Performance enhanced protease variants vii

Abstract

The present invention relates to a protease comprising an amino acid sequence having at least 70% sequence identity over its entire length to the amino acid sequence listed in SEQ ID NO: 1 and in all cases having: (a) at the positions corresponding to positions 9, 130, 133, 144, 217, 224, 252 and 271 the amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T and 271E; and (b) at least one further amino acid substitution at at least one position corresponding to positions 89, 131 and 189. The present invention also relates to the preparation and use of said protease. Such protease exhibits very good cleaning performance.

Description

[0001] This invention belongs to the field of enzyme technology. The invention relates to proteases from *Bacillus pumilus*, whose amino acid sequences have been modified to impart better cleaning properties, particularly concerning their use in detergents and cleaning agents, and also to nucleic acids encoding said proteases and their preparation. The invention further relates to the uses of these proteases and methods of using them, as well as reagents containing them, particularly detergents and cleaning agents.

[0002] Proteases are among the most important enzymes in industry. They are the oldest established enzymes in detergents and cleaning agents and are found in almost all modern, effective detergents and cleaning agents. They degrade protein-containing stains on items to be cleaned. Of these, subtilisin-type proteases (subtilisinase, subtilisin peptidase, EC3.4.21.62) are particularly important, and are serine proteases due to their catalytic activity on serine amino acids. They act as nonspecific endopeptidases and hydrolyze any amide bonds located within peptides or proteins. Their optimal pH is typically in a distinctly alkaline range. For example, a review of this family is provided in R. Siezen's article "Subtilases: Subtilisin-like Proteases," edited by R. Bott and C. Betzel, New York, 1996, on pages 75-95 of "Subtilisin Enzymes." Subtilisinases are naturally formed by microorganisms. In particular, subtilisinases formed and secreted by Bacillus species are the most important class of subtilisinases.

[0003] Examples of subtilisin proteases preferred for use in detergents and cleaning agents include subtilisin BPN' and Carlsberg, protease PB92, subtilisin 147 and 309, proteases from Bacillus lentus, particularly proteases from Bacillus lentus DSM 5483, subtilisin DY and thermophilic protease, protease K and proteases TW3 and TW7, which can be classified as subtilisin proteases but are no longer subtilisin proteases in the narrow sense, as well as variants of the proteases whose amino acid sequences have been altered relative to the starting protease. Proteases are selectively or randomly modified by methods known in the art, and thus optimized for use in, for example, detergents and cleaning agents. These methods include site mutagenesis, deletion or insertion mutagenesis, or fusion with other proteins or protein moieties. Therefore, for most proteases known in the art, appropriately optimized variants are known.

[0004] For example, European patent application EP 2016175 A1 discloses a protease from Bacillus pumilus for use in detergents and cleaning agents. Generally, in any given case, only selected proteases are suitable for use in liquid formulations containing surfactants. Many proteases do not exhibit sufficient catalytic activity in such formulations. Therefore, for the use of proteases in detergents, high catalytic activity and high storage stability under conditions during the washing cycle are particularly desirable.

[0005] Therefore, existing liquid formulations containing proteases and surfactants have the following disadvantages: the proteases contained do not have satisfactory proteolytic activity or storage instability under standard washing conditions (e.g., in the temperature range of 20°C to 40°C), and thus the formulations do not show optimal cleaning performance for protease-sensitive stains.

[0006] Surprisingly, it has now been found that proteases from Bacillus pumilus or sufficiently similar proteases (based on sequence identity) having amino acid substitutions 9T, 130D / V, 133A, 144, 217, 224, 252, and 271 at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252, and 271 according to SEQ ID NO:1, and having at least one other amino acid substitution at at least two positions corresponding to positions 89, 131, and 189, preferably at least two of which are selected from 89A / G, 131H / Y / F, and 189T / L / I, have improved cleaning properties and are therefore particularly suitable for use in detergents or cleaning agents.

[0007] In a first aspect, the invention therefore relates to a protease comprising an amino acid sequence having at least 70% sequence identity over its entire length with the amino acid sequence listed in SEQ ID NO:1, and in all cases having, based on the numbering according to SEQ ID NO:1:

[0008] (a) Amino acid substitutions at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252, and 271, particularly amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T, and 271E; and

[0009] (b) at least one other amino acid substitution at at least two of the positions corresponding to positions 89, 131 and 189, particularly selected from 89A / G, 131H / Y / F and 189T / L / I, more preferably selected from 89A, 131H and 189T.

[0010] The present invention also relates to a method for preparing a protease as defined above, the protease comprising amino acid substitutions in an initiating protease having at least 70% sequence identity over its entire length with the amino acid sequence listed in SEQ ID NO:1, wherein the amino acid substitutions are (i) at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252, and 271 in SEQ ID NO:1, such that the protease contains an amino acid substitution at said position, particularly amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T, and 271E, and (ii) at at least one, preferably at least two, positions corresponding to positions 89, 131, and 189 in SEQ ID NO:1, having at least one other amino acid substitution, particularly selected from 89A / G, 131H / Y / F, and 189T / L / I, more preferably selected from 89A, 131H, and 189T. The protease obtained by this method has at least 70% sequence identity with the amino acid sequence listed in SEQ ID NO:1 over its entire length.

[0011] Therefore, in the sense of this patent application, protease includes both the protease itself and proteases prepared by the method according to the invention. Thus, all statements concerning proteases refer to both the protease itself and proteases prepared by the corresponding method.

[0012] Other aspects of the invention relate to nucleic acids encoding these proteases, to non-human host cells containing proteases or nucleic acids according to the invention, to reagents containing proteases according to the invention, particularly detergents and cleaning agents, to washing and cleaning methods, and to the use of proteases according to the invention in detergents or cleaning agents for removing protein-containing stains.

[0013] As used in this article, "at least one" means one or more, namely 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more.

[0014] This invention is based on the inventors’ surprising discovery that amino acid substitution at the position described herein leads to improved cleaning performance of proteases in detergents and cleaning agents.

[0015] In a preferred embodiment of the protease according to the invention, the protease has substitutions selected from amino acid substitutions 89A, 89G, 131H, 131Y, 131F, 189L, 189I, and 189T at positions corresponding to positions 89, 131, and 189. Particularly preferred are 89A, 131H, and 189T. In various embodiments, the protease has corresponding substitutions at at least two of these positions. These two substitutions may preferably be those at positions: (i) 89 and 189, (ii) 131 and 189, or (iii) 89 and 131, and optionally all substitutions at all three positions 89, 131, and 189. These are preferably 89A, 131H, and 189T.

[0016] In various embodiments, the protease has

[0017] (1) Amino acid substitutions at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252, and 271, particularly amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T, and 271E; and

[0018] (2) An amino acid substitution selected from 89A / G, 131H / Y / F and 189T / L / I at at least two positions corresponding to positions 89, 131 and 189.

[0019] In various embodiments, the protease has

[0020] (1) Amino acid substitutions at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252, and 271, particularly amino acid substitutions 9T, 130D / V, 133A, 144K, 217M, 224A, 252T, and 271E; and

[0021] (2) At the location corresponding to the following positions

[0022] (a) 131 and 189, amino acids substituted for 131H and 189T;

[0023] (b) 89 and 131, amino acids substituted for 89A and 131H;

[0024] (c)89 and 189, amino acid substitutions for 89A and 189T;

[0025] (d)89, 131 and 189, amino acid substitutions of 89A, 131H and 189T.

[0026] In various embodiments of the invention, the protease has amino acid substitutions of 9T, 130D, 133A, 144, 217, 224, 252, and 271E at positions corresponding to positions 9, 130, 133, 144, 217M, 224A, 252T, and 271E, and has at positions corresponding to the following positions...

[0027] (a) 131 and 189, amino acids substituted for 131H and 189T;

[0028] (b) 89 and 131, amino acids substituted for 89A and 131H;

[0029] (c)89 and 189, amino acid substitutions for 89A and 189T;

[0030] (d)89, 131 and 189, amino acid substitutions of 89A, 131H and 189T.

[0031] In various embodiments of the invention, the protease has amino acid substitutions 9T, 130V, 133A, 144, 217, 224, 252, and 271E at positions corresponding to positions 9, 130, 133, 144, 252, and 271, and has at positions corresponding to:

[0032] (a) 131 and 189, amino acids substituted for 131H and 189T;

[0033] (b) 89 and 131, amino acids substituted for 89A and 131H;

[0034] (c)89 and 189, amino acid substitutions for 89A and 189T;

[0035] (d)89, 131 and 189, amino acid substitutions of 89A, 131H and 189T.

[0036] In various embodiments, the protease has amino acid substitutions, particularly amino acid substitutions 9T, 130D / V, 133A, 144, 217, 224, 252, and 271, at positions corresponding to positions 9, 130, 133, 144K, 217M, 224A, 252T, and 271E; and at one or more positions corresponding to positions 89, 131, or 189, at least one, for example one, two, or three, other amino acid substitutions, preferably selected from 89A / G, 131H / Y / F, and 189T / L / I, more preferably selected from 89A, 131H, and 189T.

[0037] The remaining portion of the sequence of the protease described above has sufficient sequence identity with SEQ ID NO:1, such that the total 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 protease sequence, i.e., the sequence other than the possible mutation sites mentioned herein, 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, except for the substitutions mentioned, the sequence of the protease can correspond to the sequence of SEQ ID NO:1.

[0038] In one embodiment, the protease according to the invention has a sequence identity of greater than 70% and less than 100% with SEQ ID NO:1. Preferably, the protease according to the invention has a sequence identity of greater 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% with SEQ ID NO:1. Except for the substitutions mentioned herein, the sequence of the protease may correspond to the sequence of SEQ ID NO:1.

[0039] The protease according to the invention has improved cleaning performance. In various embodiments, the protease according to the invention has at least 101%, preferably at least 102%, or greater protein hydrolysis activity based on the wild type (SEQ ID NO:1). This improved protease can achieve improved washing results for protein hydrolysis-sensitive stains over different temperature ranges, particularly between 20°C and 40°C.

[0040] Regardless of or in addition to improved cleaning performance, the proteases according to the invention may also have improved storage stability in detergents or cleaning agents. This means that, compared with wild-type enzymes, particularly the starting variants of said proteases (SEQ ID NO:2), they have comparable (i.e. ±10%) or improved stability in detergents or cleaning agents, 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.

[0041] The proteases according to the invention exhibit enzymatic catalytic activity, i.e., they are capable of hydrolyzing peptides and proteins, particularly in detergents or cleaning agents. Therefore, the proteases according to the invention are enzymes that catalyze the hydrolysis of amide / peptide bonds in protein / peptide substrates and thus are capable of cleaving proteins or peptides. Furthermore, the proteases according to the invention are preferably mature proteases, i.e., molecules without catalytically active signal peptides and / or propeptides. Unless otherwise stated, the sequences in each case also refer to mature (processed) enzymes.

[0042] In various embodiments of the invention, the protease is a free enzyme. This means that the protease can interact directly with all components of the reagent, and if the reagent is a liquid reagent, the protease is in direct contact with the solvent of the reagent (e.g., water). In other embodiments, the reagent may contain a protease that forms an interacting complex with other molecules or a protease containing a "coating layer". In this case, a single protease molecule or multiple protease molecules can be separated from other components of the reagent by the surrounding structure. Such a separating structure can be derived from, but is not limited to, vesicles, such as micelles or liposomes. The surrounding structure can also be a viral particle, a bacterial cell, or a eukaryotic cell. In various embodiments, the reagent may comprise cells of Bacillus pumilus or Bacillus subtilis expressing the protease according to the invention, or cell culture supernatants of such cells.

[0043] In various embodiments of the invention, the protease comprises an amino acid sequence that 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 in its entire length to the amino acid sequence listed in SEQ ID NO:1 (where "at least" means each of the values), and has, in each case, amino acid substitutions based on the numbers given above according to SEQ ID NO:1. Within the scope of this invention, a protease characterized by a given substitution means that it contains one of the (given) substitutions at the relevant position, i.e., at least the given position is not otherwise mutated or deleted, for example, by fragmentation of the protease. In various embodiments, in addition to the substitutions explicitly mentioned, the proteases described herein have the sequence of SEQ ID NO:1, i.e., they are 100% identical to the sequence according to SEQ ID NO:1 except for the positions of the substitutions.

[0044] The identity of nucleic acid or amino acid sequences is determined by sequence comparison. This sequence comparison is based on the BLAST algorithm, which has been established and is commonly used in the prior art (see, 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, pp. 3389-3402), and is in principle performed by assigning similar nucleotides or amino acids in the nucleic acid or amino acid sequence to each other. The tabular assignment of relevant positions is called alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence alignment, especially multiple sequence alignment, is performed using computer programs. For example, the Clustal series (see, for example, Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500), T-Coffee (see, 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 are frequently used. Vector, a computer program with predefined default parameters, is also used. Sequence alignment using Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) and the ClustalW-based AlignX module for sequence alignment are also acceptable. Unless otherwise stated, sequence identity presented herein is determined using the BLAST algorithm.

[0045] This comparison also allows for conclusions regarding the similarity of the compared sequences. It is typically given as a percentage of identity, i.e., the proportion of identical nucleotide or amino acid residues in the alignment of the sequence or corresponding position. A broader concept of homology, in the case of amino acid sequences, considers conserved amino acid exchanges, i.e., amino acids with similar chemical activities, as they generally perform similar chemical activities in proteins. Therefore, the similarity between compared sequences can also be expressed as a percentage of homology or similarity. Information on identity and / or homology can be provided about the entire polypeptide or gene or only about individual regions. Thus, homologous or identical regions of different nucleic acid or amino acid sequences are defined by matching within the sequence. Such regions typically have the same function. They can be small and contain only a few nucleotides or amino acids. Often, such small regions play a significant role in the overall activity of the protein. Therefore, it may be advantageous for sequence matching to involve only a single, optionally small region. However, unless otherwise stated, the identity or homology information in this application refers to the total length of the specific nucleic acid or amino acid sequence indicated.

[0046] Within the scope of this invention, the statement that an amino acid position corresponds to the position of the numerical marker in SEQ ID NO:1 therefore means that, in the alignment as defined above, the corresponding position is associated with the position of the numerical marker in SEQ ID NO:1.

[0047] In another embodiment of the invention, the protease is characterized in that its cleaning performance (after storage, e.g., more than 4 weeks) is not significantly reduced compared to a protease containing an amino acid sequence corresponding to the amino acid sequence given in SEQ ID NO:2, i.e., it has at least 80%, preferably at least 100%, and more preferably at least 110% or more of the reference washing performance. The cleaning performance can be determined in a washing system containing a detergent at a dosage between 4.5 and 7.0 grams per liter of washing liquid, and the protease and the protease to be compared are used at the same concentration (based on active protein), and the cleaning performance on stains on cotton fabric is determined by measuring the degree of cleanliness of the washed textiles. For example, the washing process can be carried out at a temperature of 40°C for 60 minutes, and the water has a water hardness between 15.5 and 16.5 (German hardness). The concentration of the protease in the detergent used in this washing system is 0.001-0.1% by weight, preferably 0.01-0.06% by weight, based on active purified protein.

[0048] The liquid reference detergent used in this washing system may have the following composition (all data are by weight percentage): 4.4% alkylbenzene sulfonic acid, 5.6% other anionic surfactants, 2.4% C 12 -C 18The detergent comprises: fatty acid sodium salt (soap), 4.4% nonionic surfactant, 0.2% phosphonate, 1.4% citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerin, 0.08% preservative, 1% ethanol, and the balance being softened water. Preferably, the liquid detergent is administered at a concentration of 4.5 to 6.0 g / L of detergent solution, for example, 4.7, 4.9, or 5.9 g / L. Washing is preferably performed at a pH range of 7 to 10.5, more preferably 7.5 to 8.5.

[0049] Within the scope of this invention, cleaning performance is measured, for example, at 20°C or 40°C, using the liquid detergent described above, wherein the washing process is preferably performed at 600 rpm for 60 minutes.

[0050] Whiteness, a measure of cleaning performance, i.e., the degree of stain reduction, is determined by optical measurement methods, preferably photometry. A suitable apparatus for this purpose is, for example, the Minolta CM508d spectrometer. Typically, the apparatus used for measurement is pre-calibrated with a white standard, preferably a provided white standard.

[0051] Using corresponding proteases with comparable activity ensures that the properties of the corresponding enzymes, such as their cleaning performance on certain stains, can be compared even when there are differences in the ratio of active substance to total protein (specific activity value). Typically, low specific activity can be compensated for by adding a larger amount of protein.

[0052] Furthermore, methods for determining protease activity are well-known and frequently used by those skilled in the art of enzyme technology. For example, such methods are disclosed in Tenside, Vol. 7 (1970), pp. 125–132. Alternatively, protease activity can be determined by releasing the chromophore p-nitroaniline (pNA) from the substrate Suc-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroaniline (AAPF). The protease cleaves the substrate and releases pNA. The release of pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzyme activity (see Del Mar et al., 1979). Measurements are performed at 25 °C, pH 8.6, and a wavelength of 410 nm. Measurements are taken over 5 min at intervals of 20 to 60 s. Protease activity is typically expressed in protease units (PE). For example, a suitable total protease activity is 2.25, 5, or 10 PE / ml wash buffer. However, protease activity is not equal to zero.

[0053] An alternative test for determining the proteolytic activity of the protease according to the invention is an optical measurement method, preferably a photometric method. Suitable tests include protease-dependent cleavage of the substrate protein casein. Casein is cleaved by the protease into many smaller fractional products. Compared to uncleaved casein, these fractional products collectively exhibit increased absorbance at 290 nm, and this increased absorbance can be measured using a photometer to draw conclusions about the enzymatic activity of the protease.

[0054] Protein concentrations can be determined using known methods, such as the BCA method (dioctoctanic acid; 2,2'-bisquinolino-4,4'-dicarboxylic acid) or the biuret method (AGGornall, C.S. Bardawill and M.M. David, J. Biol. Chem., 177 (1948), pp. 751-766). In this regard, the concentration of active proteins can be determined by titrating the active site with a suitable irreversible inhibitor and determining the residual activity (see M. Bender et al., J. Am. Chem. Soc. 88, 24 (1966), pp. 5890-5913).

[0055] In addition to the amino acid alterations discussed above, the proteases according to the invention can have other amino acid alterations, particularly amino acid substitutions, insertions, or deletions. For example, such proteases are developed by targeting genetic alterations, i.e., by mutagenesis, and optimized for specific applications or for specific properties (e.g., their catalytic activity, stability, etc.). Furthermore, the nucleic acids according to the invention can be introduced into recombinant methods, thus enabling the generation of entirely new types of proteases or other polypeptides.

[0056] The aim is to introduce targeted mutations, such as substitutions, insertions, or deletions, into known molecules to, for example, improve the cleaning properties of enzymes according to the invention. For this purpose, in particular, the surface charge and / or isoelectric point of the molecule can be altered, thereby changing their interaction with the substrate. For example, the net charge of the enzyme can be altered to affect substrate binding, particularly for detergents and cleaning agents. Alternatively, one or more corresponding mutations can increase the stability or catalytic activity of the protease, thereby improving its cleaning properties. The advantageous properties of a single mutation, such as a single substitution, can be complementary. Therefore, within the scope of the invention, proteases already optimized in terms of specific properties, such as stability during storage, can also be developed.

[0057] To describe substitutions (amino acid replacements) involving exactly one amino acid position, the following convention is used: First, the naturally occurring amino acid is represented by its internationally used single-letter code, followed by the relevant sequence position, and finally the inserted amino acid. Multiple or alternative substitutions within the same polypeptide chain are separated by slashes. Thus, "130D / V" indicates that the residue at position 130 can be either D or V. For insertions, the additional amino acid is named according to its sequence position. In the case of deletions, the deleted amino acid is replaced by a symbol such as an asterisk or dash, or indicated by Δ before the corresponding position. For example, P9T describes the substitution of proline at position 9 by threonine, P9TH describes the insertion of histidine after threonine at position 9, and P9* or ΔP9 describes the deletion of proline at position 9. This nomenclature is known to those skilled in the art of enzyme technology.

[0058] Therefore, the present invention also relates to a protease, characterized in that it can be obtained from a protease as described above, which serves as a starting molecule, by one or more conserved amino acid substitutions, said protease having the aforementioned amino acid substitutions in SEQ ID NO:1. The term "conserved amino acid substitution" refers to the replacement (substitution) of one amino acid residue with another amino acid residue, such replacement not resulting in a change in the polarity or charge of the substituted amino acid position, for example, the replacement of one nonpolar amino acid residue with another nonpolar amino acid residue. Conserved amino acid substitutions within the scope of the present 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.

[0059] Alternatively or additionally, the protease is characterized in that it can be obtained from the protease according to the invention as a starting molecule by fragmentation or deletion, insertion or substitution mutagenesis, and contains an amino acid sequence that matches the length of the starting molecule for at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 or 274 consecutive amino acids, still containing the aforementioned amino acid substitutions, namely substitutions 9T, 130D / V, 133A, 144K, 217M, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 217, 252 and 271, and other amino acid substitutions at at least one, preferably at least two, positions corresponding to positions 6, 89, 131, 166, 189, 211 or 224.

[0060] For example, a single amino acid can be deleted from the end or loop of an enzyme without loss or reduction of proteolytic activity. Furthermore, such fragmentation or deletion, insertion, or substitution mutagenesis can, for example, reduce the allergenicity of the relevant enzymes, thereby improving their overall suitability. Advantageously, the enzymes retain their proteolytic activity after mutagenesis, i.e., their proteolytic activity corresponds at least to the proteolytic activity 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 starting enzyme activity. Other substitutions can also show beneficial effects. Single and multiple consecutive amino acids can be replaced by other amino acids.

[0061] In this case, other amino acid positions are defined by comparing the amino acid sequence of the protease according to the invention with the amino acid sequence of a protease from *Bacillus pumilus* (as given in SEQ ID NO:1). Furthermore, the allocation of positions depends on the mature protein. This allocation is also used in particular if the amino acid sequence of the protease according to the invention contains a greater number of amino acid residues than the protease from *Bacillus pumilus* according to SEQ ID NO:1. Starting from the aforementioned positions in the amino acid sequence of the protease from *Bacillus pumilus*, the altered positions in the protease according to the invention are those precisely assigned to these positions in the alignment.

[0062] Therefore, for sequence alterations, particularly substitutions, in proteases from *Bacillus pumilus*, advantageous positions are those corresponding to the positions described herein in the alignment (i.e., in the numbering according to SEQ ID NO:1), which are particularly important when transferred to homologous positions of the proteases according to the invention and confer advantageous functional properties upon the proteases. At the mentioned positions, the wild-type molecule of the proteases from *Bacillus pumilus* contains the following amino acid residues: P9, N130, T133, N144, Y217, S224, N252, and Q271, as well as S89, G131, and S189.

[0063] Comparative experiments can further confirm the correct allocation of the amino acids to be modified, specifically their functional correspondence. According to this comparative experiment, two positions based on the matched allocation are altered in the same manner in two compared proteases, and the enzyme activity is observed to change in the same way in both cases. For example, if a specific amino acid substitution at a particular position in a protease from *Bacillus pumilus* according to SEQ ID NO:1 is accompanied by a change in enzyme parameters, such as K... M The value increases, and corresponding changes in enzyme parameters, such as K, are observed in protease variants according to the invention that have achieved amino acid substitutions through the same introduction of amino acids. M An increase in the value can be considered a confirmation of a correct allocation.

[0064] All these aspects are also applicable to the method for preparing proteases according to the present invention. Therefore, the method according to the present invention further comprises one or more of the following method steps:

[0065] a) Introducing one or more conserved amino acid substitutions into a protease, the protease having substitutions 9T, 130D / V, 133A, 144, 217, 224, 252T, and 271E at positions corresponding to positions 9, 130, 133, 144, 217M, 224A, 252T, and 271E; and having at least one other amino acid substitution at at least two positions corresponding to positions 89, 131, and 189;

[0066] b) The amino acid sequence is altered by fragmentation or deletion, insertion or substitution mutagenesis such that the protease comprises an amino acid sequence matching the length of the starting molecule for at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 or 274 consecutive amino acids, the protease comprising substitutions of 9T, 130D / V, 133A, 144K, 217M, 224A, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252T and 271, and at least one other amino acid substitution at at least one or at least two positions corresponding to positions 89, 131 and 189.

[0067] All implementation methods are also applicable to the method according to the present invention.

[0068] In other embodiments of the invention, the protease or the protease prepared by the method according to the invention is identical in length to 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% of the amino acid sequence given in SEQ ID NO:1. Alternatively, the protease or the protease prepared by the method according to the invention is identical in its entire length to 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%, 97%, 97.5%, 98%, 98.5%, or 99%. The protease or the protease prepared by the method according to the invention has amino acid substitutions of 9T, 130D / V, 133A, 144, 224, 252, and 271E at positions corresponding to positions 9, 130, 133, 144, 224, 252, and 271; and at least one, preferably at least two, other amino acid substitutions at at least one position corresponding to positions 89, 131, and 189, in each case based on the numbering according to SEQ ID NO:1. Examples of such amino acid substitution variants are P9T, N130D, T133A, N144K, Y217M, S224A, N252T, and Q271E in combination with one of the following: (i) S89A and S189T; (ii) S89A and G131H; and (iii) S89A, G131H, and S189T. Alternatively, examples are those of P9T, N130V, T133A, N144K, Y217M, S224A, N252T, and Q271E in combination with S89A, G131H, and S189T, each based on the numbering according to SEQ ID NO:1, and the variants described in the examples. Generally, in various embodiments, each of the proteases described herein may also contain a substitution at least at position 89, particularly S89A, in addition to 9T, 130D / V, 133A, 144K, 217M, 224A, 252T, and 271E at positions corresponding to positions 9, 130, 133, 144, 224, 252, and 271.

[0069] The present invention also relates to the proteases described above, which are further stabilized, particularly by one or more mutations, such as substitution, or by coupling with a polymer. Improved stability during storage and / or during use (e.g., during washing) results in longer-lasting enzyme activity, thus improving cleaning performance. In principle, all stabilization options described and / or suitable in the prior art can be considered. Those stabilizations achieved by mutation of the enzyme itself are preferred because such stabilization does not require any further working steps after enzyme recovery. Examples of sequence alterations suitable for this purpose are as described above. Other suitable sequence alterations are known in the prior art.

[0070] Other possibilities for stabilization include:

[0071] - Altering the binding of metal ions, particularly calcium binding sites, for example by replacing one or more amino acids involved in calcium binding with one or more negatively charged amino acids and / or by introducing sequence alterations in at least one of the sequences of the two amino acids arginine and glycine.

[0072] - Preventing the effects of denaturing agents such as surfactants by causing mutations that alter the amino acid sequence on or at the surface of proteins;

[0073] - Replace the amino acids near the N-terminus with those that can come into contact with the rest of the molecule through non-covalent interactions, thus helping to maintain the globular structure.

[0074] The preferred embodiments are those in which the enzyme is stable in several ways, because several stable mutations are additive or synergistic.

[0075] The present invention also relates to a protease as described above, characterized in that it has at least one chemical modification. A protease having such a modification is called a derivative, i.e., the protease is derivatized.

[0076] Within the scope of this application, derivatives are therefore understood to refer to proteins whose pure amino acid chains have been chemically modified. This derivatization can be achieved in vivo, for example, through host cells expressing the protein. In this regard, conjugation with low molecular weight compounds such as lipids or oligosaccharides is particularly noteworthy. However, derivatization can also be carried out in vitro, for example, through the chemical transformation of amino acid side chains or through the covalent binding of another compound to the protein. For example, an amine can be conjugated to the carboxyl group of an enzyme to alter its isoelectric point. Another such compound can also be another protein, which, for example, binds to the protein according to the invention through a bifunctional compound. Derivatization can also be understood as covalently binding to a macromolecular carrier or non-covalently incorporating it into a suitable macromolecular cage structure. For example, when the conjugated substance is an inhibitor, derivatization can affect the specificity or binding strength of the substrate, or result in a temporary blockade of enzyme activity. This may be advantageous, for example, for storage duration. Such modifications can further affect stability or enzyme activity. They can also be used to reduce the allergenicity and / or immunogenicity of proteins, thereby, for example, improving their skin compatibility. For example, coupling with macromolecular compounds such as polyethylene glycol can improve protein stability and / or skin compatibility.

[0077] The protein derivatives according to the invention can also be understood in the broadest sense as referring to formulations of these proteins. Depending on the recycling, processing, or preparation, the protein can be combined with a variety of other substances, such as cultures from producing microorganisms. The protein can also be intentionally added to other substances, for example, to increase its storage stability. Therefore, all formulations of the proteins according to the invention are also according to the invention. This is also unrelated to whether it actually exhibits this enzymatic activity in a particular formulation. This is because it may be desirable for it to be inactive or have only low activity during storage and to exhibit its enzymatic function only upon use. This can be controlled, for example, by suitable concomitant substances. In particular, in this respect, formulations combining proteases with specific inhibitors are possible.

[0078] Of all the above-described proteases or protease variants and / or derivatives, within the scope of the invention, those whose storage stability corresponds at least to at least one of the proteases according to SEQ ID NO:2 or the variants tested in the examples, and / or whose cleaning performance corresponds at least to at least one of the proteases according to SEQ ID NO:2 or the variants tested in the examples, wherein the cleaning performance is determined in the washing system as described above.

[0079] The present invention also relates to nucleic acids encoding proteases according to the invention, and vectors containing such nucleic acids, particularly cloning vectors or expression vectors.

[0080] These can be DNA or RNA molecules. They can exist as single-stranded, as a single-stranded complement to that single-stranded, or as double-stranded. Especially in the case of DNA molecules, the sequences of the two complementary strands must be considered in all three possible reading frames. Furthermore, it should be noted that different codons, i.e., base triplets, can encode the same amino acid, such that a particular amino acid sequence can be encoded by multiple different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences that can encode any of the aforementioned proteases are included in this subject matter of the invention. Those skilled in the art can determine these nucleic acid sequences without question because, despite the degeneracy of the genetic code, defined amino acids can be assigned to individual codons. Thus, those skilled in the art can readily determine the nucleic acid encoding the amino acid sequence starting from the 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 particularly relates to the heterologous expression of enzymes according to the invention. Thus, each organism, such as the host cell of the producing strain, has a specific codon usage. Codon usage is understood to refer to the relevant organism translating the genetic code into amino acids. If the codons located on the nucleic acids in an organism face a relatively small number of loaded tRNA molecules, a bottleneck may occur in protein biosynthesis. Although they encode the same amino acid, this results in codons being translated less efficiently in organisms than synonymous codons that encode the same amino acid. Because there are more tRNA molecules associated with synonymous codons, they can be translated more efficiently in organisms.

[0081] Using well-known methods, such as chemical synthesis or polymerase chain reaction (PCR), combined with standard methods of molecular biology and / or protein chemistry, those skilled in the art can prepare corresponding nucleic acids or even complete genes based on known DNA and / or amino acid sequences. Such methods are known, for example, from Sambrook, J., Fritsch, EF, and Maniatis, T. 2001. *Molecular cloning: a laboratory manual*, 3rd edition, Cold Spring Laboratory Press.

[0082] Within the meaning of this invention, a vector is understood to refer to an element composed of nucleic acids containing, as characteristic nucleic acid regions, the nucleic acids according to the invention. These are capable of establishing themselves as stable genetic elements within a species or cell line through several reproductions or cell divisions. Vectors are special plasmids, i.e., circular genetic elements, particularly when used in bacteria. Within the scope of this invention, nucleic acids according to the invention are cloned into vectors. Vectors include, for example, those derived from bacterial plasmids, viruses, or bacteriophages, or primarily synthetic vectors or plasmids containing elements from multiple sources. Because additional genetic elements are present in each case, vectors are capable of establishing themselves as stable units in the corresponding host cells after several reproductions. They can exist as separate units in an extrachromosomal manner or be integrated into chromosomes or chromosomal DNA.

[0083] Expression vectors contain nucleic acid sequences that enable them to replicate in host cells containing them, preferably microorganisms, particularly bacteria, and express the contained nucleic acids therein. Expression is particularly influenced by promoters that regulate transcription. In principle, expression can be carried out by a natural promoter initially located before the nucleic acid to be expressed, by a promoter provided on the host cell in the expression vector, or by a modified or entirely different promoter from another organism or another host cell. In the case of the present invention, at least one promoter for expressing the nucleic acid according to the invention is provided and used for expression. Furthermore, expression vectors can be adjustable, for example by changing culture conditions or when a specific cell density of host cells containing them is reached, or by adding specific substances, particularly activators of gene expression. An example of such a substance is the galactose derivative isopropyl-β-D-thiogalactoside (IPTG), which is used as an activator of the bacterial lactose operon (lac operon). In contrast to expression vectors, the contained nucleic acids are not expressed in cloning vectors.

[0084] This invention also relates to non-human host cells containing nucleic acids or vectors according to the invention, or proteases according to the invention, particularly host cells that secrete proteases into a culture medium surrounding the host cell. Preferably, the nucleic acid or vector according to the invention is transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, a single component, i.e., a nucleic acid portion or fragment of the nucleic acid according to the invention, can be introduced into a host cell, such that the resulting host cell contains the nucleic acid or vector according to the invention. This procedure is particularly suitable when the host cell already contains one or more components of the nucleic acid or vector according to the invention and is subsequently supplemented accordingly. Methods for transforming cells have been established in the prior art and are well known to those skilled in the art. In principle, all cells, i.e., prokaryotic or eukaryotic cells, are suitable as host cells. Preferred host cells are those that can be treated in a genetically advantageous manner, for example, in terms of transformation with nucleic acids or vectors and their stable establishment, such as single-celled fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological manageability. For example, this involves ease of culture, high growth rates, low requirements for fermentation media, and good production and secretion rates of exogenous proteins. According to the preferred host cells of the invention, the (transgenic) expressed protein is secreted into a culture medium surrounding the host cell. Furthermore, proteases can be modified by the cells that produce them after they are produced, such as through sugar linkage, formylation, and amination. These post-translational modifications can functionally affect proteases.

[0085] Other preferred embodiments are those host cells whose activity is moduloable due to genetic regulatory elements, which may be derived from a vector, but may also be present in these cells from the outset. These host cells can be induced to express the protein by, for example, the controlled addition of a compound acting as an activator, by altering culture conditions, or by reaching a specific cell density. This makes it possible to economically produce the protein according to the invention. An example of such a compound, as described above, is IPTG.

[0086] The preferred host cells are prokaryotic or bacterial cells. Bacteria are characterized by short generation times and low requirements for culture conditions. Therefore, cost-effective culture or production methods can be established. Furthermore, those skilled in the art have extensive experience with bacteria used in fermentation technologies. For specific production processes, Gram-negative or Gram-positive bacteria may be suitable, due to various reasons that need to be determined experimentally in individual cases, such as nutrient sources, product formation rates, and time requirements.

[0087] In the case of Gram-negative bacteria such as *Escherichia coli*, large amounts of protein are secreted into the periplasmic space, that is, into the compartment between the two membranes surrounding the cell. This can be advantageous for specific applications. Furthermore, Gram-negative bacteria can be engineered to not only expel expressed proteins into the periplasmic space but also into the culture medium surrounding the bacteria. In contrast, Gram-positive bacteria such as *Bacillus* or *Actinomycetes*, or other representatives of actinomycetes, lack an outer membrane, so the secreted proteins are immediately released into the culture medium surrounding the bacteria, typically a nutrient medium, from which the expressed proteins can be purified. They can be isolated directly from the medium or further processed. Moreover, Gram-positive bacteria are biologically related to or identical to most sources of industrially important enzymes, and often even form comparable enzymes, indicating similar codon usage and correspondingly natural alignment of protein synthesizers.

[0088] The host cells according to the invention can have their culture requirements modified, can have different or additional selection markers, or can express other or additional proteins. In particular, this may also involve those host cells that are transgenic and express several proteins or enzymes.

[0089] In principle, the present invention can be used with all microorganisms, particularly all fermentable microorganisms, especially those of the genus Bacillus, and results in the preparation of proteins according to the invention by using these microorganisms. This microorganism then represents a host cell in the sense of the present invention.

[0090] In another embodiment of the invention, the host cell is characterized in that it is a bacterium, preferably selected from the group consisting of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas, and Pseudomonas, more preferably selected from the group consisting of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, and Bacillus coccidioidomyces. It is a member of the group consisting of *Bacillus globigii*, *Bacillus gibsonii*, *Bacillus clausii*, *Bacillus halodurans*, *Bacillus pumilus*, *Staphylococcus carnosus*, *Corynebacterium glutamicum*, *Arthrobacter oxidans*, *Streptomyces lividans*, *Streptomyces coelicolor*, and *Stenotrophomonas maltophilia*.

[0091] However, the host cell can also be a eukaryotic cell, characterized by having a nucleus. Therefore, the invention also relates to host cells characterized by having a nucleus. Unlike prokaryotic cells, eukaryotic cells are capable of post-translational modification of the proteins formed. Examples include fungi such as actinomycetes or yeasts such as *Saccharomyces* or *Kluyveromyces*. This can be particularly advantageous, for example, if the protein is to undergo specific modifications related to its synthesis, thus making such a system possible. Modifications performed in eukaryotic systems, particularly those related to protein synthesis, include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to reduce the allergenicity of the expressed protein. Co-expression with naturally occurring cell enzymes such as cellulase may also be advantageous. Furthermore, thermophilic fungal expression systems, for example, can be particularly suitable for expressing heat-resistant proteins or variants.

[0092] The host cells according to the invention are cultured and fermented in a conventional manner, for example in batch or continuous systems. In the first case, the host cells are inoculated into a suitable nutrient medium, and the product is harvested from the medium after an experimentally determined period of time. Continuous fermentation is characterized by reaching flow equilibrium, in which cells partially die over a relatively long period but also regrow, while the proteins formed can be removed from the medium.

[0093] Preferably, the host cell according to the invention is used to prepare the protease according to the invention. Therefore, the invention also relates to a method for preparing the protease, comprising:

[0094] a) Culturing host cells according to the present invention, and

[0095] b) Isolate the protease from the culture medium or host cell.

[0096] This subject matter of the invention preferably includes fermentation methods. Fermentation methods are known in the art and represent a practical large-scale production step, typically followed by a suitable purification method for the prepared product, such as the protease according to the invention. All fermentation methods based on the corresponding methods for preparing the protease according to the invention constitute embodiments of this subject matter of the invention.

[0097] Fermentation methods characterized by a feed-based fermentation strategy should be given special consideration. In this case, the medium components consumed in continuous culture are added. This can significantly increase cell density and cell quality or dry weight, and / or particularly the activity of proteases of interest. Furthermore, fermentation can be designed to filter out or neutralize unwanted metabolites by adding buffers or suitable counterions.

[0098] The prepared protease can be harvested from the fermentation medium. This fermentation method is superior to isolating the protease from the host cell, i.e., preparing the product from cell masses (dry matter), but requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems to enable the host cell to secrete the protease into the fermentation medium. Alternatively, in the absence of secretion, the protease can be isolated from the host cell, i.e., purified from the cell mass, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

[0099] All of the above aspects can be combined into a method to prepare the protease according to the invention.

[0100] The present invention also relates to a reagent characterized in that it contains the protease according to the invention as described above. The reagent is preferably a detergent or cleaning agent.

[0101] This invention covers all possible types of detergents or cleaning agents, including concentrates and undiluted agents, for commercial use, for use in washing machines, or for hand washing or cleaning. These include, for example, detergents for textiles, carpets, or natural fibers, for which the term detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents, or cleaning agents for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood, or leather, for which the term cleaning agent is used, i.e., in addition to manual and machine dishwashing detergents, including, for example, scrubbing agents, glass cleaners, toilet air fresheners, etc. The detergents and cleaning agents according to the invention also include auxiliary detergents added to the actual detergent during manual or machine textile washing to achieve further effects. Furthermore, the detergents and cleaning agents according to the invention also include textile pretreatment agents and posttreatment agents, i.e., those agents that come into contact with the laundry before the actual washing cycle, such as agents for loosening stubborn dirt, and those agents that impart further desired properties to the laundry in steps after the actual textile washing, such as a comfortable feel, wrinkle resistance, or low static charge. In particular, the reagents mentioned last include softeners.

[0102] The detergents or cleaning agents according to the invention can exist as powdered solids, in the form of further compressed granules, as homogeneous solutions or suspensions, and may contain all known ingredients commonly used in such reagents, except for the protease according to the invention, wherein at least one other ingredient is preferably present in the reagent. Reagents according to the invention may particularly contain surfactants, builders, peroxides, or bleaching activators. They may also contain water-miscible organic solvents, other enzymes, multivalent chelating agents, electrolytes, pH adjusters, and / or other auxiliaries such as fluorescent whitening agents, graying inhibitors, foam modifiers, dyes, and fragrances, and combinations thereof.

[0103] In particular, combinations of the protease according to the invention with one or more other components of the reagent are advantageous because, in a preferred embodiment according to the invention, such a reagent exhibits improved cleaning performance due to a synergistic effect. Specifically, such a synergistic effect can be achieved by combining the protease according to the invention with surfactants and / or detergent builders and / or peroxides and / or bleaching activators. However, in a preferred embodiment, the reagent according to the invention may be free of boric acid.

[0104] The advantageous components of the reagent according to the invention are disclosed in International Patent Application WO 2009 / 121725, beginning on page 5, second to last paragraph, and ending on page 13, after the second paragraph. This disclosure is expressly referenced and incorporated herein by reference.

[0105] The reagent according to the invention advantageously contains the protease in an amount of 2 μg to 20 mg per gram of reagent, preferably 5 μg to 17.5 mg, particularly preferably 20 μg to 15 mg, and most particularly preferably 50 μg to 10 mg. In various embodiments, the concentration of the protease (active enzyme) described herein in the reagent is >0 to 1% by weight, preferably 0.0001 or 0.001 to 0.1% by weight, based on the total weight of the reagent or composition. Furthermore, the protease contained in the reagent and / or other components of the reagent may be encapsulated in a substance impermeable to the enzyme at room temperature or in the absence of water, which becomes permeable to the enzyme under the conditions of use of the reagent. Thus, this embodiment of the invention is characterized in that the protease is encapsulated in a substance impermeable to the protease at room temperature or in the absence of water. Additionally, the detergent or cleaning agent itself may also be packaged in a container, preferably a breathable container, from which the detergent or cleaning agent is released shortly before use or during washing.

[0106] In other embodiments of the invention, the reagent is characterized in that it

[0107] (a) Existing in solid form, particularly as a free-flowing powder having a bulk density of 300 g / L to 1200 g / L, especially 500 g / L to 900 g / L, or

[0108] (b) exists in paste or liquid form, and / or

[0109] (c) Present in gel or metering pouch form, and / or

[0110] (d) Exists as a single-component system, or

[0111] (e) It is divided into multiple components.

[0112] These embodiments of the invention encompass all solid, powder, liquid, gel, or paste application forms of the reagent according to the invention, optionally also comprising a multiphase composition, and may be present in compressed or uncompressed form. The reagent may be present as a free-flowing powder, particularly having a bulk density of 300 g / L to 1200 g / L, particularly 500 g / L to 900 g / L or 600 g / L to 850 g / L. Solid application forms of the reagent also include extrudates, granules, tablets, or sachets. Alternatively, the reagent may also be liquid, gel-like, or paste-like, 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 an aqueous paste. Liquid reagents are generally preferred. Furthermore, the reagent may exist as a single-component system. Such a reagent consists of one phase. Alternatively, the reagent may also consist of multiple phases. Thus, such a reagent is divided into multiple components.

[0113] Detergents or cleaning agents according to the invention may contain only one protease. Alternatively, they may also contain other hydrolases or other enzymes at concentrations favorable to the effectiveness of the reagent. Therefore, another embodiment of the invention is a reagent that also contains one or more other enzymes. Preferably, the other enzymes that can be used are all enzymes capable of catalytic activity in the reagent according to the invention, particularly lipases, amylases, cellulases, hemicellulases, mannanases, tanninases, xylanases, xanthan gumases, xyloglucanases, β-glucosidases, pectinsases, carrageenans, peroxyhydrolases, oxidases, oxidoreductases, or other proteases different from the protease according to the invention, and mixtures thereof. Based on the active protein, the other enzymes are advantageously present in concentrations of 1 × 10⁻⁶. -8 It is present in the reagent in an amount of up to 5% by weight. More preferably, based on the active protein, each other enzyme is present in an amount of 1 × 10⁻⁶. -7 The reagent according to the invention is present in amounts of up to 3% by weight, 0.00001 to 1% by weight, 0.00005 to 0.5% by weight, 0.0001 to 0.1% by weight, and particularly preferably 0.0001 to 0.05% by weight. Particularly preferably, the enzymes exhibit synergistic cleaning properties against certain stains or blemishes, i.e., the enzymes contained in the reagent composition support each other in terms of cleaning performance. Very particularly preferably, this synergistic effect exists between the protease contained according to the invention and other enzymes in the reagent according to the invention, including, in particular, between the protease and amylase and / or lipase and / or mannanase and / or cellulase and / or pectinase. Synergistic effects can occur not only between different enzymes but also between one or more enzymes and other components of the reagent according to the invention.

[0114] In the cleaning agents described herein, the enzyme to be used may also be formulated with, for example, a byproduct of fermentation. In liquid formulations, the enzyme is preferably used as an enzyme liquid formulation.

[0115] Enzymes are typically not provided as pure proteins, but rather as stable, storable, and transportable formulations. These pre-formulated formulations include, for example, solid formulations obtained by granulation, extrusion, or lyophilization, or, particularly in the case of liquid or gel reagents, solutions of enzymes, advantageously concentrated as much as possible, with low water content, and / or mixed with stabilizers or other adjuvants.

[0116] Alternatively, for both solid and liquid application forms, the enzyme can be encapsulated, for example by spray-drying or extruding the enzyme solution together with a preferred natural polymer, or encapsulated in the form of capsules, such as those in which the enzyme is encapsulated in a solidified gel, or core-shell type in which the enzyme-containing core is covered by a protective layer that is impermeable to water, air, and / or chemicals. Other active ingredients, such as stabilizers, emulsifiers, pigments, bleaching agents, or dyes, may be additionally applied to the covering layer. Such capsules are applied by methods known per se, such as by shaking or roller granulation or in a fluidized bed process. Advantageously, such particles have low dust content, for example by the application of a polymeric film-forming agent, and exhibit storage stability due to encapsulation.

[0117] In addition, two or more enzymes can be formulated together, so that a single particle can exhibit multiple enzyme activities.

[0118] Enzymes can also be incorporated into water-soluble membranes, such as those used in the formulation of unit-dose detergents and cleaning agents. Such membranes allow the enzyme to be released upon contact with water. As used herein, "water-soluble" refers to a membrane structure that is preferably fully water-soluble. Preferably, such a membrane is composed of (fully or partially hydrolyzed) polyvinyl alcohol (PVA).

[0119] The present invention also relates to a method for cleaning textiles or hard surfaces, characterized in that a reagent according to the invention is used in at least one method step, or that a protease according to the invention is converted to catalytic activity in at least one method step, particularly such that the protease is used in an amount of 40 μg to 4 g, preferably 50 μg to 3 g, particularly preferably 100 μg to 2 g and most particularly preferably 200 μg to 1 g or at the concentration described herein.

[0120] In various embodiments, the method described above is characterized in that the protease is used at a temperature of 0 to 100°C, preferably 0 to 60°C, more preferably 20 to 40°C, and most preferably 25°C.

[0121] These include manual and mechanical methods, with mechanical methods being preferred. Methods for cleaning textiles are generally characterized by applying different cleaning active substances to the material to be cleaned in multiple process steps, washing them off after exposure time, or otherwise treating the material to be cleaned with a detergent or a solution or dilution of that reagent. This is equally applicable to methods for cleaning all materials other than textiles, particularly hard surfaces. All conceivable washing or cleaning methods can be improved in at least one process step by using a detergent or cleaning agent according to the invention or a protease according to the invention, and thus constitute embodiments of the invention. All aspects, subjects, and embodiments described for the protease according to the invention and reagents containing the protease are also applicable to the subject matter of the invention. Therefore, reference is made here explicitly to the disclosure in the corresponding location, indicating that this disclosure also applies to the methods described above according to the invention.

[0122] Because the protease according to the invention naturally possesses hydrolytic activity and also exhibits hydrolytic activity in media without cleaning power (e.g., in a simple buffer solution), a single and / or sole step of this method may involve contacting the protease according to the invention as the sole cleaning active component with the stain, preferably in a buffer solution or water. This constitutes another embodiment of this subject matter of the invention.

[0123] Alternative embodiments of this subject matter also include methods for treating textile raw materials or for textile care, wherein the protease according to the invention becomes active in at least one method step. Among these, methods for textile raw materials, fibers, or textiles having natural components are preferred, and those having wool or silk are particularly preferred.

[0124] Finally, the invention also includes the use of the proteases described herein in detergents or cleaning agents, for example, as described above, for the removal of protein-containing stains, such as from textiles or hard surfaces (modified). In a preferred embodiment of this use, the proteases in the detergent or cleaning agent are 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 prior to a washing or cleaning operation.

[0125] All aspects, topics, and embodiments described for the proteases and reagents containing proteases according to the invention are also applicable to the subject matter of the invention. Therefore, reference is made in this regard to the disclosure in the corresponding locations, indicating that such disclosure is also applicable to the above-described uses according to the invention. Example

[0126] Mutation Overview:

[0127] This invention relates to a subtilisin-type alkaline protease from Bacillus pumilus. Variants are prepared from this protease (wild-type Bacillus pumilus DSM18097 protease according to SEQ ID NO:1) by random mutagenesis, and then screened, particularly for improved washing performance and / or enzyme stability. In this manner, a performance-improved mutant (C0B [SEQ ID NO:2]) is generated from the aforementioned wild-type protease (SEQ ID NO:1) through several rounds of random mutagenesis. Further rounds of random mutagenesis are performed on this mutant, with saturation mutagenesis at specific sites. Furthermore, some mutants are prepared by targeted preparation of synthetic genes. The mutants according to the invention are generated in these mutagenesis rounds.

[0128]

[0129] Detergent base used

[0130] The table below shows the detergent bases used in the washing tests (commercially available, enzyme-free, optical brightener-free, fragrance-free, and dye-free):

[0131]

[0132] Protease activity assay

[0133] The activity of the protease was determined by the release of the p-nitroaniline chromophore from the substrate succinylalanine-alanine-proline-phenylalanine-p-nitroaniline (AAPFpNA; Bachem L-1400). The release of pNA caused an increase in absorbance at 410 nm, and the change over time was a measure of enzyme activity.

[0134] Measurements were performed at 25°C, pH 8.6, and a wavelength of 410 nm. The measurement time was 5 minutes, with measurement intervals of 20–60 seconds.

[0135] Measurement method:

[0136] 10 μL AAPF solution (70 mg / mL)

[0137] 1000 μL Tris / HCl (0.1 M, pH 8.6 with 0.1% Brij 35)

[0138] 10 μL diluted protease solution

[0139] The kinetics generated within 5 minutes at 25°C (410 nm)

[0140] Mini Wash Test and Results

[0141] Washing tests were performed using supernatant from Bacillus subtilis culture containing protease mutants selected through heterologous expression. The supernatant was used in detergents with activity equivalent to the baseline (according to SEQ ID NO:2) at commercially available standard protease concentrations. These mutants were all based on the washing performance of the wild type, which was set at 100% (the sum of 6 stains, corrected only by detergent performance).

[0142] Conditions: 40℃, 16°dH water, 1 hour

[0143] Stains:

[0144] 1.CFT CS038

[0145] 2.CFT PC-10

[0146] 3.WfK 10N

[0147] 4.CFT C-03

[0148] 5.CFT C-05

[0149] 6. H-MR-B

[0150] A perforated fabric piece (diameter = 10 mm) was placed in a microtiter plate. The detergent solution was preheated to 40°C, with a final concentration of 3.17 g / L. The liquid and enzyme were added to the stain and incubated at 40°C and 600 rpm for 1 hour. The stain was then rinsed several times with water, allowed to dry, and its brightness was measured using a colorimeter. The lighter the fabric, the better the cleaning performance. Here, the L value is equal to the brightness, with higher values ​​indicating greater brightness. The sum of the six stains, given as a percentage based on the wild type according to SEQ ID NO:2, was corrected for by the performance of a protease-free detergent.

[0151]

[0152] Compared to the starting variant according to SEQ ID NO:2 (which already has enhanced performance compared to the wild type according to SEQ ID NO:1), all variants exhibited comparable or enhanced washing performance at 20°C / 40°C.

Claims

1. From Bacillus pumilus ( Bacillus pumilus The protease comprising the amino acid sequence listed in SEQ ID NO:1, and in all cases further comprising, based on the numbering according to SEQ ID NO:1: (a) amino acid substitutions of 9T, 130D, 133A, 144K, 217M, 224A, 252T and 271E at positions corresponding to positions 9, 130, 133, 144, 217, 224, 252T and 271E; and having (b) 89 and 131, amino acid substitutions of 89A and 131H; (c) 89 and 189, amino acid substitutions of 89A and 189T; or (d) 89, 131 and 189, amino acid substitutions of 89A, 131H and 189T.

2. The nucleic acid encoding the protease according to claim 1.

3. A non-human host cell containing the nucleic acid according to claim 2 or the protease according to claim 1.

4. A method for preparing a protease, comprising: a) culturing a host cell according to claim 3; and b) isolating the protease from the culture medium or the host cell.

5. A detergent, characterized in that it contains at least one protease according to claim 1.

6. A cleaning agent, characterized in that it contains at least one protease according to claim 1.

7. A method for cleaning textiles or hard surfaces, characterized in that the detergent according to claim 5 is used in at least one method step.

8. A method for cleaning textiles or hard surfaces, characterized in that the cleaning agent according to claim 6 is used in at least one method step.

9. Use of the protease according to claim 1 in detergents for removing stains containing peptides or proteins.

10. Use of the protease of claim 1 in a cleaning agent for removing stains containing peptides or proteins.

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