Protease variants and polynucleotides encoding the same

Abstract

The present invention relates to protease variants and methods for obtaining protease variants. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

[0001] References to sequence lists

[0002] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference. Background of the Invention

[0003] Invention Field

[0004] This invention relates to novel protease variants exhibiting alterations in one or more properties relative to a parent protease, including: cleaning performance, detergent stability, and / or storage stability. The variants of this invention are suitable for use in cleaning processes and detergent compositions, such as laundry compositions and dishwashing compositions, including hand-washing and automated dishwashing compositions. This invention also relates to isolated DNA sequences encoding these variants, expression vectors, host cells, and methods for generating and using the protease variants of this invention.

[0005] Related technical specifications

[0006] Enzymes have been used as a component of detergent formulations in the detergent industry for decades. From a commercial perspective, proteases are the most relevant enzymes in such formulations, while other enzymes (including lipases, amylases, cellulases, hemicellulases, or mixtures of multiple enzymes) are also commonly used. To improve the cost and / or performance of proteases, altered properties are employed, such as increased activity at low temperatures, increased stability, increased specific activity at a given pH, and altered Ca2+. 2+ The search continues for proteases that exhibit dependence, increased stability in the presence of other detergent ingredients (such as bleach, surfactants, etc.). One family of proteases widely used in detergents is subtilisinase. This family has previously been further grouped into six distinct subgroups by Siezen RJ and Leunissen JAM, 1997, Protein Science, 6, 501-523. One of these subgroups is the subtilisinase family, which includes subtilisinases such as BPN', subtilisin 309, etc. Novozymes (A / S) and Carlsberg (a company specializing in Bacillus subtilis protease) Novozymes, subtilisin 41 (a subtilisin from psychrophilic Antarctic Bacillus TA41, Davail S et al. 1994, The Journal of Biological Chemistry, 269(26), 99.17448-17453) and subtilisin 39 (a subtilisin from psychrophilic Antarctic Bacillus TA39, Narinx E et al. 1997, Protein Engineering, 10(11), pp.1271-1279). TY-145 protease is a subtilisinase derived from the Bacillus species TY-145 (NCIMB40339), which was first described in WO 92 / 17577 (Novozymes) and later in application WO 2004 / 067737 (Novozymes) (disclosing the three-dimensional structure and the use of protein engineering to modify the functionality of a TY-145 subtilisinase). Invention Overview

[0007] The present invention further relates to a substituted protease variant comprising one or more of the following groups: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein these positions correspond to the positions in SEQ ID NO:3, wherein the variant is associated with SEQ ID NO:3. NO:3 has at least 70% sequence identity, and this variant has protease activity.

[0008] The present invention also relates to isolated polynucleotides encoding these variants, nucleic acid constructs, vectors and host cells containing these polynucleotides, and methods for generating these variants.

[0009] Sequence List Overview

[0010] SEQ ID NO:1 = is the DNA sequence of the TY-145 protease isolated from a species of Bacillus.

[0011] SEQ ID NO:2 = is the amino acid sequence as deduced from SEQ ID NO:1.

[0012] SEQ ID NO:3 = is the amino acid sequence of the mature TY-145 protease.

[0013] SEQ ID NO:4 = is the amino acid sequence of TY-145 protease + S173P + S175P.

[0014] SEQ ID NO:5 = is the amino acid sequence of TY TY-145 protease + S173P + S175P + F180Y.

[0015] definition

[0016] The term "protease" is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses: http: / / en.wikipedia.org / wiki / Category:EC_3.4). EC numbers are based on the 1992 enzyme nomenclature published by NC-IUBMB Academic Press in San Diego, California, specifically in the following publications: Eur. J. Biochem. 1994, 223, 1–5; Eur. Biochem. 1995, 232, 1–6; Eur. Biochem. 1996, 237, 1–5; Eur. Biochem. 1997, 250, 1–6; and Eur. Biochem. 1999, 264, 610–650, supplements 1–5. The term "subtilisinase" refers to the serine protease subgroup according to Essen et al., Protein Engineering, 4 (1991) 719-737 and Essen et al., Protein Science, 6 (1997) 501-523. Serine proteases, or serine peptidases, are a subgroup of proteases characterized by having a serine residue at their active site that forms a covalent adduct with the substrate. Furthermore, subtilisinases (and serine proteases) are characterized by having two active site amino acid residues in addition to serine, namely histidine and aspartic acid residues. Subtilisinases can be classified into six subgroups: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanathionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family. The term "protease activity" refers to proteolytic activity (EC 3.4). The protease of this invention is an endopeptidase (EC 3.4.21). For the purposes of this invention, protease activity is determined according to the procedure described in the following “Materials and Methods”. These protease variants of the present invention have at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% protease activity of the polypeptide of SEQ ID NO:3.

[0017] The terms "parent," "protease parent," or "precursor protease" refer to a protease that has been modified to produce the enzyme variant of the present invention. Thus, a parent is a protease having an amino acid sequence consistent with the variant but without alterations at one or more of the specified positions. It should be understood that the expression "having a consistent amino acid sequence" in this context refers to 100% sequence identity. The parent can be a naturally occurring (wild-type) polypeptide. In a specific embodiment, the parent is a protease having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 70%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% consistency with the polypeptide having SEQ ID NO:3.

[0018] The term "protease variant" means a protease having protease activity that, compared to its parent, includes a change—substitution, insertion, and / or deletion (preferably substitution)—at one or more (or one or more) positions, where the parent is a protease having an amino acid sequence consistent with the variant but without the change at one or more of the specified positions. Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that an amino acid is added adjacent to the amino acid occupying a position, for example, 1 to 10 amino acids, preferably 1 to 3 amino acids. Preferably, the variant is artificially modified. In one aspect, the variant is at least 1% pure, for example at least 5% pure, at least 10% pure, at least 20% pure, at least 40% pure, at least 60% pure, at least 80% pure, and at least 90% pure, as determined by SDS-PAGE.

[0019] The term "isolated polynucleotide" refers to a polynucleotide that has been artificially modified. In one respect, as determined by agarose gel electrophoresis, the isolated polynucleotide is at least 1% pure, for example at least 5%, at least 10%, at least 20%, at least 40%, at least 60%, at least 80%, at least 90%, and at least 95% pure. The polynucleotide can be genomic, cDNA, RNA, semi-synthetic, synthetically derived, or any combination thereof.

[0020] The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (without change in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0021] The term "substantially pure variant" means a formulation comprising, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polypeptide materials, which are naturally or recombinantly related to it. Preferably, the variant is at least 92% pure by weight of the total polypeptide material present in the formulation, for example, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and 100% pure. These variants of the invention are preferably present in substantially pure form. This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0022] The term "wild-type protease" refers to a protease expressed by a naturally occurring organism, such as bacteria, archaea, yeast, fungi, plants, or animals found in nature. An example of a wild-type protease is the TY-145 protease.

[0023] The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one respect, this mature polypeptide corresponds to the amino acid sequence having SEQ ID NO:3.

[0024] The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide with protease activity. In one respect, based on the prediction that nucleotides 1 to 81 of SEQ ID NO:1 are the signal peptide SignalP (Nielsen et al., 1997, Protein Engineering 10:1-6), the mature polypeptide coding sequence is nucleotides 331 to 1263 of SEQ ID NO:1.

[0025] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from prokaryotic or eukaryotic cells. cDNA lacks the intron sequences that are normally present in the corresponding genomic DNA. The initial RNA transcript is a precursor to mRNA, which undergoes a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0026] The term "coding sequence" refers to a polynucleotide that directly identifies the amino acid sequence of its polypeptide product. The boundaries of a coding sequence are generally defined by an open reading frame, which typically begins with an ATG start codon or an alternative start codon (such as GTG and TTG) and ends with a stop codon (such as TAA, TAG, and TGA). Coding sequences can be DNA, cDNA, synthetic, or recombinant polynucleotides.

[0027] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule isolated from naturally occurring genes, or modified in a manner not found in nature to contain nucleic acid fragments, or synthesized. When the nucleic acid construct contains the control sequence required to express the coding sequence of this invention, the term "nucleic acid construct" has the same meaning as the term "expression cassette."

[0028] The term "operably linked" refers to a configuration in which a control sequence is placed in an appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence guides the expression of the coding sequence.

[0029] The term "control sequence" refers to all components necessary for the expression of the polynucleotide encoding a variant of the present invention. Each control sequence for the polynucleotide encoding the variant may be native or exogenous, or may be native or exogenous to each other. Such control sequences include, but are not limited to: promoters, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with adapters for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the variant.

[0030] The term “expression” includes any step involved in variant generation, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0031] The term "expression vector" refers to a linear or circular DNA molecule that includes a polynucleotide encoding a variant and is operatively linked to additional nucleotides that provide expression for it.

[0032] The term "transcription promoter" is used to refer to a promoter of a DNA region that promotes the transcription of a specific gene. Transcription promoters are typically located near the gene they regulate, on the same strand, and upstream (towards the 5' region of the sense strand).

[0033] The term "transcription terminator" is used to refer to a gene sequence segment that marks the end of a gene or an operon on genomic DNA used for transcription.

[0034] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc., using nucleic acid constructs or expression vectors including the polynucleotides of this invention. The term "host cell" also encompasses any offspring of a parent cell that is inconsistent with the parent cell due to mutations occurring during replication.

[0035] The correlation between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For the purposes of this invention, the degree of sequence identity between two amino acid sequences is determined using the Needle algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 3.0.0 or later). Optional parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Nieder's annotation for "longest consistency" (obtained using the -non-simplification option) is used as the percentage consistency and is calculated as follows:

[0036] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0037] The term "highly stringent conditions" refers to following a standard DNA blotting procedure for probes at least 100 nucleotides in length, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0038] The term "very stringent conditions" refers to following standard DNA blotting procedures for probes at least 100 nucleotides in length, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0039] The term "medium-tough conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0040] The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and / or 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0041] The term "low stringency conditions" refers to following a standard DNA blotting procedure for probes of at least 100 nucleotides in length, pre-hybridizing and hybridizing at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0042] The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, a standard DNA blotting procedure is followed, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0043] The term "improved properties" refers to characteristics associated with a variant that are improved compared to the parent, or compared to a protease having SEQ ID NO:3, or compared to a protease having an amino acid sequence identical to the variant but without alteration at one or more of the specified positions. Such improved properties include, but are not limited to: washing performance, protease activity, thermal activity profile, thermal stability, pH activity profile, pH stability, substrate / cofactor specificity, improved surface properties, substrate specificity, product specificity, increased stability, improved stability under storage conditions, and chemical stability.

[0044] The term “improved protease activity” is defined herein as, for example, by increased protein conversion, an activity of a protease variant that exhibits an activity change relative to (or compared to) the parent protease, or relative to a protease having SEQ ID NO:3, or relative to a protease having an amino acid sequence consistent with the variant but without change at one or more of the specified positions (as defined above).

[0045] The term "stability" includes both storage stability and stability during use, such as stability during washing, and reflects the stability of the protease variant according to the invention as a function of time, such as how much activity it retains when the protease variant is placed in solution, particularly in detergent solution. This stability is affected by many factors, such as pH, temperature, detergent composition, such as the amount of builder, surfactant, etc. The terms "improved stability" and "enhanced stability" include "improved chemical stability," "detergent stability," or "improved detergent stability."

[0046] The term "improved chemical stability" is defined herein as the variant enzyme retaining its enzymatic activity after a period of incubation in the presence of one or more chemicals, whether naturally occurring or synthetic, that reduce the activity of the parent enzyme. Improved chemical stability may also enable these variants to catalyze reactions better in the presence of such chemicals. The term "improved thermal activity" refers to a modified temperature-dependent activity profile of the variant relative to the parent or the protease having SEQ ID NO:3 at a specific temperature.

[0047] In one embodiment, these protease variants according to the invention exhibit improved inhibitor binding compared to the parent enzyme. In one specific embodiment, these protease variants of the invention exhibit improved inhibitory activity compared to the parent, for example, compared to SEQ ID NO 3. In another specific embodiment, these protease variants of the invention exhibit increased inhibitory activity compared to the inhibition of TY-145 protease (SEQ ID NO 3) by the same inhibitor as described in Example 2 of this Material and Method.

[0048] Irreversible inhibitors lead to covalent modification of the enzyme, thus permanently reducing its activity. The interaction between these protease variants of the present invention and inhibitors suitable for these protease variants of the present invention is preferably reversible, and therefore the effect of the inhibitor can be reversed by removing the inhibitor. The inhibitor constant Ki is an indicator of the potency of the inhibitor and is the concentration required to achieve half of the maximum inhibition. Ki is a measure of the effectiveness of inhibiting a specific function. In this case, it refers to the effectiveness of inhibiting or stabilizing the protease variants of the present invention. Compared to the parental protease, these protease variants of the present invention are improved by being inhibited or stabilized by a suitable inhibitor more than the corresponding parental protease; therefore, the Ki of the protease variants according to the present invention is lower than the Ki of the corresponding parent.

[0049] The term "improved cleaning performance" is defined herein as the improved cleaning performance of the protease variant according to the invention, as measured by relevant assays (such as AMSA), relative to the parent protease, relative to the protease having SEQ ID NO:3, or relative to a protease having an amino acid sequence consistent with the variant but without alteration at one or more of the specified positions. The term "cleaning performance" includes cleaning performance in laundry and, for example, in hand washing and dishwashing. Cleaning performance can be quantified as described herein under the definition of "improved cleaning performance." The term "low-temperature performance" is defined herein as the protease variant according to the invention exhibiting cleaning performance at or below 20°C, as described above.

[0050] Unless otherwise stated, the term "detergent composition" includes all-purpose or heavy-duty cleaners in granular or powder form, especially cleaning detergents; all-purpose cleaners in liquid, gel, or paste form, especially so-called heavy-duty liquid (HDL) types; liquid delicate fabric detergents; hand-washing dishwashing agents or light-duty dishwashing agents, especially those with high foaming properties; machine-washing dishwashing agents, including various tablet, granular, liquid, and rinsing aid types for home and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning sticks, soap sticks, mouthwashes, denture cleaners, car or carpet cleaners, and bathroom cleaners; shampoos and conditioners; shower gels and body washes; metal cleaners; and cleaning aids such as bleach additives and "stain remover sticks" or pretreatment types. The terms "detergent composition" and "detergent formulation" are used with respect to mixtures in a cleaning medium intended for cleaning soiled objects. In some embodiments, the term (e.g., "laundry detergent") is used with respect to washing fabrics and / or clothing. In alternative embodiments, the term refers to other detergents such as those used for cleaning tableware, knives, etc. (e.g., "dishwashing detergent"). It is not intended to limit the invention to any particular detergent formulation or composition. The term "detergent composition" is not intended to be limited to compositions containing surfactants. It is intended that, except for variations according to the invention, the term covers detergents that may contain: for example, surfactants, builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric conditioners, foaming agents, defoaming agents, dyes, fragrances, darkening inhibitors, optical brighteners, bactericides, fungicides, dirt suspending agents, corrosion inhibitors, enzyme inhibitors or stabilizers, enzyme activators, transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0051] The term "fabric" encompasses any textile material. Therefore, it is intended that the term cover clothing, along with fabrics, yarns, fibers, non-woven materials, natural materials, synthetic materials, and any other textile material.

[0052] The term "textiles" refers to woven fabrics, together with short fibers and filaments suitable for conversion into or used as yarns, for weaving, knitting, and non-woven fabrics. This term encompasses yarns derived from both natural and synthetic (e.g., manufactured) fibers. The term "textile materials" is a general term for fibers, yarn intermediates, yarns, fabrics, and products derived from fibers (e.g., clothing and other articles).

[0053] The term "non-textile detergent composition" includes detergent compositions for non-textile surfaces, including but not limited to compositions for cleaning hard surfaces, such as dishwashing detergent compositions (manual dishwashing compositions), oral detergent compositions, denture detergent compositions, and personal hygiene compositions.

[0054] The term "effective amount of enzyme" refers to the amount of enzyme necessary to achieve the desired enzyme activity in a particular application, such as in a defined detergent composition. Such an effective amount can be readily determined by those skilled in the art and is based on a variety of factors, such as the specific enzyme used, the cleaning application, the specific composition of the detergent composition, and whether a liquid or dry (e.g., granular, stick) composition is required. The term "effective amount of protease variant" refers to, for example, the amount of the protease variant described above that achieves the desired level of enzyme activity in a defined detergent composition.

[0055] As used herein, the terms “water hardness” or “hardness” or “dH” or “°dH” refer to German hardness. It was once defined as 10 mg calcium oxide per liter of water.

[0056] The term “relevant cleaning conditions” is used here to refer to the conditions actually used in the household within the detergent segment, specifically cleaning temperature, time, cleaning mechanics, detergent concentration, detergent type, and water hardness.

[0057] The term "excipient" refers to any liquid, solid, or gaseous material selected for a specific type of detergent composition and product form (e.g., liquid, granules, powder, stick, paste, spray, tablet, gel, or foam composition), which is also preferably compatible with protease variants used in the composition. In some embodiments, the granule composition is in a "compressed" form, while in other embodiments, the liquid composition is in a "concentrated" form.

[0058] As used herein, the term "stain-removing enzyme" describes an enzyme that helps remove stains or dirt from fabrics or hard surfaces. Stain-removing enzymes act on specific substrates; for example, proteases act on proteins, amylases on starch, lipases and keratases on lipids (fat and oil), pectinases on pectin, and hemicellulases on hemicellulose. Stains are typically deposits of complex mixtures of different components, which can cause localized discoloration of the material itself or leave a sticky surface on the object that can attract dirt dissolved in cleaning solutions, thus causing discoloration of the stained area. When an enzyme acts on its specific substrate present in a stain, the enzyme degrades or partially degrades its substrate, thereby helping to remove the dirt and stain components associated with the substrate during the cleaning process. For example, when a protease acts on a blood stain, it reduces the protein component in the blood.

[0059] In this context, the term "reduced amount" means that, all else being equal, the amount of the component is less than the amount that will be used in the reference process.

[0060] The term "low detergent concentration" system includes detergents in which less than about 800 ppm of detergent components are present in the washing water. Asian (e.g., Japanese) detergents are typically considered to be low detergent concentration systems.

[0061] The term "medium detergent concentration" system includes detergents in which detergent components are present in washing water at concentrations between approximately 800 ppm and approximately 2000 ppm. North American detergents are generally considered to be in the medium detergent concentration system.

[0062] The term "high detergent concentration" system includes detergents in which detergent components are present in the washing water at concentrations greater than approximately 2000 ppm. European detergents are generally considered to be part of the high detergent concentration system.

[0063] Variant Naming Rules

[0064] For the purposes of this invention, the mature polypeptide disclosed in SEQ ID NO:3 is used to determine the corresponding amino acid residues in another protease. The amino acid sequence of the other protease is aligned with the mature polypeptide disclosed in SEQ ID NO:3, and based on this alignment, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, Journal of Molecular Biology 48:443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends in Genetics 16:276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide disclosed in SEQ ID NO:3. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0065] The identification of corresponding amino acid residues in another protease can be determined by using several computer programs that compare multiple peptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Kato et al., 2005, Nucleic Acids Research 33:511-518; Kato, 2007, Bioinformatics 23:372-374; Kato et al., 2009, Methods in Molecular Biology). Biology 537: 39-64; Kato Kazuto, 2010, Bioinformatics 26: 1899-1900) and using ClustalW's EMBOSS EMMA (1.83 or later; Thompson et al., 1994, Nucleic Acids Res. 22: 4673-4680).

[0066] When other enzymes deviate from the mature polypeptide of SEQ ID NO:3, rendering traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, Journal of Molecular Biology 295:613-615), alternative pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (characteristic curves) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple spectra through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acid Research 25:3389-3402). Even higher sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, Journal of Molecular Biology 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, Journal of Molecular Biology 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0067] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms can be used to align two or more protein structures, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747). Furthermore, implementations of these algorithms can be used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0068] In the description of variations of the invention, the following nomenclature is used for ease of reference. Recognized IUPAC single-letter or three-letter amino acid abbreviations are used. Amino acid positions are indicated as #1, #2, etc.

[0069] substitutions For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of serine at position #1 with tryptophan is represented as "Ser#1Trp" or "S#1W". Multiple mutations are separated by a plus sign ("+") or a comma (,), for example, "Ser#1Trp+Ser#2Pro" or "S#1W, S#2P", representing the substitution of serine (S) at positions #1 and #2 with tryptophan (W) and proline (P), respectively. If more than one amino acid can be substituted at a given position, these amino acids are listed in parentheses, such as [X] or {X}. Thus, if both Trp and Lys can be substituted according to the invention, replacing the amino acid occupying position #1, this is represented as X#1{W, K} or X#2[W, K], where X indicates that the amino acid residue of the different protease according to the invention can be a parent, such as a protease like SEQ ID NO:3 or a protease having at least 70% similarity to it. Therefore, in some cases, these variants are represented as #1{W,K} or X#2P, indicating that the amino acid to be substituted varies depending on the parent. Since SEQ ID NO:3 is used as the substitution number according to this application, it can be represented by the amino acid present at the corresponding position in SEQ ID NO:3.

[0070] deletions For amino acid deletions, use the following nomenclature: original amino acid, position, *. Therefore, a deletion of serine at position #1 is represented as "Ser#1*" or "S#1*". Multiple deletions are separated by a plus sign ("+") or a comma, for example, "Ser#1*+Ser#2*" or "S#1*, S#2*".

[0071] insertions The insertion of additional amino acid residues, such as the insertion of lysine after G#1, can be represented as Gly#1GlyLys or G#1GK. Alternatively, the insertion of additional amino acid residues, such as the insertion of lysine after G#1, can be represented as *#1aL. When more than one amino acid residue is inserted, such as the insertion of Lys and Ala after #1, this insertion can be represented as Gly#1GlyLysAla or G#1GKA. In such cases, the inserted amino acid residues can also be numbered by adding a lowercase letter to the amino acid residue position number before the inserted amino acid residue, in this example: *#1aK*#1bA.

[0072] various modificationsVariations with multiple changes are separated by a plus sign ("+") or a comma (,), such as "Ser#1Trp+Ser#2Pro" or "S#1W,S#2P", which represent that the serine at positions #1 and #2 is replaced by tryptophan and proline, respectively, as described above.

[0073] different alterations: When different changes can be introduced at a position, these changes are separated by commas, such as "Ser#1Trp,Lys" or S#1W, where K represents the substitution of serine at position #1 with tryptophan or lysine. Therefore, "Ser#1Trp,Lys+Ser#2Asp" represents the following variants: "Ser#1Trp+Ser#2Pro", "Ser#1Lys+Ser#2Pro", or S#1W,K+S#2D. Invention Details

[0074] This invention provides novel protease variants obtained from the genus *Bacillus*, particularly *Bacillus* TY-145. These protease variants of the invention have at least 60% sequence identity with the polypeptide having SEQ ID NO: 3 and, compared to the protease having SEQ ID NO: 3, include a substitution at at least one amino acid position selected from the group consisting of the following positions: 70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240. One embodiment of the invention relates to protease variants that are identical to the polypeptide having SEQ ID NO: 3. NO3 has at least 60% identity, possesses proteolytic activity, and comprises one or more substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein each position corresponds to SEQ ID. The position of the peptide NO:3.In a preferred embodiment, the protease variant comprises one or more substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein each position corresponds to SEQ ID. The position of the polypeptide NO:3, wherein the variant has at least 60%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99%, such as at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, but less than 100% sequence identity with SEQ ID NO:3.

[0075] Enzymes have long been used in cleaning compositions. Enzymes are beneficial because each enzyme has specific substrate specificity, which allows for the removal of a wide variety of stains. Proteases act on protein stains, and the addition of proteases helps remove stains including proteins such as coconut, blood, and egg stains. However, a common problem with proteases in cleaning compositions, especially liquid cleaning compositions, is their degradation by other enzymes in the composition and by the proteases themselves. This can lead to reduced stability of the proteases and other enzymes in the composition, and a decrease in the overall performance of the enzymes in the cleaning composition. The storage stability of enzymes in cleaning compositions can be improved by adding protease inhibitors or stabilizers, which are reversible inhibitors of protease activity (e.g., serine protease activity). Therefore, it is advantageous to stabilize proteases by protease inhibitors (preferably reversible protease inhibitors). Several protease inhibitors have been described in the art, including protease inhibitors such as peptidaldehyde, boric acid, or boronicacid; or derivatives of any of these. Some inhibitors include, but are not limited to, boric acid or derivatives thereof; preferably, phenylboronic acid or derivatives thereof. Benzylboronic acid derivatives have the following formula:

[0076]

[0077] Wherein R is selected from the group consisting of: hydrogen, hydroxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl, and substituted C1-C6 alkenyl. Preferably, R is hydrogen, CH3, CH3CH2, or CH3CH2CH2. The protease inhibitor (phenylboronic acid derivative) is 4-formyl-phenyl-boronic acid (4-FPBA). The protease inhibitor is selected from the group consisting of:

[0078] Thiophene-2-boronic acid, thiophene-3-boronic acid, acetamide phenylboronic acid, benzofuran-2-boronic acid, naphthalene-1-boronic acid, naphthalene-2-boronic acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-thiaanthraboronic acid, 4-dibenzofuranboronic acid, 5-methylthiophene-2-boronic acid, thionaphthene boronic acid, furan-2-boronic acid, furan-3-boronic acid, 4,4-biphenyl-diboronic acid, 6-hydroxy-2-naphtalene, 4-(methylthio)phenylboronic acid, 4-(trimethyl-silyl)phenylboronic acid, 3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2-naphthylboronic acid, 5-bromothiophene boronic acid (acid), 5-chlorothiopheneboronic acid, dimethylthiopheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thiaanthraboronic acid, dibenzothiopheneboronic acid, 4-carboxyphenylboronic acid, 9-anthraylboronic acid, 3,5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, p-tolylboronic acid, o-tolylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-di-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, 4-methoxyphenylboronic acid.

[0079] Other boric acid derivatives suitable as protease inhibitors in cleaning compositions are described in US 4,963,655, US 5,159,060, WO 95 / 12655, WO 95 / 29223, WO 92 / 19707, WO 94 / 04653, WO 94 / 04654, US5442100, US 5488157 and US 5472628.

[0080] This protease inhibitor can also be a peptide aldehyde with the formula X-B1-B0-H, wherein these groups have the following meanings:

[0081] a) H is hydrogen;

[0082] b) B0 is a single amino acid residue with an L- or D-configuration and the formula: NH-CHR'-CO;

[0083] c) B1 is a single amino acid residue; and

[0084] d) X consists of one or more amino acid residues (preferably one or two), optionally including an N-terminal protecting group. NH-CHR'-CO(B0) is an L- or D-amino acid residue, wherein R' can be an aliphatic or aromatic side chain, such as an aralkyl group, like benzyl, wherein R' can be optionally substituted. More specifically, the B0 residue can be bulky, neutral, polar, hydrophobic, and / or aromatic. Examples are D- or L-type Tyr (p-tyrosine), meta-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, valine (Nva), Leu, Ile, or leucine (Nle). In the above formula X-B1-B0-H, the B1 residue can be particularly small, aliphatic, hydrophobic, and / or neutral. Examples are alanine (Ala), cysteine ​​(Cys), glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), valine (Val), valine (Nva), and leucine (Nle), especially alanine, glycine, or valine.

[0085] Specifically, X can be one or two amino acid residues with an optional N-terminal protecting group (i.e., the compound is a tri- or tetrapeptide aldehyde, with or without a protecting group). Thus, X can be B2, B3-B2, Z-B2, or Z-B3-B2, where B3 and B2 each represent one amino acid residue, and Z is an N-terminal protecting group. The B2 residue can be particularly small, fatty acid, and / or neutral, such as Ala, Gly, Thr, Arg, Leu, Phe, or Val. Specifically, the B3 residue can be bulky, hydrophobic, neutral, and / or aromatic, such as Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle, or Ile.

[0086] The N-terminal protecting group Z (if present) may be selected from formyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxycarbonyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl, adamantyloxycarbonyl, p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or methylaminocarbonyl / methylurea group. In the case of a tripeptide aldehyde having one protecting group (i.e., X = Z-B2), Z is preferably a small aliphatic group, such as formyl, acetyl, fluoromethoxycarbonyl, tert-butoxycarbonyl, methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or methylaminocarbonyl / methylurea group. In the case of tripeptide aldehydes with a protecting group (i.e., X = Z-B3-B2), Z is preferably a large aromatic group, such as benzoyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP).

[0087] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO98 / 13460, WO 98 / 13461, WO 98 / 13462, WO 2007 / 141736, 2007 / 145963, WO2009 / 118375, WO 2010 / 055052 and WO 2011 / 036153. More specifically, the peptide aldehyde may be Cbz-RAY-H, Ac-GAY-H, Cbz-GAY-H, Cbz-GAL-H, Cbz-VAL-H, Cbz-GAF-H, Cbz-GAV-H, Cbz-GGY-H, Cbz-GGF -H, Cbz-RVY-H, Cbz-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGAL-H, Ac-FGAF-H, Ac-FGVY-H, Ac-FGAM-H, Ac-W LVY-H, MeO-CO-VAL-H, MeNCO-VAL-H, MeO-CO-FGAL-H, MeO-CO-FGAF-H, MeSO2-FGAL-H, MeSO2-VAL-H, PhCH2O(OH)(O)P-VAL-H, EtSO2-FGAL-H, PhCH2SO2-VAL-H, PhCH2O(OH)(O)P-LAL-H, PhCH2O(OH)(O)P-FAL-H, or MeO(OH)(O)P-LGAL-H. Here, Cbz is benzyloxycarbonyl, Me is methyl, Et is ethyl, Ac is acetyl, H is hydrogen, and the other letters indicate amino acid residues referred to by standard single-letter designations (e.g., F=Phe, Y=Tyr, L=Leu).

[0088] Alternatively, the peptide aldehyde can have the formula described in WO 2011 / 036153:

[0089] PO-(Ai-X')n-An+1-Q

[0090] Where Q is hydrogen, CH3, CX″3, CHX″2, or CH2X″, and X″ is a halogen atom; one of X' is a "double N-terminated group" CO, CO-CO, CS, CS-CS, or CS-CO, most preferably urido(CO), and the other X's are empty, where n = 1-10, preferably 2-5, and most preferably 2, and each Ai and An+1 is an amino acid residue having the following structure:

[0091] For the residues to the right of X'=-CO-, it is -NH-R″-CO-, or

[0092] For the residues to the left of X'=-CO-, it is -CO-CR″-NH-

[0093] Wherein R″ is H- or optionally substituted alkyl or alkylaryl group, which may optionally include heteroatoms and may optionally be attached to an N atom, and wherein P is hydrogen or any C-terminal protecting group. Examples of such peptide aldehydes include α-MAPI, β-MAPI, F-urea-RVY-H, F-urea-GGY-H, F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H, F-urea-GA-Nle-H, Y-urea-RVY-H, Y-urea-GAY-H, F-CS-RVF-H, F-CS-RVY-H, F-CS-GAY-H, anti-pain factor, GE20372A, GE20372B, chymotrypsin inhibitor A, chymotrypsin inhibitor B, and chymotrypsin inhibitor C. Further examples of peptide aldehydes are disclosed in the WO which are hereby incorporated by reference. 2010 / 055052 and WO 2009 / 118375, WO 94 / 04651, WO 98 / 13459, WO 98 / 13461, WO 98 / 13462, WO 2007 / 145963.

[0094] Alternatively, for peptide aldehydes, the protease inhibitor can be a hyposulfate adduct having the formula X-B1-NH-CHR-CHOH-SO3M, wherein X, B1, and R are as defined above, and M is H or an alkali metal, preferably Na or K.

[0095] This peptide aldehyde can be converted into a water-soluble hyposulfite adduct by reacting with sodium bisulfite, as described in textbooks such as March, J. Advanced Organic Chemistry, 4th Edition, Wiley-Interscience, USA, 1992, p. 895.

[0096] The aqueous solution of this bisulfite adduct can be prepared by reacting the corresponding peptide aldehyde with aqueous solutions of sodium bisulfite (NaHSO3) and potassium bisulfite (KHSO3) using known methods, for example as described in WO 98 / 47523; US 6,500,802; US 5,436,229; Journal of the American Chemical Society (J. Am. Chem. Soc.) (1978) 100, 1228; and Proceedings of the Organic Synthesis Journal (Org. Synth., Coll.) Vol. 7: 361.

[0097] The molar ratio of the aforementioned peptide aldehyde (or hyposulfate adduct) to the protease can be at least 1:1 or 1.5:1, and it can be less than 1000:1, more preferably less than 500:1, even more preferably from 100:1 to 2:1 or from 20:1 to 2:1, or most preferably, the molar ratio is from 10:1 to 2:1.

[0098] Formates (e.g., sodium formate) and formic acid have also shown good performance as inhibitors of protease activity. Formates can be used synergistically with the aforementioned protease inhibitors, as shown in WO 2013 / 004635. Formates are present in the detergent composition in an amount of at least 0.1% w / w or 0.5% w / w, for example at least 1.0%, at least 1.2%, or at least 1.5%. The amount of this salt is typically less than 5% w / w, less than 4%, or less than 3%.

[0099] WO 2007 / 141736, WO 2007 / 145963, and WO 2007 / 145964 disclose the use of reversible peptidase inhibitor-stabilized liquid detergent compositions. US 2003 / 157088 describes compositions comprising an enzyme stabilized with an inhibitor.

[0100] This invention provides protease variants stabilized by protease inhibitors (such as those mentioned above).

[0101] Therefore, specific embodiments of the present invention relate to variants of the parental protease, wherein the protease variant is consistent with SEQ ID NO. NO3 exhibits at least 60% identity, this variant possesses proteolytic activity, and comprises one or more substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein each position corresponds to SEQ ID. The position of the polypeptide at NO:3, and wherein these protease variants have enhanced binding to the inhibitor compared to the parent or compared to SEQ ID NO 3. Inhibitor binding is measured as described in Example 2. Another specific embodiment of the invention relates to variants of the parental protease, wherein the protease variant is associated with SEQ ID NO 3. NO3 exhibits at least 60% identity, this variant possesses proteolytic activity, and comprises one or more substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein each position corresponds to SEQ ID. The position of NO:3, and wherein, compared to the parent or compared to SEQ ID NO3, these variants have enhanced binding to the inhibitor, when measured as described in Example 2.

[0102] In some other embodiments, the present invention relates to protease variants having at least 60% sequence identity with SEQ ID NO 3, and when these protease variants are tested as in Example 2, these variants have Ki less than 1, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9; such as between 0.1 and 0.5, or between 0.2 and 0.7, or between 0.3 and 0.9, or between 0.4 and 0.9.

[0103] Another specific embodiment of the invention relates to a variant of the parental protease, wherein the protease variant is consistent with SEQ ID NO. 3 has at least 60% identity, this variant has proteolytic activity, and includes one or more substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein each position corresponds to SEQ ID. The position of the polypeptide at NO:3, and wherein, when measured as described in Example 2, these protease variants have enhanced binding to the inhibitor compared to proteases having SEQ ID NO 4 or SEQ ID NO 5.

[0104] In another aspect, according to the invention, the protease variant includes substitutions at one or more (e.g., several) positions corresponding to positions 70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240. In another aspect, the variant includes changes at two positions corresponding to any of positions 70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240. In yet another aspect, the variant includes changes at three positions corresponding to any of positions 70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240. On the other hand, the variants include changes at each of the positions corresponding to positions 70, 111, 114, 144, 145, 146, 146, 150, 151, 176, 178, 182, 184, 224, and 240.

[0105] In another aspect, the protease variant includes, or is composed of, a substitution at position 114 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 114 is substituted with Gln. In another aspect, the variant includes, or is composed of, the substitution S114Q of the polypeptide having SEQ ID NO:3.

[0106] In another aspect, the protease variant includes, or is composed of, a substitution at position 146 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 146 is substituted with Asn, Trp, Phe, or Ala, such as by Asn, Trp, Phe, or Ala. In another aspect, the variant includes, or is composed of, a substituted K146N polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, a substituted K146W polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, a substituted K146F polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, a substituted K146A polypeptide having SEQ ID NO:3.

[0107] In another aspect, the protease variant includes, or is composed of, a substitution at position 150 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 150 is substituted with Ala, Asn, or Arg, such as Ala, Asn, or Arg. In another aspect, the variant includes, or is composed of, the substitution I150A of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution I150N of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution I150R of the polypeptide having SEQ ID NO:3.

[0108] In another aspect, the protease variant includes, or is composed of, a substitution at position 151 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 151 is substituted with Arg. In another aspect, the variant includes, or is composed of, a substitution S151R of the polypeptide having SEQ ID NO:3.

[0109] In another aspect, the protease variant includes, or is composed of, a substitution at position 176 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 176 is substituted with Tyr. In another aspect, the variant includes, or is composed of, a polypeptide having the substitution N176Y of SEQ ID NO:3.

[0110] In another aspect, the protease variant includes, or is composed of, a substitution at position 178 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 178 is substituted with Tyr, Phe, or Pro, such as Tyr, Phe, or Pro. In another aspect, the variant includes, or is composed of, the substitution I178Y of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution I178F of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution I178P of the polypeptide having SEQ ID NO:3.

[0111] In another aspect, the protease variant includes, or is composed of, a substitution at position 184 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 184 is substituted with Phe, Tyr, Trp, or Asp, such as by Phe, Tyr, Trp, or Asp. In another aspect, the variant includes, or is composed of, a substituted peptide having SEQ ID NO:3, L184F. In another aspect, the variant includes, or is composed of, a substituted peptide having SEQ ID NO:3, L184Y. In another aspect, the variant includes, or is composed of, a substituted peptide having SEQ ID NO:3, L184W. In another aspect, the variant includes, or is composed of, a substituted peptide having SEQ ID NO:3, L184D.

[0112] In another aspect, the protease variant includes, or is composed of, a substitution at position 224 corresponding to SEQ ID NO:3. In another aspect, the amino acid at position 224 is substituted with Asp, Gly, or Ser, such as by Asp, by Gly, or by Ser. In another aspect, the variant includes, or is composed of, the substitution R224D of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution R224G of the polypeptide having SEQ ID NO:3. In another aspect, the variant includes, or is composed of, the substitution R224S of the polypeptide having SEQ ID NO:3.

[0113] On the other hand, these protease variants according to the invention comprise one or more (e.g., several) substitutions selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, or combinations thereof.

[0114] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S114Q, compared to the parent protease.

[0115] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted Q70F, compared to the parent protease.

[0116] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted Q70A, compared to the parent protease.

[0117] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted Q70N compared to the parent protease.

[0118] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S111R, compared to the parent protease.

[0119] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S111E, compared to the parent protease.

[0120] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S111D, compared to the parent protease.

[0121] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S114A compared to the parent protease.

[0122] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146T, compared to the parent protease.

[0123] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted S144R compared to the parent protease.

[0124] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted A145E, compared to the parent protease.

[0125] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I150N compared to the parent protease.

[0126] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I150S, compared to the parent protease.

[0127] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted G182A, compared to the parent protease.

[0128] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted L184F, compared to the parent protease.

[0129] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted L184Y, compared to the parent protease.

[0130] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted Y240R, compared to the parent protease.

[0131] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146T, compared to the parent protease.

[0132] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146N, compared to the parent protease.

[0133] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146W, compared to the parent protease.

[0134] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146F, compared to the parent protease.

[0135] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted K146A, compared to the parent protease.

[0136] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I150A, compared to the parent protease.

[0137] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I150N compared to the parent protease.

[0138] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted A151R, compared to the parent protease.

[0139] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted N176Y, compared to the parent protease.

[0140] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I178Y, compared to the parent protease.

[0141] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I178F, compared to the parent protease.

[0142] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted I178P, compared to the parent protease.

[0143] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted L184F, compared to the parent protease.

[0144] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted F184Y, compared to the parent protease.

[0145] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:4 or SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted L184W, compared to the parent protease.

[0146] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted L184D, compared to the parent protease.

[0147] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted R224D, compared to the parent protease.

[0148] One aspect of the invention relates to a protease variant of a protease parent, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted R224G, compared to the parent protease.

[0149] One aspect of the invention relates to a protease variant of a parent protease, wherein the parent protease is a polypeptide having SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a polypeptide having at least 60%, 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%, at least 96%, at least 97%, at least 98%, or at least 99% similarity to the polypeptide having SEQ ID NO:3, and wherein the protease variant comprises, or is composed of, a substituted R224S, compared to the parent protease.

[0150] The present invention also relates to cleaning compositions, such as detergent compositions comprising protease variants of the present invention. In one embodiment, the cleaning composition is a liquid or powder laundry detergent suitable for cleaning, for example, at high temperatures and / or high pH, ​​such as at or above 40°C and / or at or above pH 8. In another embodiment, the cleaning composition is a liquid or powder laundry detergent suitable for cleaning, for example, at low temperatures and / or low pH, such as at or below 20°C and / or pH 6. The detergent may also be formulated as a unit-dose detergent and / or optionally as a dense detergent with minimal or no water. The detergent may also be a dishwashing detergent, preferably phosphate-free. The cleaning composition may further comprise at least one additional enzyme, such as a carbohydrate-active enzyme, like carbohydratease, pectinase, mannanase, amylase, cellulase, arabinase, galactanase, xylanase, or a protease such as a metalloproteinase, lipase, keratinase, oxidase, such as laccase, and / or peroxidase.

[0151] Typically, the positions within the protease used to create variants are those where, compared to the unchanged protease, at least one substitution results in a variant exhibiting improved characteristics. These variants may further include one or more additional modifications at one or more (e.g., several) other positions. In a particularly preferred embodiment, the protease variants of the invention further include substitutions at one or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO:3, wherein the variant has at least 60% sequence identity with SEQ ID NO:3 and the variant possesses protease activity. In even more preferred embodiments, the amino acid at position 171 corresponding to SEQ ID NO:3 is selected from the group consisting of: Trp, Lys, Glu, Asn and / or the amino acid at position 173 corresponding to SEQ ID NO:3 is Pro, and / or the amino acid at position 175 corresponding to SEQ ID NO:3 is Ala, Val, Pro, and / or the amino acid at position 179 corresponding to SEQ ID NO:3 is selected from the group consisting of: Cys, Val, Gln, Ser, Thr, Glu, His, Lys, Met, Asn, Tyr and Ala and / or the amino acid at position 180 corresponding to SEQ ID NO:3 is Tyr. In another preferred embodiment, the protease variant of the present invention further comprises substitutions at two or more positions corresponding to positions 171, 173, 175, 179, or 180 of SEQ ID NO:3, wherein the variant has at least 60% and less than 100% sequence identity with SEQ ID NO:3, and the variant has protease activity at two positions corresponding to any one of positions 171, 173, 175, 179, and 180. In a preferred embodiment of the present invention, these variants comprise the mutant S173P+S175P as shown in SEQ ID NO:4. In another preferred embodiment, the protease variant of the present invention comprises the mutant S173P+S175P+F180Y as shown in SEQ ID NO:5.

[0152] In specific embodiments of the present invention, compared to SEQ ID NO 3, the protease variant of the present invention includes the following substitutions:

[0153] S114Q, S173P, S175P, F180Y

[0154] K146T, S173P, S175P, F180Y

[0155] K146N, S173P, S175P, F180Y

[0156] K146W、S173P、S175P、F180Y

[0157] K146F、S173P、S175P、F180Y

[0158] K146A、S173P、S175P、F180Y

[0159] I150A、S173P、S175P、F180Y

[0160] I150N、S173P、S175P、F180Y

[0161] S27K、I150N、S171N、S173P、G174R、S175P、F180Y、Q198E、T297P

[0162] S27K、K146P、S148R、A151R、S171N、S173P、G174R、S175P、F180Y、Q198E、N199K、T297P

[0163] S173P、S175P、N176Y、F180Y

[0164] S173P、S175P、I178Y、F180Y

[0165] S173P、S175P、I178F、F180Y

[0166] S173P、S175P、I178F、F180Y

[0167] S173P、S175P、I178P、F180Y

[0168] S173P、S175P、F180Y、L184F

[0169] S173P、S175P、F180Y、L184F

[0170] S27K、S171N、S173P、G174R、S175P、F180Y、L184F、Q198E、T297P

[0171] S27K、I121V、S171N、S173P、G174R、S175P、F180Y、L184F、Q198E、T297P

[0172] S27K、Q70N、G107N、I121V、E127Q、S173P、S175P、F180Y、L184F、Q198E、T297P

[0173] S27K、S173P、G174K、S175P、F180Y、L184F、Q198E、N199K、T297P

[0174] S27K、S173P、G174K、S175P、F180Y、L184F、Q198E、N199R、T297P

[0175] S173P、S175P、F180Y、L184Y

[0176] S27K、S171N、S173P、G174R、S175P、F180Y、L184Y、Q198E、T297P

[0177] S27K、S173P、G174K、S175P、F180Y、L184Y、Q198E、N199K、T297P

[0178] S27K、S173P、G174K、S175P、F180Y、L184Y、Q197K、Q198E、T297P

[0179] S173P、S175P、F180Y、L184W

[0180] S27K、S171N、S173P、G174R、S175P、F180Y、L184W、Q198E、T297P

[0181] S173P、S175P、F180Y、L184D

[0182] S173P、S175P、F180Y、R224D

[0183] S173P、S175P、F180Y、R224G

[0184] S27K、S171N、S173P、G174R、S175P、F180Y、Q198E、N199K、R224G、T297P

[0185] S173P、S175P、F180Y、R224S

[0186] S27K、I121V、S171N、S173P、G174R、S175P、F180Y、Q198E、R224S、T297P

[0187] S27K, S171D, S173P, G174R, S175P, G182A, L184F, Q198E, N199K, R224G, T297P

[0188] S27K, S171D, S173P, G174R, S175P, G182A, L184F, Q198E, N199K, T297P

[0189] S27K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, R224G, T297P

[0190] S27K, S171N, S173P, G174R, S175P, F180Y, G182A, L184F, Q198E, N199K, T297P

[0191] S27K, V162T, S173P, G174K, S175P, F180Y, L184F, Q197K, Q198E, T297P

[0192] S27K, V162T, S173P, G174K, S175P, F180Y, Q197K, Q198E, T297P

[0193] Q70F

[0194] Q70A

[0195] Q70N

[0196] S111R

[0197] S111E

[0198] S111D

[0199] S114A

[0200] S144R

[0201] A145E

[0202] I150N

[0203] I150S

[0204] G182A

[0205] L184F

[0206] L184Y

[0207] R224G or

[0208] Y240R

[0209] These variants may further include one or more additional alterations at one or more (e.g., several) other positions. Amino acid alterations can be minor in nature, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions of 1-5 amino acids, typically; small amino or carboxyl terminal extensions, such as N-terminal methionine residues; small linker peptides of up to 20-25 residues at the amino or carboxyl terminus; or small extensions that facilitate purification by altering net charge or another function, such as multihistidine sequences, antigenic epitopes, or binding domains.

[0210] Examples of conserved substitutions are found in the following group: 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 generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and R.R. Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes include: Ala / Ser, Val / Ile, Asp / Glu, Asn / Gln, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Glu / Gln, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0211] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the thermal stability of the peptide, change its substrate specificity, and alter its optimal pH.

[0212] Essential amino acids in peptides can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the protease activity of the resulting variant molecule is tested to identify amino acid residues essential to the activity of the molecule. See also Hilton et al., 1996, Journal of Biochemistry 271:4699-4708. The active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contact site. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from comparisons with related polypeptides. For the TY-145 protease (SEQ ID NO:3), the catalytic triad including amino acids D35, H72, and S251 is essential for the enzyme's protease activity.

[0213] In the examples, this variant exhibits improved catalytic activity compared to the parent enzyme.

[0214] The homology between two amino acid sequences, described by the parameter "identity" for the purposes of this invention, is determined using the Niedermann-Onsch algorithm as described above. The results from the program calculate the "percentage identity" between the two sequences in addition to amino acid alignment.

[0215] Based on this description, it will be fairly straightforward for those skilled in the art to identify suitable homologous proteases that can be modified according to the present invention.

[0216] Essentially homologous parental protease variants may have one or more amino acid substitutions, deletions, and / or insertions. In this context, the terms "one or more" and "several" are used interchangeably. These changes preferably have a minor nature, i.e., conserved amino acid substitutions and other substitutions as described above that do not significantly affect the three-dimensional folding or activity of the protein or polypeptide; small deletions, typically from 1 to about 30 amino acids; and small N-terminal or C-terminal extensions, such as N-terminal methionine residues, small linker peptides comprising up to about 20-25 residues, or small extensions (affinity markers) that facilitate purification, such as polyhistidine sequences (tracts) or protein A (Nilsson et al., 1985, EMBO J. 4:1075; Nilsson et al., 1991, Methods Enzymol. 198:3. See also Ford et al., 1991, Protein Expression and Purification 2:95-107.)

[0217] Although these modifications described above are preferably minor, they can also be substantial, such as the fusion of a large polypeptide consisting of up to 300 or more amino acids, each extended from an amino terminus or carboxyl terminus.

[0218] The parental protease may include, or be composed of, the amino acid sequence of SEQ ID NO:3 or its allele variants, or fragments thereof with protease activity. In one aspect, the parental protease includes, or is composed of, the amino acid sequence of SEQ ID NO:3.

[0219] The parent protease can be (a) a polypeptide having at least 60% sequence identity with the polypeptide of SEQ ID NO:3; (b) a polypeptide encoded by a polynucleotide that hybridizes under medium or high stringency conditions with (i) the sequence encoding the mature polypeptide of SEQ ID NO:1, (ii) the sequence encoding the mature polypeptide of SEQ ID NO:2, or (iii) the full-length complement of (i) or (ii); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO:1.

[0220] In one respect, the parent protease has at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity with the polypeptide having SEQ ID NO:3.

[0221] On one hand, the amino acid sequence of the parent protease is approximately 15 amino acids longer than that of the polypeptide having SEQ ID NO:3, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0222] In another aspect, the parent contains, or is composed of, the amino acid sequence of SEQ ID NO:3. In yet another aspect, the parent contains, or is composed of, amino acids 1 to 311 of SEQ ID NO:2.

[0223] On one hand, the amino acid sequence of the parent protease is approximately 15 amino acids longer than that of the polypeptide having SEQ ID NO:4, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0224] On the other hand, the parent includes, or is composed of, the amino acid sequence of SEQ ID NO:4.

[0225] On one hand, the amino acid sequence of the parent protease is approximately 15 amino acids longer than that of the polypeptide having SEQ ID NO:5, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0226] On the other hand, the parent includes, or is composed of, the amino acid sequence of SEQ ID NO:5.

[0227] On the other hand, the parental protease is encoded by a polynucleotide that hybridizes with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the sequence coding the mature polypeptide of SEQ ID NO:2, or (iii) the full-length complement of (i) or (ii) under very low stringency, low stringency, medium stringency, or high stringency, or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York).

[0228] Nucleic acid probes can be designed using the polynucleotide or a subsequence of SEQ ID NO:1, together with the polypeptide or fragment of SEQ ID NO:3, to identify and clone parental DNA encoding strains from different genera or species, according to methods well known in the art. Specifically, such probes can be hybridized with the genomic DNA or cDNA of the cells of interest, following standard DNA blotting procedures, to identify and isolate the corresponding genes therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probes are labeled (e.g., with...). 32 P, 3 H, 35 This invention covers probes that detect corresponding genes (such as S, biotin, or avidin).

[0229] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened for DNA that hybridizes to and encodes the parental DNA as described above. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred to and immobilized on nitrocellulose or other suitable carrier material. The carrier material is used in DNA blotting to identify clones or DNA or their subsequences that hybridize to SEQ ID NO:1.

[0230] For the purposes of this invention, the hybridization indicator hybridizes the polynucleotide with (i) SEQ ID NO:1; (ii) the coding sequence of the mature polypeptide of SEQ ID NO:1; (iii) the sequence encoding the mature polypeptide of SEQ ID NO:2; (iv) its full-length complement; or (v) its subsequence, corresponding to a labeled nucleic acid probe, under very low to very high stringency conditions. Molecules hybridized under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0231] In one respect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO:1. In another respect, the nucleotide probe is a long fragment of SEQ ID NO:1, ranging from 80 to 1140 nucleotides, for example, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or 1100 nucleotides long. In yet another respect, the nucleic acid probe is a polypeptide encoding SEQ ID NO:2, its mature polypeptide, or a fragment thereof, of a polynucleotide. In yet another respect, the nucleic acid probe is the sequence of SEQ ID NO:1 or the mature polypeptide encoding SEQ ID NO:2.

[0232] In another embodiment, the parent is encoded by a polynucleotide that has at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity with the sequence encoding the mature polypeptide of SEQ ID NO:1 or the sequence encoding the mature polypeptide of SEQ ID NO:2.

[0233] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0234] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused at the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide with the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are within a frame and the expression of the fusion peptide is under the control of the same promoter and terminator. Fusion peptides can also be constructed using integrin techniques, wherein the fusion peptide is generated post-translationally (Cooper et al., 1993, Journal of the European Society for Molecular Biology 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0235] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, *Journal of Industrial Microbiology and Biotechnology* (J. Ind. Microbiol. Biotechnol.) 3:568-576; Svetina et al., 2000, *Journal of Biotechnology* (J. Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, *Applied and Environmental Microbiology* (Appl. Environ. Microbiol.) 63:3488-3493; [The text abruptly ends here, likely due to an incomplete translation or missing information.] Ward et al., 1995, Biotechnology 13:498-503; Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0236] The parent can be obtained from organisms of any genus. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source should mean that the parent encoded by the polynucleotide is produced by that source or by a strain that has inserted a polynucleotide from that source. In one aspect, the parent is extracellularly secreted.

[0237] The parent can be a bacterial protease. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, or Streptomyces protease; or a Gram-negative bacterial polypeptide, such as Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Staphylococcus, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease.

[0238] On one hand, the parent is alkalophilic Bacillus, amyloliquefaciens, brevis bacillus, circular Bacillus, Clausii Bacillus, coagulant Bacillus, strong Bacillus, brilliant Bacillus, slow-release Bacillus, licheniformis, megaterium Bacillus, brevis Bacillus, thermophilic steatobacterium, Bacillus subtilis, or Bacillus thuringiensis protease.

[0239] On one hand, the parent is a Bacillus species protease, such as the protease with SEQ ID NO:3 or the mature polypeptide with SEQ ID NO:2.

[0240] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Dutch Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Agricultural Research Culture Collection (ARC).

[0241] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural environment are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening another microorganism or a library of genomic DNA or cDNA from a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with the probe, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0242] Preparation of variants

[0243] The present invention also relates to a method for obtaining a protease variant having at least one improved property compared to SEQ ID NO 3, the method comprising:

[0244] a) Introducing one or more of the following substitutions into a parental protease having at least 60% identity with SEQ ID NO:3: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein the variant has identity with SEQ ID NO:3. NO:3 has an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95% identical; and

[0245] b) Recycle the variant.

[0246] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0247] These variations of the invention can also be prepared by procedures such as those mentioned below.

[0248] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0249] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acid Research 18:7349-4966.

[0250] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0251] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques for synthesizing and assembling oligonucleotides on optically programmable microfluidic chips.

[0252] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or truncation methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0253] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0254] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments in conjunction with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenic primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0255] Polynucleotides

[0256] The present invention also relates to isolated polynucleotides encoding variants of the invention.

[0257] Nucleic acid constructs

[0258] The present invention also relates to nucleic acid constructs comprising polynucleotides operably linked to one or more control sequences encoding variants of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0259] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0260] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including variants, truncated and heterozygous promoters, and can be acquired by a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0261] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). (Microbiology) 13:97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO99 / 43835.

[0262] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator sequence is operatively linked to the 3' end of the polynucleotide encoding that variant. Any terminator that is functional in the host cell can be used.

[0263] The preferred terminator for bacterial host cells is derived from the genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0264] Control sequences can also be mRNA stabilizer regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[0265] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0266] The control sequence can also be a signal peptide coding region, encoding a signal peptide linked to the N-terminus of the variant and guiding the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to enhance the variant's secretion. However, any signal peptide coding sequence that guides the expressed variant into the host cell's secretory pathway can be used.

[0267] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of *Bacillus NCIB 11837* for maltose amylase, *Bacillus licheniformis* for subtilis protease, *Bacillus licheniformis* for calcium-lactamase, *Bacillus thermophilus* for α-amylase, *Bacillus thermophilus* for neutral proteases (nprT, nprS, nprM), and *Bacillus subtilis* for prsA. Other signal sequences are described by Simonen and Palva, 1993, *Microbiological Reviews* 57:109-137.

[0268] The control sequence can also be a propeptide-coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. Propeptide-coding sequences can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0269] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0270] It would also be desirable to add regulatory sequences that modulate the expression of the variant in relation to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include lac, tac, and the trp operon system.

[0271] expression carrier

[0272] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0273] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0274] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any elements necessary to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with the chromosome in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0275] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, heavy metal resistance, or auxotrophic prototrophs, etc.

[0276] Examples of bacterial selective markers include the dal gene of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline).

[0277] The vector preferably contains elements that allow the vector to integrate into the host cell's genome or to replicate autonomously in the cell independently of the genome.

[0278] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide the precise location within the chromosome for integration into the host cell genome via homologous recombination. To increase the likelihood of integration at a precise location, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence in the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0279] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0280] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0281] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the generation of variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectivity marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectivity marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0282] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0283] host cells

[0284] This invention also relates to recombinant host cells comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences directing the generation of the variant. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0285] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0286] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Clostridium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0287] The bacterial host cell can be any Bacillus genus cell, including but not limited to: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sturdier, Bacillus splendidus, Bacillus stagnation, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0288] The bacterial host cell can also be any streptococcal cell, including but not limited to: Streptococcus equina, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equine subsp. veterinaryis.

[0289] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces, and light blue Streptomyces cells.

[0290] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168:111-115); competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubna...). (u) and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into E. coli cells via protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acid Research 16:6127-6145). DNA can be introduced into *Streptomyces* cells via protoplast transformation, electroporation (see, for example, Gong et al., 2004, *Folia Microbiol.* (Prague) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, *Journal of Bacteriology* 171:3583-3585), or transduction (see, for example, Burke et al., 2001, *Proceedings of the National Academy of Sciences* 98:6289-6294). DNA can be introduced into *Pseudomonas* cells via electroporation (see, for example, Choi et al., 2006, *Journal of Microbiological Methods* 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, *Applied and Environmental Microbiology* 71:51-57).Introducing DNA into Streptococcus cells can be achieved through: native competent cells (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Applied and Environmental Microbiology 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0291] Production methods

[0292] The present invention also relates to a method for generating a variant, the method comprising: (a) culturing a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.

[0293] These host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. The culture occurs using procedures known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0294] The variant can be detected using methods known in the art that are specific to variants with protease activity. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, enzyme assays can be used to determine the activity of the variant.

[0295] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium through a variety of conventional procedures, including but not limited to: collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0296] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to: chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0297] Alternatively, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0298] Composition

[0299] In addition to enzymes, these detergent compositions may include other components. The selection of additional components is within the skill of a person skilled in the art and includes conventional ingredients, including the exemplary, non-limiting components listed below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as a component may include additional functionality as would be understood by a person skilled in the art.

[0300] The detergent composition of the present invention

[0301] The protease variant of the present invention can be added to a detergent composition in amounts corresponding to the following: 0.001-100 mg of protein per liter of cleaning solution, such as 0.01-100 mg of protein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and even most preferably 0.01-1 mg of protein.

[0302] Detergent compositions may include one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In specific embodiments, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. Such surfactants or these surfactants are typically present at levels 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%. Such surfactants or these surfactants are selected based on the desired cleaning application and include any conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be used.

[0303] When included therein, the detergent will typically 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% anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0304] When included therein, the detergent will typically contain from about 1% to about 40% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyltrimethylammonium bromide (CTAB), dimethyl dioctadecyl ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, and combinations thereof, alkyl quaternary ammonium compounds, and alkoxylated quaternary ammonium (AQA).

[0305] When included therein, the detergent will typically contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, particularly 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 nonionic 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), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA), or fatty acid glucosamide (FAGA)), together with products and combinations thereof available under the trade names SPAN and TWEEN.

[0306] When included therein, detergents will typically contain from about 1% to about 40% by weight of a semi-polar surfactant. Non-limiting examples of semi-polar surfactants include amine oxides (AOs) (such as alkyl dimethyl amine oxides), N-(cocoylalkyl)-N,N-dimethyl amine oxides and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl) amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0307] When included therein, the detergent will typically contain from about 1% to about 40% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethyl betaine, and sulfobetaine, and combinations thereof.

[0308] These compositions of the present invention may also include a co-hydrazone, which is a compound that dissolves a hydrophobic compound in an aqueous solution (or conversely, a polar substance in a nonpolar environment). Typically, co-hydrazones possess both hydrophilic and hydrophobic characteristics (such as the so-called amphiphilic properties known from surfactants); however, the molecular structure of co-hydrazones generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12:121-128. Co-hydrazones do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and the formation of lipid micelles, thin layers, or other well-defined intermediate phases occur. Instead, many co-hydrazones exhibit a continuous type of aggregation process, where the size of the aggregates increases with increasing concentration. However, many co-hydrazones alter the phase behavior, stability, and colloidal properties of systems containing substances with both polar and nonpolar characteristics, including mixtures of water, oils, surfactants, and polymers. Water-soluble solvents are routinely used in a variety of industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble solvents in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0309] The detergent may contain 0% to 5% by weight, such as about 0.5% to about 5%, or about 3% to about 5%, of a water-soluble solvent. Any water-soluble solvent known in the art for use in detergents may be used. Non-limiting examples of water-soluble solvents include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthoate, sodium ethylhexyl sulfonate, and combinations thereof.

[0310] Detergent compositions may contain, by weight, about 0% to 65%, such as about 5% to about 50%, of detergent builders or co-builders, or mixtures thereof. In dishwashing detergents, the level of builders is typically 40% to 65%, particularly 50% to 65%. Builders and / or co-builders may specifically be chelating agents that form water-soluble complexes having Ca and Mg. Any builders and / or co-builders known in the art for use in laundry, ADW, and hard surface cleaning detergents may be utilized. Non-limiting examples of detergent 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-aminoethanol (MEA), iminodiethanol (DEA) and 2,2',2'-triazine (TEA), and carboxymethyl inulin (CMI), and combinations thereof.

[0311] The detergent composition may also contain 0%-65% by weight, such as about 5% to about 40%, of a detergent co-builder or a mixture thereof. The detergent composition may include only a co-builder, or in combination with a builder, such as a zeolite builder. Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-dimethylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), and N-(2-sulfoethyl)aspartic acid (SEA). S), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-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), p-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), aminoethanesulfonic acid-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations thereof and salts thereof. Other exemplary builders and / or co-builders are described in, for example, WO 09 / 102854, US 5977053.

[0312] The detergent may contain 0%-10% by weight, such as about 1% to about 5%, of a bleaching system. Any bleaching system known in the art for use in laundry, ADW, and hard surface cleaning detergents can be utilized. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, pre-formed peracids, and mixtures thereof. Suitable pre-formed peracids include, but are not limited to: peroxycarboxylic acids and their salts, percarbonates and their salts, and perimidic acids. Acids and their salts, peroxymonosulfate and its salts (e.g., potassium persulfate (Oxone(R))), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may include, for example, inorganic salts that form bleaching activator combinations with peracids, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, and sodium salts of persilicates. Bleaching activator herein refers to a compound that reacts with a peroxide bleaching agent (like hydrogen peroxide) to form a peracid. The peracid formed in this way constitutes the activated bleaching agent. To be continued... Suitable bleaching activators used herein include those belonging to the ester amide, imide, or acid anhydride classes. Suitable examples are tetraacetylethylenediamine (TAED), sodium 3,5,5-trimethylhexanoyloxybenzenesulfonate, bisperoxydodecanedioic acid, 4-(dodecyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS), tetraacetylethylenediamine (TAED), and 4-(nonanoyloxy)benzenesulfonate (NOBS), and / or WO4. Those disclosed in 98 / 17767. Specific families of bleaching activators of interest are disclosed in EP 624154, and a particularly preferred family is triethyl acetylacetic acid (ATC). ATC, or short-chain triglycerides (like Triacin), has the advantage of being environmentally friendly because it ultimately degrades into citric acid and alcohol. Furthermore, triethyl acetylacetic acid and triacetyl glycerol exhibit good hydrolytic stability in the product during storage, and are effective bleaching activators. Finally, ATC provides good washing ability for laundry additives. Alternatively, the bleaching system may include, for example, amides, imides, or sulfone-type peroxy acids. The bleaching system may also include peracids such as 6-(phthaloylamino)percapanoic acid (PAP). The bleaching system may also include a bleaching catalyst. In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of: organic catalysts having the following formula:

[0313]

[0314] (iii) and its mixtures; wherein each R 1Independently, it is a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably, each R 1 Independently, it is a branched alkyl group containing 9 to 18 carbons or a straight-chain alkyl group containing 11 to 18 carbons; more preferably, each R 1 Independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isonyl, isodecyl, isotridecyl, and isopentadecanyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, and WO 2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc phthalocyanine.

[0315] The cleaning composition 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 used. The polymer may function as a co-cleaning agent as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or anti-foaming properties. Some polymers may have more than one of the properties mentioned above and / or more than one of the motifs mentioned below. 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 polyethylene glycol, copolymers of polyethylene terephthalate and polyethylene terephthalate (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the polymers mentioned above are also considered.

[0316] These cleaning compositions may also include fabric colorants, such as dyes or pigments that, when formulated in a detergent composition, can be deposited on the fabric upon contact with a cleaning liquid comprising the detergent composition, thereby altering the fabric's color through visible light absorption / reflection. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants alter the color of a surface because they absorb at least a portion of the visible light spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling under the Colour Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, as described, for example, in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276 and EP 1876226 (which are hereby incorporated by reference). The detergent composition preferably includes a fabric toner 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%. The composition may include from 0.0001 wt% to 0.2 wt% of fabric toner, which may be particularly preferred when the composition is in the form of a unit-dose packet. Suitable colorants are also disclosed, for example, in WO 2007 / 087257 and WO2007 / 087243.

[0317] In one embodiment, the variant according to the invention is combined with one or more enzymes, such as at least two enzymes, more preferably at least three, four or five enzymes. Preferably, these enzymes have different substrate specificities, such as proteolytic activity, starch-degrading activity, lipid-degrading activity, hemicellulose-dissolving activity or pectin-dissolving activity.

[0318] Detergent additives and detergent compositions may include one or more additional enzymes, such as carbohydrate-active enzymes like carbohydrateases, pectinases, mannanases, amylases, cellulases, arabinases, galactanases, xylanases, or proteases, lipases, keratinases, oxidases such as laccases, and / or peroxidases.

[0319] Generally speaking, the selected enzyme should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the enzyme should be present in an effective amount.

[0320] cellulases :

[0321] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered variants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Cladosporium*, such as the fungal cellulases produced by *Pyrophyllus*, *Thermophyllus*, and *Fusarium* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0322] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are those cellulase variants 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, and WO 99 / 001544.

[0323] Other cellulases are endoglucanases having the following sequence, which has at least 97% identity with the amino acid sequence at positions 1 to 773 of SEQ ID NO:2 of WO 2002 / 099091, or family 44 xyloglucanases having the following sequence, which has at least 60% identity with positions 40-559 of SEQ ID NO:2 of WO 2001 / 062903.

[0324] Commercially available cellulases include Celluzyme TM and Carezyme TM (Novozymes), Carezyme Premium TM (Novozymes), Celluclean TM (Novozymes), Celluclean Classic TM (Novozymes), Cellusoft TM (Novozymes), Whitezyme TM (Novozymes), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM(Kao Corporation).

[0325] mannanases:

[0326] Suitable mannanases include those of bacterial or fungal origin. This includes chemically or genetically modified variants. The mannanase can be a basic mannanase from family 5 or 26. It can be a wild-type from the genera *Bacillus* or *Pythium*, particularly *Bacillus mucosa*, *Bacillus licheniformis*, *Bacillus alkalophilus*, *Bacillus croceae*, or specific *Pythium*. Suitable mannanases are described in WO 1999 / 064619. One commercially available mannanase is Mannaway (Novozymes).

[0327] proteases:

[0328] Suitable alternative proteases include those of bacterial, fungal, plant, viral, or animal origin, such as plant or microbial sources. Microbial sources are preferred. This includes chemically modified or protein-engineered variants. It can be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases can be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from family M4 or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0329] The term "subtilisinase" refers to the serine protease subgroup according to Essen et al., Protein Engineering 4 (1991) 719-737 and Essen et al., Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by the presence of a serine residue at its active site that forms a covalent adduct with the substrate. Subtilisins can be classified into six subfamilies: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanathionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0330] Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus lentus*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and *Lentus*, *Novo*, *Carlsberg*, *Bacillus licheniformis*, *BPN'*, *309*, *147*, and *168* as described in WO 89 / 06279, and *PD138* as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., from pigs or cattle) and Fusarium proteases (described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372), as well as chymotrypsin derived from the genus Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0331] Further preferred proteases are alkaline proteases from Bacillus tarda DSM 5483 (as described in, for example, WO95 / 23221), and their variants (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0332] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0333] Examples of useful proteases are described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO Variants in 11 / 036264, especially those with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, use BPN' numbers. More preferred protease variants may include the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

[0334] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm Durazym Tm , Ultra Ultra Ultra Ultra and (Novozymes), those sold under the following product names: Purafect Purafect Purafect Purafect and (Danisco / DuPont), Axapem TM (Gist-Brocades NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0335] lipases and cutinases :

[0336] Suitable lipases and keratins include those of bacterial or fungal origin. This includes chemically modified or protein-engineered variants. Examples include lipases from the genus *Thermophilic*, such as those from *Thermophilic Hypotherium latifolium* (formerly named *Pythium latifolium*) as described in EP 258068 and EP305216; cutinases from the genus *Pythium*, such as *Pythium salivarium* (WO 96 / 13580); lipases from strains of the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *Alcaligenes* or *Alcaligenes-like* (EP 218272), *Pseudomonas cepacia* (EP331376), *Pseudomonas* strain SD705 (WO 95 / 06720 & WO 96 / 27002), *Pseudomonas wisconsinensis* (WO 96 / 12012); GDSL-type *Streptomyces* lipase (WO 10 / 065455); and cutinases from *Bacillus oryzae* (WO 10 / 065455). 10 / 107560); cutinase from Pseudomonas mendoza (US 5,389,536); lipase from Thermobifida fusca (WO 11 / 084412); lipase from Bacillus stearothermophilus (WO 11 / 084417); lipase from Bacillus subtilis (WO 11 / 084599); and lipase from Streptomyces griseus (WO11 / 150157) and Streptomyces pristinaespiralis (WO 12 / 137147).

[0337] 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 07 / 87508, and WO 09 / 109500.

[0338] Preferred commercially available lipase products include Lipolase TM Lipex TM Lipolex TM and Lipoclean TM (Novozymes), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).

[0339] Other examples are lipases sometimes called acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarctica lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and variants of Mycobacterium smegmatis perhydrolases (particularly the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 10 / 100028).

[0340] amylases:

[0341] Suitable amylases that can be used with the protease variants of the present invention can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified or protein-engineered variants. Amylases include, for example, α-amylases obtained from the genus *Bacillus*, such as α-amylases of specific strains of *Bacillus licheniformis* described in more detail in GB 1,296,839.

[0342] Suitable amylases include the amylase having SEQ ID NO:2 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO:3. Preferred variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at 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.

[0343] Suitable amylases include the amylase having SEQ ID NO:6 in WO 02 / 010355 or its variants having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193.

[0344] Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* as shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the α-amylase derived from *Bacillus licheniformis* as shown in SEQ ID NO:4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those having substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase, including residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4, is those having the following substitutions:

[0345] M197T;

[0346] H156Y+A181T+N190F+A209V+Q264S; or

[0347] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0348] Other suitable amylases are those having SEQ ID NO:6 in WO 99 / 019467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182 or positions H183 and G184.

[0349] Other amylases that can be used are those of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 under WO 96 / 023873, or variants thereof having 90% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476, numbered using SEQ ID 2 of WO 96 / 023873. More preferred variants are those with deletions at two positions selected from 181, 182, 183, and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 are those with deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0350] Other usable amylases are those having SEQ ID NO:2 of WO 08 / 153815, SEQ ID NO:10 of WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO:2 of WO 08 / 153815 or SEQ ID NO:10 of WO 01 / 66712. Preferred variants of SEQ ID NO:10 in WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0351] Other suitable amylases are the amylase of SEQ ID NO:2 having WO 09 / 061380 or its variants having 90% sequence identity with SEQ ID NO:2. Preferred variants of SEQ ID NO:2 are those having a C-terminal truncation and / or substitution, deletion or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO:2 are those having substitutions in one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or those having deletions in positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO:2 are those having the following substitutions:

[0352] N128C+K178L+T182G+Y305R+G475K;

[0353] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0354] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0355] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0356] Other suitable amylases are the amylase of SEQ ID NO:1 having WO 13184577 or its variants having 90% sequence identity with SEQ ID NO:1. Preferred variants of SEQ ID NO:1 are those having substitutions, deletions, or insertions in one or more of the following positions: K176, R178, G179, T180, G181, E187, N192, M199, I203, S241, R458, T459, D460, G476, and G477. A more preferred variant of SEQ ID NO:1 is one or more of the following positions: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K, and G477K, having a substitution and / or a deletion at position R178 and / or S179 or T180 and / or G181. The most preferred amylase variant of SEQ ID NO:1 is one having the following substitutions:

[0357] E187P+I203Y+G476K

[0358] E187P+I203Y+R458N+T459S+D460T+G476K、

[0359] These variants optionally include a substitution at position 241 and / or a deletion at positions 178 and / or 179.

[0360] Other suitable amylases are the amylase of SEQ ID NO:1 having WO 10104675 or its variants having 90% sequence identity with SEQ ID NO:1. Preferred variants of SEQ ID NO:1 are those having substitutions, deletions, or insertions at one or more of the following positions: N21, D97, V128, K177, R179, S180, I181, G182, M200, L204, E242, G477, and G478. More preferred variants of SEQ ID NO:1 are those having substitutions at one or more of the following positions: N21D, D97N, V128I, K177L, M200L, L204YF, E242QA, G477K, and G478K and / or deletions at positions R179 and / or S180 or I181 and / or G182. The most preferred amylase variant of SEQ ID NO:1 is those with the following substitutions:

[0361] N21D+D97N+V128I

[0362] These variants optionally include a substitution at position 200 and / or a deletion at positions 180 and / or 181.

[0363] Other suitable amylases are α-amylases having SEQ ID NO:12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO:12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO:12 in WO 01 / 66712: R28, R118, N174, R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314, R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, and additionally having substituted variants at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with most preferred variants having substituted variants at all of these positions.

[0364] Other examples are amylase variants, such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087.

[0365] Commercially available amylase is Duramyl TM Special amylase TM Fungaly TM Stainzyme TM StainzymePlus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc. / DuPont).

[0366] peroxidases / oxidases:

[0367] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered variants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as those from *Coprinus spp.*, and their variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0368] Commercially available peroxidases include Guardzyme. TM (Novozymes)

[0369] other enzymes:

[0370] The protease variants according to the invention can also be combined with other enzymes, such as pectin lyases (e.g., Pectawash). TM (e.g., chlorophyllase, etc.) The protease variants of the present invention can be mixed with any other enzyme.

[0371] The detergent enzyme can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combination of additives containing all of these enzymes. Detergent additives, i.e., individual or combined additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially dust-free granules; liquids, especially stabilized liquids; or slurries.

[0372] Non-dust particles can be generated, for example, as disclosed in US 4,106,991 and 4,661,452, and can optionally be coated by methods known in the art. Examples of wax-coated materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and has 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP238,216.

[0373] auxiliary materials

[0374] Any detergent component known in the art for use in laundry detergents may also be used. Other optional detergent components include corrosion inhibitors, shrinkage inhibitors, anti-fouling redeposition agents, anti-wrinkle 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, optical brighteners / brighteners, foaming agents, foam (foam) regulators, fragrances, soil suspending agents, softeners, defoaming agents, dulling inhibitors, and wicking agents, used alone or in combination. Any ingredient known in the art for use in laundry detergents may be used. The selection of such ingredients is entirely within the skill of a person of ordinary skill.

[0375] dispersants These detergent compositions may also contain dispersants. Specifically, powdered detergents may include dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc.

[0376] dye transfer inhibitors These detergent compositions may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole, or mixtures thereof. When present in the test composition, the dye transfer inhibitor 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.

[0377] optical brightenersThe detergent composition will also preferably contain additional components that can color the cleaned item, such as optical brighteners or fluorescent whitening agents. The brightening agent is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in this composition. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents include sodium salts of the following: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-diphenylamino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate; Preferred fluorescent whitening agents are 4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 2-(stilbyl)-4"-naphthalene-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Tinopal DMS and Tinopal CBS are available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate. A preferred fluorescent whitening agent is Parawhite KX, commercially available and supplied by Paramount Minerals and Chemicals, Mumbai, India. Other suitable fluorescent agents include 1,3-diarylpyrazoline and 7-alkylaminocoumarin.

[0378] Suitable levels of optical brighteners range from a low level of about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from a low level of about 0.2 wt% to a high level of 0.5 wt% or even 0.75 wt%.

[0379] soil release polymers- The detergent composition may also include one or more stain-releasing polymers that help remove stains from fabrics, such as cotton or polyester-based fabrics, particularly hydrophobic stains from polyester-based fabrics. Stain-releasing polymers can be, for example, polymers based on nonionic or anionic terephthalic acid, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, powdered detergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Decker. Another type of stain-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and multiple alkoxylated groups attached to that core structure. The core structure may include a polyalkylimide structure or a polyalkanolamine structure, as described in detail in WO 2009 / 087523 (which is hereby incorporated by reference). Furthermore, any graft copolymer is a suitable stain-releasing polymer. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856, and WO 2006 / 113314 (which are hereby incorporated by reference). Other dirt-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, as described in EP 1867808 or WO 2003 / 040279 (both of which are hereby incorporated by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose, and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, ester carboxymethylcellulose, and mixtures thereof.

[0380] anti-redeposition agents These detergent compositions may also include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under Fouling-Release Polymers may also be used as anti-redeposition agents.

[0381] other suitable adjuncts Including but not limited to: shrink-proof agents, wrinkle-proof agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, water-soluble solvents, fragrances, pigments, sodsuppressors, solvents, structurants and / or elasticizing agents for liquid detergents.

[0382] Detergent product formulations

[0383] The detergent composition can be in any conventional form, such as a bar, a uniform tablet, a tablet with two or more layers, a regular or compressed powder, granules, paste, gel, or a regular, compressed or concentrated liquid.

[0384] Detergent formulations can be in the form of layers (same or different phases), bags, or, in contrast, machine-made dispensing units.

[0385] The pouch can be configured as single-chambered or multi-chambered. It can have any form, shape, and material suitable for containing the composition, for example, preventing the composition from being released from the pouch before contact with water. The pouch is made of a water-soluble film containing an internal volume. This internal volume can be divided into chambers of the pouch. Preferred films are polymeric materials, preferably polymers formed into films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the film, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference M8630, such as those sold by Chris Craft Industrial Products, Gary, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry detergent compositions or portions thereof and / or liquid detergent compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may be structurally different from those containing solid components. Reference: (US 2009 / 0011970A1)

[0386] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets, thus avoiding undesirable storage interactions between components. In the cleaning solution, the different dissolution profiles of each compartment can also cause delayed dissolution of selected components.

[0387] These forms of definition / characteristics:

[0388] Non-unit-administered liquid or gel detergents may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70%, about 65%, about 55%, about 45%, or about 35%. Other types of liquids, including but not limited to alkanols, amines, glycols, ethers, and polyols, may be included in aqueous liquids or gels. Aqueous liquid or gel detergents may contain from 0% to 30% organic solvents.

[0389] Liquid or gel detergents can be non-aqueous.

[0390] Methods and uses

[0391] These protease variants of the present invention can be added to detergent compositions and thus become components of the detergent compositions, wherein said variants include SEQ ID. One or more of the following substitutions for NO:3: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein the variant is related to SEQ ID NO:3 has at least 60%, such as at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity.

[0392] Detergent compositions are typically used in cleaning processes such as laundry and / or hard surface cleaning, such as dishwashing.

[0393] One embodiment of the present invention relates to detergent compositions, such as laundry or dishwashing compositions, which include those containing SEQ ID NO. 3. Protease variants having at least 60% homology with a protease parent, wherein said variants comprise at least one substitution selected from the group consisting of: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R.

[0394] The detergent composition may include at least one protease variant, wherein said variant includes one or more of the following substitutions of SEQ ID NO:3: Q70F, Q70A, Q70N, S111R, S111E, S111D, S114A, S114Q, S144R, A145E, K146T, K146N, K146W, K146F, K146A, I150A, I150N, I150N, I150S, A151R, N176Y, I178Y, I178F, I178P, G182A, L184F, L184Y, L184F, L184Y, L184W, L184D, R224D, R224G, R224S, and Y240R, wherein the protease variant is consistent with SEQ ID NO:3. NO:3 has at least 60% sequence identity, such as having at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, or at least 99% sequence identity with SEQ ID NO:3, and the variant has protease activity. The at least one protease variant preferably has the inhibitory effect of an increased inhibitor as described under “Materials and Methods”.

[0395] The detergent composition can be formulated as, for example, a hand or machine wash detergent composition, including a laundry additive composition suitable for pretreating soiled fabrics and a fabric softener composition for rinsing, or a detergent composition for general household hard surface cleaning operations, or a detergent composition for hand or machine dishwashing operations.

[0396] Cleaning or textile care processes can be, for example, laundry processes, dishwashing processes, or cleaning of hard surfaces such as bathroom tiles, floors, countertops, drains, sinks, and washbasins. Laundry processes can be, for example, household laundry, but they can also be industrial laundry. A process for washing fabrics and / or clothing can be one that involves treating the fabric with a cleaning solution containing a detergent composition and at least one protease variant. For example, the cleaning or textile care process can be performed during machine washing or manual washing. The cleaning solution can be, for example, an aqueous washing solution containing a detergent composition.

[0397] Fabrics and / or garments that have undergone washing, cleaning, or textile care processes can be regular washable garments, such as household laundry. Preferably, the main component of the laundry is clothing and fabrics, including knitwear, woven fabrics, twill, non-woven fabrics, felt, yarns, and towels. These fabrics can be cellulose-based, such as natural cellulose, including cotton, linen, flax, jute, ramie, sisal, or coconut fiber; or man-made cellulose (e.g., derived from wood pulp), including cellulose gum / rayon, ramie, cellulose acetate fiber (tricell), lyocell, or blends thereof. These fabrics can also be non-cellulose-based, such as natural polyamides, including wool, camel hair, cashmere, mohair, rabbit hair, or silk; or synthetic polymers, such as nylon, aramid, polyester, acrylic, polypropylene, and spandex / elastomeric fibers; or blends thereof, as well as blends of cellulose-based and non-cellulose-based fibers. Examples of blends are blends of cotton and / or rayon / cellulose with one or more accompanying materials, such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., rayon / cellulose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers).

[0398] In recent years, there has been a growing interest in replacing components in detergents, stemming from the use of renewable biological components such as enzymes and peptides to replace petrochemicals without compromising cleaning performance. Lipases and amylases are needed to achieve similar or improved cleaning performance compared to conventional detergent compositions when the components of a detergent composition alter the activity of new enzymes or offer alternative and / or improved properties compared to commonly used detergent enzymes (such as proteases).

[0399] Proteases and their variants can be used in the removal of protein-based stains. Protein stains can be food stains, such as baby food, sebum, cocoa, eggs, blood, milk, ink, grass, or combinations thereof.

[0400] A typical detergent composition includes a variety of components besides enzymes, each with a different function. Some components, like surfactants, reduce the surface tension of the detergent, allowing the stains being cleaned to be lifted and dispersed and subsequently rinsed away. Other components, such as bleaching systems, typically remove color through oxidation, and many bleaches also have strong bactericidal properties and are used for disinfection and sterilization. Still other components, such as builders and chelating agents, soften the cleaning water, for example, by removing metal ions from the liquid.

[0401] These enzyme compositions may further include at least one or more of the following: surfactants, detergent builders, chelating agents or chelating reagents, bleaching systems or bleaching components used in laundry or dishwashing.

[0402] The amounts of surfactants, builders, chelating agents or chelating reagents, bleaching systems and / or bleaching components may be reduced compared to the amounts used without the addition of the protease variant of the present invention. Preferably, the at least one component as a surfactant, synergist, chelating agent or chelating reagent, bleaching system and / or bleaching component is present in amounts that are 1%, such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% less than the amount in the system without the addition of the protease variant of the present invention (e.g., the conventional amount of this component). The detergent composition may also be a composition that does not contain at least one component, which is a surfactant, a builder, a chelating agent or chelating reagent, a bleaching system or bleaching component and / or a polymer.

[0403] Cleaning method

[0404] The detergent composition is ideally suited for use in laundry applications. These methods include methods for washing fabrics. The method includes the step of contacting a fabric to be washed with a cleaning laundry solution comprising a detergent composition. The fabric may include any fabric capable of being washed under normal consumer use conditions. The solution preferably has a pH from about 5.5 to about 11.5. The composition may be used in the solution at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm. The water temperature typically ranges from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, and about 90°C. The water-to-fabric ratio typically ranges from about 1:1 to about 30:1.

[0405] In a specific embodiment, the cleaning method is performed at the following pH values: from about 5.0 to about 11.5, or from about 6 to about 10.5, about 5 to about 11, about 5 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, about 5.5 to about 11, about 5.5 to about 10, about 5.5 to about 9, about 5.5 to about 8, about 5.5 to about 7, about 6 to about 11, about 6 to about 10, about 6 to about 9, about 6 to about 8, about 6 to about 7, about 6.5 to about 11, about 6.5 to about 10, about 6.5 to about 9, about 6.5 to about 8, about 6.5 to about 7, about 7 to about 11, about 7 to about 10, about 7 to about 9, or about 7 to about 8, about 8 to about 11, about 8 to about 10, about 8 to about 9, about 9 to about 11, about 9 to about 10, about 10 to about 11, preferably about 5.5 to about 11.5.

[0406] In a specific embodiment, the cleaning method is performed at the following hardness levels: from about 0°dH to about 30°dH, such as about 1°dH, about 2°dH, about 3°dH, about 4°dH, about 5°dH, about 6°dH, about 7°dH, about 8°dH, about 9°dH, about 10°dH, about 11°dH, about 12°dH, about 13°dH, about 14°dH, about 15°dH, about 16°dH, about 17°dH, about 18°dH, about 19°dH, about 20°dH, about 21°dH, about 22°dH, about 23°dH, about 24°dH, about 25°dH, about 26°dH, about 27°dH, about 28°dH, about 29°dH, and about 30°dH. Under typical European cleaning conditions, the hardness is approximately 16°dH, under typical American cleaning conditions, it is approximately 6°dH, and under typical Asian cleaning conditions, it is approximately 3°dH.

[0407] The composition used in the above methods may further include at least one additional enzyme as listed in the "Other Enzymes" section above, such as enzymes selected from the group consisting of: hydrolases (such as proteases), lipases and keratinases, carbohydrate enzymes (such as amylases), cellulases, hemicellulases, xylanases, and pectinases or combinations thereof.

[0408] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0409] Example

[0410] Materials and Methods

[0411] protease activity assay

[0412] 1) Suc-AAPF-pNA activity assay:

[0413] Proteolytic activity can be determined using the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline, and it is a blocked peptide that can be cleaved by an endopeptide. Upon cleavage, a free pNA molecule is released, and it is yellow in color and can therefore be measured spectrophotometrically at a wavelength of 405 nm. This Suc-AAPF-pNA substrate is manufactured by Bachem (catalog number L1400, dissolved in DMSO).

[0414] The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay was performed by transferring 30 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / mL in 100 mM Tris, pH 8.6). The solution was mixed at room temperature and absorbance was measured at OD 405 nm over 5 minutes at 20 seconds. Under a given set of conditions, the slope (absorbance per minute) of the time-correlated absorbance curve was directly proportional to the activity of the protease in question. The protease sample should be diluted to a level where the slope is linear.

[0415] Example 1: Constructing the TY145 variant through site-directed mutagenesis

[0416] Site-directed variants are constructed from the TY145 protease (SEQ ID NO:3) including specific substitutions according to the invention. These variants are prepared by conventional cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, 1989), using PCR along with properly designed mutagenic oligonucleotides that introduce the desired mutations into the resulting sequence.

[0417] Mutagenic oligonucleotides designed to correspond to the DNA sequence flanking the desired mutation site, isolated from DNA base pairs defining insertion / deletion / substitution, and purchased from oligonucleotide suppliers such as Life Technologies, were used to construct and generate the variants listed in Table 1 below.

[0418] To test the TY145 protease variant of the present invention, mutant DNA comprising the variant of the present invention was transformed into a competent Bacillus subtilis strain and fermented using a standard protocol (liquid medium, 3–4 days, 30°C). The culture was centrifuged (26,000 x g, 20 min) and the supernatant was carefully separated from the precipitate by decanting. The supernatant was filtered through a Nalgene 0.2 μm filter to remove any remaining Bacillus host cells. The 0.2 μm filtrate was mixed 1:1 with 3.0 M (NH4)2SO4 and the mixture was fed onto a phenyl-agarose FF (high sub) column (from GE Healthcare) equilibrated with 100 mM H3BO3, 10 mM MES / NaOH, 2 mM CaCl2, and 1.5 M (NH4)2SO4 (pH 6.0). After washing the column with equilibration buffer, the protease was eluted stepwise with 100 mM H3BO3, 10 mM MES, and 2 mM CaCl2 (pH 6.0). The elution peak (containing protease activity) was collected and transferred to a bacitracin agarose column (from Upfront Chromatography) equilibrated in 100 mM H3BO3, 10 mM MES, and 2 mM CaCl2 (pH 6.0). After thoroughly washing the column with equilibration buffer, the protease was eluted with 100 mM H3BO3, 10 mM MES, 2 mM CaCl2, and 1 M NaCl (pH 6) containing 25% (v / v) 2-propanol. The elution peak (containing protease activity) was transferred to a G25 dextran gel column (from GE Healthcare) with 20 mM MES and 2 mM CaCl2 (pH 6.0). The peak transferred via G25 was the purified formulation and was used for further experiments.

[0419]

[0420]

[0421]

[0422]

[0423]

[0424] Example 2: Determination of the binding constant of an inhibitor

[0425] The purified protease variant was pre-diluted in dilution buffer to approximately 0.2 mg / mL. Then, 40 μL of the diluted protease was mixed with 40 μL of inhibitor solution (Z-Gly-Ala-NHCH(CH2C6H4pOH)C(OH)(SO3Na)-H, where Z is benzyloxycarbonyl) or 40 μL of 4-FPBA solution in the wells of a 96-well microtiter plate (Nunc F 96-MTP). For each protease variant, eight concentrations of inhibitor solution (600 μM, 200 μM, 67 μM, 22 μM, 7.4 μM, 2.47 μM, 0.82 μM, and 0 μM) and eight concentrations of 4-FPBA (120 mM, 40 mM, 13.3 mM, 4.44 mM, 1.48 mM, 0.494 mM, 0.164 mM, and 0 mM) were tested. After mixing the protease and inhibitor for 10 min at room temperature, 30 μl of the mixture was transferred to a microtiter plate (Nunc U96PP 0.5 ml) containing 270 μl of standard B detergent in each well, resulting in an inhibitor concentration of 0–30 μM and a 4-FPBA concentration of 0–6000 μM. The protease, inhibitor, and detergent were mixed with a magnetic rod for 1 hour and kept at room temperature for 1 hour to reach equilibration. Then, 20 μl was transferred to a microtiter plate (Nunc F 96-MTP). 100 μl of substrate solution was added, and after mixing for 5 seconds, the absorbance was measured at 405 nm every 20 seconds for 5 min on a SpectraMax Plus reader. The apparent binding constant was calculated using the slope of a linear regression from the initial increase in absorbance at 405 nm.

[0426]

[0427]

[0428]

[0429] calculation of Ki

[0430] Assume that the protease and inhibitor react according to the following equation:

[0431]

[0432] Where E is the protease, I is the inhibitor, and EI is the protease-inhibitor complex. It is further assumed that an equilibrium is reached between the protease, inhibitor, and protease-inhibitor complex during the 1-hour incubation in standard B, and that the addition of substrate solution during the measurement time used for linear regression does not significantly alter this equilibrium. The concentration of the enzyme-inhibitor in the detergent solution is then given as follows:

[0433] [EI]=(([E tot ]+[I tot ]+K i -sqrt(([E tot ]+[I tot ]+K i )^2-4*[E tot ]*[I tot ])) / 2

[0434]

[0435] Where E tot It is the total protein concentration ([E) tot [I] = [E] + [EI]), where Itot is the total inhibitor concentration ([I] = [E] + [EI]). tot ]=[I]+[EI]), and K i This is the equilibrium binding constant for this reaction. The measured slope V is given by:

[0436] V = V0 * (1 - [EI] / [E] tot ])=V0*(1-(([E tot ]+[I tot ]+K i -sqrt(([E tot ]+[I tot ]+K i )^2-4*[E tot ]*[I tot ])) / 2 / [E tot ])

[0437]

[0438] Where V0 is the slope without the addition of an inhibitor. To calculate the apparent binding constant K... i , with K i We used V0 as variables to perform a least-squares fit of the equation to the measured slope at various inhibitor concentrations.

[0439] As described above, calculate K for this parental protease and for each variable. i .

[0440] For each variant, Ki is available, and it is possible to calculate the effect of a single mutation if data is available for at least two variants that differ only in the mutation itself. In this case, the relative Ki for the mutation is calculated as:

[0441]

[0442] Lower K i This is desirable because satisfactory inhibition can be achieved at lower inhibitor concentrations, thus beneficial mutations have a relative K... i <1.0

[0443] The following table shows these relative Ki mutations. Each row shows:

[0444] Mutation (Column 1: "Mutation")

[0445] • A mutant protease variant (relative to SEQ ID NO:2) comprising a mutant from column 1 (column 2: “variant with mutation”).

[0446] • Another protease variant (relative to SEQ ID NO:2) differs from the protease variant in column 2 only in that it does not have the mutation found in column 1 (column 3: "variants without mutations").

[0447] • In the context of the variant in column 3, the relative Ki mutation of the mutation in column 1 measured in standard B detergent (column 4: "Inhibitor binding in standard B")

[0448] • In the context of the variant in column 3, the relative Ki mutation of the mutation in column 1 measured in Tris buffer at pH 8 (Column 5: "Inhibitor binding in Tris buffer (pH 8)").

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455] Example 4: Determination of the binding constant in standard detergent B

[0456] Ki was calculated as described in Example 3. Experimental conditions are shown in Table 5. The improvement factor (IF) or relative improvement factor against the inhibitors NNIC and 4-FPBA are also shown. As shown in Tables 6 and 7.

[0457]

[0458]

[0459]

[0460] sequence list <110> Novozymes <120> Protease variants and the polynucleotides they encode <130> 13008-WO-PCT <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 1263 <212> DNA <213> Bacillus species <220> <221> CDS <222> (1)..(1263) <220> <221> signal peptide <222> (1)..(80) <220> <221> Mature peptides <222> (331)..(1263) <400> 1 atg aag aaa ccg ttg ggg aaa att gtc gca agc acc gca cta ctc 45 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu -110 -105 -100 att tct gtt gct ttt agt tca tcg atc gca tcg gct gca ctt gca aaa 93 Ile Ser Val Wing Phe Ser Ser Ser Ile Wing Ser Wing Leu Wing Lys ‐95‐90‐85‐80 gac aaa gtt gag gta aag gaa ca gat tca tat cgt gtg cta atc ​​aaa 141 Asp Lys Val Glu Val Lys Glu Gln Asp Ser Tyr Arg Val Leu Ile Lys ‐75 ‐70 ‐65 gca cca act aca tca atc ​​agt act ttt caa tca CA tac gat gtc cgt 189 Ala Pro Thr Thr Ser Ser Ile Ser Thr Phe Gln Ser Gln Tyr Asp Val Arg ‐60 ‐55 ‐50 tgg gat ttt ggc aaa gag gga ttt aca aca gat gtt gat gcc aaa cag 237 Trp Asp Phe Gly Lys Glu Gly Phe Thr Thr Asp Val Asp Ala Lys Gln ‐45 ‐40 ‐35 ctc caa acg ctt CA agc aac aaa gac att CA att cag aag gta aat 285 Leu Gln Thr Leu Gln Serves Lys Asp With Gln Ile Gln Lys Val Asn ‐30 ‐25 ‐20 gaa atg aca gta gaa act gtt aca aca gaa aag gcg gaa gtg acg gcg 333 Glu Met Thr Val Glu Thr Val Thr Thr Glu Lys Ala Glu Val Thr Ala ‐15 ‐10 ‐5 ‐1 gta cca agt aca caa acc cct tgg ggc ata aag tca att tat aat gat 381 Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn Asp 5 10 15 caa tca att aca aaa aca act gga ggc agc gga att aag gta gct gtt 429 Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala Val 20 25 30 tta gat aca ggg gtt tat aca agc cat tta gat tta gct ggt tct gcc 477 Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser Ala 35 40 45 gag caa tgc aag gat ttt acc caa tct aat cct tta gta gat ggt tca 525 Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly Ser 50 55 60 65 tgc acc gat cgc caa ggg cat ggt aca cat gtt gcc gga act gta ttg 573 Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val Leu 70 75 80 gcg cat gga ggc agt aat gga caa ggc gtt tac ggg gtg gct ccg caa 621 Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro Gln 85 90 95 gcg aaa cta tgg gca tat aaa gta tta gga gat aac ggc agc gga tac 669 Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly Tyr 100 105 110 tct gat gat att gca gca gct atc aga cat gta gct gat gaa gct tca 717 Ser Asp Asp Ile Only Only Ile Arg His Val Only Asp Glu Only Ser 115 120 125 cgt aca ggt tcc aaa gta gta att aat atg tcg cta ggt tca tct gcc 765 Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser Ala 130 135 140 145 aag gat tca ttg att gct agt gca gta gat tat gca tat gga aaa ggt Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys Gly 150 155 160 gta tta atc ​​gtt gct gcg gct gct aat agt ggg tca gggc agc aat aca 861 Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn Thr 165 170 175 atc ggc ttt cct ggc ggg ctt gta aat gca gtg gca gta gcg gca ttg 909 Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala Leu 180 185 190 gag aat gtt cag caa aat gga act tat cga gta gct gat ttc tca tct 957 Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser Ser 195 200 205 aga ggg aat ccg gca act gct gga gat tat atc att caa gag cgt gat 1005 Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg Asp 210 215 220 225 att gaa gtt tca gct ccg gga gca agt gta gag tct aca tgg tac act 1053 Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr Thr 230 235 240 ggc ggt tat aat acg atc agc ggt aca tca atg gct aca cct cat gta 1101 Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His Val 245 250 255 gct ggg tta gct gct aaa atc tgg tca gcg aat act tca tta agt cat 1149 Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser His 260 265 270 agc caa ctg cgc aca gaa ttg caa aat cgc gct aaa gta tat gat att 1197 Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp Ile 275 280 285 aaa ggt ggt atc gga gcc gga aca ggt gac gat tat gca tca ggg ttc 1245 Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly Phe 290 295 300 305 gga tat cca aga gta aaa 1263 Gly Tyr Pro Arg Val Lys 310 <210> 2 <211> 421 <212> PRT <213> Bacillus species <400> 2 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu ‑110 ‑105 ‑100 Ile Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Leu Ala Lys ‑95 ‑90 ‑85 ‑80 Asp Lys Val Glu Val Lys Glu Gln Asp Ser Tyr Arg Val Leu Ile Lys ‑75 ‑70 ‑65 Ala Pro Thr Thr Ser Ile Ser Thr Phe Gln Ser Gln Tyr Asp Val Arg ‑60 ‑55 ‑50 Trp Asp Phe Gly Lys Glu Gly Phe Thr Thr Asp Val Asp Ala Lys Gln ‑45 ‑40 ‑35 Leu Gln Thr Leu Gln Ser Asn Lys Asp Ile Gln Ile Gln Lys Val Asn ‑30 ‑25 ‑20 Glu Met Thr Val Glu Thr Val Thr Thr Glu Lys Ala Glu Val Thr Ala ‑15 ‑10 ‑5 ‑1 1 Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn Asp 5 10 15 Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala Val 20 25 30 Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser Ala 35 40 45 Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly Ser 50 55 60 65 Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val Leu 70 75 80 Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro Gln 85 90 95 Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly Tyr 100 105 110 Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala Ser 115 120 125 Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser Ala 130 135 140 145 Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys Gly 150 155 160 Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn Thr 165 170 175 Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala Leu 180 185 190 Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser Ser 195 200 205 Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg Asp 210 215 220 225 Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr Thr 230 235 240 Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His Val 245 250 255 Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser His 260 265 270 Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp Ile 275 280 285 Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly Phe 290 295 300 305 Gly Tyr Pro Arg Val Lys 310 <210> 3 <211> 311 <212> PRT <213> Bacillus species <400> 3 Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn 1 5 10 15 Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala 20 25 30 Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser 35 40 45 Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly 50 55 60 Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro 85 90 95 Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly 100 105 110 Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala 115 120 125 Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser 130 135 140 Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys 145 150 155 160 Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Ser Gly Ser Asn 165 170 175 Thr Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala 180 185 190 Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser 195 200 205 Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg 210 215 220 Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr 225 230 235 240 Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His 245 250 255 Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser 260 265 270 His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp 275 280 285 Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly 290 295 300 Phe Gly Tyr Pro Arg Val Lys 305 310 <210> 4 <211> 311 <212> PRT <213> Bacillus species <400> 4 Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn 1 5 10 15 Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala 20 25 30 Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser 35 40 45 Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly 50 55 60 Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro 85 90 95 Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly 100 105 110 Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala 115 120 125 Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser 130 135 140 Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys 145 150 155 160 Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Pro Gly Pro Asn 165 170 175 Thr Ile Gly Phe Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala 180 185 190 Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser 195 200 205 Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg 210 215 220 Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr 225 230 235 240 Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His 245 250 255 Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser 260 265 270 His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp 275 280 285 Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly 290 295 300 Phe Gly Tyr Pro Arg Val Lys 305 310 <210> 5 <211> 311 <212> PRT <213> Bacillus species <400> 5 Ala Val Pro Ser Thr Gln Thr Pro Trp Gly Ile Lys Ser Ile Tyr Asn 1 5 10 15 Asp Gln Ser Ile Thr Lys Thr Thr Gly Gly Ser Gly Ile Lys Val Ala 20 25 30 Val Leu Asp Thr Gly Val Tyr Thr Ser His Leu Asp Leu Ala Gly Ser 35 40 45 Ala Glu Gln Cys Lys Asp Phe Thr Gln Ser Asn Pro Leu Val Asp Gly 50 55 60 Ser Cys Thr Asp Arg Gln Gly His Gly Thr His Val Ala Gly Thr Val 65 70 75 80 Leu Ala His Gly Gly Ser Asn Gly Gln Gly Val Tyr Gly Val Ala Pro 85 90 95 Gln Ala Lys Leu Trp Ala Tyr Lys Val Leu Gly Asp Asn Gly Ser Gly 100 105 110 Tyr Ser Asp Asp Ile Ala Ala Ala Ile Arg His Val Ala Asp Glu Ala 115 120 125 Ser Arg Thr Gly Ser Lys Val Val Ile Asn Met Ser Leu Gly Ser Ser 130 135 140 Ala Lys Asp Ser Leu Ile Ala Ser Ala Val Asp Tyr Ala Tyr Gly Lys 145 150 155 160 Gly Val Leu Ile Val Ala Ala Ala Gly Asn Ser Gly Pro Gly Pro Asn 165 170 175 Thr Ile Gly Tyr Pro Gly Gly Leu Val Asn Ala Val Ala Val Ala Ala 180 185 190 Leu Glu Asn Val Gln Gln Asn Gly Thr Tyr Arg Val Ala Asp Phe Ser 195 200 205 Ser Arg Gly Asn Pro Ala Thr Ala Gly Asp Tyr Ile Ile Gln Glu Arg 210 215 220 Asp Ile Glu Val Ser Ala Pro Gly Ala Ser Val Glu Ser Thr Trp Tyr 225 230 235 240 Thr Gly Gly Tyr Asn Thr Ile Ser Gly Thr Ser Met Ala Thr Pro His 245 250 255 Val Ala Gly Leu Ala Ala Lys Ile Trp Ser Ala Asn Thr Ser Leu Ser 260 265 270 His Ser Gln Leu Arg Thr Glu Leu Gln Asn Arg Ala Lys Val Tyr Asp 275 280 285 Ile Lys Gly Gly Ile Gly Ala Gly Thr Gly Asp Asp Tyr Ala Ser Gly 290 295 300 Phe Gly Tyr Pro Arg Val Lys 305 310

Claims

1. A protease variant having protease activity, wherein the variant is more susceptible to inhibition by a protease inhibitor compared to SEQ ID NO: 3, and wherein the inhibitor is NNIC or 4-FPBA, wherein NNIC is Z-Gly-Ala-NHCH(CH2C6H4pOH)C(OH)(SO3Na)-H, wherein Z is a benzyloxycarbonyl group, and the changes in the variant compared to SEQ ID NO: 3 are selected from the group consisting of: S114Q, S173P, S175P, F180Y K146T, S173P, S175P, F180Y K146N, S173P, S175P, F180Y K146W, S173P, S175P, F180Y K146F, S173P, S175P, F180Y K146A, S173P, S175P, F180Y I150A, S173P, S175P, F180Y I150N, S173P, S175P, F180Y S27K,I150N,S171N,S173P,G174R,S175P,F180Y,Q198E,T297P S27K,K146P,S148R,A151R,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,T297P S173P, S175P, N176Y, F180Y S173P, S175P, I178Y, F180Y S173P, S175P, I178F, F180Y S173P, S175P, I178P, F180Y S173P, S175P, F180Y, L184F S27K,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P S27K,I121V,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P S27K,Q70N,G107N,I121V,E127Q,S173P,S175P,F180Y,L184F,Q198E,T297P S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199K,T297P S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199R,T297P S173P, S175P, F180Y, L184Y S27K,S171N,S173P,G174R,S175P,F180Y,L184Y,Q198E,T297P S27K,S173P,G174K,S175P,F180Y,L184Y,Q198E,N199K,T297P S27K,S173P,G174K,S175P,F180Y,L184Y,Q197K,Q198E,T297P S173P, S175P, F180Y, L184W S27K,S171N,S173P,G174R,S175P,F180Y,L184W,Q198E,T297P S173P, S175P, F180Y, L184D S173P, S175P, F180Y, R224D S173P, S175P, F180Y, R224G S27K,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,R224G,T297P S173P, S175P, F180Y, R224S S27K,I121V,S171N,S173P,G174R,S175P,F180Y,Q198E,R224S,T297P S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,R224G,T297P S27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,R224G,T297P S27K,V162T,S173P,G174K,S175P,F180Y,L184F,Q197K,Q198E,T297P Q70F Q70A Q70N S111R S111E S111D S114A S144R A145E I150N I150S G182A L184F L184Y R224G and Y240R, These positions correspond to the position of SEQ ID NO:

3.

2. A detergent composition comprising the protease variant according to claim 1 and one or more detergent components.

3. The detergent composition according to claim 2, further comprising one or more additional enzymes selected from the group consisting of: Protease, amylase, lipase, keratinase, cellulase, endoglucanase, xyloglucanase, pectinase, pectin lyase, xanthan gumase, peroxidase, halogenated peroxidase, catalase, and mannanase, or any mixture thereof.

4. The detergent composition according to claim 2 or 3, wherein it is in the form of a bar, a uniform tablet, a tablet having two or more layers, a bag having one or more chambers, a regular or compressed powder, granules, paste, gel, or a regular, compressed or concentrated liquid.

5. Use of the detergent composition according to any one of claims 2-4 in a cleaning process.

6. The use according to claim 5, wherein the cleaning process is laundry or hard surface cleaning.

7. The use according to claim 5 or 6, wherein the cleaning process is dishwashing.

8. A method for obtaining a protease variant, the method comprising introducing a modification into a parent protease comprising SEQ ID NO: 3, said modification being selected from the group consisting of: S114Q, S173P, S175P, F180Y K146T, S173P, S175P, F180Y K146N, S173P, S175P, F180Y K146W, S173P, S175P, F180Y K146F, S173P, S175P, F180Y K146A, S173P, S175P, F180Y I150A, S173P, S175P, F180Y I150N, S173P, S175P, F180Y S27K,I150N,S171N,S173P,G174R,S175P,F180Y,Q198E,T297P S27K,K146P,S148R,A151R,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,T297P S173P, S175P, N176Y, F180Y S173P, S175P, I178Y, F180Y S173P, S175P, I178F, F180Y S173P, S175P, I178P, F180Y S173P, S175P, F180Y, L184F S27K,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P S27K,I121V,S171N,S173P,G174R,S175P,F180Y,L184F,Q198E,T297P S27K,Q70N,G107N,I121V,E127Q,S173P,S175P,F180Y,L184F,Q198E,T297P S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199K,T297P S27K,S173P,G174K,S175P,F180Y,L184F,Q198E,N199R,T297P S173P, S175P, F180Y, L184Y S27K,S171N,S173P,G174R,S175P,F180Y,L184Y,Q198E,T297P S27K,S173P,G174K,S175P,F180Y,L184Y,Q198E,N199K,T297P S27K,S173P,G174K,S175P,F180Y,L184Y,Q197K,Q198E,T297P S173P, S175P, F180Y, L184W S27K,S171N,S173P,G174R,S175P,F180Y,L184W,Q198E,T297P S173P, S175P, F180Y, L184D S173P, S175P, F180Y, R224D S173P, S175P, F180Y, R224G S27K,S171N,S173P,G174R,S175P,F180Y,Q198E,N199K,R224G,T297P S173P, S175P, F180Y, R224S S27K,I121V,S171N,S173P,G174R,S175P,F180Y,Q198E,R224S,T297P S27K,S171D,S173P,G174R,S175P,G182A,L184F,Q198E,N199K,R224G,T297P S27K,S171N,S173P,G174R,S175P,F180Y,G182A,L184F,Q198E,N199K,R224G,T297P S27K,V162T,S173P,G174K,S175P,F180Y,L184F,Q197K,Q198E,T297P Q70F Q70A Q70N S111R S111E S111D S114A S144R A145E I150N I150S G182A L184F L184Y R224G and Y240R, These positions correspond to the position of SEQ ID NO:

3. Compared to SEQ ID NO: 3, this variant exhibits increased inhibition by a protease inhibitor, wherein the inhibitor is NNIC or 4-FPBA, wherein NNIC is Z-Gly-Ala-NHCH(CH2C6H4pOH)C(OH)(SO3Na)-H, and Z is a benzyloxycarbonyl group; and Recycle this variant.

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