Protease variants and compositions comprising same

Abstract

The present invention relates to protease variants having improved stability at low pH values, e.g., in low pH liquid detergent compositions. The present invention further relates to polynucleotides encoding said variants; nucleic acid constructs and expression vectors comprising said polynucleotides; recombinant host cells transformed with said polynucleotides; compositions comprising said variants; use of said variants in cleaning processes; and methods for producing said variants.

Description

[0001] PROTEASE VARIANTS AND COMPOSITIONS COMPRISING SAME

[0002] Reference to a Sequence Listing

[0003] This application contains a sequence listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to protease variants having improved stability at low pH values, e.g., in low pH detergent compositions. The invention further relates to polynucleotides to compositions comprising the variants, and to methods of using the variants and compositions.

[0006] BACKGROUND OF THE INVENTION

[0007] In the detergent industry, enzymes have been implemented in washing formulations for many decades. Enzymes used in such formulations include proteases, lipases, amylases, cellulases, mannosidases as well as other enzymes or mixtures thereof. Commercially, the most important enzymes are proteases.

[0008] An increasing number of commercially used proteases for e.g., laundry and dishwashing detergents are protein engineered variants of naturally occurring wild type proteases. Further, other protease variants have been described in the art with alterations relative to a parent protease resulting in improvements such as better wash performance, thermal stability, storage stability or catalytic activity.

[0009] However, various factors make further improvement of proteases advantageous. For example, washing conditions such as temperature and pH tend to change over time, and are also different in different countries or regions of the world, and many stains are still difficult to completely remove under conventional washing conditions. Another challenge in detergent compositions is enzyme stability, since the chemical components of these compositions as well as conditions of pH, temperature and humidity often tend to inactivate enzymes. Further, in-wash conditions can also result in inactivation of the enzymes (due to e.g., pH, temperature or chelation instability), resulting in loss of wash performance during the wash cycle. Thus, despite the intensive research in protease development there remains a need for new and improved proteases that have improved stability, for example in low pH detergents, without compromising wash performance.

[0010] The present invention addresses these challenges by providing protease variants with improved stability at low pH values, in particular in low pH detergent compositions, while maintaining wash performance. SUMMARY OF THE INVENTION

[0011] The present invention relates to protease variants having improved stability at low pH values, in particular improved storage stability in low pH liquid detergent compositions.

[0012] In one aspect, the present invention relates to a protease variant of a parent protease, wherein the variant has protease activity and comprises substitutions in three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T {e.g., S3T), X59V / R e.g., G59V / R), X198D {e.g., N198D or G198D), X209K / M / I / R {e.g., A209K / M / I / R), and X212S / T {e.g., N212S / T), and wherein position numbers are based on the numbering of SEQ ID NO:1 ; and wherein the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three- dimensional structure of the parent protease; and / or wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease.

[0013] In other aspects, the present invention relates to polynucleotides encoding said variants, nucleic acid constructs and expression vectors comprising said polynucleotides, recombinant host cells transformed with said polynucleotides, compositions comprising said variants, use of said variants in cleaning processes, and methods for producing said variants.

[0014] BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:2.

[0016] Figure 2 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:3.

[0017] Figure 3 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:4.

[0018] Figure 4 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:5.

[0019] Figure 5 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:6.

[0020] Figure 6 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:7.

[0021] Figure 7 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:8.

[0022] OVERVIEW OF SEQUENCES

[0023] SEQ ID NO:1 is a stabilized variant of the protease of SEQ ID NO:2.

[0024] SEQ ID NO:2 is the subtilisin 309 protease (Savinase®) from Bacillus lentus. SEQ ID NO:3 is the subtilisin BPN’ protease from Bacillus amyloliquefaciens. SEQ ID NO:4 is the subtilisin Carlsberg protease (Alcalase®) from Bacillus licheniformis. SEQ ID NO:5 is the Bacillus lentus alkaline protease (BLAP) from Bacillus lentus DSM 5483. SEQ ID NO:6 is a subtilisin protease from Bacillus gibsonii DSM 14391.

[0025] SEQ ID NO:7 is a subtilisin protease from Bacillus gibsonii DSM 8722.

[0026] SEQ ID NO:8 is a subtilisin protease from Bacillus pumilus DSM 18097.

[0027] DEFINITIONS

[0028] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0029] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0030] Protease: The term “protease” means an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several sub-groups, including EC 3.4.21 (serine endopeptidase), which further contains several sub-groups, including EC. 3.4.21.62 (subtilisin). The terms “protease” and the expression “polypeptide having protease activity” are used interchangeably herein. For purpose of the present invention, protease activity may be determined according to the Suc-AAPF-pNA protease activity assay described in the Examples herein.

[0031] AlphaFold structure calculation: AlphaFold is a computational method for calculating the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., 2021 , Nature 596: 583-589). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 algorithm (Varadi et al., 2021 , Nucleic Acids Res. 50(D1):D439-D444). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.

[0032] In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and predicting structures of new polypeptides at source code repositories such as Github.com under deepmind / alphafold / , using notebooks / AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokrypton / ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).

[0033] AlphaFold produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy {e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0034] Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0035] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the variant, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

[0036] Expression: The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0037] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0038] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a variant, wherein the “extended” variant has protease activity.

[0039] Fragment: The term “fragment” means a polypeptide having one or more amino acids absent from the amino and / or carboxyl terminus of a mature polypeptide; wherein the fragment has protease activity. Such a fragment may preferably contain at least 85%, at least 90% or at least 95% of the number of amino acids in the polypeptide of SEQ ID NO:1. Or such a fragment may preferably contain at least 85%, at least 90% or at least 95% of the number of ammo acids in the polypeptide of SEQ ID NO:4.

[0040] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0041] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0042] Improved property: The term “improved property” means a characteristic associated with a variant that is improved compared to a reference protease, e.g., a parent protease, e.g., SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3. SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. Such improved properties include, but are not limited to, storage and in-wash stability.

[0043] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0044] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component with which it is naturally associated as found in nature, including but not limited to, for example, other proteins, nucleic acids, cells, etc. An isolated polypeptide will typically be purified, but also includes a culture broth containing the secreted polypeptide.

[0045] Low pH value: The term “low pH” as used herein means a pH value of less than about 7, typically a pH of less than about 6.5, such as from about 4 to about 6.5. The low pH value may for example be in the range of from about 4.0 to about 6.0, such as a pH of about 6, about 5.5, about 5, about 4.5 or about 4. For example, the low pH may be used to describe a property of a detergent composition, such as, e.g., a liquid laundry detergent composition.

[0046] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing e.g., removal of signal peptide).

[0047] Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity.

[0048] Mutant: The term “mutant” means a polynucleotide encoding a variant. Native: The term native means a nucleic acid or polypeptide naturally occurring in a host cell..

[0049] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a variant. Nucleic acids may be single stranded or double stranded or may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0050] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

[0051] Parent or parent protease: The term “parent” or “parent protease” means a protease to which an alteration is made to produce the protease variants of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof. The parent may also be referred to herein as a “reference protease”, where the reference protease is a parent protease having the same amino acid sequence as a variant of the invention other than any claimed substitutions, in particular other than any claimed substitutions in positions 3, 37, 59, 76, 106, 113, 198, 205, 206, 209, 210 and / or 212 of SEQ ID NO:1. In one embodiment, the parent protease is the protease of SEQ ID NO:1. In another embodiment, the parent protease is the protease of SEQ ID NO:4.

[0052] Protease activity: The term “protease activity” means a proteolytic activity (EC 3.4), in particular endopeptidase activity (EC 3.4.21). There are several protease activity types, the three main activity types being: trypsin-like, where there is cleavage of amide substrates following Arg or Lys at P1 , chymotrypsin-like, where cleavage occurs following one of the hydrophobic amino acids at P1, and elastase-like with cleavage following an Ala at P1. Protease activity may be determined according to the procedure described in WO 2016 / 087619.

[0053] Purified: The term “purified” means a nucleic acid, variant or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0054] In one aspect, the term "purified" as used herein refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0055] Accordingly, the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The variant may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0056] It is, therefore, preferred that the substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The variant of the present invention is preferably in a substantially pure form i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.

[0057] Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0058] Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hydro-cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0059] Sequence difference: The term "sequence difference" means the percent of amino acid differences between a variant of the invention and the parent protease, e.g., SEQ ID NO:1 or SEQ ID NO:4, and is calculated as follows:

[0060] (Number of different residues x 100) / (Number of residues in parent protease) wherein the term “different residues” means the total number of amino acid residues that have been substituted, deleted, and / or inserted in the variant compared to the parent.

[0061] For a variant of SEQ ID NO:1 having the substitutions S3T, G59V, S76E, N198D, A209K, S210I, and N212S ( / .e., seven different residues), the sequence difference is calculated as follows:

[0062] (7 x 100) / 269 = 2.6%

[0063] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0064] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobnef option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0065] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0066] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0067] Stability: The term “stability” includes storage stability and stability during use, e.g., during a wash process, and reflects the stability of the protease variant according to the invention as a function of time, e.g., how much protease activity is retained when the protease variant is kept in a detergent composition, in particular in a liquid detergent solution, or otherwise in a solution. The stability is influenced by many factors such as pH, temperature, and the particular detergent composition, e.g., the amount and nature of the builders, surfactants, etc. Protease stability may be measured as described in the examples and expressed e.g., as a half-life improvement factor (abbreviated HIF or simply IF) compared to the parent protease or a reference sequence, such as SEQ ID NO:1 or SEQ ID NO:4. The term “improved stability” or “increased stability” in the context of the present invention means that a protease variant displays an increased stability in at low pH, e.g., in a low pH liquid detergent composition, relative to the stability of the parent protease without the substitutions in the variant and / or relative to SEQ ID NO:1 or SEQ ID NO:4. The terms “improved stability” and “increased stability” include detergent stability.

[0068] Structural Similarity: For purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.

[0069] A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g., X-ray crystallography or using in silico methods such as AlphaFold (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):

[0070] TM-score where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, d, is the distance between pair / of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.

[0071] For the purposes of the present invention, LN is the length of the reference polypeptide: TM-score

[0072] A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, Protein Eng., 11 , 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).

[0073] For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website (zhanggroup.org / TM-score / ). The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:

[0074] TMalign <query . pdb> <ref erence . pdb> -L <length of reference>

[0075] Where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, and <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.

[0076] The maximal TM-score is 1 , e.g., 1.0, corresponding to identical three-dimensional structures.

[0077] Variant: The term “variant” means a polypeptide having protease activity comprising an alteration, i.e., a substitution, insertion, and / or deletion, at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0078] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).

[0079] Conventions for Designation of Variants

[0080] For purposes of the present invention, the polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid positions in another protease. The amino acid sequence of another protease is aligned with the polypeptide disclosed in SEQ ID NO:1 , and based on the alignment, the ammo acid position number corresponding to any ammo acid residue in the polypeptide disclosed in SEQ ID NO:1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0081] Alignments of SEQ ID NO:1 and SEQ ID NOs: 2, 3, 4, 5, 6, 7, and 8, respectively, are provided as Figures 1-7.

[0082] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted III PAG single letter or three letter amino acid abbreviation is employed. The terms “alteration” or “mutation” may be used interchangeably herein to refer to substitutions, insertions and deletions.

[0083] Substitutions: For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. For example, the substitution of a threonine (T) at position 220 with alanine (A) is designated as “T220A”. Multiple substitutions may be listed with individual substitutions separated by a space (“ ”), e.g., “T220A G229V”, representing substitutions at positions 220 and 229 of threonine (T) with alanine (A) and glycine (G) with valine (V), respectively. Alternatively, multiple substitutions may be listed with individual substitutions separated by an addition mark (“+”) or a comma (“,”).

[0084] Alternative substitutions in a particular position may be indicated with a slash (“ / ”). For example, substitution of threonine (T) in position 220 with either alanine (A), valine (V), or leucine (L) many be designated “T220A / V / L”.

[0085] Substitutions may also be indicated with an “X” preceding a position number, which means that any original amino acid in a parent protease other than original amino acid found in SEQ ID NO:1. For example, “X9E” means that any amino acid residue other than E at position 9 of a parent protease is substituted with E.

[0086] Deletions: For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of threonine (T) at position 220 is designated “T220*”. Multiple deletions may be listed with individual deletions separated by a space (“ ”), e.g., “T220* G229*”, or alternatively separated by an addition mark (“+”) or a comma (“,”). The use of an “X” preceding a position number is as described above for substitutions, e.g., “X131*” means that any amino acid residue at position 131 is deleted.

[0087] Insertions: For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after threonine at position 220 is designated “T220TK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted ammo acid #2; etc.]. For example, the insertion of lysine and alanine after threonine at position 220 is indicated as “T220TKA”. Multiple insertions may be listed with individual insertions separated by a space (“ ”), e.g., “T220TK G229GV”, or alternatively separated by an addition mark (“+”) or a comma (“,”)■

[0088] In such cases the inserted amino acid residue(s) are numbered by the addition of lowercase letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

[0089] Multiple alterations: Variants comprising multiple alterations are separated by a space (“ ”), e.g., “R170Y G195E” representing a substitution of arginine (R) and glycine (G) at positions 170 and 195 with tyrosine (Y) and glutamic acid (E), respectively. Multiple alterations may alternatively be listed with individual mutations separated by an addition mark (“+”) or a comma (“,”)■

[0090] A combination of, e.g., a substitution and an insertion may be denoted as follows: S99AD, which represents substitution of a serine residue in position 99 with an alanine residue as well as insertion of an aspartic acid residue.

[0091] Different alterations: Where different alterations can be introduced at a position, the different alterations may be separated by a comma, e.g., “R170Y.E” represents a substitution of arginine (R) at position 170 with tyrosine (Y) or glutamic acid (E). Thus, “Y167G.A R170G.A” designates the following variants:

[0092] “Y167G+R170G”, “Y167G+R170A”, “Y167A+R170G”, and “Y167A+R170A”.

[0093] Different alterations in a position may also be indicated with a slash (“ / ”), for example “T220A / V / L” as explained above. Alternatively, different alterations may be indicated using brackets, e.g., R170[Y,G].

[0094] Numbering of amino acid positions / residues: Amino acid position numbers as used herein are based on the numbering of the polypeptide of SEQ ID NO:1. Thus, amino acid positions of a parent protease polypeptide having, e.g., SEQ ID NO:3, 4 or 5 are those of the corresponding positions of SEQ ID NO:1 as explained above.

[0095] DETAILED DESCRIPTION OF THE INVENTION

[0096] The present invention relates to protease variants having certain substitutions that have been found to provide improved stability at low pH values, in particular improved storage stability in low pH detergent compositions. Importantly, the improved stability at low pH values is achieved without compromising proteolytic activity, in particular without compromising wash performance. Protease Variants

[0097] In one aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 ; wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R, preferably X209K (e.g., A209K / M / I / R, preferably A209K), and X212S / T (e.g., N212S / T); wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.

[0098] In an embodiment, the variant has a TM-score of at least 0.90, e.g., at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0099] In an embodiment, the variant has a TM-score of at least 0.95, e.g., at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three- dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0100] In an embodiment, the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0101] In an embodiment, the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.

[0102] In one embodiment, the parent protease is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In a preferred embodiment, the parent protease is SEQ ID NO:1. In another preferred embodiment, the parent protease is SEQ ID NO:4.

[0103] In one embodiment, the parent protease is an otherwise identical protease without substitutions at three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R, preferably X209K (e.g., A209K / M / I / R, preferably A209K), and X212S / T (e.g., N212S / T).

[0104] In one embodiment, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ I D NO: 1 , and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:1 , and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.

[0105] In one embodiment, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:2, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:2, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.

[0106] In one embodiment, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:3, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:3, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.

[0107] In one embodiment, the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:4, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:4, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.

[0108] In one embodiment, the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:5, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:5, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.

[0109] In one embodiment, the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:6, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:6, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.

[0110] In one embodiment, the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:7, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:7, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.

[0111] In one embodiment, the parent is SEQ ID NO:8 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:8, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:8, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:8, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:8, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:8, wherein the three-dimensional structure is calculated using AlphaFold.

[0112] For some parent proteases, a three-dimensional structure is publicly available. A three- dimensional structure of SEQ ID NO:2 (Savinase®) is available under UniProt accession number P29600 or, alternatively, PDB accession number 1SVN. A three-dimensional structure of SEQ ID NO:3 (BPN’) is available under UniProt accession number P00782. A three-dimensional structure of SEQ ID NO:4 (Alcalase®) is available under UniProt accession number P00780.

[0113] In one embodiment, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Preferably, the parent is SEQ ID NO:2, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Most preferably, the parent is SEQ ID NO:2, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.

[0114] In one embodiment, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Preferably, the parent is SEQ ID NO:3, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Most preferably, the parent is SEQ ID NO:3, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.

[0115] In one embodiment, the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Preferably, the parent is SEQ ID NO:4, and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Most preferably, the parent is SEQ ID NO:4, and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.

[0116] In one aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 ; wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R, preferably X209K (e.g., A209K / M / I / R, preferably A209K), and X212S / T (e.g., N212S / T); wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variant has protease activity.

[0117] In one embodiment, the parent protease is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In a preferred embodiment, the parent protease is SEQ ID NO:1. In another preferred embodiment, the parent protease is SEQ ID NO:4.

[0118] In one embodiment, the parent protease is an otherwise identical protease without substitutions at three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1.

[0119] In another embodiment, the parent is SEQ ID NO:1 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1.

[0120] In another embodiment, the parent is SEQ ID NO:2 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:2.

[0121] In another embodiment, the parent is SEQ ID NO:3 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3.

[0122] In another embodiment, the parent is SEQ ID NO:4 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:4. In another embodiment, the parent is SEQ ID NO:5 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:5.

[0123] In another embodiment, the parent is SEQ ID NO:6 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:6.

[0124] In another embodiment, the parent is SEQ ID NO:7 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:7.

[0125] In another embodiment, the parent is SEQ ID NO:8 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:8.

[0126] In one aspect, the number of substitutions in the variants of the present invention is 3-30, e.g., 3-25, 3-20, 3-15 and 3-10, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, substitutions. In a preferred embodiment, the number of substitutions in the variants of the present invention is 3-10, such as 3, 4, 5, 6, 7, 8, 9, or 10 substitutions.

[0127] The variants of the invention may comprise a substitution at a position corresponding to position 3 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 3 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Thr.

[0128] The variants of the invention comprise a substitution at a position corresponding to position 59 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 59 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Arg or Vai.

[0129] The variants of the invention may comprise a substitution at a position corresponding to position 198 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 198 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.

[0130] The variants of the invention may comprise a substitution at a position corresponding to position 209 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 209 of SEQ ID NO:1 is substituted with Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Arg, lie, Lys, or Met, most preferably with Lys. The variants of the invention may comprise a substitution at a position corresponding to position 212 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 212 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Vai, preferably with Ser or Thr.

[0131] In a preferred embodiment, the substitutions in the at least three positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 are selected from the group consisting of X3T {e.g., S3T), X59V {e.g., G59V), X198D {e.g., N198D or G198D), X209K {e.g., A209K), and X212S {e.g., N212S).

[0132] In another preferred embodiment, the protease variant comprises substitutions in at least four positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein the at least four substitutions are selected from the group consisting of X3T {e.g., S3T), X59V / R {e.g., G59V / R), X198D {e.g., N198D or G198D), X209K / M / I / R {e.g., A209K / M / I / R), and X212S / T {e.g., N212S / T); preferably wherein the at least four substitutions are selected from the group consisting of X3T {e.g., S3T), X59V {e.g., G59V), X198D {e.g., N198D or G198D), X209K {e.g., A209K), and X212S {e.g., N212S).

[0133] In another preferred embodiment, the protease variant comprises substitutions in five positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein the five substitutions are selected from the group consisting of X3T {e.g., S3T), X59V / R {e.g., G59V / R), X198D {e.g., N198D or G198D), X209K / M / I / R {e.g., A209K / M / I / R), and X212S / T {e.g., N212S / T); preferably wherein the five substitutions are selected from the group consisting of X3T {e.g., S3T), X59V {e.g., G59V), X198D {e.g., N198D or G198D), X209K {e.g., A209K), and X212S {e.g., N212S).

[0134] In one aspect, the variant further comprises one or more substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210IA / / Y / F / E / T / M).

[0135] In one aspect, the variant further comprises one or more substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N), X76E / D {e.g., S76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I), X205P {e.g., G205P), X206G / A / D / F {e.g., S206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M).

[0136] The variants of the invention may further comprise a substitution at a position corresponding to position 37 of SEQ ID NO: 1. In one embodiment, the amino acid at a position corresponding to position 37 of SEQ ID NO:1 is substituted with Arg, Asn, Asp, Cys, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asn. The variants of the invention may further comprise a substitution at a position corresponding to position 76 of SEQ ID NO: 1. In one embodiment, the amino acid at a position corresponding to position 76 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp or Glu.

[0137] The variants of the invention may further comprise a substitution at a position corresponding to position 106 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 106 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Vai.

[0138] The variants of the invention may further comprise a substitution at a position corresponding to position 113 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 113 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Vai or lie.

[0139] The variants of the invention may further comprise a substitution at a position corresponding to position 205 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 205 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Pro.

[0140] The variants of the invention may further comprise a substitution at a position corresponding to position 206 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 206 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Gly, Ala, Asp, or Phe.

[0141] The variants of the invention may further comprise a substitution at a position corresponding to position 210 of SEQ ID NO:1. In one embodiment, the amino acid at a position corresponding to position 210 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with lie, Vai, Tyr, Phe, Glu, Thr, or Met.

[0142] In one embodiment, the variant further comprises at least two substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D (e.g., S76E / D or T76E / D), X106V (e.g., A106V), X113V / I (e.g., G113V / I or T113V / l), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210IA / / Y / F / E / T / M).

[0143] In one embodiment, the variant further comprises at least two substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N (e.g., T37N), X76E / D (e.g., S76E / D), X106V (e.g., A106V), X113V / I (e.g., G113V / I), X205P (e.g., G205P), X206G / A / D / F (e.g., S206G / A / D / F), and X210I / V / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M). In one embodiment, the variant further comprises at least three substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210IA / / Y / F / E / T / M).

[0144] In one embodiment, the variant further comprises at least three substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N), X76E / D {e.g., S76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I), X205P {e.g., G205P), X206G / A / D / F {e.g., S206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M).

[0145] In one embodiment, the variant further comprises at least four substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210IA / / Y / F / E / T / M).

[0146] In one embodiment, the variant further comprises at least four substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N), X76E / D {e.g., S76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I), X205P {e.g., G205P), X206G / A / D / F {e.g., S206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M).

[0147] In one embodiment, the variant further comprises at least five substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210IA / / Y / F / E / T / M).

[0148] In one embodiment, the variant further comprises at least five substitutions in positions corresponding to any of positions 37, 76, 106, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0149] In a preferred embodiment, the protease variant comprises two, three, four, or five substitutions in positions corresponding to any of positions 76, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein the substitutions are selected from the group consisting of X76E / D {e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises two, three, four, or five substitutions selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / l), X205P (e.g., G205P or T205P), X206G (e.g., S206G or N206G), and X210I / V (e.g., S2101 / V or T2101 / V).

[0150] In a preferred embodiment, the protease variant comprises at least two substitutions in positions corresponding to any of positions 76, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein the substitutions are selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises at least two substitutions selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G (e.g., S206G or N206G), and X210I / V (e.g., S2101 / V or T2101 / V).

[0151] In a preferred embodiment, the protease variant comprises at least three substitutions in positions corresponding to any of positions 76, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein the substitutions are selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises at least three substitutions selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G (e.g., S206G or N206G), and X210I / V (e.g., S2101 / V or T2101 / V).

[0152] In a preferred embodiment, the protease variant comprises at least four substitutions in positions corresponding to any of positions 76, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein the substitutions are selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises at least four substitutions selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T1131 / V), X205P (e.g., G205P or T205P), X206G (e.g., S206G or N206G), and X210I / V (e.g., S2101 / V or T2101 / V).

[0153] In a preferred embodiment, the protease variant comprises five substitutions in positions corresponding to positions 76, 113, 205, 206, and 210 of SEQ ID NO:1 , wherein the substitutions are selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises five substitutions selected from the group consisting of X76E / D {e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / I), X205P {e.g., G205P or T205P), X206G {e.g., S206G or N206G), and X210I / V e.g., S210I / V or T210l / V).

[0154] Preferably, the protease variant comprises a substitution in a position corresponding to position 76 of SEQ ID NO:1 , preferably the substitution X76E / D {e.g., S76E / D or T76E / D).

[0155] Preferably, the protease variant comprises a substitution in a position corresponding to position 210 of SEQ ID NO:1 , preferably the substitution X210I / V {e.g., S210I / V or T210l / V).

[0156] Preferably, the protease variant comprises one or more substitutions in a position corresponding to positions 113, 205, 206, and / or 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X113V / I {e.g., G113V / I or T113V / I), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0157] More preferably, the protease variant comprises substitutions in two positions corresponding to positions 76 and 210 of SEQ ID NO:1 , in particular the substitution X76E / D {e.g., S76E / D or N76E / D) and the substitution X210I / V {e.g., S210I / V or T210I / V). The variant may thus comprise the substitutions X76E+X210I {e.g., S76E+S210I or N76E+T210I), X76E+X210V {e.g., S76E+S210V or N76E+T210V), X76D+X210I {e.g., S76D+S210I or N76D+T210I), or X76D+X210V {e.g., S76D+S210V or N76D+T210V).

[0158] In other preferred embodiments, the protease variant comprises two, three, four, or five substitutions selected from the group consisting of X76E / D {e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M). More preferably, the variant comprises two, three, four, or five substitutions selected from the group consisting of X76E / D {e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G {e.g., S206G or N206G), and X210I / V {e.g., S210I / V or T210l / V).

[0159] In one embodiment, the variant comprises substitutions in positions corresponding to each of positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein the substitutions are X3T {e.g., S3T), X59V {e.g., G59V), X198D {e.g., N198D or G198D), X209K {e.g., A209K), and X212S {e.g., N212S), and at least one substitution in a position corresponding to any one of positions 37, 76, 106, 113, 205, 206, or 210 of SEQ ID NO:1 , wherein the at least one substitution is selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0160] In one embodiment, the variant comprises a substitution in three or more of positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 and a substitution in at least one position corresponding to any one of positions 37, 76, 106, 113, 205, 206, or 210 of SEQ ID NO:1.

[0161] In a preferred embodiment, the variant comprises a substitution in three or more, preferably four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , and preferably a substitution in a position corresponding to positions 76 and / or210 of SEQ ID NO:1.

[0162] Preferred variants comprise substitutions in positions corresponding to positions 3, 209, and 212 of SEQ ID NO:1 , wherein the substitutions are selected from X3T (e.g., S3T), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T). Preferred variants may further comprise substitutions in one or two positions corresponding to positions 59 and / or 198 of SEQ ID NO:1 , wherein the substitutions are X59V (e.g., G59V) and / or X198D (e.g., N198D or G198D). Preferred variants may even further comprise substitutions in one or two positions corresponding to positions 76 and / or 210 of SEQ ID NO:1 , wherein the substitutions are X76D (e.g., S76D or T76D) or X76E (e.g., S76E or T76E) and / or X210I (e.g., S210I or T210l) or X210V (e.g., S210V or T210V).

[0163] Preferred variants comprise substitutions in positions corresponding to positions 3, 209, and 212 of SEQ ID NO:1 , substitutions in one or two positions corresponding to positions 59 and / or 198 of SEQ ID NO:1 , and substitutions in one or two positions corresponding to positions 76 and / or 210 of SEQ ID NO: 1 , in particular X76D (e.g., S76D or T76D) or X76E (e.g., S76E or T76E) and / or X210I (e.g., S210I or T210l) or X210V (e.g., S210V or T210V).

[0164] Non-limiting examples of preferred protease variants of the invention include those comprising one of the following sets of substitutions compared to SEQ ID NO:1 :

[0165] • X3T X59V X76E X198D X209K X210I X212S

[0166] • X3T X59V X76E X198D X205P X206G X209K X210V X212S

[0167] • X3T X59V X76E X198D X205P X206G X209K X210I X212S

[0168] • X3T X59V X76E X198D X205P X209K X210V X212S

[0169] • X3T X59V X76E X113V X198D X205P X206G X209K X210V X212S

[0170] • X3T X59 X76E X113I X198D X205P X206G X209K X210V X212S

[0171] • X3T X76D X106V X198D X209K X210I X212T

[0172] • X3T X59V X76D X198D X206D X209K X210I X212S

[0173] • X3T X59V X76D X198D X209K X210I X212S

[0174] • X3T X59V X76D X198D X209K X212S

[0175] • X3T X76D X209K X210I X212S

[0176] • X3T X59V X76E X198D X209K X212S

[0177] • X3T X59R X76D X198D X206D X209K X210I X212S

[0178] • X3T X37N X59 X76D X209K X210I X212S

[0179] • X3T X59R X198D X209K X212S In particular, based on the amino acid residues present in the relevant positions of SEQ ID NO:1 , the variants listed above may be described as variants comprising one of the following sets of substitutions compared to SEQ ID NO:1 :

[0180] • S3T G59V S76E N198D A209K S210I N212S

[0181] • S3T G59V S76E N198D G205P S206G A209K S210V N212S

[0182] • S3T G59V S76E N198D G205P S206G A209K S210I N212S

[0183] • S3T G59V S76E N198D G205P A209K S210V N212S

[0184] • S3T G59V S76E G113V N198D G205P S206G A209K S210V N212S

[0185] • S3T G59V S76E G113I N198D G205P S206G A209K S210V N212S

[0186] • S3T S76D A106V N198D A209K S210I N212T

[0187] • S3T G59V S76D N198D S206D A209K S210I N212S

[0188] • S3T G59V S76D N198D A209K S210I N212S

[0189] • S3T G59V S76D N198D A209K N212S

[0190] • S3T S76D A209K S210I N212S

[0191] • S3T G59V S76E N198D A209K N212S

[0192] • S3T G59R S76D N198D S206D A209K S210I N212S

[0193] • S3T T37N G59V S76D A209K S210I N212S, or

[0194] • S3T G59R N198D A209K N212S

[0195] In particular, based on the amino acid residues present in the relevant positions of SEQ ID NO:4, the variants listed above may be described as variants comprising one of the following sets of substitutions compared to SEQ ID NO:4:

[0196] • G59V T76E G198D T205P N206G A209K T210V N212S

[0197] • G59V T76E G198D T205P N206G A209K T210I N212S

[0198] • G59V T76E G198D T205P A209K T210V N212S

[0199] • G59V T76E T113V G198D T205P N206G A209K T210V N212S

[0200] • S37N G59V T76D A209K T210I N212S

[0201] • G59R G198D A209K N212S

[0202] • T76D G198D A209K T210I N212T

[0203] • G59V T76E G198D A209K T210I N212S

[0204] • G59V T76D G198D N206D A209K T210I N212S

[0205] • G59V T76D G198D A209K T210I N212S

[0206] • G59V T76D G198D A209K N212S

[0207] • T76D A209K T210I N212S

[0208] • G59V T76E T1131 G198D T205P N206G A209K T210V N212S

[0209] • G59V T76E G198D A209K N212S, or

[0210] • G59R T76D G198D N206D A209K T210I N212S In preferred embodiments, any of the protease variants disclosed above or elsewhere herein preferably have at least 70%, e.g., at least 75%, at least 80%, at least 85%, or at least 90%, but less than 100%, sequence identity to SEQ ID NO:1 . The variants may for example have at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, but less than 100%, sequence identity to SEQ ID NO:1.

[0211] In other preferred embodiments, any of the protease variants disclosed above or elsewhere herein preferably have at least 70%, e.g., at least 75%, at least 80%, at least 85%, or at least 90%, but less than 100%, sequence identity to SEQ ID NO:4. The variants may for example have at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, but less than 100%, sequence identity to SEQ ID NO:4.

[0212] Alternatively, any of the protease variants disclosed herein may have a sequence identity of at least 70% but less than 100% to one of SEQ ID NOs: 2, 3, 5, 6, 7, or 8. For example, a protease variant may have at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96%, but less than 100%, sequence identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0213] In one aspect, the variant has a sequence difference of at most 10%, e.g., at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, or at most 2%, but more than 1 %, compared to the parent, e.g., the polypeptide of SEQ ID NO:1 , wherein the variant has protease activity.

[0214] In one aspect, the variant has a sequence difference of at most 10%, e.g., at most 9%, at most 8%, at most 7%, at most 6%, at most 5%, at most 4%, at most 3%, or at most 2%, but more than 1 %, compared to the parent, e.g., the polypeptide of SEQ ID NO:4, wherein the variant has protease activity.

[0215] As noted above, the protease variants of the invention have an improved detergent stability at low pH values, in particular an improved storage stability in a low pH liquid detergent composition. The variants of the invention thus preferably have an improved storage stability, compared to a parent protease not having the substitutions in the variant e.g., a reference protease having the same amino acid sequence as the variant other than any claimed substitutions in positions 3, 37, 59, 76, 106, 113, 198, 205, 206, 209, 210, and / or 212), as measured in a liquid detergent composition having a pH of below about 7, such as a pH of about 6.5, about 6, about 5 or about 4. Preferably, the variants of the invention have an improved storage stability compared to the protease of SEQ ID NO:1 , as measured in a liquid detergent composition having a pH of below about 7, such as a pH of about 6.5, about 6, about 5.5, about 5, about 4.5 or about 4. Or preferably, the variants of the invention have an improved storage stability compared to the protease of SEQ ID NO:4, as measured in a liquid detergent composition having a pH of below about 7, such as a pH of about 6.5, about 6, about 5.5, about 5, about 4.5 or about 4.

[0216] In one embodiment, the variants of the invention have an improved storage stability, compared to a parent protease not having the substitutions in the variant {e.g., a reference protease having the same amino acid sequence as the variant other than any claimed substitutions in positions 3, 37, 59, 76, 106, 113, 198, 205, 206, 209, 210, and / or 212), as measured in a liquid detergent composition having a pH of between 4 and 5. The variants of the invention may, e.g., have an improved storage stability compared to the protease of SEQ I D NO: 1 , as measured in a liquid detergent composition having a pH of between 4 and 5. Or the variants of the invention may, e.g., have an improved storage stability compared to the protease of SEQ ID NO:4, as measured in a liquid detergent composition having a pH of between 4 and 5.

[0217] Storage stability may be determined as described in the examples and may be expressed as residual protease activity after storage.

[0218] The protease variants of the invention preferably have an improved low pH stability, expressed as an improvement factor (IF or HIF) compared to the parent protease or compared to the protease of SEQ ID NO:1 or SEQ ID NO:4, as determined after storage in a relevant low pH detergent.

[0219] In one embodiment, the protease variants of the invention have an improved low pH stability, expressed as an improvement factor (IF) compared to the parent protease or compared to the protease of SEQ ID NO:1 , of at least 1.1 , such as at least 1.2, at least 1.3, at least 1.4 or at least 1.5, determined in liquid model detergent 2 having a pH between 4 and 5 at 25°C for 18 hours.

[0220] In another embodiment, the protease variants of the invention have an improved low pH stability, expressed as an improvement factor (IF) compared to the parent protease or compared to the protease of SEQ ID NO:1 , of at least 10, such as at least 20, at least 30, at least 40 or at least 50, determined in liquid model detergent 2 having a pH between 4 and 5 at 37°C for 18 hours.

[0221] In one embodiment, the protease variants of the invention have improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 1.01 , such as at least 1.05, at least 1.1 , at least 1.5 or at least 2.0, determined in a liquid model detergent having a pH value between 4 and 5 at 37°C for 8 hours.

[0222] In another embodiment, the protease variants of the invention have improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 2.0, such as at least 2.2, at least 2.7, at least 3.0 or at least 3.5, determined in a liquid model detergent having a pH value between 4 and 5 at 25°C for 8 hours. Since the improvement factor is defined as the ratio of the median activity of a protease variant divided by the median activity of a reference protease, it will be clear that in the case of a reference protease having no residual activity after storage in a detergent, it will not be possible to calculate an improvement factor. On the other hand, if the reference protease has no residual activity and the variant has some residual activity, it can be concluded that the stability of the variant is improved.

[0223] In addition to the amino acid alterations specifically disclosed herein, a protease variant of the invention may comprise additional alterations at one or more other positions. These additional alterations may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an aminoterminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0224] Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, in The Proteins, Academic Press, New York. Common conservative substitution groups include but are not limited to: G=A=S; l=V=L=M; D=E; Y=F; and N=Q (where e.g., “G=A=S” means that these three amino acids may be substituted for each other).

[0225] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0226] Parent Proteases

[0227] Protease variants of the invention may be based on any parent protease. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0228] In a preferred aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:1 of at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:1. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ I D NO: 1 .

[0229] In another preferred aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:4 of at least 80%, e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:4. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:4.

[0230] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:2 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:2. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:2.

[0231] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:3 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:3. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:3. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or at least 96% sequence identity to SEQ ID NO:3.

[0232] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:4 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:4. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:4. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:4. In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:5 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:5. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:5.

[0233] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:6 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:6. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:6. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:6.

[0234] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:7 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:7. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:7. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:7.

[0235] In one aspect, the parent protease has a sequence identity to the polypeptide of SEQ ID NO:8 of at least 80%, e.g., at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and has protease activity. In an embodiment, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:8. In an embodiment, the parent comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:8. In this aspect, the resulting protease variant preferably has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or at least 96% sequence identity to SEQ ID NO:8.. Detergent Compositions

[0236] The invention also relates to a composition comprising a variant of the invention, e.g., a detergent or cleaning composition. Examples of detergent or cleaning compositions include granular or powder-form all-purpose or heavy-duty washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so-called heavy- duty liquid (HDL) types; unit dose compositions such as pods, capsules, tabs, etc. with one or more chambers; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; and machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use. Preferred compositions are of the liquid type, i.e., liquids and gels including HDL and unit dose compositions.

[0237] The invention also relates to a composition comprising a variant of the invention and further comprising: one or more detergent components; and / or one or more additional enzymes. In a preferred embodiment, the composition is a detergent composition comprising one or more detergent components, in particular one or more non-naturally occurring detergent components.

[0238] The present invention also relates to a composition comprising a variant of the present invention and further comprising one or more additional enzymes selected from the group consisting of amylases {e.g., alpha-amylases), catalases, cellulases e.g., endoglucanases), cutinases, DNases, hexosaminidases haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, lichenases and xyloglucanases, or any mixture thereof.

[0239] The choice of additional components for a detergent composition is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.

[0240] In a particular embodiment, a detergent composition comprises a variant of the invention and one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, antiredeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0241] In one embodiment, the variant of the invention may be added to a detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.

[0242] An automatic dish wash (ADW) composition may for example include 0.001 %-30%, such as 0.01 %-20%, such as 0.1-15%, such as 0.5-10% of enzyme protein by weight of the composition.

[0243] A granulated composition for laundry may for example include 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0244] A liquid composition for laundry may for example include 0.0001 %-10%, such as 0.001- 7%, such as 0.1 %-5% or 0.2%-2% of enzyme protein by weight of the composition.

[0245] The enzymes such as the variant of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708 or the variants according to the invention may be stabilized using peptide aldehydes or ketones such as described in WO 2005 / 105826 and WO 2009 / 118375.

[0246] The variants of the invention may be formulated in liquid laundry compositions such as a liquid laundry compositions composition comprising: a) at least 0.01 mg of active variant per liter detergent, b) 2 wt% to 60 wt% of at least one surfactant c) 5 wt% to 50 wt% of at least one builder.

[0247] The detergent composition may be formulated into a granular detergent for laundry. Such detergent may comprise; a) at least 0.01 mg of active protease variant per gram of composition b) anionic surfactant, preferably 5 wt % to 50 wt % c) nonionic surfactant, preferably 1 wt % to 8 wt % d) builder, preferably 5 wt % to 40 wt %, such as carbonates, zeolites, phosphate builder, calcium sequestering builders or complexing agents.

[0248] Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the person skilled in the art.

[0249] The detergent compositions of the invention preferably have a pH of less than about 6.5, typically from about 4 to about 6.5. The pH may, for example, be from about 4.0 to about 6.0, from about 4.0 to about 5.5 or from about 4.0 to about 5.0, or from about 4.5 to about 6.5, from about 4.5 to about 6.0 or from about 4.5 to about 5.5. The pH may thus e.g., be about 6, about 5.5, about 5, about 4.5 or about 4. Surfactants

[0250] The detergent composition may comprise one or more surfactants, which may be anionic and / or cationic and / or non-ionic and / or semi-polar and / or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The one or more surfactants are typically present in the composition in a total amount of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactants are chosen based on the desired cleaning application, and may include any conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be utilized. Surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away.

[0251] When included therein, the detergent will usually contain from about 1 % to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.

[0252] When included therein, the detergent will usually contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0253] When included therein, the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[0254] When included therein, the detergent will usually contain from about 0% to about 10% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N- (tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0255] When included therein, the detergent will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

[0256] Builders and Co-Builders

[0257] The detergent composition may contain about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. Builders and chelators soften, e.g., the wash water by removing the metal ions form the liquid. The builder and / or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and / or co-builder known in the art for use in laundry detergents may be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates e.g., SKS-6 from Hoechst), ethanolamines such as 2- aminoethan-1-ol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2’,2”-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0258] The detergent composition may also contain 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid. Additional specific examples include 2, 2’, 2”- nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N’-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane- 1 ,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetnammepentakis (methylenephosphomc acid) (DTPMPA or DTMPA), N-(2- hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid- N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2- sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), a-alanine-N, N-diacetic acid (a-ALDA), serine-N, N-diacetic acid (SEDA), isoserine- N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TLIDA) and sulfomethyl-N, N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N, N’, N’-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described in, e.g., WO 2009 / 102854 and US 5,977,053.

[0259] The variants of the invention may also be formulated into a dish wash composition, preferably an automatic dish wash composition (ADW), comprising: a) at least 0.01 mg of active protease variant according to the invention, and b) 10-50 wt % builder preferably selected from citric acid, methylglycine-N, N-diacetic acid (MGDA) and / or glutamic acid-N, N-diacetic acid (GLDA) and mixtures thereof, and c) at least one bleach component.

[0260] Bleaching Systems

[0261] The detergent may contain 0-50% by weight, such as about 0.1 % to about 25%, of a bleaching system. Bleach systems remove discolor often by oxidation, and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra- hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator.

[0262] The term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4- (dodecanoyloxy) benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4- (decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A particular family of bleach activators of interest was disclosed in EP 624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmentally friendly as it eventually degrades into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are efficient bleach activators. Finally, ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst or a booster.

[0263] Some non-limiting examples of bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1 ,4,7-trimethyl-1 ,4,7-triazacyclononane (Me3- TACN) or 1 ,2,4,7-tetramethyl-1 ,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2,2',2"-nitrilotris(ethane-1 ,2-diylazanylylidene-KN-methanylylidene) triphenolato-

[0264] K3O]manganese(lll). The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.

[0265] In some embodiments, the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula:

[0266] (iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n- tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl. Other exemplary bleaching systems are described, e.g., in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine.

[0267] Hydrotropes

[0268] A hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characters (so-called amphiphilic properties as known from surfactants); however, the molecular structures of hydrotropes generally do not favor spontaneous self-aggregation, see, e.g., review by Hodgdon and Kaier, 2007, Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micellar, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care and food to technical applications. Use of hydrotropes in detergent compositions allows for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

[0269] The detergent may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p- toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0270] Polymers

[0271] The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and / or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006 / 130575. Salts of the above-mentioned polymers are also contemplated.

[0272] Fabric Hueinq Agents

[0273] The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when the fabric is contacted with a wash liquor comprising the detergent compositions and thus altering the tint of the fabric through absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Color Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO 2005 / 003274, WO 2005 / 003275, WO 2005 / 003276 and EP 1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05 wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g., WO 2007 / 087257 and WO 2007 / 087243.

[0274] Additional Enzymes

[0275] A detergent additive or detergent composition comprising the variant of the invention may comprise one or more enzymes such as an amylase {e.g., alpha-amylase), arabinase, carbohydrase, cellulase e.g., endoglucanase), cutinase, DNase, hexosaminidase, galactanase, haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and / or peroxidase, pectinase, pectin lyase, protease, xylanase, xanthanase or xyloglucanase.

[0276] The properties of the selected enzyme(s) should be compatible with the selected detergent {e.g., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.). Cellulases

[0277] The term “cellulase” means one or more e.g., several) enzymes that hydrolyze a cellulosic material. The terms “cellulase” and the expression “polypeptide having cellulase activity” are used interchangeably. Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172. Such enzymes include endoglucanase(s) {e.g., EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.

[0278] Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. The cellulase may for example be a mono-component or a mixture of mono-component endo-1 ,4- beta-glucanase also referred to as endoglucanase.

[0279] Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, Myceliophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., T. reesei or T. viride. Other suitable cellulases are from Thielavia, e.g., Thielavia terrestris as described in WO 96 / 29397, or the fungal cellulases produced from Myceliophthora thermophila and Fusarium oxysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89 / 09259 and WO 91 / 17244. Also relevant are cellulases from Bacillus as described in WO 02 / 099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471 , WO 98 / 12307.

[0280] Other cellulases are endo-beta-1 , 4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2 of WO 2002 / 099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO:2 of WO 2001 / 062903.

[0281] Yet another group of suitable cellulases comprise a stabilized linker between the core and the CBM. Particularly useful are such cellulase having at least 80% identity to SEQ ID NO:397, SEQ ID NO:398 or SEQ ID NO:399 of WO 2023 / 061928.

[0282] Commercially available cellulases include Carezyme®, Carezyme® Premium, Celluzyme®, Carezyme Elite®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme® Celluclean® Classic, and Cellusoft® (Novozymes A / S); Puradax®, Puradax HA, Puradax EG, Revitalenz 1000, Revitalenz 200, and Revitalenz 2000 (Dupont Industrial Biosciences); KAC- 500(B)™ (Kao Corporation); and Biotouch DCL and Biotouch FLX1 (AB Enzymes).

[0283] The two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

[0284] Proteases

[0285] The composition may comprise one or more additional proteases including those of bacterial, fungal, plant, viral or animal origin, e.g., vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as subtilisin. A metalloprotease may for example be a thermolysin from, e.g., family M4 or other metalloprotease such as those from M5, M7 or M8 families.

[0286] Examples of metalloproteases are the neutral metalloproteases as described in WO 2007 / 044993 (Genencor I nt.) such as those derived from Bacillus amyloliquefaciens.

[0287] Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress® Excel, Progress® Key, and Progress® Uno (Novozymes A / S), those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, Preferenz® P200, Preferenz® P300, and Preferenz® P400 (DuPont / IFF), Axapem™ (Gist-Brocades N.V.), BLAP (sequence shown in Figure 29 of US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0288] Lipases and Cutinases

[0289] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO 96 / 13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95 / 06720 & WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 2010 / 065455), cutinase from Magnaporthe grisea (WO 2010 / 107560), cutinase from Pseudomonas mendocina (US 5,389,536), lipase from Thermobifida fusca (WO 2011 / 084412), Geobacillus stearothermophilus lipase (WO 2011 / 084417), lipase from Bacillus subtilis (WO 2011 / 084599), and lipase from Streptomyces griseus (WO 2011 / 150157) and S. pristinaespiralis (WO 2012 / 137147).

[0290] Other examples are lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541 , WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381 , WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 2007 / 87508 and WO 2009 / 109500.

[0291] Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

[0292] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g., acyltransferases with homology to Candida antarctica lipase A (WO 2010 / 111143), acyltransferase from Mycobacterium smegmatis (WO 2005 / 056782), perhydrolases from the CE 7 family (WO 2009 / 067279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010 / 100028).

[0293] Amylases

[0294] Suitable amylases which can be used together with the protease variants of the invention may be an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alphaamylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.

[0295] Suitable amylases include amylases having SEQ ID NO:2 in WO 95 / 10603 or variants having 90% sequence identity to SEQ ID NO:1 thereof. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO:4 of WO 99 / 019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.

[0296] Other suitable amylases include amylases having SEQ ID NO:6 in WO 02 / 010355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a deletion in positions 181 and 182 and a substitution in position 193.

[0297] Still other amylases which are suitable are hybrid alpha-amylase comprising residues 1- 33 of the alpha-amylase obtained from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006 / 066594 or variants having 90% sequence identity thereof. Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181 , N190, M197, 1201 , A209 and Q264.

[0298] Other examples are amylase variants such as those described in WO 2011 / 098531 , WO 2013 / 001078 and WO 2013 / 001087.

[0299] Other examples are amylase variants such as those described in WO 2011 / 098531 , WO 2013 / 001078 and WO 2013 / 001087. Commercially available amylases include Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A / S), and Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont).

[0300] Peroxidases / Oxidases

[0301] Suitable peroxidases / oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0302] Commercially available peroxidases include Guardzyme™ (Novozymes A / S).

[0303] DNases

[0304] The term “DNase” means a polypeptide having DNase (deoxyribonuclease) activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in a DNA backbone, thus degrading DNA. Preferred DNases may be selected from any of the enzyme classes E.C. 3.1.21.X, where X = 1 , 2, 3, 4, 5, 6, 7, 8 or 9, e.g., Deoxyribonuclease I, Deoxyribonuclease IV, Type I site-specific deoxyribonuclease, Type II site-specific deoxyribonuclease, Type III site-specific deoxyribonuclease, CC-preferring endo-deoxyribonuclease, Deoxyribonuclease V, T(4) deoxyribonuclease II or T(4) deoxyribonuclease IV, or from E.C. 3.1.22.Y, where Y = 1 , 2, 4 or 5, e.g., Deoxyribonuclease II, Aspergillus deoxyribonuclease K(1), Crossover junction endo- deoxyribonuclease or Deoxyribonuclease X.

[0305] DNase polypeptides have been found to be useful for deep cleaning of microbial biofilm that may be present on surfaces such as textiles or dishware or other hard surfaces, and which consists of a matrix of extracellular polymeric substance (EPS) composed of extracellular DNA, proteins, and polysaccharides. When present on textiles, microbial biofilm can result in e.g., malodor and redeposition of soil during laundering.

[0306] The DNase polypeptide is typically a microbial enzyme, preferably of fungal or bacterial origin, or a genetically engineered variant of a microbial DNase.

[0307] Suitable bacterial DNases may, for example, be obtained from species of Bacillus and related genera (cf. Patel and Gupta, Int. J. Syst. Evol. Microbiol. 2020; 70:406-438, who proposed six new Bacillaceae genera from species formerly classified as belonging to the genus Bacillus), e.g., from Bacillus, Cytobacillus, Metabacillus, Alkalihalobacillus, Rossellomorea or Mesobacillus. Examples of species from which DNases may be obtained include Bacillus licheniformis, Bacillus subtilis, Bacillus horikoshii, Cytobacillus horneckiae, Metabacillus indicus, Alkalihalobacillus algicola, Rossellomorea vietnamensis, Alkalihalobacillus hwajinpoensis, Metabacillus indicus, Mesobacillus campisalis, Bacillus idriensis, Bacillus algicola, Bacillus marisflavi and Bacillus luciferensis. Preferred bacterial DNases include those obtained from Metabacillus indicus (previously known as Bacillus cibi) and variants thereof.

[0308] DNases may also be obtained from a fungal species. Examples of preferred fungal DNases are those obtained from Aspergillus, for example from Aspergillus oryzae, from Trichoderma, for example from Trichoderma harzianum, from Vibressa, for example from Vibressea flavovirens, from Morchella, for example from Morchella costata, and from Rhizoctonia, for example from Rhizoctonia solani, as well as variants thereof. Preferred fungal DNases include those obtained from Aspergillus oryzae and variants thereof.

[0309] Suitable DNases, DNase variants, and use thereof in detergent compositions are disclosed, for example, in WO 2014 / 087011 , WO 2015 / 155350, WO 2015 / 155351 , WO 2017 / 060475, WO 2017 / 060493, WO 2017 / 060505, WO 2017 / 064269, WO 2018 / 011277, WO 2018 / 177203, WO 2018 / 177936, WO 2018 / 177938, WO 2019 / 081724, WO 2019 / 081721 , WO 2022 / 189521 , WO 2022 / 194668, WO 2022 / 194673 and WO 2023 / 165950.

[0310] Hexosaminidases

[0311] The term hexosaminidase includes “dispersin’’ and the abbreviation “Dsp”, which means a polypeptide having hexosaminidase activity, EC 3.2.1.- that catalyzes the hydrolysis of (3-1 ,6- glycosidic linkages of N-acetyl-glucosamine polymers found e.g., in biofilm. The term hexosaminidase includes polypeptides having N-acetylglucosaminidase activity and -N- acetylglucosaminidase activity.

[0312] A polypeptide having hexosaminidase activity may be obtained from microorganisms of any genus, in particular from bacteria or fungi, or a genetically engineered variant of a microbial hexosaminidase. Preferred hexosaminidases, e.g., dispersins, include those obtained from Terribacillus, Curtobacterium, Aggregatibacter, Haemophilus or Actinobacillus. The hexosaminidase may also be a variant of a polypeptide obtained from any of these or other organisms.

[0313] Suitable hexosaminidases, hexosaminidase variants, and use thereof in detergent compositions are disclosed, for example, in WO 2017 / 186936, WO 2017 / 186937, WO 2017 / 186943, WO 2019 / 086528, WO 2019 / 086530, WO 2020 / 207944 and WO 2023 / 194204.

[0314] Other enzymes

[0315] In addition to a DNase and / or a hexosaminidase, compositions of the invention may include one or more additional enzymes that have activity on biofilm, for example one or more of a PelA enzyme, a PsIG enzyme or an RNase enzyme, or another nuclease or glycosyl hydrolase such as those mentioned below. PelA: The term “PelA” refers to a Glyco_hydro_114 glycosyl hydrolase enzyme (Pfam domain id PF03537, Pfam version 31.0 Finn (2016). Nucleic Acids Research, Database Issue 44:D279-D285), which is active towards the Pel polysaccharide present in many biofilms. PelA enzymes belong to the glycosyl hydrolases (EC 3.2.1.-), which are a widespread group of enzymes that hydrolyse the glyosidic bond between two or more carbohydrates or between a carbohydrate and a non-carbohydrate moiety. Examples of suitable PelA enzymes are disclosed e.g., in WO 2018 / 185181 and WO 2020 / 070011.

[0316] PsIG: The term “PsIG” or GH39 polypeptide refers to an enzyme comprising a GH39 domain that is able to degrade the exopolysaccharide Psi, a pentasaccharide comprising D- glucose, L-rhamnose and D-mannose which is found in microbial EPS and which acts as a glue in e.g., bacterial surface interactions. GH39 polypeptides also belong to the glycosyl hydrolases (EC 3.2.1.-). Examples of suitable GH39 polypeptides are disclosed e.g., in WO 2018 / 185150.

[0317] RNase: The term “RNase” ” is an abbreviation of the term ribonuclease, which means a nuclease having RNase activity (Pfam PF00545, including EC 3.1.27) that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, with especially endoribonucleases having potential interest for degradation of components of biofilm. Examples of suitable RNases are disclosed e.g., in WO 2018 / 178061 and WO 2020 / 057510.

[0318] Other nucleases: Other nucleases suitable for use in compositions for degrading biofilm components include those belonging to the Pfam families PF02265 (S1-P1_nuclease), PF01223 (Endonuclease_NS) and PF13930 (Endonuclea_NS_2). Such nucleases are disclosed in WO 2020 / 069670.

[0319] GH114 glycosyl hydrolase: GH114 glycosyl hydrolase enzymes having alpha-1 ,4- polygalactosaminidase activity and which are suitable for cleaning and removal or reduction of biofilm are disclosed in WO 2019 / 228448.

[0320] GHL13 glycosyl hydrolase: GHL13 glycosyl hydrolase enzymes comprising a GHL13 pFam domain (PF14883) may also be used for cleaning and removal or reduction of biofilm. Examples of such enzymes are the BpsB and PgaB homologs comprising a GHL13 domain disclosed in WO 2018 / 185152.

[0321] Adjunct materials

[0322] Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric conditioners including clays, fillers / processing aids, fluorescent whitening agents / optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.

[0323] Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant Science Series, volume 71 , Marcel Dekker, Inc., 1997.

[0324] Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01% to about 5% or even from about 0.1 % to about 3% by weight of the composition.

[0325] Fluorescent whitening agent: The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 05%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6- ylamino) stilbene-2,2'-disulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'- disulphonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2, 1 ,3-triazol-2-yl)stilbene-2,2'-disulphonate; 4,4'- bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate and 2-(stilbyl-4"-naptho-1.,2':4,5)-1 ,2,3-trizole-2"-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %. Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester- based fabrics. The soil release polymers may for example be nonionic or anionic terephthalate- based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc. Another type of soil release polymers is amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009 / 087523 (hereby incorporated by reference). Furthermore, random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 03 / 040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.

[0326] Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and / or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose-based polymers described under soil release polymers above may also function as anti-redeposition agents.

[0327] Other suitable adjunct materials include, but are not limited to, anti-shrink agents, antiwrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and / or structure elasticizing agents.

[0328] Formulation of Detergent Products

[0329] The protease variant of the invention and optionally one or more additional enzymes, may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes. A detergent additive comprising one or more enzymes can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations include granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.

[0330] The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit. Preferably, however, detergent compositions comprising the protease variant of the invention are in the form of a liquid, optionally where the liquid is a component in a unit dose composition.

[0331] Pouches can be configured as single or multiple compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g., without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. The inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials, preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water-soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod, of Gary, Indiana, US) plus plasticizers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry detergent composition or part components and / or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids. See, e.g., US 2009 / 0011970.

[0332] Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

[0333] A liquid or gel detergent which is not unit dosed may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

[0334] Liquid Formulations

[0335] The present invention also relates to liquid compositions comprising a protease variant of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).

[0336] In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

[0337] In an aspect, the liquid formulation comprises 20-80% w / w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative.

[0338] In another embodiment, the invention relates to liquid formulations comprising:

[0339] (A) 0.001-25% w / w of a variant of the present invention;

[0340] (B) 20-80% w / w of polyol;

[0341] (C) optionally 0.001-2% w / w preservative; and

[0342] (D) water.

[0343] In another embodiment, the invention relates to liquid formulations comprising:

[0344] (A) 0.001-25% w / w of a variant of the present invention;

[0345] (B) 0.001-2% w / w preservative;

[0346] (C) optionally 20-80% w / w of polyol; and

[0347] (D) water.

[0348] In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0349] In another embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

[0350] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w / w preservative, e.g., 0.05-1% w / w preservative or 0.1-0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative ( / .e., total amount of preservative), e.g., 0.02- 1.5% w / w preservative, 0.05-1% w / w preservative, or 0.1-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0351] In another embodiment, the liquid formulation further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, DNase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, hexosaminidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.

[0352] Uses and Cleaning Methods

[0353] The present invention is also directed to methods for using the variants of the invention or compositions comprising said variants in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing. The invention is also directed to methods for using the variants according to the invention or compositions thereof in cleaning hard surfaces such as floors, tables, walls, roofs, etc., as well as surfaces of hard objects such as cars (car wash) and dishes (dishwashing).

[0354] The variants of the present invention may be added to and thus become a component of a detergent composition. Thus, one aspect of the invention relates to the use of a variant of the invention in a cleaning process such as laundering and / or hard surface cleaning.

[0355] A detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations or be formulated for hand or machine dishwashing operations.

[0356] The cleaning process or the textile care process may for example be a laundry process, a dishwashing process, or cleaning of hard surfaces such as bathroom tiles, floors, tabletops, drains, sinks and washbasins. Laundry processes can for example be household laundering but may also be industrial laundering. Furthermore, the invention relates to a process for laundering of fabrics and / or garments, where the process comprises treating fabrics with a washing solution containing a detergent composition and at least one protease variant of the invention. The cleaning process or a textile care process can for example be carried out in a machine washing or manually. The washing solution can for example be an aqueous washing solution containing a detergent composition.

[0357] In one aspect, the variants of the invention are used in a cleaning process, e.g., a laundry process, that comprises a short wash cycle, typically a wash cycle of not more than about 30 minutes, such as not more than about 20 minutes, e.g., not more than about 15 minutes or not more than about 10 minutes. It has surprisingly been found that the protease variants of the invention are remarkably effective in short wash cycles lasting, for example, only about 10-20 minutes. This may be useful in, e.g., top-loading washing machines that often have short wash cycles or for hand-washing of laundry.

[0358] In another aspect, the variants of the invention are used in a cleaning process, e.g., a laundry process, where the wash water is used for more than one portion of laundry. In this case, the wash water containing a detergent with a variant of the invention may be used in a first wash cycle for a first portion of laundry, and then reused one or more times for additional wash cycles with new portions of laundry. It has been found that detergents containing a variant of the invention are able to substantially maintain cleaning performance on protease-sensitive stains even after three wash cycles or more. This may for example be useful for laundry washed by hand and / or in regions with water scarcity.

[0359] The last few years there has been an increasing interest in replacing components in detergents that are derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising the wash performance. When the components of detergent compositions change, new enzyme activities or new enzymes having alternative and / or improved properties compared to the previously used detergent enzymes such as proteases, lipases and amylases may be needed to achieve a similar or improved wash performance when compared to the traditional detergent compositions.

[0360] The invention further concerns the use of variants of the invention in a proteinaceous stain removing process. The proteinaceous stains may be stains such as food stains, e.g., baby food, cocoa, egg or milk, or other stains such as sebum, blood, ink or grass, or a combination hereof.

[0361] Washing Method

[0362] The present invention provides a method of cleaning a fabric, dishware or a hard surface with a detergent composition comprising a variant of the invention.

[0363] The method of cleaning comprises contacting an object with a detergent composition comprising a protease variant of the invention under conditions suitable for cleaning the object.

[0364] In a preferred embodiment the detergent composition is used in a laundry or a dish wash process.

[0365] Another embodiment relates to a method for removing stains from fabric or dishware which comprises contacting the fabric or dishware with a composition comprising a protease of the invention under conditions suitable for cleaning the object. In the method of cleaning of the invention, the object being cleaned may be any suitable object such as a textile or a hard surface such as dishware or a floor, table, wall, etc.

[0366] The protease variant of the detergent composition of the invention may be stabilized using conventional stabilizing agents and protease inhibitors, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, different salts such as NaCI; KCI; lactic acid, formic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, or a peptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehyde analogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X (X=halogen), BO is a single amino acid residue (preferably with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two or three), optionally comprising an N-terminal protection group, or as described in WO 2009 / 118375, WO 98 / 13459) or a protease inhibitor of the protein type such as RASI, BASI, WASI (bifunctional alpha-amylase / subtilisin inhibitors of rice, barley and wheat) or CI2 or SSI. The composition may be formulated as described in, e.g., \NQ) 92 / 19709, WO 92 / 19708 and US 6,472,364. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and / or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)). Polynucleotides

[0367] The present invention also relates to polynucleotides encoding a protease variant as disclosed herein.

[0368] The polynucleotide encoding a protease variant as disclosed herein may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.

[0369] In an aspect, the polynucleotide is isolated, preferably purified.

[0370] Nucleic Acid Constructs

[0371] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a protease variant as disclosed herein operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Examples of control sequences that may be used are promoters, terminators, mRNA stabilizers, leader sequences, polyadenylation sequences, signal peptides, propeptides, regulatory sequences and transcription factors, all of which are well known in the art.

[0372] The polynucleotide may be manipulated in a variety of ways to provide for expression of a polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0373] Expression Vectors

[0374] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a protease variant as disclosed herein, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0375] The recombinant expression vector may be any vector {e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid. Expression vectors suitable for recombinant expression are well known in the art, as are e.g., methods for introducing them into a host cell. Host Cells

[0376] The present invention also relates to recombinant host cells, comprising a polynucleotide encoding a protease variant as disclosed herein operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0377] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.

[0378] The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

[0379] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Grampositive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0380] The bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0381] In a preferred embodiment, the recombinant host cell is Bacillus subtilis cell.

[0382] In a preferred embodiment, the recombinant host cell is Bacillus licheniformis cell.

[0383] A fungal host cell may be a yeast cell or a filamentous fungal cell.

[0384] The filamentous fungal host cell may e.g., be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

[0385] In an aspect, the host cell is isolated, preferably purified. Methods of Production

[0386] The present invention also relates to methods of producing a protease variant as disclosed herein, comprising

[0387] (a) cultivating a recombinant host cell of the invention under conditions conducive for production of the polypeptide; and

[0388] (b) optionally, recovering the polypeptide.

[0389] The host cell is cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions {e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0390] The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the polypeptide.

[0391] The polypeptide may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered.

[0392] The polypeptide may be purified by a variety of procedures known in the art to obtain substantially pure polypeptides and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0393] In an alternative aspect, the polypeptide is not recovered.

[0394] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0395] EXAMPLES

[0396] Materials and methods of protease variants

[0397] Mutation and introduction of expression cassettes into Bacillus subtilis were performed by standard methods known in the art. DNA manipulations were performed by PCR (e.g., as described by Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, 4thEdition) using standard methods known to the skilled person.

[0398] Bacillus subtilis cells each transformed with a protease variant were grown in a suitable medium for 48 days in deep-well plates at 37°C. The pellet was spun down, and the supernatant was recovered. Controls were prepared similarly by transforming Bacillus subtilis cells with the relevant protease backbone without the relevant mutations.

[0399] Purification of culture supernatants was performed as follows: The culture broth was centrifuged at 26,000 x g for 60 minutes and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 pm filtration unit to remove the remains of the host cells. The pH value of the filtrate was adjusted to pH 8 with 3 M Tris base, and the pH-adjusted filtrate was applied to a MEP HyperCel™ column (Pall Corporation) equilibrated in 20 mM Tris / HCI, 1 mM CaCh, pH 8.0. After washing the column with the equilibration buffer, the column was step-eluted with 20 mM CHsCOOH / NaOH, 1 mM CaCh, pH 4.0. The eluted samples from the MEP HyperCel™ column were further purified and buffer- exchanged on a Sepahdex-G25 column. The purified proteases were eluted by adding 20 mM MES / NaOH, pH 6.0, and samples were analyzed by SDS-PAGE.

[0400] Suc-AAPF-pNA protease activity assay

[0401] Proteolytic activity can be determined by colorimetric assays employing Suc-AAPF-pNA as substrate. Suc-AAPF-pNA is an abbreviation of N-Succinyl-Alanine-Alanine-Proline- Phenylalanine-p-Nitroanilide, which is a blocked peptide that can be cleaved by endo-proteases. Following cleavage, a free pNA molecule is liberated which has a yellow color and can be measured by visible spectrophotometry at wavelength 405 nm.

[0402] The protease sample to be analyzed was diluted in assay buffer (100 mM Tris, pH 8.0, with 0.1 mM CaCh and 0.01 % Tween® 20). The assay was performed by transferring 10 pL of diluted sample to a 384-well microtiter plate and adding 40 pL of substrate working solution (2.5 mg / ml Suc-AAPF-pNA in 100 mM Tris pH 8.0 with 0.01% Tween® 20). The solutions were mixed at room temperature and absorption was measured every 20 seconds over 5 minutes at OD 405 nm (kinetics) or every 5 minutes over at least 30 minutes (stacked kinetics).

[0403] The slope (absorbance per minute) of the time-dependent absorption curve is directly proportional to the activity of the protease in question under the given set of conditions. The protease sample was diluted to a level where the slope is linear.

[0404] Model Detergents

[0405] The composition of two liquid model detergents used in the examples is given in Table 1 and Table 2 below. Model detergent 1 represents a low pH liquid detergent of the type used in the Asia Pacific region and in Latin America. Model detergent 2 represents a low pH liquid detergent of the type used in Europe. Model detergent 1 was used for testing single substitution variants, and model detergents 1 and 2 were used for testing combination variants. Storage stability assay

[0406] The storage stability of protease variants at low pH was determined as follows. To test up to 84 protease variants, four 96-well plates were prepared with 90 pL of model detergent per well. To each well with 90 pL model detergent was added 10 pL of a sample of either a variant or a reference protease. After addition of the protease samples, the wells were mixed thoroughly for 10 minutes before the plates were sealed tightly using a plate sealer {e.g., an Agilent PlateLoc

[0407] Thermal Microplate Sealer). Four identical 96-well plates were prepared in this way. Three of the plates were then incubated at the stress temperatures (40°C, 43°C, or 45°C for the single mutation variants, and up to 60°C for the combination variants) for 18 hours (referred to as “stress plates”), with the fourth plate being stored at -20°C as a reference plate.

[0408] After incubation, the plates (stressed and reference) were unsealed and each sample was diluted 2000 times in assay buffer before being measured in the Suc-AAPF-pNA protease activity assay. The three stressed plates were measured in the same 384-well plate as the reference plate.

[0409] The residual activity of a protease variant was evaluated as the ratio of the median activity of the three replicates in a well on a stressed plate divided by the median activity of the corresponding well on the reference plate. This ratio is referred to as an improvement factor (IF). Example 1 : Low pH detergent stability of single mutation variants

[0410] Variants of the protease of SEQ ID NO:1 each having a single substitution were screened as supernatants in the storage stability assay described above in liquid model detergent 1, resulting in the improvement factors shown in Table 3. The results were collected over several experiments, with the incubation temperature being 40°C in most cases, although 43°C or 45°C was used in some. The substitutions indicated in Table 3 are relative to SEQ ID NO:1.

[0411] Example 2: Low pH detergent stability of combination variants

[0412] Based on the results of the screening of single mutation variants described in Example 1 , several rounds of combination variants were designed and screened. Combination variants with improved stability at low pH identified in this manner were isolated, and the protease enzyme variants were purified as described above. These purified protease samples were tested at a concentration of 400 ppm in the storage stability assay described above. The samples were incubated for 18 hours in liquid model detergent 2 at a temperature of 25°C, 37°C, 45°C, or 55°C as indicated in Tables 4, 5, 6 and 7. Table 8 shows samples incubated in liquid model detergent 1 at a temperature of 25°C. The elevated temperatures of 37°C, 45°C, and 55°C for model detergent 2, as well as the lower temperature of 25°C for model detergent 1 , show stability of the protease variants at stressed conditions.

[0413] The results are shown as residual activities (RA), where the residual activity is the activity after storage of the enzyme determined using the Suc-AAPF-pNA protease activity assay described above, after incubation under the given conditions, compared to the activity of the same enzyme after storage at -20°C. A residual activity of 1 corresponds to 100% maintained enzyme activity after storage, whereas a residual activity of 0 corresponds to no enzyme activity after storage.

[0414] In addition, Tables 4 and 5 show an improvement factor (IF) for the variants, calculated as the residual activity of the variant divided by the residual activity of the reference protease, here SEQ ID NO:1. An improvement factor of 1 thus corresponds to the variant having the same residual activity as the reference protease.

[0415] Under storage in the stress conditions of Tables 6, 7 and 8, the reference protease of SEQ ID NO:1 had no measurable activity, i.e., the residual activity was zero, thus an improvement factor could not be calculated for the variants compared to SEQ ID NO:1. The substitutions indicated are relative to SEQ ID NO:1.

[0416]

[0417] It may be seen that all of the variants have improved storage stability (measured as improved residual activity after storage) compared to the reference protease of SEQ ID NO:1. This is quite noteworthy, given that the protease of SEQ ID NO:1 is itself a stabilized variant of the Savinase® protease (SEQ ID NO:2), where SEQ ID NO:1 is known to be highly stabile under a variety of different stress conditions, for example in harsh detergent formulations and at elevated temperatures.

[0418] Example 3: Low pH detergent stability of combination variants Similar to the stability studies described in Example 1 , additional combination variants were designed and screened as supernatants for low pH detergent stability. The combination variants were screened similarly to the single mutation variants, but at a stress temperature of 60°C and in model detergent 2.

[0419] Since the purified protease of SEQ ID NO:1 was found to be unstable in model detergent 1 , even at a temperature of only 25°C, a combination variant having the substitutions S3T+G59V+S76E+N198D+A209K+S210I+N212S compared to SEQ ID NO:1 and which had been found to be quite stable in both model detergents (see Tables 4-8) was used as the reference in this example.

[0420] The results, expressed as improvement factors (IF) compared to the reference protease, are shown in Table 9.

[0421] The results in Table 9 show that the low pH stability of the reference sequence could be further improved by means of the additional substitutions in positions 113, 205, 206 and 210.

[0422] Example 4: Low pH detergent stability of combination variants of SEQ ID NO:4

[0423] Combination variants of SEQ ID NO:4 were designed and screened as purified protease variants for low pH detergent stability. The combination variants were screened in a storage stability assay as described above with incubation for 8 hours in liquid model detergents 1 or 2 (see Table 1 and Table 2, respectively) at stress temperatures of 25°C or 37°C.

[0424] The results in Tables 10, 11 , 12, and 13 are shown as half-life improvement factor (HIF), where HIF correlates the stability half-life of a variant protease with that of the reference protease (SEQ ID N0:4). Half-life improvement factor (H IF) was calculated as: Half-life improvement Factor (HIF) of variant = (half-life (T%) of the variant / half-life (T%) of the reference protease).

[0425] A HIF that is greater than 1 (HIF>1) indicates an improved stability of a variant as compared to the reference, while HIF of 1 (HIF=1) identifies a variant which is on par with the reference, and a HIF of less than 1 (HIF<1) identifies a variant that is less stable than the reference.

[0426] The substitutions indicated in Tables 10-13 are relative to SEQ ID NO:4. The position numbering indicated in Tables 10-13 is according to SEQ ID NO:1.

[0427]

[0428] The results in Tables 10-13 show that the low pH detergent stability of the reference sequence according to SEQ ID NO:4 could be significantly improved by means of the substitutions according to the invention.

[0429] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0430] The invention is further defined by the following numbered paragraphs:

[0431] 1. A protease variant of a parent protease, wherein the variant has protease activity and comprises substitutions in three or more positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T), and wherein position numbers are based on the numbering of SEQ ID NO:1 ; and wherein the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease; and / or wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease.

[0432] 2. The protease variant according to paragraph 1 , which has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease.

[0433] 3. The protease variant according to paragraph 1 or 2, which has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease.

[0434] 4. The protease variant according to any of the preceding paragraphs, wherein the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0435] 5a. The protease variant according to any of the preceding paragraphs, wherein the parent protease is SEQ ID NO:1.

[0436] 5b. The protease variant according to any of paragraphs 1-4, wherein the parent protease is SEQ ID NO:4.

[0437] 6. The protease variant according to any of the preceding paragraphs, wherein the substitutions in three or more positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 are selected from the group consisting of X3T (e.g., S3T), X59V (e.g., G59V), X198D (e.g., N198D or G198D), X209K (e.g., A209K), and X212S (e.g., N212S).

[0438] 7. The protease variant according to any of the preceding paragraphs, wherein the variant comprises substitutions in at least the positions corresponding to positions 3, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected X3T (e.g., S3T), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T); preferably wherein the variant comprises the substitutions X3T (e.g., S3T), X209K (e.g., A209K), and X212S (e.g., N212S).

[0439] 8. The protease variant of any of the preceding paragraphs, which comprises substitutions in at least four positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T); preferably wherein said substitutions are selected from X3T (e.g., S3T), X59V (e.g., G59V), X198D (e.g., N198D or G198D), X209K (e.g., A209K), and X212S (e.g., N212S).

[0440] 9. The protease variant of any of the preceding paragraphs, wherein the variant comprises a substitution in each of positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T); preferably wherein said substitutions are X3T (e.g., S3T), X59V (e.g., G59V), X198D (e.g., N198D or G198D), X209K (e.g., A209K), and X212S (e.g., N212S).

[0441] 10. The protease variant according to any of paragraphs 1-9, which further comprises one or more substitutions in a position corresponding to positions 37, 76, 106, 113, 205, 206, and / or 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N (e.g., T37N or S37N), X76E / D (e.g., S76E / D or T76E / D), X106V (e.g., A106V), X113V / I (e.g., G113V / I or T113V / l), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0442] 11. The protease variant according to any of paragraphs 1-9, which further comprises one or more substitution in a position corresponding to positions 113, 205, 206, and / or 210 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X113V / I (e.g., G113V / I or T113V / l), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0443] 12. The protease variant according to any of paragraph 1-9, which further comprises the substitution X76E / D (e.g., S76E / D or T76E / D), and optionally the substitution X210I / V (e.g., S2101 / V or T2101 / V)

[0444] 13. The protease variant according to any of paragraphs 1-9, which comprises further two, three, four, or five substitutions selected from the group consisting of X76E / D (e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

[0445] 14. The protease variant according to any of paragraphs 1-9, which further comprises two, three, four or five substitutions selected from the group consisting of X76E / D e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / I), X205P {e.g., G205P or T205P), X206G {e.g., S206G or N206G), and X210I / V {e.g., S210I / V or T210l / V).

[0446] 15. The protease variant of any of the preceding paragraphs, which comprises one of the following sets of substitutions compared to the parent protease:

[0447] • X3T X59V X76E X198D X209K X210I X212S

[0448] • X3T X59V X76E X198D X205P X206G X209K X210V X212S

[0449] • X3T X59V X76E X198D X205P X206G X209K X210I X212S

[0450] • X3T X59V X76E X198D X205P X209K X210V X212S

[0451] • X3T X59V X76E X113V X198D X205P X206G X209K X210V X212S

[0452] • X3T X59 X76E X113I X198D X205P X206G X209K X210V X212S

[0453] • X3T X76D X106V X198D X209K X210I X212T

[0454] • X3T X59V X76D X198D X206D X209K X210I X212S

[0455] • X3T X59V X76D X198D X209K X210I X212S

[0456] • X3T X59V X76D X198D X209K X212S

[0457] • X3T X76D X209K X210I X212S

[0458] • X3T X59V X76E X198D X209K X212S

[0459] • X3T X59R X76D X198D X206D X209K X210I X212S

[0460] • X3T X37N X59V X76D X209K X210I X212S, or

[0461] • X3T X59R X198D X209K X212S.

[0462] 16a. The protease variant of paragraph 15, wherein the parent protease is SEQ ID NO:1, and the variant comprises one of the following sets of substitutions compared to SEQ ID NO:1 :

[0463] • S3T G59V S76E N198D A209K S210I N212S

[0464] • S3T G59V S76E N198D G205P S206G A209K S210V N212S

[0465] • S3T G59V S76E N198D G205P S206G A209K S210I N212S

[0466] • S3T G59V S76E N198D G205P A209K S210V N212S

[0467] • S3T G59V S76E G113V N198D G205P S206G A209K S210V N212S

[0468] • S3T G59 S76E G113I N198D G205P S206G A209K S210V N212S

[0469] • S3T S76D A106V N198D A209K S210I N212T • S3T G59V S76D N198D S206D A209K S210I N212S

[0470] • S3T G59V S76D N198D A209K S210I N212S

[0471] • S3T G59V S76D N198D A209K N212S

[0472] • S3T S76D A209K S210I N212S

[0473] • S3T G59V S76E N198D A209K N212S

[0474] • S3T G59R S76D N198D S206D A209K S210I N212S

[0475] • S3T T37N G59V S76D A209K S210I N212S, or

[0476] • S3T G59R N198D A209K N212S.

[0477] 16b. The protease variant of paragraph 15, wherein the parent protease is SEQ ID NO:4, and the variant comprises one of the following sets of substitutions compared to SEQ ID NO:4:

[0478] • G59V T76E G198D T205P N206G A209K T210V N212S

[0479] • G59V T76E G198D T205P N206G A209K T210I N212S

[0480] • G59V T76E G198D T205P A209K T210V N212S

[0481] • G59V T76E T113V G198D T205P N206G A209K T210V N212S

[0482] • S37N G59V T76D A209K T210I N212S

[0483] • G59R G198D A209K N212S

[0484] • T76D G198D A209K T210I N212T

[0485] • G59V T76E G198D A209K T210I N212S

[0486] • G59V T76D G198D N206D A209K T210I N212S

[0487] • G59V T76D G198D A209K T210I N212S

[0488] • G59V T76D G198D A209K N212S

[0489] • T76D A209K T210I N212S

[0490] • G59V T76E T1131 G198D T205P N206G A209K T210V N212S

[0491] • G59V T76E G198D A209K N212S, or

[0492] • G59R T76D G198D N206D A209K T210I N212S.

[0493] 17. The protease variant of any of paragraphs 1-16b, wherein the variant has improved storage stability compared to an otherwise identical reference protease without the substitutions in three or more positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 and, optionally, without the one or more substitution in a position corresponding to positions 37, 76, 106, 113, 205, 206, and / or 210 of SEQ ID NO:1 , wherein storage stability is measured as residual activity in a liquid detergent composition having a pH value of between 4 and 5.

[0494] 18. The protease variant of any of paragraphs 1-16b, wherein the variant has improved storage stability compared to a parent protease selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, wherein storage stability is measured as residual activity in a liquid detergent composition having a pH value of between 4 and 5.

[0495] 19a. The protease variant of any of paragraphs 1-16a, wherein the variant has improved storage stability compared to SEQ ID NO:1 , wherein storage stability is measured as residual activity in a liquid detergent composition having a pH value of between 4 and 5.

[0496] 19b. The protease variant of any of paragraphs 1-16 or 16b, wherein the variant has improved storage stability compared to SEQ ID NO:4, wherein storage stability is measured as residual activity in a liquid detergent composition having a pH value of between 4 and 5.

[0497] 20. The protease variant of any of paragraphs 1-16b, wherein the variant has improved storage stability compared to an otherwise identical reference protease without the substitutions in three or more positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 and, optionally, without the one or more substitution in a position corresponding to positions 37, 76, 106, 113, 205, 206, and / or 210 of SEQ ID NO:1 , wherein storage stability is measured as residual activity in liquid model detergent 2 described herein.

[0498] 21 . The protease variant of any of paragraphs 1-16b, wherein the variant has improved storage stability compared to a parent protease selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, wherein storage stability is measured as residual activity in liquid model detergent 2 described herein.

[0499] 22. The protease variant of any of paragraphs 1-16a, wherein the variant has improved storage stability compared to SEQ ID NO:1 , wherein storage stability is measured as residual activity in liquid model detergent 2 described herein.

[0500] 23. The protease variant of any of paragraphs 1-16a, wherein the variant has improved low pH stability, expressed as an improvement factor (IF) compared to the parent protease or compared to the protease of SEQ ID NO:1 , of at least 1.1 , such as at least 1.2, at least 1.3, at least 1.4 or at least 1.5, wherein low pH stability is determined as residual activity following storage at 25°C for 18 hours in liquid model detergent 2 as described herein.

[0501] 24. The protease variant of any of paragraphs 1-16a, wherein the variant has improved low pH stability, expressed as an improvement factor (IF) compared to the parent protease or compared to the protease of SEQ ID NO:1 , of at least 10, such as at least 20, at least 30, at least 40 or at least 50, wherein low pH stability is determined as residual activity following storage at 37°C for 18 hours in liquid model detergent 2 as described herein.

[0502] 25a. The protease variant of any of paragraphs 1-16 or 16b, wherein the variant has improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 1.01, such as at least 1.05, at least 1.1, at least 1.5 or at least 2.0, wherein low pH stability is determined as residual activity following storage at 37°C for 8 hours in a liquid model detergent having a pH value between 4 and 5.

[0503] 25b. The protease variant of any of paragraphs 1-16 or 16b, wherein the variant has improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 2.0, such as at least 2.2, at least 2.7, at least 3.0 or at least 3.5, wherein low pH stability is determined as residual activity following storage at 25°C for 8 hours in a liquid model detergent having a pH value between 4 and 5.

[0504] 26a. The protease variant of any of paragraphs 1-16 or 16b, wherein the variant has improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 1.01 , such as at least 1.05, at least 1.1 , at least 1.5 or at least 2.0, wherein low pH stability is determined as residual activity following storage at 37°C for 8 hours in a liquid model detergent as described herein.

[0505] 26b. The protease variant of any of paragraphs 1-16 or 16b, wherein the variant has improved low pH stability, expressed as a half-life improvement factor (HIF) compared to the parent protease or compared to the protease of SEQ ID NO:4, of at least 2.0, such as at least 2.2, at least 2.7, at least 3.0 or at least 3.5, wherein low pH stability is determined as residual activity following storage at 25°C for 8 hours in a liquid model detergent as described herein.

[0506] 27. A composition comprising the protease variant of any of paragraphs 1-26; preferably wherein the composition is a detergent composition.

[0507] 28. The composition according to paragraph 27, wherein the composition is in the form of a liquid, a gel, or a unit dose detergent composition. 29. The composition according to paragraph 27 or 28, which is in the form of a liquid laundry detergent composition.

[0508] 30. The composition according to any of paragraphs 27-29, wherein the composition has a pH value of from about 4 to about 6.5.

[0509] 31. The composition according to any of paragraphs 27-30, wherein the composition has a pH value of from about 4 to about 5.

[0510] 32. Use of a protease variant according to any of paragraphs 1-26b or a composition according to any of paragraphs 27-31 in a cleaning process.

[0511] 33. Use according to paragraph 32, wherein the cleaning process is selected from laundry and hard surface cleaning such as dishwashing; preferably wherein the cleaning process is laundry.

[0512] 34. A method of cleaning an item, wherein the item is a textile, dishware, or a hard surface, comprising contacting the item with a composition according to any of paragraphs 27-31 ; preferably wherein the item is a textile.

[0513] 35. A polynucleotide encoding a protease variant according to any of paragraphs 1-26b.

[0514] 36. A nucleic acid construct or expression vector comprising the polynucleotide according to paragraph 35.

[0515] 37. A recombinant host cell transformed with the polynucleotide according to paragraph 35, or the nucleic acid construct or expression vector according to paragraph 36.

[0516] 38. A method of producing a protease variant, comprising:

[0517] (a) cultivating the recombinant host cell of paragraph 37 under conditions suitable for expression of the variant, and

[0518] (b) recovering the variant.

[0519] 39. A method for obtaining a protease variant according to any of paragraphs 1-26b, the method comprising:

[0520] (a) introducing into a parent protease substitutions in three or more of positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T (e.g., S3T), X59V / R (e.g., G59V / R), X198D (e.g., N198D or G198D), X209K / M / I / R (e.g., A209K / M / I / R), and X212S / T (e.g., N212S / T); and optionally one or more substitutions in a position corresponding to positions 37, 76, 106, 113, 205, 206, and / or 210 or SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X37N (e.g., T37N or S37N), X76E / D (e.g., S76E / D orT76E / D), X106V (e.g., A106V), X113V / I (e.g., G113V / I or T113V / I), X205P (e.g., G205P or T205P), X206G / A / D / F (e.g.,

[0521] S206G / A / D / F or N206G / A / D / F), and X210IA / / Y / F / E / T / M (e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M); wherein the variant has protease activity; and optionally

[0522] (b) recovering the variant.

Claims

CLAIMS1. A protease variant of a parent protease, wherein the variant has protease activity and comprises substitutions in three or more, e.g., four or more, or five, positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 , wherein said substitutions are selected from the group consisting of X3T {e.g., S3T), X59V / R e.g., G59V / R), X198D {e.g., N198D or G198D), X209K / M / I / R {e.g., A209K / M / I / R), and X212S / T {e.g., N212S / T), and wherein position numbers are based on the numbering of SEQ ID NO:1 ; and wherein the variant has a TM-score of at least 0.80, e.g., at least 0.81, at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease; and / or wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease.

2. The protease variant according to claim 1 , wherein the parent protease is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; preferably wherein the parent protease is SEQ ID NO:1 or SEQ ID NO:4.

3. The protease variant according to any of claims 1-2, wherein the at least three substitutions in positions corresponding to positions 3, 59, 198, 209, and 212 of SEQ ID NO:1 are selected from the group consisting of X3T {e.g., S3T), X59V {e.g., G59V), X198D {e.g., N198D or G198D), X209K {e.g., A209K), and X212S {e.g., N212S).

4. The protease variant according to any of claims 1-3, which further comprises one or more substitutions in a position corresponding to positions 37, 76, 106, 113, 205, 206 and / or 210 of SEQ ID NO:1, wherein said substitutions are selected from the group consisting of X37N {e.g., T37N or S37N), X76E / D {e.g., S76E / D or T76E / D), X106V {e.g., A106V), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M).

5. The protease variant according to claim 4, which comprises the substitution X76E / D {e.g., S76E / D or T76E / D) and / or the substitution X210I / V {e.g., S210I / V or T210I / V).

756. The protease variant according to claim 4, which comprises two, three, four, or five substitutions selected from the group consisting of X76E / D {e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G / A / D / F {e.g., S206G / A / D / F or N206G / A / D / F), and X210I / V / Y / F / E / T / M {e.g., S210I / V / Y / F / E / T / M or T210I / V / Y / F / E / T / M); preferably two, three, four, or five substitutions selected from the group consisting of X76E / D e.g., S76E / D or T76E / D), X113V / I {e.g., G113V / I or T113V / l), X205P {e.g., G205P or T205P), X206G {e.g., S206G or N206G), and X210I / V {e.g., S210I / V or T210l / V).

7. The protease variant according to any of claims 1-6, wherein the variant has improved storage stability compared to the parent protease, wherein storage stability is measured as residual activity in a liquid detergent composition having a pH value of between 4 and 5; preferably wherein the parent protease is SEQ ID NO:1 or SEQ ID NO:4.

8. The protease variant according to any of claims 1-7, wherein the variant comprises one of the following substitution sets compared to the parent protease:• X3T X59V X76E X198D X209K X210I X212S• X3T X59V X76E X198D X205P X206G X209K X210V X212S• X3T X59V X76E X198D X205P X206G X209K X210I X212S• X3T X59V X76E X198D X205P X209K X210V X212S• X3T X59V X76E X113V X198D X205P X206G X209K X210V X212S• X3T X59 X76E X113I X198D X205P X206G X209K X210V X212S• X3T X76D X106V X198D X209K X210I X212T• X3T X59V X76D X198D X206D X209K X210I X212S• X3T X59V X76D X198D X209K X210I X212S• X3T X59V X76D X198D X209K X212S• X3T X76D X209K X210I X212S• X3T X59V X76E X198D X209K X212S• X3T X59R X76D X198D X206D X209K X210I X212S• X3T X37N X59 X76D X209K X210I X212S, or• X3T X59R X198D X209K X212S; for example one of the following substitution sets compared to SEQ ID NO:1 :• S3T G59V S76E N198D A209K S210I N212S• S3T G59V S76E N198D G205P S206G A209K S210V N212S• S3T G59V S76E N198D G205P S206G A209K S210I N212S• S3T G59V S76E N198D G205P A209K S210V N212S• S3T G59V S76E G113V N198D G205P S206G A209K S210V N212S76• S3T G59V S76E G113I N198D G205P S206G A209K S210V N212S• S3T S76D A106V N198D A209K S210I N212T• S3T G59V S76D N198D S206D A209K S210I N212S• S3T G59V S76D N198D A209K S210I N212S• S3T G59V S76D N198D A209K N212S• S3T S76D A209K S210I N212S• S3T G59V S76E N198D A209K N212S• S3T G59R S76D N198D S206D A209K S210I N212S• S3T T37N G59V S76D A209K S210I N212S, or• S3T G59R N198D A209K N212S.

9. A polynucleotide encoding a protease variant according to any of the preceding claims.

10. A nucleic acid vector or expression vector comprising the polynucleotide of claim 9.

11. A recombinant host cell transformed with the polynucleotide of claim 9, or a nucleic acid construct or expression vector comprising said polynucleotide; preferably wherein the recombinant host cell is a Bacillus cell.

12. A composition comprising the protease variant of any of claims 1-8; preferably wherein the composition is a detergent composition.

13. The composition according to claim 12, which is a liquid laundry detergent composition.

14. Use of a protease variant according to any of claims 1-8 or a composition according to any of claims 12-13 in a cleaning process.

15. A method for producing a protease variant, comprising(a) cultivating a recombinant host cell according to claim 11 under conditions suitable for expression of the variant, and(b) optionally, recovering the variant; preferably wherein the recombinant host cell is a Bacillus cell.77

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