacteria. 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 origins are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements from a wide variety of 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 exist extrachromosomally as separate units or integrate into a chromosome or chromosomal DNA.

Expression vectors comprise nucleic acid sequences which enable them to replicate in the host cells containing them, preferably microorganisms, particularly preferably bacteria, and there to express a nucleic acid contained therein. Expression is influenced in particular by the promoter or promoters that regulate transcription. In principle, the expression can be effected by the natural promoter originally located in front of the nucleic acid to be expressed, but also by a promoter of the host cell provided on the expression vector or by a modified or a completely different 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 is made available and used for its expression. Expression vectors can also be regulated, for example by changing the cultivation conditions or when a certain cell density of the host cells they contain has been reached or by adding certain substances, in particular activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-thiogalactopyranoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the nucleic acid contained in cloning vectors is not expressed.

A further object of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention, or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. A nucleic acid according to the invention or a vector according to the invention is preferably transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, individual components, i.e. nucleic acid parts or fragments of a nucleic acid according to the invention can also be introduced into a host cell in such a way that the resulting host cell then contains a nucleic acid according to the invention or a vector according to the invention. This procedure is particularly suitable when the host cell already contains one or more components of a nucleic acid according to the invention or of a vector according to the invention and the other components are then supplemented accordingly. Methods for transforming cells are established in the prior art and well known to those skilled in the art. In principle, all cells are suitable as host cells, ie prokaryotic or eukaryotic cells. Such host cells are preferred which can be handled genetically in an advantageous manner, for example with regard to the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenically) expressed protein into the medium surrounding the host cells. Furthermore, the proteases can be modified by the cells producing them after their production, for example by attachment of sugar molecules, formylation, amination, etc. Such post-translational modifications can affect the protease functionally.

Further preferred embodiments are those host cells whose activity can be regulated on the basis of genetic regulatory elements which are made available, for example, on the vector but which can also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the cultivation conditions or when a certain cell density is reached, expression can be stimulated. This enables the proteins according to the invention to be produced economically. An example of such a connection is IPTG as described above.

Preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low demands on the cultivation conditions. Inexpensive cultivation processes or manufacturing processes can be established as a result. In addition, the subject mann has a wealth of experience with bacteria in fermentation technology. Gram-negative or Gram-positive bacteria can be suitable for a special production for a wide variety of reasons, which are to be determined experimentally in the individual case, such as nutrient sources, product formation rate, time requirement, etc.

In gram-negative bacteria such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, i.e. into the compartment between the two membranes enclosing the cells. This can be advantageous for special applications. Furthermore, gram-negative bacteria can also be designed in such a way that they smuggle out the expressed proteins not only into the periplasmic space, but also into the medium surrounding the bacterium. In contrast, Gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of the Actinomycetales do not have an outer membrane, so that secreted proteins are immediately released into the medium surrounding the bacteria, usually the nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or processed further. In addition, gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form comparable enzymes themselves, so that they have a similar codon usage and their protein synthesis apparatus is naturally aligned accordingly.

Host cells according to the invention can be modified in terms of their requirements for the culture conditions, have other or additional selection markers or express other or additional proteins. In particular, the host cells can also be those which transgenically express a number of proteins or enzymes.

The present invention can in principle be applied to all microorganisms, in particular to all fermentable microorganisms, particularly preferably to those of the genus Bacillus, and results in proteins according to the invention being able to be produced through the use of such microorganisms. Such microorganisms then represent host cells within the meaning of the invention.

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

However, the host cell can also be a eukaryotic cell, which is characterized in that it has a cell nucleus. A further object of the invention is therefore a host cell which is characterized in that it has a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are able to post-translationally modify the formed protein. Examples are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This can be particularly advantageous, for example, if the proteins are to undergo specific modifications in connection with their synthesis, which make such systems possible. The modifications that eukaryotic systems carry out, particularly in connection with protein synthesis, include, for example, the binding of low-molecular compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of an expressed protein. Co-expression with the enzymes naturally formed by such cells, such as cellulases, can also be advantageous. Furthermore, for example, thermophilic fungal expression systems can be particularly suitable for expressing temperature-stable proteins or variants.

The host cells according to the invention are cultivated and fermented in the usual way, for example in discontinuous or continuous systems. In the first case, a suitable nutrient medium is inoculated with the host cells and the product is harvested from the medium after a period of time to be determined experimentally. Continuous fermentations are characterized by the achievement of a steady state in which cells partially die but also regrow over a comparatively long period of time and at the same time the protein formed can be removed from the medium.

1. a) Kultivieren einer erfindungsgemäßen Wirtszelle, und
2. 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. A further subject matter of the invention is therefore a method for the production of a protease

1. a) culturing a host cell according to the invention, and
2. b) isolating the protease from the culture medium or from the host cell.

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

Fermentation processes which are characterized in that the fermentation is carried out using a feed strategy are particularly suitable. Here, the media components that are consumed by the ongoing cultivation are fed. As a result, considerable increases can be achieved both in the cell density and in the cell mass or dry matter and/or in particular in the activity of the protease of interest. Furthermore, the fermentation can also be designed in such a way that unwanted metabolites are filtered out or neutralized by adding buffers or appropriate counterions.

The protease produced can be harvested from the fermentation medium. Such a fermentation process is preferred to isolating the protease from the host cell, i.e. processing the product from the cell mass (dry matter), but requires the provision of suitable host cells or of one or more suitable secretion markers or mechanisms and/or transport systems so that the host cells can Secrete protease into the fermentation medium. Alternatively, without secretion, the protease can be isolated from the host cell, i.e. it can be purified from the cell mass, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

All of the above issues can be combined into methods to produce the protease of the invention.

In a further embodiment of the invention, the protease is characterized in that its cleaning performance (after storage, e.g. over 4 weeks) compared to that of a protease which comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 or compared to a starting variant , is not significantly reduced, i.e. has at least 80% of the reference wash performance, preferably at least 100%, more preferably at least 110% or more.

With regard to detergents, cleaning performance in the context of the invention is understood as meaning the ability of an agent to partially or completely remove existing soiling, in particular the lightening performance on one or more soilings on textiles. Examples of such stains are blood on cotton or chocolate milk/soot on cotton, cocoa on cotton or porridge on cotton. Within the scope of the invention, both the detergent which comprises the protease, or the washing liquor formed by this agent, and the protease itself have a respective cleaning performance. The cleaning performance of the protease thus contributes to the cleaning performance of the agent or the wash liquor formed by the agent. The cleaning performance is preferably determined as indicated below.

Washing liquor is understood to mean the use solution containing the detergent which acts on the textiles or fabrics and thus comes into contact with the soiling present on the textiles or fabrics. The washing liquor is usually created when the washing process begins and the detergent is diluted with water, e.g. in a washing machine or in another suitable container.

The cleaning performance can be determined in a washing system that contains a detergent in a dosage of between 2.0 and 8.0 grams per liter of wash liquor and the protease. The proteases to be compared are used in the same concentration (based on active protein).

Using the respective protease with the same activity ensures that even if the ratio of active substance to total protein (the values of the specific activity) differs, the respective enzymatic properties, ie eg the cleaning performance on certain types of soiling, are compared. Generally speaking, a low specific activity due to addition can be compensated for with a larger amount of protein. Furthermore, the enzymes to be examined can also be used in the same amount or weight if the enzymes to be examined have a different affinity for the test substrate in an activity test. In this context, the expression “same amount of substance” refers to the use of the same moles of the enzymes to be examined. The expression "equal weight" refers to an equal weight of the enzymes to be examined.

The concentration of the protease in the detergent intended for this washing system is 0.0001 to 0.1% by weight, in particular 0.001 to 0.1% by weight, more preferably 0.01 to 0.06% by weight and particularly preferred 0.001 to 0.02% by weight based on active protein.

A preferred liquid detergent for such a washing system is composed as follows (all data in percent by weight): <1% anti-foam agent, 1-4% citric acid, 0.5-3% glycerol, 0.3-2% NaOH, 4-8% 1,2-propanediol, 2-5% FAEOS (fatty alcohol ether sulfate), 5-9% nonionic surfactants (e.g. FAEO), 7-12% anionic surfactants (e.g. LAS), 0.5-1.5% protease stabilizer , 1-3% palm kernel oil fatty acids, 0.5-2% phosphonate (e.g. HEDP (1-hydroxyethane-(1,1-diphosphonic acid))), 2-6% monoethanolamine (MEA), 0.2-1% soil release polymer, remainder demineralised water. The dosage of the liquid detergent is preferably between 2.0 and 8.0 grams per liter of wash liquor, e.g. 2.5 g/L, 3.2 g/L, 3.5 g/L, 4.0 g/L, 4 .7 g/L, 4.9 g/L, 5.5 g/L or 5.9 g/L wash liquor or about 55 g/job. Washing is preferably carried out in a pH range from about 9 to about 12, preferably from about 10 to 11, particularly preferably at pH 10.

Another example of a liquid reference detergent for such a washing system can be composed as follows (all data in percent by weight): 10% anionic surfactants (e.g. LAS), 2.4% C ₁₂ -C ₁₈ Na salts of fatty acids (soaps ), 4.4% non-ionic surfactants (e.g. FAEO), 0.2% phosphonates (e.g. HEDP), 1.4% citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerol, 0.08 % preservatives, 1% ethanol, balance demineralized water. The dosage of the liquid detergent is preferably between 4.5 and 6.0 grams per liter of wash liquor, for example 4.7, 4.9 or 5.9 grams per liter of wash liquor. Washing is preferably carried out in a pH value range between pH 7 and pH 10.5, preferably between pH 7.5 and pH 8.5.

The cleaning performance is determined against soiling on cotton by measuring the degree of cleaning of the washed textiles. For example, the washing process can take place for 60 minutes at a temperature of 40° C. and the water can have a water hardness between 15.5° dH and 16.5° dH (German hardness).

The degree of whiteness, i.e. the lightening of the soiling, as a measure of the cleaning performance, is determined using optical measuring methods, preferably photometrically. A suitable device for this is, for example, the Minolta CM508d spectrometer. The devices used for the measurement are usually calibrated beforehand with a white standard, preferably a supplied white standard.

Preferred embodiments of proteases according to the invention achieve such advantageous cleaning performance even at low temperatures, in particular in the temperature ranges between 10°C and 60°C, preferably between 15°C and 50°C and particularly preferably between 20°C and 40°C.

The cleaning performance of a dishwashing detergent can be determined in a system that contains an automatic dishwashing detergent in a dosage as specified herein and the enzyme combination of protease and amylase according to the invention, the proteases to be compared being used in the same concentration (based on active protein) and the cleaning performance compared to one Soiling of tea, meat, spaghetti and/or crème brûlée is determined using the IKW method in a Miele GSL (program 45°C, 21°dH). The concentration of the enzymes in the agent intended for this washing system is in each case 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active, purified protein.

Liquid dishwashing detergent (two-component formulation): Enzyme Phase (EP) - Preparation A Active substance content in % by weight Phosphonate (e.g. HEDP), if permitted by regulation 0-7.5 CaCl ₂ 0.05-1.5 Amylase containing enzyme composition (tq) 0-4.0 Protease containing enzyme composition (tq) 0.00001-10 sorbitol 2.0-10.0 Polymer containing sulfonic acid groups 0-12.0 Thickener (based on acrylate or xanthan gum) 0.01-6.0 GLDA or MGDA 3.0-25.0 KOH 0.50-4.0 nonionic surfactants 1.0-6.0 sodium citrate 2.0-20.0 zinc salt 0-1.0 Leftovers (perfume, colorants, preservatives, water, enzyme stabilizer) (% by weight) to 100 Alkaline Phase (AP) Preparation B Phosphonate where permitted by regulation 0-7.5 thickener (acrylate or xanthan) 0.01-6.0 GLDA or MGDA 3.0-25.0 KOH 0.50-4.0 soda 5.0-20.0 monoethanolamine 0-5.0 acrylate polymer 0-3.0 sodium citrate 2.0-20.0 Leftovers (perfume, dyes, preservatives, water, etc.) (% by weight) to 100

Solid dishwashing detergent: wt% citrate, sodium salt 15-20 Phosphonate (e.g. HEDP) If permitted by regulation 0-7.5 (2.5-7.5) MGDA, Na salt 0-25 Disilicate, Na salt 5-35 soda 10-25 silver protection 0-1.0 Percarbonate, Na salt 10-15 Bleach catalyst (preferably Mn-based) 0.02-0.5 Bleach activator (e.g. TAED) 1-3 Nonionic surfactant(s), eg fatty alcohol alkoxylate, preferably 20-40 EO, optionally endcapped 2.5-10 polycarboxylate 4-10 cationic copolymer 0-0.75 Disintegrant - (ex. Crosslinked PVP) 0-1.5 Protease Preparation (tq) 0-5 Amylase Preparation (tq) 0-3 Perfume 0.05-0.25 dye solution 0.0-1 zinc salt 0.1-0.3 sodium sulfate 0-10 water 0-1.5 pH adjusting agent (e.g. citric acid) 0-1.5 process aids 0-5

Methods for determining the protease activity are familiar to the person skilled in the field of enzyme technology and are routinely used by him. For example, such methods are disclosed in Tenside, Vol. 7 (1970), pp. 125-132. 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 is carried out at a temperature of 25° C., at pH 8.6 and at a wavelength of 410 nm. The measurement time is 5 minutes and the measurement interval is 20 s to 60 s. The protease activity is usually given in protease units (PU). . Suitable protease activities are, for example, 2.25, 5 or 10 PU per ml of wash liquor. However, the protease activity is not zero.

An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measurement method, preferably a photometric method. The test suitable for this involves the protease-dependent cleavage of the substrate protein casein. This is split by the protease into a large number of smaller partial products. All of these partial products have an increased absorption at 290 nm compared to uncleaved casein, with this increased absorption being determined using a photometer and thus a conclusion being drawn as to the enzymatic activity of the protease.

The protein concentration can be determined using known methods, for example the BCA method (bicinchoninic acid; 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the Biuret method (A.G. Gornall, C.S. Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766). In this regard, the active protein concentration can be determined by titrating the active centers using a suitable irreversible inhibitor and determining the residual activity (cf. M. Bender et al., J. Am. Chem. Soc. 88, 24 (1966), p. 5890 -5913).

Another object of the invention is an agent which is characterized in that it contains a protease according to the invention as described above. The agent is preferably a washing or cleaning agent.

This subject of the invention includes all conceivable types of washing or cleaning agents, both concentrates and agents to be used undiluted, for use on a commercial scale, in the washing machine or for hand washing or hand cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which the term detergent is used. This also includes, for example, dishwashing detergents for dishwashers (automatic dishwashing detergents) or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tile, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, i.e in addition to manual and machine dishwashing detergents, for example, also scouring agents, glass cleaners, toilet deodorizers, etc. The detergents and cleaning agents within the scope of the invention also include washing aids that are added to the actual detergent in manual or machine washing in order to achieve a further effect . Furthermore, detergents and cleaning agents within the scope of the invention also include textile pre-treatment and after-treatment agents, i.e. those agents with which the item of laundry is brought into contact before the actual wash, for example to loosen stubborn dirt, and also those agents that are in one of the actual Textile washing step downstream of the laundry other desirable properties such as pleasant feel, creases confer freedom or low static charge. Fabric softeners are counted among the latter.

The washing or cleaning agents according to the invention, which can be in the form of powdered or granular solids, in compacted or post-compacted particle form, as homogeneous solutions or suspensions, can contain, in addition to a protease according to the invention, all known ingredients that are customary in such detergents, with at least one further ingredient preferably being present in the funds are available. The agents according to the invention can contain, in particular, surfactants, builders (builders), polymers, glass corrosion inhibitors, corrosion inhibitors, bleaching agents such as peroxygen compounds, bleaching activators or bleaching catalysts. They can also contain water-miscible organic solvents, other enzymes, enzyme stabilizers, sequestering agents, electrolytes, pH regulators and/or other auxiliaries such as optical brighteners, graying inhibitors, color transfer inhibitors, foam regulators and dyes and fragrances and combinations thereof.

In particular, a combination of a protease according to the invention with one or more other ingredient(s) of the agent is advantageous, since such an agent in preferred configurations according to the invention has an improved cleaning performance due to the resulting synergisms. Such a synergism can be achieved in particular by combining a protease according to the invention with a surfactant and/or a builder (builder) and/or a peroxygen compound and/or a bleach activator. However, in preferred embodiments, the agent according to the invention cannot contain any boric acid.

Advantageous ingredients of agents according to the invention are disclosed in the international patent application WO 2009/121725 , starting there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. Reference is expressly made to this disclosure and the disclosure content there is included in the present patent application.

An agent according to the invention advantageously contains the protease in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg and very particularly preferably 50 μg to 10 mg per g of the agent. In various embodiments, the concentration of the protease (active enzyme) described herein in the agent is >0 to 1% by weight, preferably 0.0001 or 0.001 to 0.1% by weight, based on the total weight of the agent or composition.

An agent according to the invention contains the protease more preferably in an amount of from 1×10 ^-8 to 5% by weight, from 0.0001 to 1% by weight, from 0.0005 to 0.5% by weight, from 0.001 to 0.1% by weight, based in each case on active protein and based on the total weight of the detergent.

Furthermore, the protease contained in the agent and/or other ingredients of the agent can be coated with a substance which is impermeable to the enzyme at room temperature or in the absence of water and which becomes permeable to the enzyme under the conditions in which the agent is used. Such an embodiment of the invention is thus characterized in that the protease is coated with a substance which is impermeable to the protease at room temperature or in the absence of water. Furthermore, the washing or cleaning agent itself can also be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.

1. (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
2. (b) in pastöser oder in flüssiger Form vorliegt, und/oder
3. (c) in gelförmiger oder dosierbeutelförmiger (Pouches) Form vorliegt, und/oder
4. (d) als Einkomponentensystem vorliegt, oder
5. (e) in mehrere Komponenten aufgeteilt ist.

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

1. (a) is in solid form, in particular as a free-flowing powder with a bulk density of 300 g/l to 1200 g/l, in particular 500 g/l to 900 g/l, or
2. (b) is in paste or liquid form, and/or
3. (c) in gel or pouch form, and/or
4. (d) is present as a one-component system, or
5. (e) is divided into several components.

These embodiments of the present invention include all solid, powdery, liquid, gel-like or pasty dosage forms of the invention, which can optionally also consist of several phases and can be in compressed or non-compressed form. The agent can be in the form of a free-flowing powder, in particular with a bulk density of 300 g/l to 1200 g/l, esp particularly 500 g/l to 900 g/l or 600 g/l to 850 g/l. The solid dosage forms of the agent also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gel-like 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. Liquid agents are generally preferred. Furthermore, the agent can be in the form of a one-component system. Such funds consist of one phase. Alternatively, a remedy can also consist of several phases. Such a remedy is therefore divided into several components.

If the detergents or cleaning agents according to the invention are in liquid form, they preferably contain more than 40% by weight, preferably 50 to 90% by weight and particularly preferably 60 to 80% by weight of water, based on their total weight.

Detergents or cleaning agents according to the invention can contain only one protease. Alternatively, they can also contain further hydrolytic enzymes or other enzymes in a concentration appropriate for the effectiveness of the agent. A further embodiment of the invention is thus represented by agents which also comprise one or more further enzymes. All enzymes which can develop a catalytic activity in the agent according to the invention can preferably be used as further enzymes, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase, Oxidase, oxidoreductase or other protease--which can be distinguished from the proteases according to the invention--and mixtures thereof. Other enzymes are advantageously contained in the agent in an amount of 1 × 10 ^-8 to 5 percent by weight based on active protein. Increasingly preferred is any additional enzyme in an amount of from 1×10 ^-7 to 3%, from 0.00001 to 1%, from 0.00005 to 0.5%, from 0.0001% by weight up to 0.1% by weight and particularly preferably from 0.0001 to 0.05% by weight in agents according to the invention, based on active protein. The enzymes particularly preferably show synergistic cleaning performance against certain types of soiling or stains, ie the enzymes contained in the agent composition support each other in their cleaning performance. Such a synergism is very particularly preferred between the protease contained according to the invention and another enzyme of an agent according to the invention, including in particular between said protease and an amylase and/or a lipase and/or a mannanase and/or a cellulase and/or a pectinase . Synergistic effects can occur not only between different enzymes, but also between one or more enzymes and other ingredients in the

In the cleaning agents described herein, the enzymes to be used can also be formulated together with accompanying substances, for example from the fermentation. In liquid formulations, the enzymes are preferably used as enzyme liquid formulation(s).

As a rule, the enzymes are not provided in the form of the pure protein, but rather in the form of stabilized preparations that can be stored and transported. These ready-made preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, particularly in the case of liquid or gel-like preparations, solutions of the enzymes, advantageously as concentrated as possible, low in water and/or mixed with stabilizers or other auxiliaries.

Alternatively, the enzymes can be encapsulated for both the solid and the liquid dosage form, for example by spray drying or extrusion of the enzyme solution together with a preferably natural polymer, or in the form of capsules, for example those in which the enzymes are enclosed as in a set gel or in those of the core-shell type, in which an enzyme-containing core is coated with a water, air and/or chemical impermeable protective layer. Additional active substances, for example stabilizers, emulsifiers, pigments, bleaching agents or dyes, can also be applied in superimposed layers. Such capsules are applied by methods known per se, for example by shaking or rolling granulation or in fluid-bed processes. Advantageously, such granules, for example due to the application of polymeric film formers, produce little dust and are stable in storage due to the coating.

Furthermore, it is possible to formulate two or more enzymes together, so that a single granulate has several enzyme activities.

The enzymes can also be incorporated into water-soluble films such as are used, for example, in the formulation of detergents and cleaning agents in unit dose form. Such a film allows the enzymes to be released upon contact with water. As used herein, refers “water soluble” refers to a film structure that is preferably completely water soluble. Such a film preferably consists of (completely or partially hydrolyzed) polyvinyl alcohol (PVA).

Another subject of the invention is a method for cleaning textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one method step, or that a protease according to the invention becomes catalytically active in at least one method step, in particular such that the protease in an amount of 40 μg to 4 g, preferably from 50 μg to 3 g, particularly preferably from 100 μg to 2 g and very particularly preferably from 200 μg to 1 g, or in the concentrations described herein.

In various embodiments, the method described above is characterized in that the protease is at a temperature of 0°C to 100°C, preferably 0°C to 60°C, more preferably 20°C to 40°C and most preferably at 25°C is used.

This includes both manual and machine methods, with machine methods being preferred due to their more precise controllability, e.g. with regard to the quantities used and exposure times. Processes for cleaning textiles are generally characterized in that various cleaning-active substances are applied to the items to be cleaned in several process steps and washed off after the exposure time, or that the items to be cleaned are treated in some other way with a detergent or a solution or dilution of this agent.

The same applies to processes for cleaning all materials other than textiles, especially hard surfaces. All conceivable washing or cleaning methods can be enriched in at least one of the method steps by the use of a washing or cleaning agent according to the invention or a protease according to the invention and then represent embodiments of the present invention.

Since proteases according to the invention naturally already have hydrolytic activity and also develop this in media that otherwise have no cleaning power, such as in mere buffer, an individual and / or the only step of such a method can consist in using a protease according to the invention as the only cleaning-active component is contacted with the soil, preferably in a buffer solution or in water. This represents a further embodiment of this subject matter of the invention.

Alternative embodiments of this subject matter of the invention also represent processes for the treatment of textile raw materials or for textile care, in which a protease according to the invention becomes active in at least one process step. Among these, processes for textile raw materials, fibers or textiles with natural components are preferred, and particularly for those with wool or silk.

Finally, the invention also covers the use of the proteases described herein in detergents or cleaning agents, for example as described above, for the (improved) removal of protein-containing soiling, for example from textiles or hard surfaces. In preferred embodiments of this use, the protease in the washing or cleaning agent is 3 or more days, 4 or more days, 7 or more days, 10 or more days, 12 or more days, 14 or more days, 21 or more days or 28 or more days stored.

• - eine Verwendung eines erfindungsgemäßen Mittels zur Reinigung von Textilien und/oder harten Oberflächen, und/oder
• - eine Verwendung einer Protease, die eine Aminosäuresequenz umfasst, die mindestens 70% Sequenzidentität mit der in SEQ ID NO:1 angegebenen Aminosäuresequenz über deren Gesamtlänge aufweist und jeweils bezogen auf die Nummerierung gemäß SEQ ID NO:1 (i) an den Positionen, die den Positionen 9, 130, 133, 144, 217, 252 und 271 entsprechen, Aminosäuresubstitutionen, die Aminosäuresubstitutionen 9T, 130D, 133A, 144K, 217M, 252T und 271E, und (ii) an mindestens einer, vorzugsweise mindestens zwei, der Positionen, die den Positionen 6 oder 166 entsprechen, mindestens eine weitere Aminosäuresubstitution, insbesondere ausgewählt aus 6W und 166M, aufweist, in einem Wasch- oder Reinigungsmittel, zur Entfernung von proteasesensitiven Anschmutzungen, insbesondere eihaltigen Anschmutzunge, auf Textilien und/oder harten Oberflächen,

insbesondere derart, dass die Protease jeweils in einer Menge von 2 µg bis 20 mg, vorzugsweise von 5 µg bis 17,5 mg, besonders bevorzugt von 20 µg bis 15 mg und ganz besonders bevorzugt von 50 µg bis 10 mg pro Gramm des Mittels eingesetzt wird.Further objects of the invention are therefore

• - A use of a composition according to the invention for cleaning textiles and/or hard surfaces, and/or
• - A use of a protease which comprises an amino acid sequence which has at least 70% sequence identity with the amino acid sequence specified in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO: 1 (i) at the positions which corresponding to positions 9, 130, 133, 144, 217, 252 and 271, amino acid substitutions corresponding to amino acid substitutions 9T, 130D, 133A, 144K, 217M, 252T and 271E, and (ii) at at least one, preferably at least two, of the positions, which correspond to positions 6 or 166, has at least one further amino acid substitution, in particular selected from 6W and 166M, in a detergent or cleaning agent for removing protease-sensitive soiling, in particular soiling containing eggs, on textiles and/or hard surfaces,

in particular such that the protease is used in an amount of 2 μg to 20 mg, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg and very particularly preferably 50 μg to 10 mg per gram of the agent will.

In a preferred embodiment, the invention also relates to the use of proteases according to the invention, i.e. proteases which comprise an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO :1 (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, in particular amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, and (b) at least one, preferably both, of the positions corresponding to positions 6 and 166, has at least one further amino acid substitution, in particular selected from 6W and 166M, for removing egg-containing soils from textiles and/or hard surfaces, in particular Dishes.

All facts, objects and embodiments that are described for the protease according to the invention and agents containing it are also expressly applicable to this object of the invention. Therefore, at this point, reference is expressly made to the disclosure at the appropriate point, with the indication that this disclosure also applies to the above use according to the invention.

examples

Overview of the mutations:

This invention relates to a subtilisin-type alkaline protease from Bacillus pumilus. Variants of this protease (wild-type Bacillus pumilus DSM18097 protease according to SEQ ID NO:1) were produced by random mutagenesis, which were then screened, inter alia, for improved washing performance and/or enzyme stability. In this way, performance-improved mutants (SEQ ID NO:1 with P9T-N130D-T133A-N144K-Y217M-S224AN252T-Q271E; starting variant) were generated from the above-mentioned wild-type protease (SEQ ID NO:1) in several rounds by random mutagenesis. Another round of random mutagenesis was set up on this mutant, and some saturation mutagenesis was carried out at certain positions. Furthermore, some mutants have been created by the targeted generation of synthetic genes. The mutants according to the invention were generated in this round of mutations. variant Amino acid substitutions relative to SEQ ID NO:1 E1 P9T-N130D-T133A-N144K-Y217M-S224A-N252T-Q271E Y6W E2 P9T-N130D-T133A-N144K-Y217M-S224A-N252T-Q271E G166M

Detergent matrix used

The following table shows the detergent matrix (commercially available, without enzymes, optical brighteners, perfume and dyes) that was used for the washing test: chemical name % by weight of active substance in the raw material % by weight of active substance in the formulation water demin. 100 rest alkyl benzene sulfonic acid 96 12-18 anionic surfactants 70 4-8 C ₁₂ -C ₁₈ fatty acid Na salt 30 2-4 Non-ionic surfactants 100 8-14 phosphonate 60 0.5-2 citric acid 100 3-5 NaOH 50 0.5-2 defoamer 100 <1% glycerin 99.5 1-3 1,2-propanediol 100 8-12 monoethanolamine 100 4-8 Soil repellent polymer 30 0.5-1 protease stabilizer 100 0.5-1.5 Without optical brighteners, perfume, dyes and enzymes; Dosage 3.17 g/L

Protease Activity Assays

The activity of the protease is determined by the release of the chromophore para-nitroaniline from the substrate succinyl alanine-alanine-proline-phenylalanine-para-nitroanilide (AAPFpNA; Bachem L-1400). 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.

The measurement was carried out at a temperature of 25° C., at pH 8.6 and at a wavelength of 410 nm. The measurement time was 5 minutes with a measurement interval of 20 to 60 seconds.

• 10 µL AAPF-Lösung (70 mg/mL)
• 1000 µL Tris/HCl (0,1 M; pH 8,6 mit 0,1% Brij 35)
• 10 µL verdünnte Proteaselösung
• Kinetik erstellt über 5 min bei 25°C (410 nm)

measurement approach:

• 10 µL AAPF solution (70 mg/mL)
• 1000 µL Tris/HCl (0.1 M; pH 8.6 with 0.1% Brij 35)
• 10 µL diluted protease solution
• Kinetics generated over 5 min at 25°C (410 nm)

Mini wash test and results

Wash test with Bacillus subtilis culture supernatants containing the screened protease mutants by heterologous expression. The supernatants are used with the same activity as the benchmark (starting variant) with a commercially available concentration for proteases in detergents. The mutants are all related to the wash performance of the wild type, which is set equal to 100% (corrected for the performance of the detergent alone).

Conditions: 40°C, 16°dH water, 1 h

soiling: 1. CFT CS038 egg yolk 2. CFT PC-10 pigment/oil/milk 3. WfK 10N egg/pigment 4. H-MR-B milk/soot

Tissues punched out (diameter=10 mm) were placed in microtiter plates, washing lye preheated to 40° C., final concentration 3.17 g/L, lye and enzyme applied to the soiling, incubated for 1 h at 40° C. and 600 rpm, then the Rinse the soiling several times with clear water, leave to dry and determine the brightness with a colorimeter. The lighter the fabric becomes, the better the cleaning performance. The L value = brightness is measured here, the higher the brighter. The cleaning performance on 4 stains is given in % based on the starting variant corrected by the performance of the detergent without protease. exit variant E1 E2 CFT CS-038 100% 123% 125% CFT PC-10 100% 218% 268% WfK 10N 100% 108% 129% H-MR-B 100% 113% 101%

All variants show a comparable or increased washing performance compared to the starting variant (which already has an increased performance compared to the wild type according to SEQ ID NO:1).

QUOTES INCLUDED IN DESCRIPTION

This list of the documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent Literature Cited

• EP 2016175 A1 [0004]
• WO 2009121725 [0098]

Claims (10)

Protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO:1 over its entire length and in each case based on the numbering according to SEQ ID NO:1 (a) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271 has amino acid substitutions, preferably amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E; such as (b) at least one, preferably both, of the positions corresponding to positions 6 and 166, at least one other amino acid substitution, preferably selected from 6W and 166M; having. protease after claim 1 , wherein the protease (a) has the amino acid substitutions 9T, 130D, 133A, 144K, 217M, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 217, 252 and 271, and (b1) at the position corresponding to position 6, the amino acid substitution 6W; (b2) at the position corresponding to position 166, the amino acid substitution G166M; or (b3) at positions corresponding to positions 6 and 166, amino acid substitutions 6W and 166M. Protease, characterized in that (a) it consists of a protease according to any one of Claims 1 until 2 is obtainable as a starting molecule by one or more conservative amino acid substitutions, the protease at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, preferably the amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E; and at least one of the positions corresponding to positions 6 or 166 has at least one other amino acid substitution; or (b) they are derived from a protease claim 1 until 2 is obtainable as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266 , 267, 268, 269, 270, 271, 272, 273 or 274 contiguous amino acids match the starting molecule, with the protease at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271, Amino acid substitutions, preferably amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E; and at least one of the positions corresponding to positions 6 or 166 has at least one other amino acid substitution. Nucleic acid encoding a protease according to any one of Claims 1 until 3 . Non-human host cell containing a nucleic acid claim 4 or a protease according to any one of Claims 1 until 3 contains. A method for producing a protease comprising a) culturing a host cell according to claim 5 ; and 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 contains at least one protease according to one of Claims 1 until 3 contains. A method for cleaning textiles or hard surfaces, characterized in that in at least one step of the process after a means claim 7 is applied. Use of a protease according to any one of Claims 1 until 3 in a washing or cleaning agent to remove soiling containing peptides or proteins. Use of a protease which comprises an amino acid sequence which has at least 70% sequence identity with the amino acid sequence specified in SEQ ID NO: 1 over its entire length and in each case based on the numbering according to SEQ ID NO: 1 (a) at the positions which correspond to the positions 9, 130, 133, 144, 217, 224, 252 and 271, amino acid substitutions, particularly amino acid substitutions 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E, and (b) on at least one, preferably both, of the Positions corresponding to positions 6 and 166 at least one more Amino acid substitution, in particular selected from 6W and 166M, for removing egg-containing stains from textiles and/or hard surfaces, in particular dishes.