DE102018004207A1 - Performance-enhanced variants of the alkaline protease from B. lentus - Google Patents

Abstract

The invention is in the field of enzyme technology. The invention relates to proteases from Bacillus lentus whose amino acid sequence, in particular with regard to the use in detergents and cleaners has been modified to give them in combination with conventional detergent ingredients a better cleaning performance on various soils, and the nucleic acids coding for them as well as their production. The invention further relates to the uses of these proteases and processes in which they are used as well as agents containing them, in particular washing and cleaning agents.

Description

The invention is in the field of enzyme technology. The invention relates to proteases from Bacillus lentus, whose amino acid sequence, in particular with regard to the use in detergents and cleaners have been changed to give them in combination with conventional detergent ingredients a better cleaning performance on various soils, and encoding them nucleic acids as well as their production. The invention further relates to the uses of these proteases and methods in which they are used, as well as agents containing them, in particular washing and cleaning agents.

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

Examples of the subtilisin-type proteases preferably used in detergents and cleaners are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the the subtilases, but not the subtilisins in the narrower sense attributable enzyme thermitase, proteinase K and the proteases TW3 and TW7, as well as variants of said proteases, which have a relation to the parent protease modified amino acid sequence. Proteases are selectively or randomly modified by methods known from the prior art and thus optimized, for example, for use in detergents and cleaners. These include point mutagenesis, deletion or insertion mutagenesis or fusion with other proteins or protein parts. Thus, correspondingly optimized variants are known for most proteases known from the prior art.

In general, only selected proteases are suitable for use in liquid surfactant-containing preparations. Many proteases do not show sufficient catalytic performance in such formulations. For the use of proteases in detergents, therefore, a high catalytic activity under conditions as they are during a wash cycle, and a high storage stability is particularly desirable.

Although the prior art discloses protease and surfactant-containing formulations which exhibit satisfactory proteolytic activity under standard washing conditions and are storage stable and which show improved cleaning performance on protease-sensitive soils, there is a need for further improved proteases similar to those already known and enzymes used in detergents and cleaning agents, for example, are superior in terms of performance.

Surprisingly, it has now been found that certain variants of the protease from Bacillus lentus with the amino acid sequence according to SEQ ID NO: 1 or a sufficiently similar protease (based on the sequence identity), each based on the numbering according to SEQ ID NO: 1 ( a) at the position corresponding to position 99, the amino acid substitution 99E; and (b) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 or 245, each based on the numbering of SEQ ID NO: 1, has at least one further amino acid substitution when used in conventional washing - And cleaning agents with respect to the cleaning performance compared to the wild-type and / or reference mutants, in particular a reference mutant, which has only the substitution of 99E, is improved and is therefore particularly suitable for use in detergents or cleaners.

1. a) an der Position, die der Position 99 entspricht, die Aminosäuresubstitution 99E; und
2. b) an mindestens einer der Positionen, die den Positionen 76, 77, 188, 238, 242 und 245, jeweils bezogen auf die Nummerierung gemäß SEQ ID NO:1, entsprechen, mindestens eine weitere Aminosäuresubstitution aufweist.

The invention therefore in a first aspect is a protease comprising an amino acid sequence which has at least 80% 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 the position corresponding to position 99, the amino acid substitution 99E; and
2. b) at least one of the positions corresponding to the positions 76, 77, 188, 238, 242 and 245, each based on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution.

A further subject of the invention is a process for the preparation of a protease as defined above comprising the substitution of amino acids in an initial protease which has at least 80% sequence identity to the amino acid sequence given in SEQ ID NO: 1 over its entire length (i) at the position, corresponds to position 99 in SEQ ID NO: 1 such that the protease at position 99 comprises amino acid substitution 99E, and (ii) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 and 245, in each case based on the numbering according to SEQ ID NO: 1, having at least one further amino acid substitution. The protease obtainable by this method has at least 80% 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 comprises both the protease as such and a protease produced by a method according to the invention. All versions of 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, to non-human host cells comprising proteases or nucleic acids, and to agents comprising proteases, in particular detergents and cleaners, to washing and cleaning processes, and to uses of the proteases according to the invention in detergents or cleaners for the removal of proteinaceous soils.

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

As used herein, when the protease is defined to include "amino acid substitutions 238A, 238S, 238R, 238L, 238P or 238W", this means that position 238 in the SEQ ID NO: 1 count is either A, S, R , L, P or W is mutated. Thus, the phrase "comprising the amino acid substitutions 99E and 238A or 238L, and optionally 3T and 4I" means that position 99 is to E, position 238 either to A or L, and positions 3 and 4 optionally to T or I mutated. The expression that the protease comprises a substitution at "at least one of the positions 76, 77, 188, 238, 242 and 245" means that the protease can have a substitution at exactly one of these positions, but also at 2, 3, 4, 5 or all 6 of said positions.

The present invention is based on the surprising discovery of the inventors that amino acid substitutions at the positions corresponding to the positions 99 and at least one of 76, 77, 188, 238, 242 and 245, each based on the numbering according to SEQ ID NO: 1 such that the protease at position 99, the amino acid substitution 99E and preferably at least one of the positions 76, 77, 188, 238, 242 and 245 one or more of the substitutions 76H, 76K, 76W, 77Q, 77R, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 242G, 242W, 245N, 245R, 245W, has improved purification performance in a protease comprising at least 80% identical amino acid sequence to the amino acid sequence set forth in SEQ ID NO: 1 modified protease in detergents and cleaners causes. This is especially surprising inasmuch as none of the amino acid substitutions at positions 76, 77, 188, 238, 242 and 245 has previously been associated with increased protease purification performance.

In preferred embodiments of the protease of the invention, the protease has at least one amino acid substitution according to (b) selected from the group consisting of 76H, 76K, 76W, 77Q, 77R, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 242G, 242W, 245N, 245R and 245W, each based on the numbering according to SEQ ID NO: 1, is selected.

In various embodiments, the protease has only one amino acid substitution according to (b), which is selected from the group consisting of 76H, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 245N and 245W, each based on the numbering according to SEQ ID NO: 1.

In various alternative embodiments, the protease has at least two amino acid substitutions according to (b) selected from 76H, 76W, 76K, 77R, 188L, 238A, 238R, 238P, 242G and 245N, preferably from 76W + 77R, 76K + 188L , 76H + 188L, 188L + 238P, 188L + 238R, 188L + 238A, 188L + 242G, 188I + 245N and 238R + 242G, more preferably 76H + 188L and 188L + 238P.

1. (i) R99E und A188L sowie optional zusätzliche eine von S76K, S76H, S76W, I77R, V238A, V238P, V238R, V238L, V238S, V238W, N242G, K245N und K245W;
2. (ii) R99E und eine von S76H, S76K und S76W sowie optional eine von I77R, V238A, V238P, V238L, V238S, V238W, N242G, K245N und K245W;
3. (iii) R99E und eine von V238A, V238S, V238R, V238L, V238W;
4. (iv) R99E und V238R sowie eine von S76K, S76H, S76W, I77R, N242G, K245N und K245W.
5. (v) R99E und N242G;
6. (vi) R99 und K245N oder K245W; und
7. (vii) R99 und 177R.

In various embodiments, the protease has one of the following amino acid substitution variants, each based on the numbering according to SEQ ID NO: 1, on:

1. (i) R99E and A188L and optionally additional ones of S76K, S76H, S76W, I77R, V238A, V238P, V238R, V238L, V238S, V238W, N242G, K245N and K245W;
2. (ii) R99E and one of S76H, S76K and S76W, and optionally one of I77R, V238A, V238P, V238L, V238S, V238W, N242G, K245N and K245W;
3. (iii) R99E and one of V238A, V238S, V238R, V238L, V238W;
4. (iv) R99E and V238R; and one of S76K, S76H, S76W, I77R, N242G, K245N and K245W.
5. (v) R99E and N242G;
6. (vi) R99 and K245N or K245W; and
7. (vii) R99 and 177R.

In the aforementioned embodiments, the remainder of the amino acid sequence, i. the positions not mentioned as substituted, which correspond to SEQ ID NO: 1. Alternatively, however, the protease may also have further substitutions. For example, it is possible and contemplated by the invention that the proteases as described above contain one (or more) additional amino acid substitutions at one or more of the positions corresponding to positions 3, 4, 154, 199, and 256, based on the numbering SEQ ID NO: 1, preferably, one or more of the amino acid substitutions 3T, 41, 154D, 1991 and 256E, more preferably 3T + 4I + 199I or 154D + 256E.

The proteases of the invention have an improved cleaning performance. This means that it has an increased cleaning performance on one or more stains in detergents or cleaning agents compared to a reference mutation variant of the protease, which has only the substitution R99E. Such performance-enhanced proteases enable improved wash results on proteolytically-sensitive soils in various temperature ranges, in particular a temperature range of 20 ° C to 40 ° C.

In various embodiments, the proteases according to the invention may have a washing performance, based on a reference mutation variant of the protease with SEQ ID NO: 1 and the substitution R99E at least 102%, at least 103%, at least 104%, at least 105%, at least 106 %, at least 107%, at least 108%, at least 110%, at least 115%, at least 120% or at least 125%. Such performance-enhanced proteases allow in a washing or cleaning agent matrix improved washing results on proteolytically sensitive soils in various temperature ranges, in particular a temperature range of 20 ° C to 40 ° C.

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

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 solvent of the agent (eg water). In other embodiments, an agent may contain proteases that form an interaction complex with other molecules or that contain a "cladding". Here, a single or multiple protease molecules may be separated from the other components of the agent by a surrounding structure. Such a separating structure may arise from, but is not limited to, vesicles such as a micelle or a liposome. The surrounding structure may also be a virus particle, a bacterial cell or a eukaryotic cell. In various embodiments, a means Cells of Bacillus lentus or Bacillus subtilis which express the proteases of the invention or cell culture supernatants of such cells.

In one embodiment of the invention, the protease comprises an amino acid sequence which corresponds to the amino acid sequence given in SEQ ID NO: 1 over at least 80% of its total length, 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 98.8%, and in each case based on the numbering according to SEQ ID NO: 1 (a ) at the position corresponding to position 99, the amino acid substitution 99E; and (b) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 and 245, each related to the numbering of SEQ ID NO: 1, has at least one more amino acid substitution. In the context of the present invention, the feature means that a protease has the indicated substitutions that it contains at least one of the corresponding amino acids at the corresponding positions, i. not all of the indicated positions are otherwise mutated or deleted, for example by fragmentation of the protease. Preferred substitutions are those selected from the group consisting of 76H, 76K, 76W, 77Q, 77R, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 242G, 242W, 245N, 245R and 245W, each numbering according to SEQ ID NO: 1 are selected. Particularly preferred is the combination of 99E with one of 76H, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 245N and 245W, each based on the numbering according to SEQ ID NO: 1. Also particularly preferred are combinations of 99E having at least two amino acid substitutions selected from 76W, 76K, 77R, 188L, 238A, 238R, 238P, 242G and 245N, preferably 76W + 77R, 76K + 188L, 188L + 238P, 188L + 238R, 188L + 238A, 188L + 242G, 188I + 245N and 238R + 242G.

The identity of nucleic acid or amino acid sequences is determined by a sequence comparison. This sequence comparison is based on the BLAST algorithm established in the prior art and commonly used (cf., for example, US Pat Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410 and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs "; Nucleic Acids Res., 25, pp.3389-3402) and in principle occurs by assigning similar sequences of nucleotides or amino acids in the nucleic acid or amino acid sequences to one another. A tabular assignment of the respective positions is referred to as alignment. Another algorithm available in the prior art is the FASTA algorithm. Sequence comparisons (alignments), in particular multiple sequence comparisons, are created with computer programs. 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. Also possible are alignment comparisons (alignments) with the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California, USA) with the default parameters whose AlignX module for sequence comparisons is based on ClustalW. Unless otherwise specified, the sequence identity given herein is determined by the BLAST algorithm.

Such a comparison also allows a statement about the similarity of the compared sequences to each other. It is usually given in percent identity, that is, the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions. The broader concept of homology involves conserved amino acid substitutions in the consideration of amino acid sequences, that is, amino acids with similar chemical activity, as these usually perform similar chemical activities within the protein. Therefore, the similarity of the sequences compared may also be stated as percent homology or percent similarity. Identity and / or homology information can be made about whole polypeptides or genes or only over individual regions. Homologous or identical regions of different nucleic acid or amino acid sequences are therefore defined by matches in the sequences. Such areas often have identical functions. They can be small and comprise only a few nucleotides or amino acids. Often, such small regions exert essential functions for the overall activity of the protein. It may therefore be useful to relate sequence matches only to individual, possibly small areas. Unless otherwise indicated, identity or homology information in the present application, however, refers to the total length of the particular nucleic acid or amino acid sequence indicated.

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

In a further embodiment of the invention, the protease is characterized in that its purification performance is increased over that of a protease comprising an amino acid sequence corresponding to the amino acid sequence given in SEQ ID NO: 1 with the additional replacement R99E, i. at least 101%, more preferably at least 110% or more. The cleaning performance can be determined in a washing system containing a detergent in a dosage between 2.5 and 7.0 grams per liter of wash liquor and the protease, wherein the proteases to be compared in concentration (based on active protein) are used and the cleaning performance compared a soiling on cotton is determined by measuring the degree of cleaning of the washed textiles. For example, the washing process for 60 minutes at a temperature of 40 ° C and the water have a water hardness between 15.5 and 16.5 ° (German hardness). The concentration of the protease in the detergent intended for this washing system is 0.001 to 0.1% by weight, preferably 0.01 to 0.06% by weight, based on active, purified protein.

A liquid reference detergent for such a washing system may be composed as follows (all figures in weight percent): 4.4% alkyl benzene sulfonic acid, 5.6% further anionic surfactants, 2.4% C12-C18 Na salts of fatty acids (soaps) , 4.4% nonionic surfactants, 0.2% phosphonates, 1.4% citric acid, 0.95% NaOH, 0.01% defoamer, 2% glycerol, 0.08% preservatives, 11% polyacrylate (e.g. Polyacrylic acid; Acusol 445N), 1% ethanol, balance demineralized water. Preferably, the dosage of the liquid detergent is between 2.0 and 6.0 grams per liter of wash liquor, for example, 2.5, 2.8, 3.1, 3.4 or 3.9 grams per liter of wash liquor. Preference is given to washing in a pH range between pH 7 and pH 10.5, preferably between pH 7.5 and pH 8.5.

In the context of the invention, the determination of the cleaning performance, for example, at 20 ° C or 40 ° C using a liquid detergent as stated above, wherein the washing process is preferably carried out for 60 minutes at 600 rpm.

The whiteness, i. the brightening of the stains, as a measure of the cleaning performance is determined by optical measurement methods, preferably photometrically. A suitable device for this purpose is for example the spectrometer Minolta CM508d. Usually, the devices used for the measurement are previously calibrated with a white standard, preferably a supplied white standard.

The activity-equivalent use of the respective protease ensures that even if the ratio of active substance to total protein (the values of the specific activity) diverge, the respective enzymatic properties, for example the cleaning performance of certain soils, are compared. In general, a low specific activity can be compensated by adding a larger amount of protein.

Otherwise, methods for determining protease activity are well known and used routinely by those skilled in the art of enzyme technology. 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 pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of enzymatic activity (see Del Mar et al., 1979). The measurement is carried out at a temperature of 25 ° C, at pH 8.6, and a wavelength of 410 nm. The measuring time is 5 min and the measuring interval 20 s to 60 s. The protease activity is usually indicated in protease units (PE). Suitable protease activities are, for example, 2.25, 5 or 10 PE per ml wash liquor. However, the protease activity is not equal to zero.

An alternative test for determining the proteolytic activity of the proteases according to the invention is an optical measuring method, preferably a photometric method. The appropriate test involves the protease-dependent cleavage of the substrate protein casein. This is split by the protease into a multitude of smaller partial products. The totality of these partial products has an increased absorption at 290 nm compared to uncleaved casein, wherein this increased absorption can be determined using a photometer and thus a conclusion can be drawn on the enzymatic activity of the protease.

The protein concentration can be determined by known methods, for example the BCA method (bicinchoninic acid, 2,2'-biquinolyl-4,4'-dicarboxylic acid) or the biuret method ( Gornall AG, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766 ). The determination of the active protein concentration can in this regard via a titration of the active sites using a suitable irreversible inhibitor and determination of the residual activity (see. Bender, M., et al., J. Am. Chem. Soc. 88, 24 (1966), p. 5890-5913 ) respectively.

In addition to the amino acid changes discussed above, proteases of the invention may have other amino acid changes, especially amino acid substitutions, insertions or deletions. Such proteases are, for example, by targeted genetic modification, i. by mutagenesis, further developed and optimized for specific applications or specific properties (for example, in terms of catalytic activity, stability, etc.). Furthermore, nucleic acids according to the invention can be introduced into recombination approaches and thus used to generate completely novel proteases or other polypeptides.

The goal is to introduce into the known molecules targeted mutations such as substitutions, insertions or deletions, for example, to improve the cleaning performance of enzymes of 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. Thus, for example, the net charge of the enzymes can be changed in order to influence the substrate binding, in particular for use in detergents and cleaners. Alternatively or additionally, one or more corresponding mutations can increase the stability or catalytic activity of the protease and thereby improve its purification performance. Advantageous properties of individual mutations, e.g. individual substitutions can complement each other. A protease which has already been optimized with regard to certain properties, for example with respect to its stability during storage, can therefore be further developed within the scope of the invention.

For the description of substitutions concerning exactly one amino acid position (amino acid substitutions), the following convention is used herein: first, the naturally occurring amino acid is designated in the form of the international one-letter code, followed by the associated sequence position and finally the inserted amino acid. Several exchanges within the same polypeptide chain are separated by slashes. For insertions, additional amino acids are named after the sequence position. In the case of deletions, the missing amino acid is replaced by a symbol, for example a star or a dash, or a Δ is specified in front of the corresponding position. For example, R99E describes the substitution of arginine at position 99 by glutamic acid, R9RH the insertion of histidine after the amino acid arginine at position 9 and R99 * or ΔR99 the deletion of arginine at position 99. This nomenclature is known to those skilled in the art of enzyme technology.

Another object of the invention is therefore a protease which is characterized in that it is obtainable from a protease as described above as the starting molecule by one or multiple conservative amino acid substitution, wherein the protease in the count according to SEQ ID NO: 1 amino acid substitutions (a ) at the position corresponding to position 99 in SEQ ID NO: 1, namely 99E, and (b) at least one amino acid substitution at one or more of the positions 76, 77, 188, 238, 242 and 245 in SEQ ID NO: 1, as described above. The protease thus obtainable preferably also fulfills the feature that its amino acid sequence has at least 80% identity with the sequence according to SEQ ID NO: 1 over its entire length. The term "conservative amino acid substitution" means the replacement of one amino acid residue with another amino acid residue, which exchange does not result in a change in polarity or charge at the position of the exchanged amino acid, e.g. the replacement of a nonpolar amino acid residue with another nonpolar 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 is obtainable from a protease according to the invention as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is over a length of at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267 or 268, the protease at the position corresponding to position 99 having the amino acid substitution 99E; and at least one of the positions corresponding to the positions 76, 77, 188, 238, 242 or 245, in each case based on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution, ie, the (n ) Amino acid substitution (s) at the positions corresponding to positions 99, 76, 77, 188, 238, 242 and 245 in SEQ ID NO: 1 are still present.

Thus, 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 diminished. Furthermore, such fragmentation, deletion, insertion or substitution mutagenesis can also reduce, for example, the allergenicity of the enzymes concerned and thus improve their overall applicability. Advantageously, the enzymes retain their proteolytic activity even after mutagenesis, i. their proteolytic activity is at least equal to that of the parent enzyme, i. in a preferred embodiment, the proteolytic activity is at least 80%, preferably at least 90% of the activity of the starting enzyme. Other substitutions can also show beneficial effects. Both single and multiple contiguous amino acids can be substituted for other amino acids.

Alternatively or additionally, the protease is characterized in that it is obtainable from a protease according to the invention as the starting molecule by mono- or multiple conservative amino acid substitution, wherein the protease in the count according to SEQ ID NO: 1 (a) at the position, the position 99 in SEQ ID NO: 1 corresponds to the amino acid substitution 99E, and (b) has at least one further amino acid substitution at one or more of the positions corresponding to positions 76, 77, 188, 238, 242 and 245 in SEQ ID NO: 1, as in described above.

1. (i) an der Position, die der Position 99 entspricht, die Aminosäuresubstitution 99E; und
2. (ii) an mindestens einer der Positionen, die den Positionen 76, 77, 188, 238, 242 und 245, jeweils bezogen auf die Nummerierung gemäß SEQ ID NO:1, entsprechen, mindestens eine weitere Aminosäuresubstitution aufweist.

In further embodiments, the protease is characterized in that it is obtainable from a protease according to the invention as the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which has a length of at least that over a length of at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267 or 268 contiguous amino acids with the parent molecule, wherein the protease

1. (i) at the position corresponding to position 99, the amino acid substitution 99E; and
2. (ii) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 and 245, each based on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution.

The further amino acid positions are hereby defined by an alignment of the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as indicated 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 if the amino acid sequence of a protease according to the invention comprises a higher number of amino acid residues than the protease from Bacillus lentus according to SEQ ID NO: 1. Starting from the mentioned positions in the amino acid sequence of the protease from Bacillus lentus, the alteration positions in a protease according to the invention are those which are just assigned to these positions in an alignment.

Advantageous positions for sequence changes, in particular substitutions, of the protease from Bacillus lentus, which are preferably transferred to homologous positions of the proteases according to the invention and which confer advantageous functional properties on the protease, are therefore, besides the position 99, the positions which are aligned with the positions 76 , 77, 188, 238, 242 and 245 in SEQ ID NO: 1, ie in the count according to SEQ ID NO: 1. At the positions mentioned, the following amino acid residues are present in the wild-type molecule of the protease from Bacillus lentus: S76, 177, A188, V238, N242 and K245.

Further confirmation of the correct assignment of the amino acids to be changed, ie in particular their functional correspondence, can provide comparative experiments, according to which the two positions assigned to each other on the basis of an alignment are changed in the same way in both compared proteases, and if both are observed the enzymatic activity is changed in the same way. If, for example, an amino acid exchange in a specific position of the protease from Bacillus lentus according to SEQ ID NO: 1 is accompanied by a change in an enzymatic parameter, for example by an increase in the K _M value, then a corresponding change in the enzymatic parameter, for example likewise one Increasing the K _M value, observed in a protease variant according to the invention, whose amino acid exchange has been achieved by the same introduced amino acid, it is to be seen here a confirmation of the correct assignment.

1. a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution, wobei die Protease
   1. (i) die Aminosäuresubstitution R99E an der Position, die der Position 99 gemäß SEQ ID NO:1 entspricht, und
   2. (ii) mindestens eine der Aminosäuresubstitutionen S76H, S76K, S76W, I77Q, I77R, A188L, V238A, V238S, V238R, V238L, V238P, V238W, N242G, N242W, K245N, K245R und K245W an den Positionen, die den Positionen 76, 77, 18, 238, 242, und 245 gemäß SEQ ID NO:1 entsprechen, umfasst; und/oder
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 oder 268 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die Protease
   1. (i) die Aminosäuresubstitution R99E an der Position, die der Position 99 gemäß SEQ ID NO:1 entspricht, und
   2. (ii) mindestens eine der Aminosäuresubstitutionen S76H, S76K, S76W, I77Q, I77R, A188L, V238A, V238S, V238R, V238L, V238P, V238W, N242G, N242W, K245N, K245R und K245W an den Positionen, die den Positionen 76, 77, 18, 238, 242, und 245 gemäß SEQ ID NO:1 entsprechen, umfasst.

All these facts are also applicable to the method of producing a protease according to the invention. Accordingly, a method according to the invention further comprises one or more of the following method steps:

1. a) introducing a single or multiple conservative amino acid substitution, wherein the protease
   1. (i) the amino acid substitution R99E at the position corresponding to position 99 according to SEQ ID NO: 1, and
   2. (ii) at least one of the amino acid substitutions S76H, S76K, S76W, I77Q, I77R, A188L, V238A, V238S, V238R, V238L, V238P, V238W, N242G, N242W, K245N, K245R and K245W at positions corresponding to positions 76, 77 , 18, 238, 242, and 245 as shown in SEQ ID NO: 1; and or
2. b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis in such a way that the protease has an amino acid sequence which is over a length of at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266, 267 or 268 contiguous amino acids with the parent molecule, wherein the protease
   1. (i) the amino acid substitution R99E at the position corresponding to position 99 according to SEQ ID NO: 1, and
   2. (ii) at least one of the amino acid substitutions S76H, S76K, S76W, I77Q, I77R, A188L, V238A, V238S, V238R, V238L, V238P, V238W, N242G, N242W, K245N, K245R and K245W at positions corresponding to positions 76, 77 , 18, 238, 242, and 245 as shown in SEQ ID NO: 1.

All embodiments also apply to the inventive method.

In further embodiments of the invention, the protease or the protease prepared by a method according to the invention is still at least 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 98.8% identical to the amino acid sequence given in SEQ ID NO: 1 over their entire length.

The protease or the protease produced by a method according to the invention preferably has the amino acid substitutions or combinations of amino acid substitutions already disclosed above.

Another object of the invention is a protease described above, 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 in the washing process, leads to longer enzymatic activity and thus improves cleaning performance. In principle, all stabilization options described in the prior art and / or appropriate considerations come into consideration. Preference is given to those stabilizations which are achieved by mutations of the enzyme itself, since such stabilizations do not require any further working steps following the recovery of the enzyme. Examples of sequence changes suitable for this purpose are mentioned above. Other suitable sequence changes are known from the prior 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.

Further possibilities of stabilization are, for example:

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

Preferred embodiments are those in which the enzyme is stabilized in several ways, as several stabilizing mutations act additive or synergistic.

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 called a derivative, ie the protease is derivatized.

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

Derivatives of a protein according to the invention can also be understood in the broadest sense to mean preparations of these proteins. Depending on the extraction, processing or preparation, a protein can be combined with various other substances, for example from the culture of the producing microorganisms. A protein may also have been deliberately added to other substances, for example to increase its storage stability. Therefore, all preparations of a protein according to the invention are also according to the invention. This is also independent of whether or not it actually exhibits this enzymatic activity in a particular preparation. Because it may be desired that it has no or only low activity during storage, and unfolds its enzymatic function only at the time of use. This can be controlled, for example, via appropriate accompanying substances. In particular, the joint preparation of proteases with specific inhibitors is possible in this regard.

Of all proteases or protease variants and / or derivatives described above, particular preference is given in the context of the present invention to those whose enzyme performance is at least one of those of the proteases exemplarily tested in the examples, the purification performance being determined in a washing system as described above.

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

These may be DNA or RNA molecules. They can be present as a single strand, as a single strand that is complementary to this single strand, or as a double strand. Especially in the case of DNA molecules, the sequences of both complementary strands must be taken into account in all three possible reading frames. Furthermore, it should be noted that different codons, so base triplets, can code for the same amino acids, so that a particular amino acid sequence can be encoded by several different nucleic acids. Due to this degeneracy of the genetic code, all nucleic acid sequences are included in this subject of the invention which can encode any of the proteases described above. The person skilled in the art is able to determine these nucleic acid sequences unequivocally since, despite the degeneracy of the genetic code, individual codons are assigned defined amino acids. Therefore, the person skilled in the art can easily determine nucleic acids coding for this amino acid sequence on the basis of an amino acid sequence. Furthermore, in the case of nucleic acids according to the invention, one or more codons may be replaced by synonymous codon (s). This aspect relates in particular to the heterologous expression of the enzymes according to the invention. Thus, each organism, for example a host cell of a production strain, has a particular codon usage. Codon usage is understood to mean the translation of the genetic code into amino acids by the particular organism. There may be bottlenecks in protein biosynthesis if the codons lying on the nucleic acid in the organism of a comparatively small number of loaded tRNA Facing molecules. Although coding for the same amino acid, this results in a codon being translated less efficiently in the organism than a synonymous codon encoding the same amino acid. Due to the presence of a higher number of tRNA molecules for the synonymous codon, it can be more efficiently translated in the organism.

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

For the purposes of the present invention, vectors are understood as consisting of nucleic acids which contain a nucleic acid according to the invention as a characteristic nucleic acid region. They can establish these in a species or cell line over several generations or cell divisions as a stable genetic element. Vectors, especially when used in bacteria, are special plasmids, ie circular genetic elements. In the context of the present invention, a nucleic acid according to the invention is cloned into a vector. The vectors include, for example, those whose origin are bacterial plasmids, viruses or bacteriophages, or predominantly synthetic vectors or plasmids with elements of various origins. With the other genetic elements present in each case, vectors are able to establish themselves as stable units in the relevant host cells over several generations. They may be extrachromosomal as separate units or integrated 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 to express a contained nucleic acid there. In particular, expression is influenced by the promoter (s) that regulate transcription. In principle, the expression may 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 also by a modified or 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. Furthermore, expression vectors can be regulatable, for example by changing the culturing conditions or when a specific cell density of the host cells contained therein is reached or by addition of specific 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 is not expressed in cloning vectors.

A further subject of the invention is a non-human host cell which contains a nucleic acid according to the invention or a vector according to the invention or which contains a protease according to the invention, in particular one which secretes the protease into the medium surrounding the host cell. Preferably, a nucleic acid according to the invention or a vector according to the invention is transformed into a microorganism, which then represents a host cell according to the invention. Alternatively, individual components, i. Nucleic acid parts or fragments of a nucleic acid according to the invention are introduced into a host cell such that the resulting host cell 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 constituents of a nucleic acid according to the invention or a vector according to the invention and the further constituents are then supplemented accordingly. Methods of transforming cells are well established in the art and well known to those skilled in the art. In principle, all cells, that is to say prokaryotic or eukaryotic cells, are suitable as host cells. Preference is given to those host cells which can be handled genetically advantageously, for example as regards the transformation with the nucleic acid or the vector and its stable establishment, for example unicellular fungi or bacteria. Furthermore, preferred host cells are characterized by good microbiological and biotechnological handling. This concerns, for example, easy culturing, high growth rates, low demands on fermentation media and good production and secretion rates for foreign proteins. Preferred host cells according to the invention secrete the (transgenially) expressed protein into the medium surrounding the host cells. Furthermore, the proteases can be modified by the cells producing them after their production, for example by attachment of sugar molecules, formylations, aminations, etc. Such post-translational modifications can functionally influence the protease.

Further preferred embodiments are those host cells which are regulatable in their activity due to genetic regulatory elements which are provided, for example, on the vector, but may also be present in these cells from the outset. For example, by controlled addition of chemical compounds that serve as activators, by changing the culture conditions or when reaching a specific cell density, these can be excited for expression. This enables an economical production of the proteins according to the invention. An example of such a compound is IPTG as described above.

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

In Gram-negative bacteria, such as Escherichia coli, a large number of proteins are secreted into the periplasmic space, ie 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 such that they eject the expressed proteins not only into the periplasmic space but into the medium surrounding the bacterium. In contrast, gram-positive bacteria such as Bacilli or Actinomycetes or other representatives of Actinomycetales have no outer membrane, so that secreted proteins are released immediately 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 further processed. In addition, Gram-positive bacteria are related or identical to most of the organisms of origin for technically important enzymes and usually form even comparable enzymes, so they have a similar codon use and their protein synthesizer is naturally aligned accordingly.

Host cells according to the invention may be altered in their requirements of the culture conditions, have different or additional selection markers or express other or additional proteins. In particular, it may also be those host cells which express several proteins or enzymes transgene.

The present invention is applicable in principle to all microorganisms, in particular to all fermentable microorganisms, particularly preferably those of the genus Bacillus, and results in the production of proteins according to the invention by the use of such microorganisms. Such microorganisms then represent host cells in the sense 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 the genera of Escherichia, Klebsiella, Bacillus, Staphylococcus, Corynebacterium, Arthrobacter, Streptomyces, Stenotrophomonas and Pseudomonas, more preferably one selected from the group consisting of Escherichia coli, Klebsiella planticola, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus alcalophilus, Bacillus globigii, Bacillus gibsonii, Bacillus clausii, Bacillus halodurans, Bacillus pumilus, Staphylococcus carnosus, Corynebacterium glutamicum , Arthrobacter oxidans, Streptomyces lividans, Streptomyces coelicolor and Stenotrophomonas maltophilia.

The host cell may also be a eukaryotic cell, which is characterized by having a cell nucleus. A further subject of the invention therefore represents a host cell, which is characterized in that it has a cell nucleus. In contrast to prokaryotic cells, eukaryotic cells are capable of post-translationally modifying the protein formed. Examples thereof are fungi such as Actinomycetes or yeasts such as Saccharomyces or Kluyveromyces. This may be particularly advantageous, for example, if the proteins are to undergo specific modifications in the context of their synthesis that enable such systems. Modifications that eukaryotic systems perform, especially in connection with protein synthesis, include, for example, the binding of low molecular weight compounds such as membrane anchors or oligosaccharides. Such oligosaccharide modifications may be desirable, for example, to lower the allergenicity of an expressed protein. Also co-expression with the enzymes naturally produced by such cells, such as cellulases, may be advantageous. Furthermore, for example, thermophilic fungal expression systems may be particularly suitable for the expression of temperature-resistant proteins or variants.

The host cells according to the invention are cultured 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 an experimentally determined period of time. Continuous fermentations are characterized by achieving a flow equilibrium, in which over a relatively long period of time cells partly die out but also regrow 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. Another object of the invention is therefore a method for preparing a protease comprising

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

This subject 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 of the product produced, for example the proteases according to the invention. All fermentation processes which are based on a corresponding process for the preparation 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 via a feed strategy, come in particular into consideration. Here, the media components 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 mass and / or in particular in the activity of the protease of interest. Furthermore, the fermentation can also be designed so that undesired metabolic products are filtered out or neutralized by the addition of buffer or suitable counterions.

The protease produced can be harvested from the fermentation medium. Such a fermentation process is resistant to isolation of the protease from the host cell, i. however, requires the provision of suitable host cells or one or more suitable secretion markers or mechanisms and / or transport systems for the host cells to secrete the protease into the fermentation medium. Alternatively, without secretion, the isolation of the protease from the host cell, i. a purification of the same from the cell mass, carried out, for example by precipitation with ammonium sulfate or ethanol, or by chromatographic purification.

All of the above-described facts can be combined to form methods for producing proteases according to the invention.

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

The washing or cleaning agents according to the invention, which may be in the form of homogeneous solutions or suspensions in the form of powdered solids, may contain, in addition to a protease according to the invention, all known ingredients conventionally used in such agents, preferably at least one further ingredient being present in the composition , The agents according to the invention may in particular contain surfactants, builders, peroxygen compounds or bleach activators. In addition, they may contain water-miscible organic solvents, further enzymes, sequestering agents, electrolytes, pH regulators and / or further auxiliaries such as optical brighteners, grayness inhibitors, foam regulators, as well as dyes and fragrances, and combinations thereof.

In particular, a combination of a protease according to the invention with one or more further ingredients of the composition is advantageous, since in preferred embodiments according to the invention such an agent has an improved cleaning performance by virtue of resulting synergisms. In particular, the combination of a protease according to the invention with an anionic or polyanionic component of the washing or cleaning agent, such a synergism can be achieved.

The term "anionic or polyanionic component" as used herein refers to substances that carry at least one, and preferably several, negative charges. It is preferably a polymer having at least one negatively charged monomer, preferably having a plurality of negatively charged monomers. According to the invention, this polymer is therefore a negatively charged polymer. Preferred are, for example, polymers of organic acids or their salts, in particular polyacrylates and / or poly-sugar acids and / or polyacrylate copolymers and / or poly-sugar copolymers. Of the aforementioned compounds, either the corresponding salts, in particular alkali metal salts, or else the free acids can be used. Further preferred compounds are polyacrylsulfonates or polycarboxylates and their salts, copolymers or salts of the copolymers. Preference is given, for example, to (poly) anionic polymers, in particular those which contain carboxylic acid monomers, in particular those which contain units derived from acrylic acid or methacrylic acid, very particularly polyacrylic or polymethacrylic acid or copolymers thereof and the corresponding salts. Likewise preferred are polymers based on aminocarboxylic acid monomers, for example polyglutamic acid / polyaspartic acid and salts thereof.

Examples of particularly preferable substances to be used are Acusol 587D (polyacrylic sulfonate, Rohm & Haas / Dow Chemical Company), Acusol 445N (polycarboxylate sodium salt, Rohm & Haas / Dow Chemical Company), Acusol 590 (polyacrylate copolymer; Rohm & Haas / Dow Chemical ), Acusol 916 (polyacrylate sodium salt, Rohm & Haas / Dow Chemical), Sokalan CP42 (modified polycarboxylate sodium salt, BASF), Sokalan PA 30CL (polycarboxylate sodium salt, BASF), Dequest P 9000 (polymaleic acid, Thermphos), alginic acid , Poly-2-acrylamido-2-methyl-1-propanesulfonic acid, poly-4-styrenesulfonic acid-co-maleic acid sodium salt, poly-acrylamido-co-acrylic acid sodium salt, polymethacrylic acid sodium salt, poly-methylvinylether-old maleic acid, polyvinylsulfonic acid sodium salt, polyglutamic acid sodium salt , Polyaspartic acid sodium salt.

Such polymers may preferably be used in amounts of 0.5 to 30% by weight, more preferably 1 to 20% by weight, more preferably 2 to 15% by weight, based on the total weight of the composition.

Further advantageous ingredients of compositions according to the invention are disclosed in the international patent application WO2009 / 121725 starting there on page 5, penultimate paragraph, and ending on page 13 after the second paragraph. This disclosure is incorporated herein by reference and the disclosure is incorporated herein by reference.

An agent of the invention advantageously contains the protease in an amount of from 2 μg to 20 mg, preferably from 5 μg to 17.5 mg, more preferably from 20 μg to 15 mg and most preferably from 50 μg to 10 mg per gram of the agent. Further, the protease contained in the agent, and / or other ingredients of the agent, may be coated with a substance impermeable to the enzyme at room temperature or in the absence of water, which becomes permeable to the enzyme under conditions of use of the agent. 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 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 having 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 pasty or liquid form, and / or
3. (c) is in the form of a gel or pouch, 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, powdered, liquid, gelatinous or paste-like administration forms of compositions according to the invention, which if appropriate can also consist of several phases and can be present in compressed or uncompressed form. The agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. The solid dosage forms of the composition also include extrudates, granules, tablets or pouches. Alternatively, the agent can also be liquid, gelatinous or pasty, for example in the form of a non-aqueous liquid detergent or a non-aqueous paste or in the form of an aqueous liquid detergent or a water-containing paste. Furthermore, the agent may be present as a one-component system. Such funds consist of one phase. Alternatively, an agent can also consist of several phases. Such an agent is therefore divided into several components.

Detergents or cleaning agents according to the invention may contain only one protease. Alternatively, they may also contain other hydrolytic enzymes or other enzymes in a concentration effective for the effectiveness of the agent. A further embodiment of the invention thus represents agents which further comprise one or more further enzymes. Other enzymes which can be used as further enzymes are all enzymes which can display catalytic activity in the agent according to the invention, in particular a lipase, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, xyloglucanase, .beta.-glucosidase, pectinase, carrageenase, perhydrolase, Oxidase, oxidoreductase or other - distinguishable from the proteases proteases - and their mixtures. Further enzymes are advantageously contained in the agent in each case in an amount of 1 × 10 ^-8 to 5 wt .-% based on active protein. More preferably, each further enzyme is in an amount of 1 x 10 ^-7 to 3 wt%, from 0.00001 to 1 wt%, from 0.00005 to 0.5 wt%, of 0.0001 to 0.1 wt .-% and particularly preferably from 0.0001 to 0.05 wt .-% in inventive compositions, based on active protein. The proteases according to the invention can also be used in such amounts. Particularly preferably, the enzymes show synergistic cleaning performance against certain stains or stains, ie the enzymes contained in the middle composition mutually support each other in their cleaning performance. Very particular preference is given to such a synergism between the protease according to the invention and a further 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 of the composition according to the invention.

In the cleaning agents described herein, the enzymes to be used may 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).

The enzymes are usually not provided in the form of the pure protein, but rather in the form of stabilized, storable and transportable preparations. Such prefabricated preparations include, for example, the solid preparations obtained by granulation, extrusion or lyophilization or, especially in the case of liquid or gel-form detergents, solutions of the enzymes, advantageously as concentrated as possible, low in water and / or added with stabilizers or further auxiliaries.

Alternatively, the enzymes may be encapsulated for both the solid and liquid dosage forms, for example by spray-drying or extruding the enzyme solution together with a preferably natural polymer or in the form of capsules, for example those in which the enzymes are entrapped as in a solidified gel or in those of the core-shell type, in which an enzyme-containing Core is coated with a water, air and / or chemical impermeable protective layer. In deposited layers, further active ingredients, for example stabilizers, emulsifiers, pigments, bleaches or dyes, may additionally be applied. Such capsules are applied by methods known per se, for example by shaking or rolling granulation or in fluid bed processes. Advantageously, such granules, for example by applying polymeric film-forming agent, low in dust and storage stable due to the coating.

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

The enzymes can also be incorporated into water-soluble films, such as those used in the formulation of detergents and cleaners in unit dosage form. Such a film allows the release of the enzymes after contact with water. As used herein, "water-soluble" refers to a film structure that is preferably completely water-soluble. Preferably, such a film consists of (fully or partially hydrolyzed) polyvinyl alcohol (PVA).

A further subject of the invention is a process for the purification of textiles or hard surfaces, which is characterized in that an agent according to the invention is used in at least one process step, or in at least one process step, a protease of the invention becomes catalytically active, in particular such that the protease in an amount of from 40 μg to 4 g, preferably from 50 μg to 3 g, more preferably from 100 μg to 2 g and most preferably from 200 μg to 1 g is used.

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

These include both manual and mechanical processes, with mechanical processes being preferred. Processes for cleaning textiles are generally distinguished by the fact that various cleaning-active substances are applied to the items to be cleaned and washed off after the action time, or that the items to be cleaned are otherwise treated with a detergent or a solution or dilution of this agent. The same applies to processes for cleaning all other materials than textiles, especially hard surfaces. All conceivable washing or cleaning methods can be enriched in at least one of the method steps to the application of a detergent or protease according to the invention and then represent embodiments of the present invention. All facts, objects and embodiments, which protease according to the invention and containing them Means are described are also applicable to this subject invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above inventive method.

Since proteases according to the invention naturally already have a hydrolytic activity and also unfold them in media which otherwise have no cleaning power, for example in bare buffer, a single and / or the sole step of such a method can consist in that the protease which is the only active component of the invention is contacted with the soiling, preferably in a buffer solution or in water. This represents a further embodiment of this subject of the invention.

Alternative embodiments of this subject matter of the invention are also 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, methods for textile raw materials, fibers or textiles with natural components are preferred, and especially for those with wool or silk.

Finally, the invention also encompasses the use of the proteases described herein in detergents or cleaners, for example as described above, for the (improved) removal of proteinaceous soils, for example textiles or hard surfaces.

All aspects, objects, and embodiments described for protease and agents containing it are also applicable to this subject of the invention. Therefore it will be on expressly refer to the disclosure in the appropriate place with the note that this disclosure also applies to the above inventive use.

Examples

Example 1: Detergent matrix

This is the detergent matrix (commercially available, without enzymes, opt. Brightener, perfume and dyes) used for the wash test: Chemical name Wt .-% active substance in the raw material Wt .-% active ingredient in the formulation Water demin. 100 rest alkyl benzene sulfonic acid 96 4.4 Anionic surfactants 70 5.6 C12-C18 fatty acid Na salt 30 2.4 Nonionic surfactants 100 4.4 phosphonates 40 0.2 citric acid 100 1.4 NaOH 50 0.95 defoamers tq 0.01 glycerin 100 2 preservative 100 0.08 ethanol 93 1 polyacrylic acid 100 11 Without opt. Brightener, perfume, dye and enzymes. Dosage 3.1 g / L

Example 2: 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 pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity.

• 10 µL AAPF-Lösung (70 mg/mL)
• 1000 µL Tris/HCI (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)

The measurement was carried out at a temperature of 25 ° C, at pH 8.6 and a wavelength of 410 nm. The measuring time was 5 min at a measuring interval of 20 to 60 seconds.
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 created over 5 min at 25 ° C (410 nm)

Example 3: mini wash test

The miniwash test was performed on the shake flask supernatant of the proteases produced.

Conditions: 40 ° C, 16 ° dH water, 1h
Detergent concentration: 3.1 g / L (composition according to Example 1)
Enzyme Concentration: The supernatants are washed to the same activity as compared to a benchmark with the protease according to SEQ ID NO: 1 and the substitution R99E, with a market-standard concentration for proteases in detergent or the detergent without protease

soils:

10N (whole egg, pigment)
C-03 (chocolate milk and soot)
C-05 (blood, milk, ink)

Execution:

• • Place excised tissue (diameter = 10 mm) in a microtiter plate, preheat the wash liquor to 40 ° C, final concentration 4.52 g / L in 16 ° dH water
• • apply lye, enzyme and polymer to the soil, incubate for 1 h at 40 ° C and 600 rpm,
• • then rinse the soiling several times with clean water, leave to dry and determine the brightness with a colorimeter.

The lighter the tissue, the better the cleaning performance. The L-value = brightness is measured, the higher the brighter. It is washed with a common liquid detergent without enzymes. Table 1 shows the measured L values for the matrix without enzymes, the matrix with the R99E variant and the matrix with further variants according to the invention. Table 2 shows the differences of the L value from the respective variant tested according to the invention minus the L value measured for the R99E variant. Table 1: Results of the mini wash test (L values) 10 N C 03 C05 Detergent matrix without protease 4.5 -0.4 2.3 R99E 11.9 6.8 21.6 R99E + S76H 12.0 7.9 20.4 R99E + A188L 13.3 10.3 24.3 R99E + V238A 13.1 10.3 23.3 R99E + V238S 12.7 9.8 24.4 R99E + V238R 12.8 7.4 21.4 R99E + V238L 11.0 8.7 22.5 R99E + V238P 13.7 9.9 24.3 R99E + V238W 12.8 9.1 24.8 R99E + K245N 11.4 10.5 22.8 R99E + K245W 13.6 8.9 17.8 Table 2: Results of the mini-wash test (L value (variant) - L value (R99E)) variant 10 N C 03 C05 R99E + K245N + A188L 1.4 5.1 1.1 R99E + V238P + A188L 1.8 4.1 7.0 R99E + V238R + A188L 3.1 3.0 2.4 R99E + S76H + A188L 0.1 4.0 7.7 R99E + V238A + A188L 0.1 3.7 -1.9 R99E + + V238R N242G 1.8 4.4 7.6 R99E + + S76W I77R 0.1 3.7 4.0 R99E + A188L + N242G 6.2 6.6 4.7

It is clear from the results of the wash tests that all the proteases according to the invention listed above the known variant R99E lead to a better washing performance.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been 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.

Cited patent literature

• WO 2009/121725 [0083]

Cited non-patent literature

• Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410 [0024]
• Gornall, C.S. Bardawill and M.M. David, J. Biol. Chem., 177 (1948), pp. 751-766 [0034]
• Bender, M., et al., J. Am. Chem. Soc. 88, 24 (1966), pp. 5890-5913 [0034]
• Sambrook, J., Fritsch, E.F. and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press [0059]

Claims (10)

Protease comprising an amino acid sequence which has at least 80% 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 position corresponding to position 99, the amino acid substitution 99E; and (B) at least one of the positions corresponding to the positions 238, 188, 76, 77, 242 or 245, each based on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution. Protease after Claim 1 wherein said at least one amino acid substitution according to (b) is selected from the group consisting of 76H, 76K, 76W, 77Q, 77R, 188L, 238A, 238S, 238R, 238L, 238P, 238W, 242G, 242W, 245N, 245R and 245W, respectively based on the numbering according to SEQ ID NO: 1, is selected. Protease after one of Claims 1 or 2 wherein the protease has only one amino acid substitution according to (b), which is selected from the group consisting of 238P, 76H, 188L, 238A, 238S, 238R, 238L, 238W, 245N and 245W, each based on the numbering according to SEQ ID NO :1. Protease after one of Claims 1 to 3 wherein the protease has at least two amino acid substitutions according to (b), these being selected from 76H, 76W, 76K, 77R, 188L, 238A, 238R, 238P, 242G and 245N, preferably from 76W + 77R, 76K + 188L, 76H + 188L, 188L + 238P, 188L + 238R, 188L + 238A, 188L + 242G, 188I + 245N and 238R + 242G, most preferably 76H + 188L and 238P + 188L. Protease after one of Claims 1 to 4 wherein the protease has an additional amino acid substitution at one or more of the positions corresponding to positions 3, 4, 154, 199 and 256, based on the numbering according to SEQ ID NO: 1, preferably one or more of the amino acid substitutions 3T, 41, 154D, 1991 and 256E, more preferably 3T + 4I + 199I or 154D + 256E. Protease after one of Claims 1 to 5 in which the protease has one of the following amino acid substitution variants, in each case based on the numbering according to SEQ ID NO: 1: (i) R99E and A188L and optionally additional ones of S76K, S76H, S76W, I77R, V238A, V238P, V238R, V238L, V238S, V238W, N242G, K245N and K245W; (ii) R99E and one of S76H, S76K and S76W, and optionally one of I77R, V238A, V238P, V238L, V238S, V238W, N242G, K245N and K245W; (iii) R99E and one of V238A, V238S, V238R, V238L, V238W; (iv) R99E and V238R; and one of S76K, S76H, S76W, I77R, N242G, K245N and K245W. (v) R99E and N242G; (vi) R99 and K245N or K245W; and (vii) R99 and I77R. Protease, characterized in that (a) it consists of a protease according to one of the Claims 1 to 6 as starting molecule is obtainable by single or multiple conservative amino acid substitution, wherein the protease (i) at the position corresponding to the position 99, the amino acid substitution 99E; and (ii) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 or 245, based in each case on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution; and / or (b) from a protease according to any one of Claims 1 to 6 is obtainable as a starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and an amino acid sequence over a length of at least 200, 210, 220, 230, 240, 250, 260, 261, 262, 263, 264, 265, 266 , 267 or 268 contiguous amino acids matches the parent molecule, wherein the protease (i) at the position corresponding to position 99, the amino acid substitution 99E; and (ii) at least one of the positions corresponding to positions 76, 77, 188, 238, 242 or 245, based in each case on the numbering according to SEQ ID NO: 1, has at least one further amino acid substitution. Agent, in particular a washing or cleaning agent, characterized in that it comprises at least one protease according to one of Claims 1 to 7 contains and optionally at least one anionic polymer. Process for the cleaning of textiles or hard surfaces, characterized in that in at least one process step, an agent Claim 8 is applied. Use of a protease according to one of Claims 1 to 7 in a washing or cleaning agent which optionally contains at least one anionic polymer for the removal of peptide- or protein-containing stains.