DE102013219467A1 - Stabilized inhibitor protease variants - Google Patents

Abstract

The invention relates to proteases comprising an amino acid sequence which has at least 70% sequence identity to that shown in SEQ ID NO. 1 amino acid sequence having the total length thereof, which has the amino acid residues 160G and 185R and in which the amino acids in at least one, preferably 2, preferably 3, especially 4, more preferably 5, most preferably six of the positions corresponding to the positions 39, 74 , 253 and 3, 4, 199 according to SEQ ID NO. 1, substituted for 39E, 74D, 253D, 3T, 4I and 199I and their preparation and use. Such proteases show a very good stability, in particular surfactant stability, at the same time good inhibitor performance, in particular with benzylmalonic acid.

Description

The invention is in the field of enzyme technology. More particularly, the invention relates to proteases and their preparation whose amino acid sequence has been modified, in particular with regard to use in detergents and cleaners, all sufficiently similar proteases with a corresponding change and nucleic acids coding for them. The invention further relates to methods and uses of these proteases and 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 offers for example the Article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 in "Subtilisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 , Subtilases are naturally produced by microorganisms. Of these, in particular, the subtilisins formed and secreted by Bacillus species are to be mentioned as the most important group within the subtilases.

Examples of the subtilisin-type proteases preferably used in detergents and cleaners are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the protease from Bacillus lentus, in particular from Bacillus lentus DSM 5483, subtilisin DY and the the subtilases, but not the subtilisins in the narrower sense attributable enzyme thermitase, proteinase K and the proteases TW3 and TW7, 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 the international patent applications WO 95/23221 and WO 92/21760 Variants of the alkaline protease from Bacillus lentus DSM 5483 are disclosed which are suitable for use in detergents or cleaners. Further, in the international patent application WO 2011/032988 Detergents and cleaning agents disclosed which also variants of the alkaline protease from Bacillus lentus DSM 5483 included. The protease variants disclosed in these references may be altered, among other positions at positions 3, 4, 99 and 199, in the alkaline protease counting method of Bacillus lentus DSM 5483 and, for example, at said positions, amino acids 3T, 4I, 99D, 99E or 199I exhibit.

Such modifications serve to increase the stability and purification performance of the proteases. Detergents require proteases that show good washing performance. However, enzymes are not indefinitely stable when stored in protease-containing liquid detergents because the proteolytic activity of the proteases leads to degradation of the enzymes. In this case, not only by the degradation of the protease different enzymes, such as amylases, lipases, etc. affected by the degradation, but also the proteases themselves.

It is known in the art to stabilize the enzymes in such agents by reversibly inhibiting the protease by means of appropriate inhibitors, which inhibitors reduce proteolytic degradation during storage. In order not to rely on specific, established inhibitors such as boric acid and 4-formylphenylboronic acid (4-FPBA), the enzyme may also be linked to an otherwise inferior inhibitor, e.g. Benzylmalonic acid (BMA), to be adjusted. Herein, such an adaptation means that the enzyme is mutated so as to better enhance the inhibitor, i. for example, with higher binding affinity.

Although some such adjustments lead to better inhibitability, but to a higher sensitivity to surfactants, although the temperature stability remains substantially unchanged. In a detergent matrix this leads to a rapid degradation of the protease.

Surprisingly, it has now been discovered that a protease of the alkaline protease type from Bacillus lentus DSM 5483 or a sufficiently similar protease (in terms of sequence identity) which, in addition to the inhibitory mutations S160G and Q185R, enhance the binding of BMA Substitution of the amino acids at positions 39, 74 and / or 253 by glutamic acid (39E) or aspartic acid (74D or 253D) or at positions 3, 4 and 199 by threonine (3T) or isoleucine (4I, 199I) in has the method of counting the alkaline protease from Bacillus lentus DSM 5483, is particularly suitable for its use in detergents and advantageously improved, in particular with regard to the surfactant stability with further improved inhibitor performance with BMA over the wild-type enzyme or an enzyme, the above Does not exhibit inhibitor mutations. This allows the use of the corresponding modified protease in surfactant-containing matrices such as detergents and cleaners.

The invention therefore in a first aspect is a protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO.1 over the entire length thereof, which at the positions 160 and 185 of SEQ ID NO. 1 having amino acid residues 160G and 185R and having at least one, preferably two, more preferably three, especially four, more preferably five, most preferably six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I in each case based on the numbering according to SEQ ID NO.1.

Another object of the invention is a method for producing a protease comprising substituting the amino acids at least one, preferably 2, more preferably 3, especially four, more preferably five, most preferably six positions, the positions 39, 74 and 253 3, 4 and 199 in SEQ ID NO.1, in an initial protease having at least 70% sequence identity to that shown in SEQ ID NO. 1 at its positions corresponding to positions 160 and 185 in SEQ ID NO.1, having amino acid residues 160G and 185R such that the protease at the corresponding positions comprises amino acids 39E and / or 74D and / or 253D and / or 3T and / or 4I and / or 199I.

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 statements on the protease therefore relate both to the protease as a substance and to the corresponding processes, in particular production processes of the protease.

As further subjects of the invention, proteases encoding the proteases according to the invention or protease-producing nucleic acids, proteases or nucleic acids containing non-human host cells and proteases according to the invention are defined comprising agents, in particular detergents and cleaners, washing and cleaning processes, and proteases according to the invention Uses connected.

The present invention is based on the surprising finding of the inventors that an inventive change in the positions corresponding to the positions 39, 74, 253, 3, 4 or 199 of the alkaline protease from Bacillus lentus DSM 5483 according to SEQ ID NO.1, in a Proteases which have one to the in SEQ ID NO. 1 amino acid sequence comprises at least 70% identical amino acid sequence and the inhibitory mutations has 160G and 185R, such that at the corresponding positions of the amino acids 39E, 74D and / or 253D are present, an improved surfactant stability of this modified protease in detergents and cleaners causes.

In particular, the inventors have found that stability to surfactants can be improved by introducing mutations besides the inhibitory mutations 160G and 185R at positions 3, 4 and / or 199, in particular substitutions 3T, 4I and / or 199I , This is particularly surprising inasmuch as this combination of mutations acts synergistically without adversely affecting inhibitor performance over BMA.

It was particularly surprising that the stability of the improved proteases could be improved by selecting at least one amino acid substitution at positions 3, 4 and / or 199, in particular substitutions 3T, 4I and / or 199I, in combination with at least one mutation at the Positions 39, 74 and / or 253, in particular by the substitution (s) 39E, 74D and / or 253D, a still higher stability could be achieved without reducing the inhibitor performance over BMA.

The proteases of the invention have a particular stability in detergents or cleaners, in particular to surfactants but also to bleaching agents and / or to temperature influences, in particular to high temperatures, for example between 50 and 65 ° C, especially 60 ° C, and / or acidic or alkaline conditions and / or to pH values. Changes and / or to denaturing or oxidizing agents and / or to proteolytic degradation and / or to a change in the redox ratios. Thus, with particularly preferred embodiments of the invention, performance-enhanced protease variants are provided. Such advantageous embodiments of proteases according to the invention consequently enable improved wash results on protease-sensitive soiling in a wide temperature range.

A protease according to the invention has a proteolytic activity, that is, it is capable of hydrolysing peptide bonds of a polypeptide or protein, in particular in a washing or cleaning agent. A protease according to the invention is therefore an enzyme which catalyzes the hydrolysis of peptide bonds and thereby is able to cleave peptides or proteins. Furthermore, a protease of the invention is preferably a mature protease, i. to the catalytically active molecule without signal and / or propeptide (s). Unless otherwise stated, the sequences given refer to each mature enzyme.

In a further embodiment of the invention, the protease comprises an amino acid sequence which corresponds to the amino acid sequence shown in SEQ ID NO. 1 at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, of their total length. 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% and 98.8% is identical to those at the positions the positions 160 and 185 in the count according to SEQ ID NO. 1, amino acids 160G and 185R as inhibitor mutations and at least one of the positions corresponding to positions 39, 74 and 253, and 3, 4 and 199 in the count of SEQ ID NO.1, amino acids 39E, N74D and / or 253D or 3T, 4I and / or 199I.

Preferably, the protease has one of the mutations 39E, 74D or 253D, more preferably a combination of two mutations, i. For example, 39E and 74D, 39E and 253D or 74D and 253D, and most preferably all three mutations, i. 39E + 74D + 253D, up.

In particular, the corresponding protease has at least one, preferably two, more preferably three, in particular four, more preferably five, most preferably six amino acid substitutions selected from P39E, N74D, S253D, S3T, V4I and V199I based in each case on the numbering according to SEQ ID NO.1.

In another preferred embodiment, in addition to 160G and 185R, the protease has as inhibitor mutations, one of the 3T, 4I or 199I mutations, more preferably a combination of two mutations, i. For example, 3T and 4I, 3T and 199I or 4I and 199I, and most preferably all three mutations, i. 3T + 4I + 199I, up.

In a further most preferred embodiment, the protease, 160G and 185R as inhibitor mutations, has at least two amino acid substitutions, wherein at least one amino acid substitution is selected from the group consisting of 39E, 74D and 253D, and at least one further amino acid substitution is selected from the group consisting from 3T, 4I and 199I, positions in each case based on the numbering according to SEQ ID NO.1, on.

Particularly preferred is the protease having 160G and 185R as inhibitor mutations, one of the following two amino acid substitutions: 39E + 3T, 39E + 4I, 39E + 199I, 74D + 3T, 74D + 4I, 74D + 199I, 253D + 3T, 253D + 4I, 253D + 199I,

Further preferred is the protease which, in addition to 160G and 185R as inhibitor mutations, has one of the following three amino acid substitutions: 39E + 74D + 3T, 39E + 74D + 4I, 39E + 74D + 199I, 74D + 253D + 3T, 74D + 253D + 4I , 74D + 253D + 199I, 39E + 253D + 3T, 39E + 253D + 4I, 39E + 253D + 199I,

Particularly preferred is the protease which, in addition to 160G and 185R as inhibitor mutations, has one of the following four amino acid substitutions: 39E + 74D + 253D + 3T, 39E + 74D + 253D + 4I, 39E + 74D + 253D + 199I, 39E + 253D + 3T + 4I, 39E + 253D + 3T + 199I, 39E + 253D + 4T + 199I, 74D + 253D + 3T + 4I, 74D + 253D + 3T + 199I, 74D + 253D + 4I + 199I,

Most preferred is the protease which has 160G and 185R as inhibitor mutations, one of the following five amino acid substitutions: 39E + 74D + 253D + 3T + 4I, 39E + 74D + 253D + 3T + 199I, 39E + 74D + 253D + 4I + 199I, 39E + 74D + 3T + 4I + 199I, 39E + 253D + 3T + 4I + 199I, 74D + 253D + 3T + 4I + 199I.

For example, the protease which, besides 160G and 185R as inhibitor mutations, has all of the following amino acid substitutions is preferred: 39E + 74D + 253D + 3T + 4I + 199I.

Especially preferred according to the invention is a protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO.1 over its entire length, which correspond to the amino acid residues at the positions corresponding to positions 160 and 185 of SEQ ID NO.1 160G and 185R, and having at least one, preferably two, more preferably three, especially four, more preferably five, most preferably six amino acid substitutions selected from the group consisting of P39E, N74D, S253D, S3T, V4I and V199I in each case based on the numbering according to SEQ ID NO.1.

The abovementioned preferred combinations apply in particular to the abovementioned amino acid substitutions which are selected from the group consisting of P39E, N74D, S253D, S3T, V4I and V199I, each based on the numbering according to SEQ ID NO.1.

Particularly preferred is a protease which comprises besides 160G and 185R as inhibitor mutations at least one, two or three amino acid substitutions of P39E, N74D and S253D, and at least one, two or three further amino acid substitutions selected from the group consisting of S3T, V4I and V199I, positions in each case based on the numbering according to SEQ ID NO.1.

Particularly preferred is the protease which, in addition to 160G and 185R as inhibitor mutations, has the amino acid substitutions P39E, N74D, S253D, S3T, V4I and V199I.

In a preferred embodiment, in the context of the present invention, the feature that a protease has the indicated substitutions means that it contains all the corresponding amino acids at the corresponding positions, i. none of the indicated positions is otherwise mutated or deleted, for example by fragmentation of the protease.

Proteases of this type which are preferred according to the invention are shown in SEQ ID NOs. 2-15. The proteases of the invention may comprise or consist of one of the amino acid sequences given in SEQ ID NOs 1-15.

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

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 statement that an amino acid position corresponds to a numerically designated position in SEQ ID NO. 1 therefore means that the corresponding position is assigned to the numerically designated position in SEQ ID NO. 1 in an alignment as defined above.

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, the stability of the protease can be further increased by one or more corresponding mutations, thereby improving 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 towards surfactants and / or bleaches and / or other components, can therefore be further developed within the scope of the invention.

For the description of substitutions that concern exactly one amino acid position (amino acid substitutions), the following convention is used: 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. If no amino acid is indicated before the sequence position (for example 3T), the replacement of any amino acid at the named position to the amino acid named after the position (ie at position 3 a substitution of any amino acid in threonine) takes place. 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, A95G describes the substitution of alanine at position 95 by glycine, A95AG the insertion of glycine after the amino acid alanine at position 95 and A95 * or ΔA95 the deletion of alanine at position 95. 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 counting according to SEQ ID NO. Figure 1 also depicts the amino acid substitutions of the invention at positions corresponding to positions 160 and 185 and at least one of 39, 74, 253 and 3, 4 and 199 in SEQ ID NO.1, as described above. The term "conservative amino acid substitution" means the substitution of one amino acid residue for another amino acid residue, which substitution does not result in a change in polarity or charge at the position of the exchanged amino acid, e.g. Example, the replacement of a nonpolar amino acid residue against 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 the starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence which is over a length of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 or 266 are contiguous amino acids with the parent molecule, the mutant amino acid residues present in the parent molecule are still present at the positions corresponding to positions 160 and 185 and at least one of 39, 74, 253 and 3, 4 and 199 in SEQ ID NO.1.

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 starting molecule by one or more amino acid substitutions in positions which correspond to the positions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268 of the Protease from Bacillus lentus according to SEQ ID NO. 1 are assigned in an alignment, wherein the protease in the count according to SEQ ID NO. 1, the substitutions at the positions corresponding to positions 160 and 185 as well as at least one of 39, 74, 253 as well as 3, 4 and 199 in SEQ ID NO.1, as described above. The further amino acid positions are determined by an alignment of the amino acid sequence of a protease according to the invention with the amino acid sequence of the protease from Bacillus lentus, as described in SEQ ID NO. 1 is defined. 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 said positions in the amino acid sequence of the protease from Bacillus lentus, the alteration positions in a protease according to the invention are those which 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 the positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268, to be assigned in an alignment with SEQ ID NO. 1 and thus in the count according to SEQ ID NO. 1. In the positions mentioned, the following amino acid residues are present in the wild-type molecule of the protease from Bacillus lentus (SEQ ID NO.16) or also the protease according to SEQ ID NO.1: S36, N42, A47, T56, G61, T69, E87 , A96, A101, I102, S104, N114, H118, A120, S130, S139, T141, S142, S154, S157, V193, G205, L211, A224, K229, S236, N237, N242, H243, N255 and T268.

Substances G61A, S154D, S154E and V193M, for example, are furthermore advantageous if the correspondingly homologous positions in a protease according to the invention are not already naturally taken up by one of these preferred amino acids.

The abovementioned positions for changing the sequence, in particular substitution, apply according to the invention to all of the protease variants explicitly mentioned above. In particular, these additional sequence changes, in particular substitutions, can be carried out starting from one of the proteases which have an amino acid sequence selected from the sequence according to SEQ ID No.1-15, the resulting proteases are also particularly preferred.

In all of the proteases of the invention described above, in a preferred embodiment, the amino acid residue at the position corresponding to position 99 in SEQ ID NO.1 is E, i. 99E. This residue is preferably retained in all possible modifications described above. Examples of such proteases are those with the in SEQ ID NOs. 1-15 indicated amino acid sequences.

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 to the same Way is changed. If, for example, an amino acid exchange in a specific position of the protease from Bacillus lentus according to SEQ ID NO. 1 associated with a change in an enzymatic parameter, for example, with the increase of the K _M value, and a corresponding change in the enzymatic parameter, for example, also an increase in the K _M value, observed in a protease variant according to the invention, the amino acid exchange the same introduced amino acid has been achieved, a confirmation of the correct association is to be seen herein.

In a further embodiment of the invention, the protease is characterized in that its purification performance compared to that of a protease comprising an amino acid sequence which corresponds to that shown in SEQ ID NOs. 1-15 corresponds to the amino acid sequence indicated, is not significantly reduced, i. has at least 80% of the reference washing performance.

Methods for the determination of protease activity are familiar to the expert in the field of enzyme technology and are routinely used by him. For example, such methods are disclosed in Surfactants, Vol. 7 (1970), pp. 125-132 , Alternatively, the protease activity can be determined via the release of the chromophore para-nitroaniline (pNA) from the substrate suc-L-Ala-L-Ala-L-Pro-L-Phe-p-Nitroanilide (AAPF). The protease cleaves the substrate and releases pNA. The release of the pNA causes an increase in absorbance at 410 nm, the time course of which is a measure of the enzymatic activity (cf. Del Mar et al., 1979 ). The measurement 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 20s to 60s. 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.

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 (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766) be determined. The determination of the active protein concentration can in this regard via a titration of the active sites using a suitable irreversible inhibitor (for proteases, for example, phenylmethylsulfonyl fluoride (PMSF)) and determination of the residual activity (See M. Bender et al., J. Am. Chem. Soc., 88, 24 (1966), pp. 5890-5913) respectively.

• (a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution, wobei die Protease die Aminosäurereste 160G und 185R an den Positionen, die den Positionen 160 und 185 gemäß SEQ ID NO.1 entsprechen, sowie mindestens eine, vorzugsweise zwei, noch bevorzugter drei, insbesondere vier, besonders bevorzugt fünf, ganz besonders bevorzugt sechs Aminosäuresubstitutionen aufweist, die ausgewählt werden aus der Gruppe bestehend aus 39E, 74D, 253D, 3T, 4I und 199I, jeweils bezogen auf die Nummerierung gemäß SEQ ID NO.1;
• (b) Veränderung der Aminosäuresequenz durch Fragmentierung, Deletions-, Insertions- oder Substitutionsmutagenese derart, dass die Protease eine Aminosäuresequenz umfasst, die über eine Länge von mindestens 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 oder 266 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die Protease die Aminosäurereste 160G und 185R an den Positionen, die den Positionen 160 und 185 gemäß SEQ ID NO.1 entsprechen, sowie mindestens eine, vorzugsweise 2, noch bevorzugter drei, insbesondere vier, besonders bevorzugt fünf, ganz besonders bevorzugt sechs Aminosäuresubstitutionen aufweist, die ausgewählt werden aus der Gruppe bestehend aus 39E, 74D, 253D, 3T, 4I und 199I, jeweils bezogen auf die Nummerierung gemäß SEQ ID NO.1;
• (c) Einbringen einer ein- oder mehrfachen Aminosäuresubstitution in eine oder mehrere der Positionen, die den Positionen 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255 und 268, der Protease gemäß SEQ ID NO. 1 entsprechen, wobei die Protease die Aminosäurereste 160G und 185R an den Positionen, die den Positionen 160 und 185 gemäß SEQ ID NO.1 entsprechen, sowie mindestens eine, vorzugsweise 2, noch bevorzugter drei, insbesondere vier, besonders bevorzugt fünf, ganz besonders bevorzugt sechs Aminosäuresubstitutionen aufweist, die ausgewählt werden aus der Gruppe bestehend aus 39E, 74D, 253D, 3T, 4I und 199I, jeweils bezogen auf die Nummerierung gemäß SEQ ID NO.1.

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:

• (a) introducing a single or multiple conservative amino acid substitution, said protease comprising amino acid residues 160G and 185R at the positions corresponding to positions 160 and 185 of SEQ ID NO.1 and at least one, preferably two, more preferably three, in particular four, more preferably five, most preferably has six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering of SEQ ID NO.1;
• (b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease has an amino acid sequence of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 or 266 contiguous amino acids matches the parent molecule, the protease having amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, and at least one, preferably 2, more preferably 3, especially 4, more preferably 5, most preferably 6 amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1;
• (c) introducing a single or multiple amino acid substitution into one or more of the positions corresponding to positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268, the protease according to SEQ ID NO. 1, wherein the protease the amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, and at least one, preferably 2, more preferably three, especially four, more preferably five, most preferably has six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1.

• (a) Einbringen einer ein- oder mehrfachen konservativen Aminosäuresubstitution, wobei die Protease in der Zählung gemäß SEQ ID NO. 1 die Aminosäurereste 160G und 185R sowie mindestens eine, vorzugsweise 2, noch bevorzugter drei Aminosäuresubstitutionen ausgewählt aus 39E, 74D und S253D aufweist;
• b) Veränderung der Aminosäuresequenz durch Fragmentierung, Deletions-, Insertions- oder Substitutionsmutagenese derart, dass die Protease eine Aminosäuresequenz umfasst, die über eine Länge von mindestens 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 oder 266 zusammenhängenden Aminosäuren mit dem Ausgangsmolekül übereinstimmt, wobei die im Ausgangsmolekül enthaltenen Aminosäurereste 160G und 185R sowie mindestens eine, vorzugsweise 2, noch bevorzugter drei Aminosäuresubstitutionen ausgewählt aus 39E, 74D und S253D noch vorhanden sind;
• (c) Einbringen einer ein- oder mehrfachen Aminosäuresubstitution in eine oder mehrere der Positionen, die den Positionen 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 und 268 der Protease aus Bacillus lentus gemäß SEQ ID NO. 1 in einem Alignment zugeordnet sind, wobei die Protease in der Zählung gemäß SEQ ID NO. 1 die Aminosäurereste 160G und 185R sowie mindestens eine, vorzugsweise 2, noch bevorzugter drei Aminosäuresubstitutionen ausgewählt aus 39E, 74D und S253D aufweist.

In particular, a method according to the invention comprises one or more of the following method steps:

• (a) introducing a single or multiple conservative amino acid substitution, wherein the protease in the count according to SEQ ID NO. 1 has amino acid residues 160G and 185R and at least one, preferably 2, more preferably three amino acid substitutions selected from 39E, 74D and S253D;
• b) alteration of 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 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 or 266, the amino acid residues 160G and 185R contained in the starting molecule and at least one, preferably 2, more preferably, three amino acid substitutions selected from 39E, 74D and S253D are still present;
• (c) introducing a single or multiple amino acid substitution into one or more of the positions corresponding to positions 3, 4, 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268 of the protease from Bacillus lentus according to SEQ ID NO. 1 are assigned in an alignment, wherein the protease in the count according to SEQ ID NO. 1 has amino acid residues 160G and 185R and at least one, preferably 2, more preferably 3 amino acid substitutions selected from 39E, 74D and S253D.

All statements also apply to the inventive method.

In further embodiments of the invention, the protease or the protease produced by a method according to the invention is still 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 98.8% identical to one of the amino acid sequences indicated in SEQ ID NOs: 1-15 over their entire length. The protease or the protease produced by a method according to the invention has the amino acid residues 160G and 185R, preferably also 99E, and at least one, preferably 2, more preferably 3, the substitutions 39E, 74D and 53D and / or, in preferred embodiments, at least one, preferably 2, more preferably 3, of substitutions 3T, 4I and 199I.

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. For an increase in stability during storage and / or during use, for example during the washing process, causes the enzymatic activity to last longer and thus improve the 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 via 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. For example, proteases can also be stabilized by replacing one or more tyrosine residues with other amino acids.

• – 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, i. 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 may be associated 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 protease inhibitors is possible in this regard.

With regard to all proteases or protease variants and / or derivatives described above, particular preference is given in the context of the present invention to those whose stability and / or activity is at least equal to that of the protease according to any one of SEQ ID NOs: 1-15 and / or their purification performance at least equal to that the protease according to any one of SEQ ID NOs. 1-15 corresponds.

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, with nucleic acids according to the invention, one or more codons may be replaced by synonymous codons. 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. Bottlenecks in protein biosynthesis can occur if the codons lying on the nucleic acid in the organism face a comparatively small number of loaded tRNA molecules. Although coding for the same amino acid, this results in a codon being less efficiently translated 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 can use well-known methods such as chemical synthesis or the polymerase chain reaction (PCR) in combination with molecular biological and / or proteinchemical standard methods, using known DNA and / or amino acid sequences, the corresponding nucleic acids to complete genes manufacture. Such methods are for example Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press , known.

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 Cultivation conditions or upon reaching a certain cell density, these can be stimulated 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 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 in that it has 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, coexpression with the enzymes naturally produced by such cells, such as cellulases or lipases, 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 removed from the medium after an experimentally determined period of time harvested. 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.

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

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

• a) cultivating a host cell according to the invention; and
• 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 protease 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 subject matter of the invention includes all conceivable types of detergents or cleaners, both concentrates and undiluted agents, for use on a commercial scale, in the washing machine or in hand washing or cleaning. These include detergents for textiles, carpets, or natural fibers, for which the term detergent is used. These include, for example, dishwashing detergents for dishwashers or manual dishwashing detergents or cleaners for hard surfaces such as metal, glass, porcelain, ceramics, tiles, stone, painted surfaces, plastics, wood or leather, for which the term detergent is used, ie in addition to manual and machine Dishwashing agents, for example, scouring agents, glass cleaners, toilet scenters, etc. The washing and cleaning agents in the invention also include washing aids which are added to the actual detergent in the manual or machine textile laundry to achieve a further effect. 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 to give the laundry further desirable properties such as comfortable grip, crease resistance or low static charge. Among the latter, i.a. calculated the fabric softener.

The washing or cleaning agents according to the invention, which can be in the form of powdered solids, in densified particle form, as homogeneous solutions or suspensions, can be used in addition to a protease according to the invention contain all known ingredients customary in such agents, wherein preferably at least one further ingredient is 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 preferred embodiments, these agents contain an inhibitor for the proteases according to the invention, preferably benzylmalonic acid. Benzylmalonic acid can be used in an amount of from 1 g / L to 100 g / L, preferably 5 g / L-50 g / L, in each case based on the volume of the (liquid) washing or cleaning agent. In addition to benzylmalonic acid, it is also possible to use other known protease inhibitors, for example boric acid or 4-formylphenylboronic acid. Boric acid is also contained in specific embodiments of the invention in an amount of from 1 g / L to 100 g / L, preferably 5 g / L-50 g / L, in each case based on the volume of the (liquid) washing or cleaning agent.

A combination of a protease of the invention with one or more other ingredients of the composition is advantageous because such agent in preferred embodiments of the present invention has improved cleaning performance by resulting synergisms. In particular, by combining a protease according to the invention with a surfactant and / or a builder (builder) and / or a peroxygen compound and / or a bleach activator, such a synergism can be achieved.

Advantageous ingredients of agents 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 according to 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 very particularly preferably from 50 μg to 10 mg per g of the composition. 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 may be packaged in a container, preferably an air-permeable container, from which it is released shortly before use or during the washing process.

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

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

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

These embodiments of the present invention include all solid, powdered, liquid, gelatinous or paste-like administration forms of compositions according to the invention, which if appropriate can also consist of several phases and can be present in compressed or uncompressed form. The agent can be present as a free-flowing powder, in particular with a bulk density of 300 g / l to 1200 g / l, in particular 500 g / l to 900 g / l or 600 g / l to 850 g / l. The solid dosage forms of the composition also include extrudates, granules, tablets or pouches. Alternatively, the agent 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. Preferred enzymes which can be used as enzymes are all enzymes which can develop a catalytic activity in the agent according to the invention, in particular a protease, amylase, cellulase, hemicellulase, mannanase, tannase, xylanase, xanthanase, Xyloglucanase, β-glucosidase, pectinase, carrageenase, perhydrolase, oxidase, oxidoreductase or a lipase, and mixtures thereof. Additional enzymes are advantageously included in the agent each in an amount of 1 × 10 ^-8 to 5 weight percent based on active protein. More preferably, each further enzyme is in an amount of 1 × 10 ^-7 -3 wt%, from 0.00001-1 wt%, from 0.00005-0.5 wt%, from 0.0001 to 0.1 wt .-% and particularly preferably from 0.0001 to 0.05 wt .-% in inventive compositions, based on active protein. 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 another enzyme of an agent according to the invention, including in particular between said protease and the 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.

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 40μg to 4g, preferably from 50μg to 3g, more preferably from 100μg to 2g and most preferably from 200μg to 1g is used.

These include both manual and mechanical processes, with mechanical processes being preferred. Methods 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 contact time, or that the items to be cleaned are otherwise treated with a detergent or a solution or dilution of this product. The same applies to processes for cleaning all other materials than textiles, especially hard surfaces. All conceivable washing or cleaning processes can be enriched in at least one of the process steps by the use of a washing or cleaning agent or a protease according to the invention and then represent embodiments of the present invention. All facts, objects and embodiments, which proteases 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 may be that, if desired, the only cleaning-active component is an inventive Protease is brought into contact 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.

Another subject of the invention is the use of an agent according to the invention for the cleaning of textiles or hard surfaces, or a protease according to the invention for the purification of textiles or hard surfaces, in particular such that the protease in an amount of 40μg to 4g, preferably from 50μg to 3g , more preferably from 100μg to 2g and most preferably from 200μg to 1g is used.

All aspects, objects, and embodiments described for proteases of the invention and agents containing them are also applicable to this subject of the invention. Therefore, reference is made at this point expressly to the disclosure in the appropriate place with the statement that this disclosure also applies to the above inventive use.

Examples

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

Example 1: Stability of the Starting Molecule - Variant A.

The protease variant adapted to the inhibitor BMA with a sequence according to SEQ ID NO.1 (variant A) was compared to established proteases, such as the protease with a sequence according to SEQ ID NO.17 (B) and the protease, which has a with the sequence according to variant F49 WO9523221 has identical amino acid sequence (C), used in a test at 60 ° C / pH 10 for temperature stability and at 40 ° C / pH8 and presence of Texapon or linear alkylbenzenesulfonate (LAS). At regular intervals the activity was determined by AAPF test. The half-life was calculated assuming a pseudo 1st order after linearization. t½ [min.] Protease (B) Protease (A) Protease (C) 60 ° C / pH10 14 8th 7 40 ° C / pH8, Texapon 410 10 nd

It is clear that the temperature stability of the protease variant with a sequence according to SEQ ID NO.1 (variant A) is within the usual stabilities, while the stability in the presence of surfactants is drastically reduced.

Example 2: Mutation Strategy

Starting from a protease which has an amino acid sequence according to SEQ ID NO. 1, a protease variant according to the invention was prepared by site-directed mutagenesis in the nucleic acid coding for the protease by means of the "PHUSION site-directed mutagenesis kit" (Finnzyme, F541). In this case, the codons for the specified amino acid positions were changed so that during translation based on the amino acid sequence, a substitution of the amino acids was carried out as indicated. The expression of the protease variant was carried out in a customary manner by transformation of Bacillus subtilis DB 104 (Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444) with a corresponding expression vector and subsequent culture of the protease variant expressing transformants. The proteases were purified by ion exchange chromatography from the appropriate cultures.

Example 3: Stability tests at high temperatures in the presence of a detergent matrix

The parallel fermented in screening tests variants were added in the same concentration as fermentation supernatant from minimized culture in a product-near detergent matrix and incubated at 50 and 55 ° C. At regular intervals the activity was determined by AAPF test. The half-life was determined assuming a pseudo 1st order after linearization. variant T50 [min] T50 [min] 50 ° C 55 ° C Protease variant with a sequence according to SEQ ID NO.17 (B) 75 Protease variant with a sequence according to SEQ ID NO.1 (variant A) 10 5 Variant A + P39E (protease variant with a sequence according to SEQ ID NO.2) 17 Variant A + N74D (protease variant with a sequence according to SEQ ID NO.3) 27 Variant A + S253D (protease variant with a sequence according to SEQ ID NO.4) 47 Variant A + P39E + S253D (protease variant with a sequence according to SEQ ID NO.6) 55 Variant A + P39E + N74D (protease variant with a sequence according to SEQ ID NO.5) 60 Variant A + N74D + S253D (protease variant with a sequence according to SEQ ID NO.7) 200 Variant A + P39E + N74D + S253D (protease variant with a sequence according to SEQ ID NO.8) 230 48 Variant A + P39E + N74D + S253D + S3T + V4I (protease variant with a sequence according to SEQ ID NO.12) 76 Variant A + P39E + N74D + S253D + V199I (protease variant with a sequence according to SEQ ID NO.11) 99 Variant A + P39E + N74D + S253D + S3T + V4I + V199I (protease variant with a sequence according to SEQ ID NO.15, variant D) 144

It is clear that the reduced half-life of the variants in the presence of surfactants compared to protease (B) is restored by the introduced mutations and even increased by synergistic use.

Example 4 Purification by Ion Exchange Chromatography

The supernatant of the proteases obtained according to Example 2 was dialyzed against a 5 mM HEPES buffer system at pH 7.8 for high isoelectric point protons and oh 7.2 with low isoelectric point. The purification was then carried out in the appropriate, 50 mM HEPES buffer via an anion and a cation exchanger and subsequent elution of the cation exchanger by a NaCl gradient.

The isolated protease fractions were pooled, stabilized by 20% PG and stored at 4 ° C.

Example 5: Inhibition Measurements in Biochemical Tests

Increasing amounts of inhibitor (benzylmalonic acid) were added to a concentration of the purified molecules in detergent matrix measurable by AAPF assay, and the residual activity was determined by means of the AAPF assay. The conductivity of the individual batches was corrected to the highest value of the conductivity increased by the inhibitor using NaCl.

The following results were achieved: c _inhibitor [mM] 255 204 170 127 85 51 25 3 0 Activity variant B [%] 50% 52% 53% 57% 88% 96% 102% 101% 100% Activity Variant A [%] 16% 18% 19% 24% 33% 45% 78% 98% 100% Activity Variant D [%] 28% 32% 33% 38% 43% 81% 83% 110% 100%

It is clear that the protease variant D can be better inhibited similar to the starting molecule protease variant A than the protease variant B.

Example 6: Storage tests in detergent matrices

The protease purified protease variants A, B and D were added in equal concentrations to detergent matrix formulations with 1% BMA. The mixtures were stored at 30 ° C. and the protease activity was determined regularly. This resulted in the following half-lives (determined by linearization assuming pseudo 1st order: Half-life [d] Variant B option A Variant D 1% BMA 12 0.5 39

It becomes clear that the protease variant D, in contrast to variant A, has a markedly improved storage stability and also shows higher stabilities compared to protease variant B.

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 95/23221 [0004]
• WO 92/21760 [0004]
• WO 2011/032988 [0004]
• WO 2009/121725 [0086]
• WO 9523221 [0098]

Cited non-patent literature

• Article "Subtilases: Subtilisin-like Proteases" by R. Siezen, pages 75-95 [0002]
• "Subtiltisin enzymes", edited by R. Bott and C. Betzel, New York, 1996 [0002]
• Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (1990) "Basic local alignment search tool." Biol. 215: 403-410 [0034]
• Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs "; Nucleic Acids Res., 25, p.3389-3402 [0034]
• Chenna et al. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497-3500 [0034]
• Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217 [0034]
• Surfactants, Vol. 7 (1970), pp. 125-132 [0050]
• Del Mar et al., 1979 [0050]
• (AG Gornall, CS Bardawill and MM David, J. Biol. Chem., 177 (1948), pp. 751-766) [0051]
• (See M. Bender et al., J. Am. Chem. Soc., 88, 24 (1966), pp. 5890-5913) [0051]
• Sambrook, J., Fritsch, EF and Maniatis, T. 2001. Molecular cloning: a laboratory manual, 3rd Edition Cold Spring Laboratory Press [0065]
• Fritsch, Sambrook and Maniatis "Molecular cloning: a laboratory manual", Cold Spring Harbor Laboratory Press, New York, 1989 [0097]
• (Kawamura and Doi (1984), J. Bacteriol., Vol. 160 (1), pp. 442-444) [0100]

Claims (14)

A protease comprising an amino acid sequence which has at least 70% sequence identity with the amino acid sequence given in SEQ ID NO.1 over its entire length, corresponding at positions 160 and 185 of SEQ ID NO.1 having amino acid residues 160G and 185R , and which has at least one, preferably 2, more preferably 3, especially 4, more preferably 5, most preferably 6 amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, based in each case on Numbering according to SEQ ID NO.1. A protease, characterized in that (i) it is obtainable from a protease according to claim 1 as starting molecule by single or multiple conservative amino acid substitution, the protease comprising amino acid residues 160G and 185R at the positions corresponding to positions 160 and 185 according to SEQ ID NO .1 and at least one, preferably 2, more preferably 3, especially 4, more preferably 5, most preferably 6 amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, respectively to the numbering according to SEQ ID NO.1; and / or (ii) it is obtainable from a protease according to claim 1 as starting molecule by fragmentation, deletion, insertion or substitution mutagenesis and comprises an amino acid sequence of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265, 266, 267, or 268 are correlated with the parent molecule, wherein the protease comprises Amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, as well as at least one, preferably 2, more preferably three, especially four, more preferably five, most preferably six amino acid substitutions are selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1; and / or (iii) it is obtainable from a protease according to claim 1 as starting molecule by one or more amino acid substitutions in positions corresponding to the positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268 of the protease according to SEQ ID NO. 1, wherein the protease the amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, and at least one, preferably 2, more preferably three, especially four, more preferably five, most preferably has six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1. Protease according to claim 1 or 2, characterized in that the protease has at least one, preferably 2, more preferably three amino acid substitutions selected from the group consisting of 39E, 74D and 253D, each based on the numbering according to SEQ ID NO.1 , Protease according to one of claims 1 to 3, characterized in that the protease has at least two amino acid substitutions, wherein at least one amino acid substitution is selected from the group consisting of 39E, 74D and 253D, and at least one other is selected from the group consisting of 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1. Protease according to one of claims 1 to 4, characterized in that the protease comprises or consists of the amino acid sequence according to one of SEQ ID NO. 2-15. A process for the preparation of a protease according to any one of claims 1 to 5, which comprises substituting the amino acids for at least one, preferably 2, more preferably 3 especially four, more preferably five, most preferably six positions, positions 39, 74, 253, 3 , 4 and 199 in SEQ ID NO.1, in a parent protease having at least 70% sequence identity to that shown in SEQ ID NO. 1 at its positions corresponding to positions 160 and 185 in SEQ ID NO.1, having amino acid residues 160G and 185R such that the protease at the corresponding positions comprises amino acids 39E and / or 74D and / or 253D and / or 3T and / or 4I (isoleucine) and / or 199I (isoleucine). The method of claim 6, further comprising one or more of the following steps: (a) introducing a single or multiple conservative amino acid substitution, wherein the protease has amino acid residues 160G and 185R at positions corresponding to positions 160 and 185 of SEQ ID NO.1 and at least one, preferably two, more preferably three, especially four, more preferably five, most preferably six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1; (b) altering the amino acid sequence by fragmentation, deletion, insertion or substitution mutagenesis such that the protease has an amino acid sequence of at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 265 or 266 contiguous amino acids matches the parent molecule, the protease having amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, and at least one, preferably 2, more preferably 3, especially 4, more preferably 5, most preferably 6 amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1; (c) introducing a single or multiple amino acid substitution into one or more of the positions corresponding to positions 36, 42, 47, 56, 61, 69, 87, 96, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 205, 211, 224, 229, 236, 237, 242, 243, 255 and 268, the protease according to SEQ ID NO. 1, wherein the protease the amino acid residues 160G and 185R at the positions corresponding to the positions 160 and 185 according to SEQ ID NO.1, and at least one, preferably 2, more preferably three, especially four, more preferably five, most preferably has six amino acid substitutions selected from the group consisting of 39E, 74D, 253D, 3T, 4I and 199I, each based on the numbering according to SEQ ID NO.1. A nucleic acid encoding a protease according to any of claims 1-5 or encoding a protease obtained by a method of claims 6 or 7. Vector containing a nucleic acid according to claim 8, in particular a cloning vector or an expression vector. A non-human host cell comprising a nucleic acid according to claim 8 or a vector according to claim 9, or which comprises a protease according to any of claims 1-5, or which comprises a protease obtained by a method of claims 6 or 7, in particular one which secretes the protease into the medium surrounding the host cell. A method for producing a protease comprising (a) culturing a host cell according to claim 10; and (b) isolating the protease from the culture medium or from the host cell. Agent, in particular a washing or cleaning agent, characterized in that it contains at least one protease according to any one of claims 1-5 or a protease obtained by a method of claims 6 or 7. Composition according to claim 12, characterized in that the agent (i) contains the protease in an amount of 2μg to 20mg per g of the agent; and / or (ii) the protease is enveloped in the composition with a substance impermeable to the protease at room temperature or in the absence of water; and / or (iii) the agent further contains an inhibitor for the protease, in particular benzylmalonic acid (BMA). Process for the cleaning of textiles or hard surfaces, characterized in that in at least one process step, an agent according to claim 12 or 13 is applied, or that in at least one process step, a protease according to any one of claims 1-5 or one according to a method of claims 6 or 7 protease obtained is catalytically active, in particular such that the protease is used in an amount of 40μg to 4g per application.