Subtilase variants

Abstract

The present invention relates to novel protease variants exhibiting improved stability and or improved wash performance in liquid detergents. The variants of the invention are suitable for use in, for example, cleaning or detergent compositions, such as laundry detergent compositions and dishwashing compositions, including automatic dishwashing compositions. The invention also relates to isolated DNA sequences encoding the variants, expression vectors, host cells, and methods for producing and using the variants of the invention.

Description

[0001] References to sequence lists

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

[0004] Invention Field

[0005] This invention relates to novel Bacillus subtilis enzyme variants that exhibit increased stability and / or improved washing performance in liquid detergent compositions. These variants of the invention are suitable for use in, for example, cleaning or detergent compositions, such as laundry detergent compositions and dishwashing compositions, including automatic dishwashing compositions. The invention also relates to isolated DNA sequences encoding these variants, expression vectors, host cells, and methods for generating and using the variants of the invention.

[0006] Related technical specifications

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

[0008] The wild-type Bacillus subtilis enzyme that has been used in laundry is the BLAP protease disclosed in WO 91 / 02792.

[0009] An increasing number of commercially available proteases are protein-engineered variants of naturally occurring wild-type proteases. and (Novozymes a / s) Purafect and (Genencor International, Inc.). Furthermore, many variants have been described in the art, such as those described in WO 2004 / 041979 (Novozymes) which exhibit alterations in, for example, detergent performance, thermal stability, stability during washing, or catalytic activity relative to the parental *Bacillus subtilis* enzyme. These variants are suitable for use in, for example, cleaning or detergent compositions.

[0010] Variants of the BLAP protease and their suitability for cleaning or detergent compositions have been disclosed, for example, in EP 701 605.

[0011] WO 99 / 57155 discloses detergent enzymes, such as proteases modified by attaching cellulose-binding domains to the enzyme. It is suggested that detergent enzymes, such as proteases, be conjugated to cellulose-containing fabrics, which would enhance washing performance.

[0012] However, several factors make further improvements to proteases advantageous. In particular, liquid detergent compositions remain a challenge for many detergent proteases, and loss of activity during storage remains a problem for many good detergent proteases. Therefore, despite ongoing research into protease development, new and improved proteases with satisfactory detergent performance and increased stability are still needed. Invention Overview

[0014] This invention relates to variants of *Bacillus subtilis* enzyme that exhibit improved stability and / or improved detergent performance in liquid detergents compared to the parental *Bacillus subtilis* enzyme. The invention specifically relates to a *Bacillus subtilis* enzyme variant having at least 90% sequence identity with SEQ ID NO:3, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, and more preferably at least 98% sequence identity, wherein the variant has a glutamic acid residue (E) at position 101, and wherein the variant exhibits increased stability in liquid detergent compositions compared to the *Bacillus subtilis* enzyme having the amino acid sequence of SEQ ID NO:3. Subtilisase further includes substitutions selected from the group consisting of: S156D, L262E, Q137H, S3T, R45E, D, Q, P55N, T58W, Y, L, Q59D, M, N, T, G61D, R, S87E, G97S, A98D, E, R, S106A, W, N117E, H120V, D, K, N, S124M, P129D, E136Q, S143W, S161T, S163A, G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T, D, and L262N, Q, D.

[0015] definition

[0016] Allelic variants: 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 from mutations 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.

[0017] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be 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.

[0018] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly identifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0019] Control sequences: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. These regulatory sequences include, but are not limited to, pro-leaders, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotides encoding the variant.

[0020] Detergent composition: Unless otherwise specified, the term "detergent composition" includes general-purpose or heavy-duty detergents in granular or powder form, especially cleaning detergents; all-purpose detergents in liquid, gel, or paste form, especially so-called heavy-duty liquid (HDL) types; liquid de-icing detergents; hand or light-duty dishwashing agents, especially those with high foaming properties; machine dishwashers, including various tablet, granular, liquid, and rinsing aids for use in homes and public institutions; liquid cleaners and disinfectants, including antibacterial hand washes, cleaning strips, soap bars, mouthwashes, denture cleaners, car or carpet shampoos, bathroom cleaners; shampoos and hairsprays; shower gels, foaming bath liquids; metal cleaners; and cleaning aids such as bleach additives and "stain-sticks" or pretreatment additives. The terms "detergent composition" and "detergent formulation" are used with respect to mixtures in a washing medium intended for cleaning soiled objects. In some embodiments, the term "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., "dishware 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 the Bacillus subtilis enzyme variant 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, one or more transferases, hydrolases, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, and solubilizers.

[0021] Dishwashing: The term "dishwashing" refers to all forms of dishwashing, such as manual or automatic dishwashing. Dishwashing includes, but is not limited to, cleaning all forms of earthenware, such as plates, cups, glasses, and bowls; all forms of tableware, such as spoons, knives, and forks; and serving utensils, including ceramic, plastic (e.g., melamine), metal, porcelain, glass, and acrylic.

[0022] Dishwashing Composition: The term "dishwashing composition" refers to all forms of compositions used for cleaning hard surfaces. This invention is not limited to any specific type of dishwashing composition or any specific detergent.

[0023] Expression: 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.

[0024] Expression vector: The term “expression vector” refers to a linear or circular DNA molecule that includes a polynucleotide encoding a variant and that the polynucleotide is operatively linked to a control sequence provided for its expression.

[0025] Hard Surface Cleaning: The term "hard surface cleaning" is defined herein as cleaning hard surfaces, which can include floors, tables, walls, ceilings, etc., along with surfaces of hard objects such as cars (car washing) and tableware (dishwashing). Dishwashing includes, but is not limited to, cleaning plates, cups, glasses, bowls, and utensils (e.g., spoons, knives, forks), serving utensils, ceramics, plastics (e.g., melamine), metals, porcelain, glass, and acrylics.

[0026] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using a nucleic acid construct or expression vector containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of the parent cell that is not identical to its parent cell due to mutations that occur during replication.

[0027] Improved washing performance: The term "improved washing performance" is defined herein as relative to a parental subtilisin (i.e., relative to a subtilisin having the same amino acid sequence as the variant but excluding the alterations in the variant), such as relative to the mature polypeptide of SEQ ID NO:2, such as a subtilisin variant exhibiting altered washing performance through increased detergency. The term "washing performance" includes washing performance in dishwashing as well as in laundry. Washing performance can be determined by calculating a so-called strength value (Int) as defined in the Automated Mechanical Stress Measurement (AMSA) for Automatic Dishwashing as described in the Materials and Methods section herein.

[0028] Isolated: The term “isolated” means a substance in a form or environment not naturally present. Non-limiting examples of isolated substances include (1) any substance not naturally present, (2) any substance including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor, which is at least partially removed from one or all of the naturally present components associated with it; (3) any substance artificially modified relative to a naturally found substance; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0029] Laundry: The term "laundry" encompasses both household and industrial laundry and refers to the process of treating textiles and / or fabrics with a solution containing the detergent composition of the present invention. The laundry process can be performed, for example, using a household or industrial washing machine or manually.

[0030] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized and includes one or more control sequences.

[0031] Operable ligation: The term “operable ligation” refers to a construction in which a control sequence is positioned relative to the coding sequence of a polynucleotide so that the control sequence directs the expression of the coding sequence.

[0032] Parent: The term "parent" refers to a protease that has been modified to produce the enzyme variant of the present invention. Therefore, a parent is a protease having the same amino acid sequence as the variant but without alteration at one or more (e.g., two or more) of the specified positions. It should be understood that the expression "having the same amino acid sequence" in the context involves 100% sequence identity. The parent can be a naturally occurring (wild-type) polypeptide. In one specific embodiment, the parent is a protease having at least 60% identity with the mature polypeptide having SEQ ID NO:2, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity.

[0033] Protease: The term “protease” is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to EC 3.4 (including each of its 13 subclasses). EC numbers are based on the 1992 enzyme nomenclature published by NC-IUBMB Academic Press in San Diego, California, and include those published in 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, Supplement 1–5.

[0034] Protease activity: The term "protease activity" refers to proteolytic activity (EC 3.4). The protease of the present invention is an endopeptidase (EC 3.4.21). Several types of protease activity exist: the three main types are: trypsin-like, wherein cleavage of the amide substrate occurs at P1 after Arg or Lys; chymotrypsin-like, wherein cleavage occurs at P1, after one of the hydrophobic amino acids; and elastase-like, wherein cleavage occurs at P1 after Ala. For the purposes of the present invention, protease activity is determined according to the procedure described in the following "Materials and Methods". These Bacillus subtilis enzyme 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%, and at least 100% protease activity of the mature polypeptide of SEQ ID NO:2.

[0035] Sequence identity: The parameter “sequence identity” is used to describe the correlation between two amino acid sequences or two nucleotide sequences. For the purposes of this invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The Needle output, marked as "Longest Consistency" (obtained using the -nobrief 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] For the purposes of this invention, the Niedle-Onsch algorithm (Niedleman and Onsch, 1970, see above), implemented in the Niedle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Niedle output labeled “Longest Consistency” (obtained using the -nobrief option) is used as the percentage consistency and is calculated as follows:

[0038] (Consistent deoxyribonucleotides x 100) / (Alignment length - total number of vacancies in the alignment)

[0039] Stability: The term "stability" includes both storage stability and stability during use, such as stability during washing (stability in washing), and reflects the stability of the Bacillus subtilis enzyme variant according to the invention as a function of time, for example, how much activity the protease retains when 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. Protease stability can be measured using the 'stability assay' as described in the Materials and Methods section herein. The terms "improved stability" or "enhanced stability" are defined herein as a variant protease exhibiting increased stability in solution relative to the parent protease. The terms "improved stability" and "enhanced stability" include "improved chemical stability," "detergent stability," or "improved detergent stability."

[0040] The term "improved chemical stability" is defined herein as the ability of a variant enzyme to retain its enzymatic activity after incubation for a period of time in the presence of one or more chemicals, whether naturally occurring or synthetic, that reduce the activity of the parent enzyme. Improved chemical stability also allows these variants to catalyze reactions better in the presence of such chemicals. In a particular aspect of the invention, this improved chemical stability is an improved stability of detergents, particularly liquid detergents. The term "detergent stability" or "improved detergent stability" specifically refers to the improved stability of protease activity when a protease variant of the invention is mixed into a liquid detergent formulation (particularly according to Table 1) and then stored at a temperature between 15°C and 50°C (e.g., 20°C, 30°C, or 40°C).

[0041] The term "improved thermal activity" refers to a variant exhibiting a modified temperature-dependent activity profile relative to the parent or relative to the protease having SEQ ID NO:3 at a specific temperature. Thermal activity values ​​provide a measure of the efficiency with which a variant enhances the catalytic activity of a hydrolytic reaction within a given temperature range. A variant with higher thermal activity will result in an increase in the rate of substrate hydrolysis of an enzyme composition, thereby reducing the required time and / or the enzyme concentration required for the activity. Alternatively, a variant with reduced thermal activity will enhance the enzymatic reaction at a temperature lower than the optimal temperature of the parent as defined by the parent's temperature-dependent activity profile.

[0042] Strict conditions: Different strict conditions are defined as follows.

[0043] 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 of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0044] The term "low stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 60°C for 15 minutes each time with 1X SSC and 0.2% SDS.

[0045] 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. Vector material is finally washed three times at 65°C for 15 minutes each time with 1X SSC and 0.2% SDS.

[0046] The term "medium-high stringent conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / mL of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 0.5X SSC and 0.2% SDS.

[0047] The term "highly stringent conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector materials are finally washed three times at 65°C for 15 minutes each time with 0.3X SSC and 0.2% SDS.

[0048] The term "very 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 35% formamide, following standard DNA blotting procedures. Vector materials are finally washed three times at 65°C for 15 minutes each time with 0.15X SSC and 0.2% SDS.

[0049] Substantially Pure Variants: The term "substantially pure variant" refers to a formulation containing, 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 material naturally or recombinantly associated with 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 a substantially pure form. This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0050] Substantially Pure Polynucleotides: The term "substantially pure polynucleotide" refers to a polynucleotide formulation that does not contain other foreign or unwanted nucleotides and is present in a form suitable for use within a genetically engineered peptide production system. Thus, substantially pure polynucleotides contain, 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 polynucleotide material naturally or recombinantly associated with the polynucleotide. However, substantially pure polynucleotides may include naturally occurring 5'- and 3'-untranslated regions, such as promoters and terminators. Preferably, substantially pure polynucleotides are at least 90% pure by weight, for example, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. These polynucleotides of the present invention are preferably present in a substantially pure form.

[0051] Variants: The term "variant" refers to a polypeptide with protease activity that includes alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding one or more (e.g., several) amino acids (e.g., 1, 2, 3, 4, or 5 amino acids) adjacent to and immediately following an amino acid occupying a position.

[0052] Washing performance: The term "washing performance" is used as the ability of an enzyme to remove stains present on an object to be cleaned during a washing process, such as laundry or hard surface cleaning. Improvements in washing performance can be quantified by calculating the so-called intensity value (Int) as defined in the AMSA assay described in the Materials and Methods section.

[0053] Wild-type subtilisin: The term "wild-type subtilisin" refers to a protease expressed by a naturally occurring organism (e.g., bacteria, archaea, yeast, fungi, plants, or animals found in nature). An example of wild-type subtilisin is BLAP, which is a subtilisin having the amino acid sequence SEQ ID NO:2.

[0054] Variant Naming Rules

[0055] For the purposes of this invention, the mature polypeptide BPN' disclosed in SEQ ID NO:1 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:1, and based on this alignment, the Niederman-Onsch algorithm, implemented in the Nieder program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite) (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:1. 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.

[0056] The identification of the corresponding amino acid residues in another protease can be determined by comparing multiple polypeptide sequences using several computer programs with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparison by log-expectation value; version 3.5 or later); MAFFT (version 6.857 or later); and EMBOSS EMMA with ClustalW (version 1.83 or later).

[0057] When other enzymes deviate from the mature polypeptide of SEQ ID NO:1, making traditional sequence-based comparison methods unable to detect their relationship, alternative pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (profiles) of polypeptide families to search a database. For example, the PSI-BLAST program generates multiple profiles through an iterative database search process and can detect distant homologs. Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER() use information from various sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potential) as input to a neural network that predicts the structural folding of a query sequence. Similarly, the method described by Gough et al. (2000, J.Mol.Biol.) 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These comparisons can then be used to generate homology models of peptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0058] 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, such as distance alignment matrices or combined extensions, can be used to align two or more protein structures, and implementations of these algorithms can be further used to query structure databases containing structures of interest to discover potential structural homologs.

[0059] In the description of variations of the invention, the following nomenclature is used for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are adopted.

[0060] replace For amino acid substitutions, the following nomenclature is used: initial amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) for arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) for phenylalanine (F).

[0061] MissingFor amino acid deletions, use the following nomenclature: initial amino acid, position, *. Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), e.g., "Gly195" * +Ser411 * "or "G195 * +S411 * ".

[0062] insert. The insertion of additional amino acid residues, such as the insertion of lysine after G195, can be represented as Gly195GlyLys or G195GK. Alternatively, the insertion of additional amino acid residues, such as the insertion of lysine after G195, can be represented as *195aL. When more than one amino acid residue is inserted, such as the insertion of Lys and Ala after G195, it can be represented as Gly195GlyLysAla or G195GKA. In such cases, the inserted amino acid residues can also be numbered by adding a lowercase letter to the position number of the amino acid residue preceding the inserted amino acid residue, in this example: * 195aK * 195bA. In the above example, sequences 194 to 196 are therefore:

[0063]

[0064] When substitution and insertion occur in the same location, this can be represented as S99SD+S99A, or simply S99AD. The same modification can also be represented as S99A+ * 99aD.

[0065] When an amino acid residue identical to an existing amino acid residue is inserted, degeneracy clearly occurs in the nomenclature. If, for example, glycine is inserted after glycine in the above example, it is represented as G195GG or *195aGbG. For the following variations, the same practical variation can also be represented simply as A194AG or... * 194aG, from:

[0066]

[0067] arrive:

[0068]

[0069] Such cases are obvious to those skilled in the art, and therefore the expression G195GG and the corresponding expression for this type of insertion are intended to include such equivalent degenerate expressions.

[0070] Multiple changes Variations involving multiple changes are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent arginine and glycine at positions 170 and 195 being replaced by tyrosine and glutamic acid, respectively. Alternatively, spaces or commas can be used to separate multiple changes, such as A170Y G195E or A170Y, G195E.

[0071] Different changes When different changes can be introduced at a single position, these changes are separated by a comma. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0072] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0073] Different alternatives or optional substitutions can be indicated by parentheses, such as Arg170[Tyr, Glu] or Arg170{Tyr, Glu} or simply R170[Y, E] or R170{Y, E}.

[0074] Amino acid position / residue numbering

[0075] Unless otherwise specified, the amino acid numbers used herein correspond to the sequence number of the BPN' (BASBPN) enzyme. For a further description of the BPN' sequence, see SEQ ID NO:1 or Siezen et al., Protein Engineering 4 (1991) 719-737.

[0076] Detailed Description of the Invention

[0077] This invention relates to a subtilisin enzyme variant having improved stability and / or improved detergent performance in liquid detergents. Preferably, the subtilisin enzyme variant also has good detergent performance; more preferably, the variant has improved detergent performance compared to the parent subtilisin enzyme, SEQ ID NO:2, or SEQ ID NO:3.

[0078] The parental subtilisin can in principle be any naturally occurring subtilisin, or it can be a modified subtilisin produced by methods known in the art, such as preparing a hybrid or two or more separate subtilisins, or by substituting, deleting or inserting one or more amino acid residues in a given subtilisin.

[0079] Many subtilisinases have well-documented and proven washing properties, and numerous publications describe their use in laundry washing or cleaning processes. However, for use in detergents, it is also important that the subtilisinase exhibits satisfactory stability in detergent compositions, such as liquid detergents. It is preferable to use parental subtilisinases with good washing properties to provide variants of this subtilisinase that offer improved stability in liquid detergents.

[0080] The preferred parental subtilisin according to the present invention is a BLAP protease having the amino acid sequence of SEQ ID NO:2, or a subtilisin having at least 60%, for example 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%, at least 99%, or 100% sequence identity with SEQ ID NO:2. The preferred parental subtilisin may be a subtilisin having the amino acid sequence of SEQ ID NO:2, wherein amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 have been modified compared to SEQ ID NO:2, and wherein each modification is independently a substitution of one amino acid residue by another, a deletion of an amino acid residue, or an insertion of an amino acid residue.

[0081] In another aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO:2. In another aspect, the parent comprises or consists of the mature polypeptide of SEQ ID NO:2. In another aspect, the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO:2. In yet another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:2.

[0082] A preferred parental subtilisin is a subtilisin having the amino acid sequence of SEQ ID NO:2, wherein the arginine residue at position 101 corresponding to SEQ ID NO:1 is replaced with a glutamic acid residue (R101E). The sequence of this preferred subtilisin is shown in SEQ ID NO:3.

[0083] Other preferred parental subtilisin according to the invention is a protease having the amino acid sequence of SEQ ID NO:3 or a subtilisin having at least 60%, for example 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%, at least 99%, or 100% sequence identity with SEQ ID NO:3. In another aspect, the parent comprises or is composed of the amino acid sequence of SEQ ID NO:3. In another aspect, the parent comprises or is composed of the mature polypeptide of SEQ ID NO:3. In another aspect, the parent comprises or is composed of amino acids 1 to 269 of SEQ ID NO:3. In yet another embodiment, the parent is an allelic variant of the mature polypeptide of SEQ ID NO:3.

[0084] The preferred parental subtilisin can be a subtilisin having the amino acid sequence of SEQ ID NO:3, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids have been modified compared to SEQ ID NO:3, and wherein each modification is independently a substitution of one amino acid residue by another amino acid residue, a deletion of an amino acid residue, or an insertion of an amino acid residue.

[0085] Further variants of the Bacillus subtilis enzyme of the present invention include those with SEQ ID NO. NO:3 has a variant with at least 90% sequence identity, which includes one or more of the following substitutions: substitution S156D; L262E; Q137H; S3T; R45E, D, Q; P55N; T58W, Y, L; Q59D, M, N, T; G61D, R; S87E; G97S; A98D, E, R; S106A, W; N117E; H120V, D, K, N; S124M; P129D; E136Q; S143W; S161T; S163A, G; Y171L; A172S; N185Q; V199M; Y209W; M222Q; N238H; V244T; N261T, D; L262N, Q, D, where these positions are numbered according to SEQ ID NO 1.

[0086] In a preferred embodiment, the subtilisin variant of the present invention exhibits improved stability and improved washing performance in liquid detergents compared to the subtilisin having the sequence of SEQ ID NO:3. Examples of such preferred subtilisin variants in this embodiment include subtilisin variants having at least 90% sequence identity with SEQ ID NO:3 and comprising one or more of the following substitutions: substitutions for R45E, D, Q; T58L; G61D; S87E; G97S; A98E; S106A; N117E; H120D, K, V; P129D; E136Q, Q137H; S156D; S161T; S163A, G; V199M; M222Q; N261T; L262E, Q, N.

[0087] The subtilisinase variants of the present invention may have other substitutions, such as those known in the art, to impart specific beneficial properties to the subtilisinase variants. Numerous substitutions exist in subtilisins known in the art, and it is contemplated that such known substitutions may be used in the present invention to impart these known beneficial effects to the subtilisinase variants of the present invention. The subtilisinase variants of the present invention may include one or more additional substitutions that may be used in the present invention to impart additional beneficial effects and / or improve existing effects, such as stability and washability.

[0088] Preferred additional mutations include one or more of the following substitutions: V4I, N76D, V104T, N128Q, S141H, R145H, A194P, G195E, V205I, N218Q, A228V, N238E, or S265H.

[0089] Particularly preferred examples of the Bacillus subtilis enzyme variants of the present invention that have improved stability and / or improved washing performance in liquid detergents, compared to the Bacillus subtilis enzyme having the amino acid sequence of SEQ ID NO:3, include the following variants comprising the following amino acid sequence:

[0090] SEQ ID NO:3+S3T

[0091] SEQ ID NO:3+R45E, D

[0092] SEQ ID NO:3+P55N,

[0093] SEQ ID NO:3+T58W, Y, L

[0094] SEQ ID NO:3+Q59D,M,N,T,

[0095] SEQ ID NO:3+G61D, R

[0096] SEQ ID NO:3+S87E,

[0097] SEQ ID NO:3+G97S,

[0098] SEQ ID NO:3+A98D、E、R,

[0099] SEQ ID NO:3+S106A、W,

[0100] SEQ ID NO:3+N117E,

[0101] SEQ ID NO:3+H120V、D、K、N,

[0102] SEQ ID NO:3+S124M,

[0103] SEQ ID NO:3+P129D

[0104] SEQ ID NO:3+E136Q,

[0105] SEQ ID NO:3+S143W,

[0106] SEQ ID NO:3+S161T,

[0107] SEQ ID NO:3+S163A、G,

[0108] SEQ ID NO.3+Y171L,

[0109] SEQ ID NO:3+A172S,

[0110] SEQ ID NO:3+N185Q,

[0111] SEQ ID NO:3+V199M,

[0112] SEQ ID NO:3+Y209W,

[0113] SEQ ID NO:3+M222Q,

[0114] SEQ ID NO:3+N238H,

[0115] SEQ ID NO:3+V244T,

[0116] SEQ ID NO:3+N261T,

[0117] SEQ ID NO:3+L262N、Q、D、E

[0118] SEQ ID NO:3+N76D+S163G+N238E

[0119] SEQ ID NO:3+S156D+L262E

[0120] SEQ ID NO:3+N238E+L262E

[0121] SEQ ID NO:3+S3T+N76D+S156D+Y209W

[0122] SEQ ID NO:3+H120D+S163G+N261D

[0123] SEQ ID NO:3+S163G+N128Q+N238E+L262E

[0124] SEQ ID NO:3+K27Q+H120D+S163G+N261D

[0125] SEQ ID NO:3+V104T+H120D+S156D+L262E

[0126] SEQ ID NO:3+G195E+V199M

[0127] SEQ ID NO:3+S3T+V4I+N261D

[0128] SEQ ID NO:3+A194P+G195E+V199M+V205I

[0129] SEQ ID NO:3+H120D+A228V

[0130] SEQ ID NO:3+S3T+V4I+A228V

[0131] SEQ ID NO:3+H120D+N261D

[0132] SEQ ID NO:3+H120D+S163G+N261D

[0133] SEQ ID NO:3+N76D+A228V+L262E

[0134] SEQ ID NO:3+N76D+Q137H+S141H+R145H+S163G+N238E

[0135] SEQ ID NO:3+Q137H+S141H+R145H+N238E+L262E

[0136] SEQ ID NO:3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W

[0137] SEQ ID NO:3+H120D+Q137H+S141H+R145H+S163G+N261D

[0138] SEQ ID NO:3+N76D+Q137H+S141H+R145H+A228V+N261D

[0139] SEQ ID NO:3+A194P+G195E+V199M+V205I+A228V+N261D

[0140] SEQ ID NO:3+N62D+H120D

[0141] SEQ ID NO:3+H120D+N261D

[0142] SEQ ID NO:3+N76D+N261D

[0143] SEQ ID NO:3+N76D+A228V+N261D

[0144] SEQ ID NO:3+A194P+G195E+V205I+N261D

[0145] SEQ ID NO:3+N76D+H120D+N261D

[0146] SEQ ID NO:3+H120D+S163G+N261D

[0147] SEQ ID NO:3+S3T+Q59D+N76D

[0148] SEQ ID NO:3+S3T+N76D+H120D

[0149] SEQ ID NO:3+S3T+N76D+A194P+G195E+V199M+V205I

[0150] SEQ ID NO:3+S3T+N76D+S156D

[0151] SEQ ID NO:3+S3T+N76D+Y209W+N261D

[0152] SEQ ID NO:3+S3T+N76D+H120D+Y209W

[0153] SEQ ID NO:3+S3T+N76D+S156D+Y209W

[0154] SEQ ID NO:3+S3T+V4I+N76D+A228V+N261D

[0155] SEQ ID NO:3+S3T+V4I+N76D+H120D

[0156] SEQ ID NO:3+H120D+P131F+A194P+N261D

[0157] SEQ ID NO:3+N76D+E136H+A228V+N261D

[0158] SEQ ID NO:3+N76D+N218S+A228V+N261D

[0159] SEQ ID NO:3+N76D+N218Q+A228V+N261D

[0160] SEQ ID NO:3+N76D+N218A+A228V+N261D

[0161] SEQ ID NO:3+K27Q+R45E

[0162] SEQ ID NO:3+N76D+A228V+L262E

[0163] SEQ ID NO:3+R45E+A88S

[0164] SEQ ID NO:3+S87E+K237E

[0165] SEQ ID NO:3+N261D+L262E

[0166] SEQ ID NO:3+S87E+L262E

[0167] SEQ ID NO:3+S87E+N238E

[0168] SEQ ID NO:3+K27Q+S87E

[0169] SEQ ID NO:3+N76D+N117E

[0170] SEQ ID NO:3+H120D+N238E

[0171] SEQ ID NO:3+Q59D+L262E

[0172] SEQ ID NO:3+K27Q+L262E

[0173] SEQ ID NO:3+H120D+L262E

[0174] SEQ ID NO:3+K27Q+Q59D

[0175] SEQ ID NO:3+K27Q+S156D

[0176] SEQ ID NO:3+K27Q+G61D

[0177] SEQ ID NO:3+Q59D+N261D

[0178] SEQ ID NO:3+Q59D+N117E

[0179] SEQ ID NO:3+K237E+N261D

[0180] SEQ ID NO:3+Q59D+N238E

[0181] SEQ ID NO:3+A15T+H120D+N261D

[0182] SEQ ID NO:3+N76D+S163G+N238E

[0183] SEQ ID NO:3+H120D+S163G+L262E

[0184] SEQ ID NO:3+H120D+S163G+N261D

[0185] SEQ ID NO:3+Q59D+H120D

[0186] SEQ ID NO:3+G61D+N76D

[0187] SEQ ID NO:3+S3T+N76D

[0188] SEQ ID NO:3+S3T+H120D

[0189] SEQ ID NO:3+G61D+H120D

[0190] SEQ ID NO:3+P55S+H120D

[0191] SEQ ID NO:3+S163G+A228V

[0192] SEQ ID NO:3+S163G+N261D;

[0193] SEQ ID NO:3+S3T+S163G

[0194] SEQ ID NO:3+G61D+S163G

[0195] SEQ ID NO:3+S156D+S163G

[0196] SEQ ID NO:3+Q59D+S163G

[0197] SEQ ID NO:3+N76D+S163G

[0198] SEQ ID NO:3+P55S+S163G

[0199] SEQ ID NO:3+H120D+S163G

[0200] SEQ ID NO:3+T58L+Q59D

[0201] SEQ ID NO:3+P55S+T58L

[0202] SEQ ID NO:3+T58L+G97D

[0203] SEQ ID NO:3+T58L+S106A

[0204] SEQ ID NO:3+T58L+A228V

[0205] SEQ ID NO:3+S3T+T58L

[0206] SEQ ID NO:3+T58L+S156D

[0207] SEQ ID NO:3+T58L+Y91H

[0208] SEQ ID NO:3+T58L+H120D

[0209] SEQ ID NO:3+T58L+S163G

[0210] SEQ ID NO:3+S163G+N261D;

[0211] SEQ ID NO:3+T58L+N261D;

[0212] SEQ ID NO:3+T58L+N76D

[0213] SEQ ID NO:3+S3T+N76D+H120D

[0214] SEQ ID NO:3+S3T+N76D+A228V

[0215] SEQ ID NO:3+S3T+N76D+S156D

[0216] SEQ ID NO:3+S3T+N76D+Y209W

[0217] SEQ ID NO:3+S3T+N76D+Y209W+V244T

[0218] SEQ ID NO:3+N76D+H120D

[0219] SEQ ID NO:3+N76D+S156D

[0220] SEQ ID NO:3+H120D+S156D

[0221] SEQ ID NO 3+R45E+L262E

[0222] SEQ ID NO 3+Q59D+G61D

[0223] SEQ ID NO 3+S87E+L262E

[0224] SEQ ID NO 3+G61D+L262E

[0225] SEQ ID NO 3+Q59D+L262E

[0226] SEQ ID NO 3+R45E+Q59D

[0227] SEQ ID NO 3+Q59D+S156D

[0228] SEQ ID NO 3+S156D+L262E

[0229] SEQ ID NO 3+S163G+N238E+L262E

[0230] SEQ ID NO 3+S3T+V4I+S163G+N261D

[0231] SEQ ID NO 3+H120D+S163G+N261D

[0232] SEQ ID NO 3+Y91H+N117H+N238H

[0233] SEQ ID NO 3+T58L+S163G+N261D

[0234] SEQ ID NO 3+S3T+V4I+S163G+N261D

[0235] SEQ ID NO 3+S87E+S163G+L262E

[0236] SEQ ID NO 3+S156D+S163G+L262E

[0237] SEQ ID NO 3+T58LS163G+N261D

[0238] SEQ ID NO 3+S156DS163G+L262E

[0239] SEQ ID NO 3+S3T+N76D+Y209W+N261D+L262E

[0240] The Bacillus subtilis enzyme variant of the present invention preferably has improved stability in liquid detergents compared with the parental protease, and preferably has improved stability in liquid detergents compared with the protease having SEQ ID NO:2 or SEQ ID NO:3.

[0241] It can be mentioned that, as examples of preferred Bacillus subtilis enzyme variants of the present invention, they have improved stability and / or improved detergency in liquid detergents compared to the parent enzyme:

[0242] SEQ ID NO:3+R45E, D, Q

[0243] SEQ ID NO:3+Q58L

[0244] SEQ ID NO:3+Q59D,

[0245] SEQ ID NO:3+G61D,

[0246] SEQ ID NO:3+S87E,

[0247] SEQ ID NO:3+G97S,

[0248] SEQ ID NO:3+A98E,

[0249] SEQ ID NO:3+N117E,

[0250] SEQ ID NO:3+H120D, K, V

[0251] SEQ ID NO:3+P129D

[0252] SEQ ID NO:3+E136Q

[0253] SEQ ID NO:3+Q137H,

[0254] SEQ ID NO:3+S156D,

[0255] SEQ ID NO:3+S160A,

[0256] SEQ ID NO:3+S163A,G

[0257] SEQ ID NO:3+V199M

[0258] SEQ ID NO:3+M222Q

[0259] SEQ ID NO:3+N261T or

[0260] SEQ ID NO:3+L262EQ、N.

[0261] Compared to the parental subtilisin, these preferred subtilisin variants of the present invention exhibit improved stability, such as detergent stability, and / or improved or equivalent washing performance. In this regard, improved washing performance is intended to mean that the variant exhibits superior washing performance on at least one stain compared to the parental subtilisin, wherein the washing performance is determined under suitable conditions in a given detergent composition using a suitable washing performance assay.

[0262] In a preferred embodiment, the improved washing performance is measured using the AMSA test described in the "Methods and Materials" section of this application.

[0263] The washing performance of the variant is preferably at least 1 unit higher, preferably at least 2 units higher, for example at least 3 units higher, for example at least 4 units higher, for example at least 5 units higher, for example at least 6 units higher, for example at least 7 units higher, for example at least 8 units higher, for example at least 9 units higher than the washing performance of the parent Bacillus subtilis enzyme.

[0264] These Bacillus subtilis enzyme variants may further include one or more additional modifications at one or more (e.g., several) other locations selected from the group consisting of: 3, 4, 9, 12, 14, 15, 40, 43, 68, 72, 79, 86, 88, 92, 98, 99, 101, 120, 146, 183, 184, 188, 194, 216, 218, 224, 228, 236, 245, 255, 261, 267, and 270, with preferred locations 9, 15, 68, and / or 120 (according to SEQ ID NO:1). It will be apparent to those skilled in the art that if a location has been modified once, it will not be modified a second time. In a preferred embodiment, a change at any position selected from the group consisting of the following items is a substitution: 3, 4, 9, 12, 14, 15, 40, 43, 68, 72, 79, 86, 88, 92, 98, 99, 101, 120, 146, 183, 184, 188, 194, 216, 218, 224, 228, 236, 245, 255, 261, 267, and 270. In a more preferred embodiment, the Bacillus subtilis enzyme variant further comprises one or more substitutions selected from the group consisting of: 3{D, E, L}, 4I, 9{H, K, R, G}, 12{D, E}, 14T, 15{G, M, S, T}, 40{A, G, M, S, T}, 43{D, E}, 63G, 68{A, G, I, L, M, S, T}, 72{V, L}, N76{D, E}, 79T, 86H, 88V, 92S 98T, 99{E, T, A, G, M, D}, 101L, 120{I, N}, 146S, 183{E, D}, 184{E, D}, 188G, 194P, 216{D, E}, 218{E, D}, 224{S, A, T, G, M}, 228T, 236D, 245{H, K, R}, 255{D, E}, 261{E}, 267{I, L, V} and / or 270{G, M, S, T} (according to SEQ ID NO:1).In a more preferred embodiment, the Bacillus subtilis enzyme variant further comprises one or more substitutions selected from the group consisting of: the mature polypeptide of SEQ ID NO:3, or a polypeptide having at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 95% sequence identity with it, including S3{D, E, L}, V4I, S9{H, K, R, G}, Q12{D, E}, P14T, A15{G, M, S, T}, P40{A, G, M, S, T}, N43{D, E}, V68{A, G, I, L, M, S, T}, I72{V, L}, N76{D, E}, I79T, P86H, A88V, A92S A98T, S99{E, T, A, G, M, D}, S101L, H120{I, N}, G146S, N183{E, D}, N184{E, D}, S188G, A194P, S216{D, E}, N218{E, D}, T224{S, A, T, G, M}, A228T, S236D, Q245{H, K, R}, T255{D, E}, N261{, E}, L267{I, L, V} and / or A270{G, M, S, T}, wherein each position corresponds to the corresponding position of the mature polypeptide of SEQ ID NO:1.

[0265] One embodiment further relates to a method for producing a subtilisase variant having improved stability and / or improved washability compared to subtilisase having the amino acid sequences of SEQ ID NO:2 and / or SEQ ID NO:3, the method comprising the following steps

[0266] a) In the subtilisin having at least 90% sequence identity with SEQ ID NO:2, the amino acid at position 101 corresponding to SEQ ID NO:1 is replaced by a glutamate residue (E).

[0267] b) Further introduce any of the following substitution positions: S156D, L262E, Q137H, S3T, R45E, D, Q, P55N, T58W, Y, L, Q59D, M, N, T, G61D, R, S87E, G97S, A98D, E, R, S106A, W, N117E, H120V, D, K, N, S124M, P129D, E136Q, S143W, S161T, S163A, G, Y171L, A172S, N185Q, V199M, Y209W, M222Q, N238H, V244T, N261T, D or L262N, Q, D.

[0268] c) Recycle the variant.

[0269] According to one embodiment and / or any of the embodiments described above, the present invention relates to a subtilisin variant having at least 90% sequence identity with SEQ ID NO:3, wherein the variant has a glutamate residue (E) at position 101 corresponding to SEQ ID NO:1, and wherein the variant has reduced cellulose binding compared to a subtilisin having the amino acid sequence of SEQ ID NO:3. One embodiment relates to a subtilisin variant having at least 90% sequence identity with SEQ ID NO:3, wherein the variant has a glutamate residue (E) at position 101 corresponding to SEQ ID NO:1, and wherein the variant has reduced cellulose binding compared to a subtilisin having the amino acid sequence of SEQ ID NO:3, and wherein the variant comprises replacing positively charged amino acid residues on the protease surface with neutral or negatively charged residues; or replacing neutral residues on the protease surface with negatively charged residues. According to one embodiment or any of the above embodiments, the present invention relates to a subtilisase variant having at least 90% sequence identity with SEQ ID NO:3, wherein the variant has a glutamate residue (E) at position 101 corresponding to SEQ ID NO:1, and wherein the variant is associated with SEQ ID NO:3. Compared to the subtilisin with the amino acid sequence NO:3, this variant has reduced cellulose binding, and / or includes substitutions of positively charged amino acid residues on the protease surface with neutral or negatively charged residues; or substitutions of neutral residues on the protease surface with negatively charged residues, wherein the subtilisin variant includes substitutions selected from the group consisting of: V4D, E, I, R10N, Q, D, E, S, H17D, K27S, N, Q, E, D, N43E, I44V, R45E, D, Q, N, G46D, S49N, D, P52E, G53D, E, Q59D, G61D, N62D, L75D, N76D, I79D, S87E, G97D, A98E, *103aE, I104T, N117E, H1 20D, E136K, Q, S156D, R170E, Q, N, D, S, N185D, G195E, N218A, K235L, W, N, Q, E, S, K2 37N, Q, D, E, S, N238D, E, V244D, R246Q, E, D, R247S, E, Q, D, K251S, D, Q, E, N, N261D, L 262D, E and S265H, preferably replaced by N117E, S156D, N238E, N261D and L262E, and preferably, the variant further includes replacements selected from S3T, N128Q, Q137H, S141H, R145H, S163G, A194P, V199M, V205I, N218Q or A228V.According to one embodiment and / or any of the embodiments described above, the present invention relates to a subtilisin variant having at least 90% sequence identity with SEQ ID NO:3, wherein the variant has a glutamate residue (E) at position 101 corresponding to SEQ ID NO:1, wherein the variant has reduced cellulose binding compared to a subtilisin having the amino acid sequence of SEQ ID NO:3, and wherein the subtilisin variant further comprises substitutions selected from the group consisting of: N117E+S3T, S156D+S3T, N238E+S3T, N261D+S3T, L262E+S3T, N117E+N128Q, S156D+N128Q, N238E+N128Q, N261D+N128Q, L262E+N128Q, N117E+Q137H. , S156D+Q137H, N238E+Q137H, N261D+Q137H, L262E+Q137H, N117E+S141H, S156D+S141H, N238E+S141H, N261D +S141H, L262E+S141H, N117E+R145H, S156D+R145H, N238E+R145H, N261D+R145H, L262E+R145H, N117E+S163G , S156D+S163G, N238E+S163G, N261D+S163G, L262E+S163G, N117E+A194P, S156D+A194P, N238E+A194P, N261D +A194P、L262E+A194P、N117E+V199M、S156D+V199M、N238E+V199M、N261D+V199M、L262E+V199M、N117E+V205I , S156D+V205I, N238E+V205I, N261D+V205I, L262E+V205I, N117E+N218Q, S156D+N218Q, N238E+N218Q, N261D +N218Q, L262E+N218Q, N117E+A228V, S156D+A228V, N238E+A228V, N261D+A228V, L262E+A228V, S156D+N262E.According to one embodiment and / or any of the above embodiments, the present invention relates to a subtilisin variant having at least 90% sequence identity with SEQ ID NO:3, wherein the variant has a glutamic acid residue (E) at position 101 corresponding to SEQ ID NO:1, wherein the variant has reduced cellulose binding compared to the subtilisin having the amino acid sequence of SEQ ID NO:3, and wherein the subtilisin variant further comprises substitutions selected from the group consisting of:

[0270] SEQ ID NO:3+V4D, E, I

[0271] SEQ ID NO:3+R10N, Q, D, E, S,

[0272] SEQ ID NO:3+H17D,

[0273] SEQ ID NO:3+K27S, N, Q, E, D,

[0274] SEQ ID NO:3+R45E, D, Q, N,

[0275] SEQ ID NO:3+G53D,

[0276] SEQ ID NO:3+Q59D,

[0277] SEQ ID NO:3+G61D,

[0278] SEQ ID NO:3+L75D,

[0279] SEQ ID NO:3+N76D,

[0280] SEQ ID NO:3+I79D,

[0281] SEQ ID NO:3+S87E,

[0282] SEQ ID NO:3+G97D,

[0283] SEQ ID NO:3+A98E,

[0284] SEQ ID NO:3+*103aE,

[0285] SEQ ID NO:3+N117E,

[0286] SEQ ID NO:3+H120D,

[0287] SEQ ID NO:3+E136K, Q

[0288] SEQ ID NO.3+S156D,

[0289] SEQ ID NO:3+R170E、Q、N、D,

[0290] SEQ ID NO:3+N185D,

[0291] SEQ ID NO:3+G195E,

[0292] SEQ ID NO:3+K235L、W、N、Q、E、S,

[0293] SEQ ID NO:3+K237N、Q、D、E、S,

[0294] SEQ ID NO:3+N238D、E,

[0295] SEQ ID NO:3+V244D

[0296] SEQ ID NO:3+R246Q、E、D,

[0297] SEQ ID NO:3+R247S、E,

[0298] SEQ ID NO:3+K251S、D、Q、E、N,

[0299] SEQ ID NO:3+N261D,

[0300] SEQ ID NO:3+L262D、E

[0301] SEQ ID NO:3+S265H

[0302] SEQ ID NO:3+A194P+G195E

[0303] SEQ ID NO:3+G195E+V199M

[0304] SEQ ID NO:3+N76D+A228V+N261D;

[0305] SEQ ID NO:3+N76D+S163G+N238E

[0306] SEQ ID NO:3+S156D+L262E

[0307] SEQ ID NO:3+N238E+L262E

[0308] SEQ ID NO:3+S3T+N76D+S156D+Y209W

[0309] SEQ ID NO:3+K27Q+H120D+S163G+N261D

[0310] SEQ ID NO:3+V104T+H120D+S156D+L262E

[0311] SEQ ID NO:3+V104T+S156D+L262E

[0312] SEQ ID NO:3+Q137H+S141H+R145H+N238E+L262E

[0313] SEQ ID NO:3+S3T+V4I+A228V;

[0314] SEQ ID NO:3+H120D S163G N261D

[0315] SEQ ID NO:3+N76D+S101E+A228V+L262E;

[0316] SEQ ID NO:3+N76D+Q137H+S141H+R145H+S163G+N238E

[0317] SEQ ID NO:3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W

[0318] SEQ ID NO:3+H120D+Q137H+S141H+R145H+S163G+N261D

[0319] SEQ ID NO:3+A194P+G195E+V199M+V205I;

[0320] SEQ ID NO:3+S3T+N76D+A194P+G195E+V199M+V205I;

[0321] SEQ ID NO:3+A228V+N261D;

[0322] SEQ ID NO:3+N76D+A228V;

[0323] SEQ ID NO:3+S3T+V4I+N261D;

[0324] SEQ ID NO:3+H120D+A228V;

[0325] SEQ ID NO:3+N76D+N261D;

[0326] SEQ ID NO:3+A194P+G195E+V199M+V205I+A228V+N261D;

[0327] SEQ ID NO:3+A194P+G195E+V205I+A228V; or

[0328] SEQ ID NO:3+H120D+N261D.

[0329] Preferably, these variants are selected from the group consisting of the following items:

[0330] SEQ ID NO:3+N238E+L262E

[0331] SEQ ID NO:3+S156D+L262E

[0332] SEQ ID NO:3+S3T+V4I+A228V;

[0333] SEQ ID NO:3+G195E+V199M

[0334] SEQ ID NO:3+H120D S163G N261D

[0335] SEQ ID NO:3+N76D+A228V+N261D;

[0336] SEQ ID NO:3+S3T+N76D+S156D+Y209W

[0337] SEQ ID NO:3+Q137H+S141H+R145H+N238E+L262E

[0338] SEQ ID NO:3+Q137H+S141H+R145H+S156D+L262E

[0339] SEQ ID NO:3+N76D+Q137H+S141H+R145H+A228V+N261D;

[0340] SEQ ID NO:3+N76D+Q137H+S141H+R145H+S163G+N238E

[0341] SEQ ID NO:3+H120D+Q137H+S141H+R145H+S163G+N261D

[0342] SEQ ID NO:3+S3T+N76D+Q137H+S141H+R145H+S156D+Y209W,

[0343] These positions correspond to positions in SEQ ID NO:1, and the subtilisin variant has at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 90%, for example, at least 95%, for example, at least 98%, or at least 99% sequence identity with SEQ ID NO:3. One embodiment relates to a nucleotide sequence encoding a variant according to any of the above embodiments, an expression vector comprising the nucleotide sequence, and a recombinant host cell comprising the nucleotide sequence or the expression vector. One embodiment further relates to a method for producing a subtilisin variant having reduced cellulose binding compared to a subtilisin having the amino acid sequence of SEQ ID NO:2, the method comprising the following steps:

[0344] a) In the subtilisin having at least 90% sequence identity with SEQ ID NO:2, the amino acid at position 101 corresponding to SEQ ID NO:1 is replaced by a glutamate residue (E).

[0345] b) Further introduce any of the following substitution positions: 4D, E, I; 10N, Q, D, E, S; H17D; K27S, N, Q, E, D; N43E; I44V; R45E, D, Q, N; G46D; S49N, D; P52E; G53D, E; Q59D; G61D; N62D; L75D; N76D; I79D; S87E; G97D; A98E; *103aE; I104T , N117E, H120D, E136K, Q, S156D, R170E, Q, N, D, S, N185D, G195E, N218A, K235L, W, N, Q, E, S, K237 N, Q, D, E, S, N238D, E, V244D, R246Q, E, D, R247S, E, Q, D, K251S, D, Q, E, N, N261D, L262D, E and S265H;

[0346] c) Recycle the variant.

[0347] These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino or carboxyl-terminal extensions, such as N-terminal methionine residues; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine segments, antigenic epitopes, or binding domains.

[0348] 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 are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0349] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the peptide's thermal stability, change its substrate specificity, change its optimal pH, and so on.

[0350] Essential amino acids in a polypeptide can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the protease activity of the resulting mutant molecule is tested to identify amino acid residues essential for the molecule's activity. The active site of the enzyme or other biological interaction 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 contract site. For BPN' (SEQ ID NO:1), the catalytic triad comprising amino acids S221, H64, and D32 is essential for the enzyme's protease activity.

[0351] These Bacillus subtilis enzyme variants can consist of 150 to 350, such as 175 to 330, 200 to 310, 220 to 300, 240 to 290, 260 to 280, or 269, 270, 271, 272, 273, 274 or 275 amino acids.

[0352] In one embodiment, the subtilisin enzyme variant has improved washing performance. In another embodiment, the subtilisin enzyme variant has improved stability, preferably improved stability during washing.

[0353] In one embodiment, the subtilisin enzyme variant exhibits improved stability in liquid detergents compared to the parent enzyme, wherein stability is measured using a 'stability assay' as described in Example 4 of the Materials and Methods section herein. In one embodiment, the subtilisin enzyme variant exhibits improved stability compared to the peptide of SEQ ID NO:2, wherein stability is measured using a 'stability assay' as described in Example 4 of the Materials and Methods section herein. In one embodiment, the subtilisin enzyme variant exhibits improved stability compared to the peptide of SEQ ID NO:3, wherein stability is measured using a 'stability assay' as described in Example 4 of the Materials and Methods section herein.

[0354] In one embodiment, the subtilisin enzyme exhibits improved washing performance compared to the parent enzyme, wherein the washing performance is measured using an automated mechanical stress measurement (AMSA) as described in Example 7 of the Materials and Methods section herein. In one embodiment, the subtilisin enzyme variant exhibits improved washing performance compared to the peptide of SEQ ID NO:2, wherein the washing performance is measured using an automated mechanical stress measurement (AMSA) as described in Example 7 of the Materials and Methods section herein. In one embodiment, the subtilisin enzyme variant exhibits improved washing performance compared to the peptide of SEQ ID NO:3, wherein the washing performance is measured using an automated mechanical stress measurement (AMSA) as described in Example 7 of the Materials and Methods section herein.

[0355] Parental protease

[0356] Enzymes that cleave amide bonds in protein substrates are classified as proteases, or (interchangeably) peptidases.

[0357] serine protease

[0358] A serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds, and contains an essential serine residue at its active site.

[0359] Bacterial serine proteases have a molecular weight range of 20,000 to 45,000 Daltons. They are inhibited by diisopropyl fluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, which is also a serine protease. In a narrower sense, alkaline proteases, comprising a subgroup, reflect the high optimum pH of some serine proteases from pH 9.0 to 11.0.

[0360] Bacillus subtilis enzyme

[0361] Siezen et al. (1991), *Protein Engineering* 4:719-737 and Siezen et al. (1997), *Protein Science* 6:501-523 proposed a subgroup of serine proteases tentatively named subtilases. These were defined by homology analysis of more than 170 amino acid sequences of serine proteases previously known as subtilase-like proteases. Subtilases were previously generally defined as serine proteases produced by Gram-positive bacteria or fungi, but are now, according to Siezen et al., a subgroup of subtilases. A wide variety of subtilases have been identified, and the amino acid sequences of many have been determined. For a more detailed description of these subtilases and their amino acid sequences, see Siezen et al. (1997).

[0362] subtilisin

[0363] A subgroup of subtilisases is subtilis proteases, which are serine proteases from the S8 family, particularly from the S8A subfamily, as defined by the MEROPS database (http: / / merops.sanger.ac.uk / cgi-bin / famsum?family=S8).

[0364] BPN' and Savinase have MEROPS numbers S08.034 and S08.003, respectively.

[0365] Parental Bacillus subtilis enzymes

[0366] The term "parental subtilisin" describes a subtilisin as defined by Siezen et al. (1997), Protein Science 6:501-523. See the above description of "subtilisin" for more detailed information. Parental subtilisin can also be a subtilisin isolated from a natural source, wherein it has been subsequently modified (e.g., one or more substitutions, one or more replacements, one or more deletions, and / or one or more insertions of one or more amino acid side chains) while retaining the characteristics of a subtilisin. Furthermore, parental subtilisin can be a subtilisin that has already been prepared using DNA shuffling technology.

[0367] Alternatively, the term "parental subtilisin" may be referred to as "wild-type subtilisin." This parental subtilisin preferably belongs to the subtilisin subgroup. A subgroup of subtilisins, I-S1 or "true" subtilisins, includes "standard" subtilisins such as subtilisin 168 (BSS168), subtilisin BPN', and Carlsberg subtilisin (subtilisin Carlsberg) (…). Novozymes), and Bacillus subtilis protease DY (BSSDY).

[0368] Siezen et al. (see above) identified another subgroup of subtilisinases, I-S2, or strongly basic subtilisinase. The I-S2 subgroup of proteases is described as strongly basic subtilisinase and includes several enzymes such as subtilisinase PB92 (BAALKP). Genencor International, Inc., and Savinase 309 (Savinase) Novozymes (A / S), Bacillus subtilis protease 147 (BLS147) (ESPERASE) Novozymes (A / S) and alkaline elastase YaB (BSEYAB). BPN' is a subtilisin BPN' derived from Bacillus amyloliquefaciens, and BPN' has the amino acid sequence SEQ ID NO:1.

[0369] For reference, Table 1 below lists some acronyms for the different Bacillus subtilis enzymes mentioned above. For other acronyms, see Siezen et al. (1991 and 1997).

[0370] Table 1: Acronyms of different Bacillus subtilis enzymes

[0371]

[0372]

[0373] Homologous Bacillus subtilis enzyme sequence

[0374] 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.

[0375] Based on this description, it is routine for those skilled in the art to identify suitable homologous subtilisases that can be modified according to the present invention.

[0376] Essentially homologous parental Bacillus subtilis 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 amino- or carboxyl-terminal extensions, such as N-terminal methionine residues, small linker peptides up to about 20-25 residues, or small extensions (affinity tags) that facilitate purification, such as polyhistidine fragments, or proteins.

[0377] While the aforementioned changes are preferably of less significant nature, such changes can also have substantial nature, such as the fusion of larger polypeptides of up to 300 amino acids or more as extensions of amino or carboxyl ends.

[0378] Nucleic acid probes can be designed using the polypeptide or fragment thereof 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 genomic DNA or cDNA of cells of interest, following standard DNA blotting procedures, to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the complete sequence, but should be at least 15 nucleotides long, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, 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 containing biotin (or avidin) to detect corresponding genes.

[0379] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened based on DNA that hybridizes to and encodes the parental DNA with the probes 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 nitrocellulose or other suitable carrier materials and immobilized thereon.

[0380] 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.

[0381] 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 that the expression of the fusion peptide is under the control of the same promoter and terminator. Fusion peptides can also be constructed using integrin technology, wherein the fusion peptide is generated post-translational.

[0382] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved during the secretion of the fusion protein, thereby releasing both peptides.

[0383] The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0384] The parent can be a bacterial protease. For example, the parent can be a Gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. Tomyces protease; or a Gram-negative bacterial polypeptide, such as Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma protease.

[0385] On one hand, the parent is a protease of Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus scoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis.

[0386] In one respect, the parent is amylolytic protease, such as the protease of SEQ ID NO:1 or its mature polypeptide.

[0387] On the other hand, the parent is a Bacillus retarder protease, such as the protease of SEQ ID NO:2 or its mature polypeptide.

[0388] 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).

[0389] 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 a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, it can be isolated or cloned using a variety of techniques known to those skilled in the art.

[0390] Preparation of variants

[0391] The present invention also relates to a method for obtaining Bacillus subtilis enzyme variants with protease activity.

[0392] 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.

[0393] 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.

[0394] 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 fragment to ligate to each other.

[0395] Site-directed mutagenesis can also be achieved in vivo using methods known in the art.

[0396] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0397] 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 multi-channel microchip-based techniques and similar techniques that synthesize and assemble oligonucleotides on optically programmable microfluidic chips.

[0398] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by relevant screening procedures. Other methods that can be used include error-prone PCR, phage display, and region-directed mutagenesis.

[0399] Mutagenesis / reorganization methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenesis-encoded peptides expressed by host cells. Mutagenesis-encoded 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.

[0400] 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.

[0401] Polynucleotides

[0402] The present invention also relates to polynucleotides encoding variants of the invention.

[0403] Nucleic acid constructs

[0404] 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.

[0405] 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.

[0406] 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 mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0407] Examples of suitable promoters guiding the transcription of the nucleic acid constructs of the present invention in bacterial host cells are promoters obtained from the following: 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, Escherichia coli lac operon, Escherichia coli trc promoter, Streptomyces agarosease gene (dagA), and prokaryotic β-lactamase gene, together with tac promoter.

[0408] 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.

[0409] The preferred terminator for bacterial host cells is obtained from genes targeting the following: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0410] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[0411] Examples of obtaining suitable mRNA stable regions from the Bacillus thuringiensis cryIIIA gene and the Bacillus subtilis SP82 gene.

[0412] 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.

[0413] The effective signal peptide coding sequences for bacterial host cells are obtained from the genes of Bacillus NCIB 11837 maltose amylase, Bacillus subtilis protease, Bacillus subtilis calc-lactamase, Bacillus thermophilus glutamyl-amylase, Bacillus thermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA.

[0414] 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, Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae sacchari-factor.

[0415] 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.

[0416] 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.

[0417] expression carrier

[0418] 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 containing 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.

[0419] 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.

[0420] 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 one or more chromosomes 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.

[0421] 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.

[0422] Examples of bacterial selective markers include the dal gene in Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (e.g., resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline).

[0423] The vector preferably contains one or more elements that allow the vector to integrate into the host cell’s genome or to replicate autonomously in the cell independently of the genome.

[0424] 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 integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, 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 within 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.

[0425] 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.

[0426] 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.

[0427] 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 selectable marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0428] The methods for connecting the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art.

[0429] host cells

[0430] 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.

[0431] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0432] 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, Fusobacterium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0433] 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 sclerosus, Bacillus splenium, Bacillus stenosis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0434] 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.

[0435] 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.

[0436] DNA can be introduced into Bacillus cells via protoplast transformation, competent cell transformation, electroporation, or conjugation. DNA can be introduced into Escherichia coli cells via protoplast transformation or electroporation. DNA can be introduced into Streptomyces cells via protoplast transformation, electroporation, conjugation, or transduction. DNA can be introduced into Pseudomonas cells via electroporation or conjugation. DNA can be introduced into Streptococcus cells via natural competence, protoplast transformation, electroporation, or conjugation. However, any method known in the art for introducing DNA into host cells may be used.

[0437] Generation method

[0438] The present invention also relates to methods for generating variants, the methods comprising: (a) culturing host cells of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant.

[0439] These host cells are cultured in a nutrient medium suitable for generating 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 containing 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.

[0440] 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.

[0441] 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 routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0442] 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.

[0443] Alternatively, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0444] Composition

[0445] In one aspect, the Bacillus subtilis enzyme variant according to the invention exhibits improved washing performance compared to the parent enzyme or compared to a protease having an amino acid sequence consistent with the variant but without alteration at one or more of the designated positions, or compared to the polypeptide of SEQ ID NO:2 or compared to the polypeptide of SEQ ID NO:3, wherein the washing performance is measured using automated mechanical stress measurement (AMSA).

[0446] In another aspect, the Bacillus subtilis enzyme variant according to the invention exhibits improved stability compared to the parent enzyme or compared to a protease having an amino acid sequence consistent with the variant but without alteration at one or more of the specified positions, or compared to the polypeptide of SEQ ID NO:2 or compared to the polypeptide of SEQ ID NO:3, preferably improved stability during washing, wherein stability is measured by a 'stability determination' as described in Example 4 of the Materials and Methods section herein.

[0447] The composition may be a detergent composition comprising a Bacillus subtilis enzyme variant according to the invention that can be used in cleaning processes such as laundry or hard surface cleaning.

[0448] The selection of other components is within the capabilities of those skilled in the art and includes conventional ingredients, including the exemplary non-limiting components described below. For fabric care, the selection of components may include considerations such as the type of fabric to be cleaned, the type and / or extent of the stain, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the components mentioned below are categorized 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 one of ordinary skill in the art.

[0449] The enzyme of the present invention

[0450] In one embodiment of the invention, the polypeptide of the invention may be added to the detergent composition in amounts corresponding to: 0.01-200 mg enzyme protein / L detergent solution, preferably 0.05-50 mg enzyme protein / L detergent solution, particularly 0.1-10 mg enzyme protein / L detergent solution.

[0451] The composition for use in an automatic dishwasher (ADW) may, for example, include 0.0001%-50%, 0.001%-30%, 0.01%-20%, or 0.5%-15% of enzyme protein by weight of the composition.

[0452] Compositions used in laundry granulation may, for example, contain 0.0001%-50%, such as 0.001%-20%, such as 0.01%-10%, such as 0.05%-5% of enzyme protein by weight of the composition.

[0453] Compositions intended for use in laundry detergents may, for example, include 0.0001%-10%, such as 0.001%-7%, such as 0.1%-5% of an enzyme protein by weight of the composition.

[0454] One or more enzymes in a detergent composition can be stabilized using conventional stabilizers, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters, or phenyl boric acid derivatives (e.g., 4-formylphenylboronic acid)). The composition can be formulated as described, for example, in WO 92 / 19709 and WO 92 / 19708, or the Bacillus subtilis enzyme variants according to the invention can be stabilized using peptide aldehydes or ketones as described in WO 2005 / 105826 and WO 2009 / 118375.

[0455] Variations of the present invention may also be incorporated into detergent formulations disclosed in WO 97 / 07202 (incorporated herein by reference).

[0456] surfactants

[0457] 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 one specific embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. Typically, the surfactants are present at levels ranging 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%. This or these surfactants are selected based on the desired cleaning application, and this or these surfactants include any one or more conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be utilized.

[0458] When included therein, the detergent will typically contain about 1% to about 40% by weight, for example about 5% to about 30%, including about 5% to about 15%, or about 20% to about 25% of 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) (e.g., 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.

[0459] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0460] 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%, for example 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 (e.g., 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 available under the trade names SPAN and TWEEN, and combinations thereof.

[0461] When included therein, the detergent will typically contain from about 0% to about 10% 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.

[0462] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

[0463] Water-soluble

[0464] A co-hydrophilic solvent is a compound that dissolves hydrophobic compounds (or conversely, polar substances in a nonpolar environment) in an aqueous solution. Typically, co-hydrophilic solvents possess both hydrophilic and hydrophobic characteristics (such as the so-called amphiphilic properties known from surfactants); however, the molecular structure of co-hydrophilic solvents generally does not favor spontaneous aggregation. Co-hydrophilic solvents do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, occurs and lipids form micelles, thin layers, or other well-defined intermediate phases. Instead, many co-hydrophilic solvents exhibit a continuous type of aggregation process, where the size of the aggregates increases with increasing concentration. However, many co-hydrophilic solvents 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). Co-hydrophilic solvents are classically used across industries from pharmaceuticals, personal care, and food to technical applications. The use of co-hydrophilic solvents in detergent compositions allows, for example, more concentrated surfactant formulations (as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0465] Detergents may contain 0-5% by weight, for example, 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.

[0466] Builders and co-builders

[0467] Detergent compositions may contain approximately 0-65% by weight, such as approximately 5% to approximately 45%, of detergent builders or co-builders, or mixtures thereof. In dishwashing detergents, the level of builders is typically 40%-65%, particularly 50%-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 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 silicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2'-meta-aminotriethanol), and carboxymethyl inulin (CMI) and combinations thereof.

[0468] The detergent composition may also contain 0-20%, for example, about 5% to about 10% by weight, a detergent co-agent or a mixture thereof. The detergent composition may include a co-agent alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of co-agents 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'-dibutanoic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta-(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), and iminodisuccinic acid (IDS). N-(2-sulfomethyl)-aspartic acid (IDA), N-(2-sulfoethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), 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), o- Aminobenzoic acid-N,N-diacetic acid (ANDA), sulfanilamide-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), and sulfonylmethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and their salts. Other exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and US 5977053.

[0469] bleaching system

[0470] The detergent may contain 0-50% by weight, such as about 0.1% to about 25%, of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborate, preformed peracids, and mixtures thereof. Suitable preformed peracids include, but are not limited to: peroxycarboxylic acids and their salts, percarbonates and their salts, perimidicacids and their salts, peroxymonosulfate and their 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 a bleaching activator combination with the peracid, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, perphosphates, and sodium salts of persilicates. The term bleaching activator herein means a bleaching agent that reacts with a peroxide bleaching agent (like peroxide...). The hydrogen peroxide reacts to form a peracid compound. The resulting peracid constitutes an activated bleaching agent. Suitable bleaching activators to be used herein include those belonging to the classes of ester amides, imides, or acid anhydrides. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), diperoxylauric acid, 4-(dodecyloxy)benzenesulfonate (LOBS), 4-(decyloxy)benzenesulfonate, 4-(decyloxy)benzoate (DOBS), 4-(nonanoyloxy)benzenesulfonate (NOBS), and / or those disclosed in WO Those in 98 / 17767. Specific families of bleaching activators of interest are disclosed in EP 624154, and in that family, acetylacetic acid triethyl ester (ATC) is particularly preferred. ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly because it eventually degrades into citric acid and alcohol. Furthermore, acetylacetic acid triethyl ester and triacetin exhibit good hydrolytic stability in the product during storage, and it is an effective bleaching activator. Finally, ATC provides good washing ability for laundry detergent 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-(phthalimide)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:

[0471]

[0472] (iii) and its mixtures; wherein each R 1 Independently, it is a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably, each R1 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, iso-tridecyl, and iso-pentadecanyl. 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.

[0473] polymer

[0474] The detergent may contain 0-10% by weight, such as 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1%, of a polymer. Any polymer known in the art for use in detergents may be used. The polymer may function as a co-adjuvant 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 silicone, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (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.

[0475] Fabric colorant

[0476] The detergent compositions of the present invention may further include fabric colorants, such as dyes or pigments, which, when formulated in the detergent composition, can deposit on the fabric when the fabric comes into contact with a detergent liquid comprising the detergent composition, and thus alter the color of the fabric by absorption / reflection of visible light. 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, for example as described in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276 and EP 1876226 (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 toners are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0477] Other enzymes

[0478] Detergent additives, together with detergent compositions, may include one or more additional enzymes, such as proteases, lipases, keratases, amylases, glycoses, cellulases, pectinases, mannanases, arabinases, galactanases, xylanases, oxidases such as laccases, and / or peroxidases.

[0479] Generally, the properties of one or more selected enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0480] Cellulase

[0481] Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Apocytozoa*, such as 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.

[0482] 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 cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0483] Examples of cellulases exhibiting endo-β-1,4-glucanase activity (EC 3.2.1.4) are those already described in WO 02 / 099091.

[0484] Other examples of cellulases include family 45 cellulases described in WO 96 / 29397, and particularly SEQ ID corresponding to WO 02 / 099091. The following positions in NO:8 contain variations of the form with substitutions, insertions, and / or deletions at one or more of the following locations: 2, 4, 7, 8, 10, 13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79, 80, 82, 84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101, 102, 103, 113, 114, 117, 119, 121, 133, 136. 137, 138, 139, 140a, 141, 143a, 145, 146, 147, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165, 168, 170, 171, 172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188, 191, 192, 195, 196, 200, and / or 20, preferably selected from P19A, G20K, Q44K, N48E, Q119H, or Q146R.

[0485] Commercially available cellulases include Celluzyme TM and Carezyme TM (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM (Kao Corporation).

[0486] protease

[0487] Suitable other proteases include those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. This includes chemically modified mutants or protein-engineered mutants. 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.

[0488] The term "subtilase" refers to the serine protease subgroup according to Siezen et al., Protein Engineering 4 (1991) 719-737 and Siezen 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. Subtilases can be divided 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.

[0489] Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus tarda*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and subtilisin protease lentus, subtilisin Novo, subtilisin Carlsberg, *Bacillus licheniformis*, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168 as described in WO 89 / 06279, and protease 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 Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0490] Other 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).

[0491] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0492] Examples of useful proteases are variants of the following: WO 92 / 19729, WO 96 / 034946, WO98 / 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 11 / 036264, especially with variants that have 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, numbered using BPN'. More preferably, these Bacillus subtilis enzyme variants may contain 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, H120 D,N,N123S,S128L,P129Q,S130A,G160D,Y167A,R170S,A194P,G195E,V199M,V205I,L217D,N218D,M222S,A232V,K235L,Q236H,Q245R,N252K,T274A (numbered using BPN').

[0493] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm Durazym Tm , Saiwei Protease Saiwei Protease as well as (Novozymes), those sold under the following product names: as well as (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0494] Lipase and keratinase

[0495] Suitable lipases and keratins include those of bacterial or fungal origin. This also includes chemically modified or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermophilic*, such as those from *Thermophilic Hypotherium* (formerly named *Pyrophyte*) as described in EP 258068 and EP305216; cutinases from the genus *Pyrophyte*, such as *Pyrophyte Specific* (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 and WO 96 / 27002), *Pseudomonas wisconsinensis* (WO 96 / 12012); and GDSL-type *Streptomyces* lipases (WO 96 / 12012). 10 / 065455); cutinase from *Oryza sativa* (WO 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* (WO 11 / 150157) and *S. pristinaespiralis* (WO 12 / 137147).

[0496] Other examples are the lipase variants described in, for example, 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.

[0497] Preferred commercially available lipase products include Lipolase TM Lipex TM Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0498] 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).

[0499] amylase

[0500] Suitable amylases that can be used with the Bacillus subtilis enzyme variants of the present invention can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified mutants or protein-engineered mutants. Amylases include, for example, α-amylases obtained from the genus Bacillus, such as from specific strains of Bacillus licheniformis as described in more detail in GB 1,296,839.

[0501] Suitable amylases include those having SEQ ID NO:3 in WO 95 / 10603 or variants 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, for example, 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.

[0502] Different suitable amylases include the amylase having SEQ ID NO:6 in WO 02 / 010355 or a variant thereof having 90% sequence identity. Preferred variants are those having a deletion at positions 181 and 182 and a substitution at position 193.

[0503] 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 variants of the hybrid α-amylase, including residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the SEQ ID NO:4 of WO 2006 / 066594, are those having the following substitutions:

[0504] M197T;

[0505] H156Y+A181T+N190F+A209V+Q264S; or

[0506] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0507] 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 with 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 with deletions at positions R181 and G182 or positions H183 and G184.

[0508] Other usable amylases are those having WO 96 / 023873 of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7, 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 having 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. More preferred variants are those having deletions at positions 181 and 182 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 in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0509] Other amylases that may be used are those having SEQ ID NO:2 in WO 08 / 153815, SEQ ID NO:10 in WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO:2 in WO 08 / 153815 or 90% sequence identity with SEQ ID NO:10 in 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.

[0510] Other suitable amylases are the amylase having SEQ ID NO:2 in WO 09 / 061380 or a variant thereof having 90% sequence identity with SEQ ID NO:2. Preferred variants of SEQ ID NO:2 are those having a truncated and / or substituted, deleted, or inserted C-terminus 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 at 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 the deletion of 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:

[0511] N128C+K178L+T182G+Y305R+G475K;

[0512] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0513] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0514] 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.

[0515] Other suitable amylases are those having SEQ ID NO:12 in WO01 / 66712 or variants thereof having 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 a variant having additional substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with the most preferred variants having additional substitutions at all of these positions.

[0516] Other examples are, for instance, those amylase variants described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0517] Commercially available amylase is Duramyl TM Terminyl TM Fungayl TM Stainzyme TM StainzymePlus TM Natalase TM Liquozyme X and BAN TM (From Novozymes) and Rapidase TM Purastar TM / Effectenz TM Powerase and Preferenz S100 (from Genencor International Inc. / DuPont).

[0518] Peroxidase / oxidase

[0519] Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified mutants or protein-engineered mutants. 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.

[0520] Commercially available peroxidases include Guardzyme. TM (Novozymes)

[0521] Detergent enzymes can be incorporated into detergent compositions by adding a single additive containing one or more enzymes, or by adding a combination of additives containing all of these enzymes. Detergent additives, whether alone or in combination, can be formulated as, for example, granules, liquids, slurries, etc., with preferred formulations being granules, particularly dust-free granules; liquids, particularly stabilized liquids; or slurries.

[0522] 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 waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated aliphatic alcohols having 15 to 80 ethylene oxide units, wherein the alcohol contains 12 to 20 carbon atoms; fatty alcohols; fatty acids; and mono-, di-, 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. The protected enzyme can be prepared according to the methods disclosed in EP 238,216.

[0523] auxiliary materials

[0524] Any detergent component known in the art for use in laundry detergents may also be used. Other optional detergent components include preservatives, shrinkage inhibitors, anti-fouling 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 finishing agents (including clays), fillers / processing aids, optical brighteners / brighteners, foaming agents, foam (foam) regulators, fragrances, soil suspending agents, softeners, defoamers, 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.

[0525] dispersant The detergent compositions of the present invention may further comprise dispersants. Specifically, powdered detergents may comprise 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.

[0526] Dye transfer inhibitors The detergent compositions of the present invention may further 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 a level 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.

[0527] Fluorescent whitening agentThe detergent compositions of the present invention 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 the compositions of the present invention. 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-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-diphenylamino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine 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-(stilbene-4"-naphthalene-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG (Basel, Switzerland). Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tianlaibao CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate. Also preferred is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-alkylaminocoumarin. Suitable fluorescent whitening agent levels range from about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from about 0.2 wt% to lower levels up to an upper limit of 0.5 wt% or even 0.75 wt%.

[0528] Fouling release polymersThe detergent compositions of the present invention may also include one or more dirt-releasing polymers that help remove dirt from fabrics, such as cotton or polyester-based fabrics, particularly hydrophobic dirt from polyester-based fabrics. The dirt-releasing polymers may 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 Dekker, Inc. Another type of dirt-releasing polymer is an amphiphilic alkoxylated oil stain cleaning polymer comprising a core structure and a plurality of alkoxylated groups attached to the 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 dirt-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, such as those 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.

[0529] Anti-redeposition agent The detergent compositions of the present invention may further 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 the category of dirt-releasing polymers may also be used as anti-redeposition agents.

[0530] Other suitable excipientsIncluding but not limited to shrink-proof agents, wrinkle-resistant agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, water-soluble agents, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastic agents used in liquid detergents.

[0531] Preparation of detergent products

[0532] The detergent composition can be in any conventional form, such as strips, homogeneous tablets, tablets with two or more layers, bags with one or more chambers, regular or compressed powders, granules, pastes, gels, or regular, compressed or concentrated liquids. Various detergent formulations exist, such as layers (same or different phases), bags, and forms for mechanical feeding devices.

[0533] The bag can be configured as a single or multiple chambers. It can have any form, shape, and material suitable for preserving the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane encapsulating an inner volume. The inner volume can be divided into chambers of the bag. Preferred membranes are polymeric materials that form membranes or sheets, preferably polymers. 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 level of the polymer (e.g., PVA) in the membrane 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, as sold by Chris Craft In. Prod., 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 cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may be structurally different from those containing solids. Reference: (US 2009 / 0011970 A1).

[0534] Detergent components can be physically separated from each other by compartments in different layers of water-soluble pouches or tablets. This avoids negative storage interactions between components. The different dissolution profiles of each compartment in the washing solution can also cause delayed dissolution of selected components.

[0535] Non-unit-volume 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 0-30% organic solvents. Liquid or gel detergents may be non-aqueous.

[0536] Laundry soap bars

[0537] The Bacillus subtilis enzyme variant of the present invention can be added to laundry soap bars and used for hand washing of fabrics and / or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, synthetic detergent bars, and detergent bars. The types of bars are generally distinguished by the type of surfactant they contain, and the term laundry soap bar includes those containing soaps derived from fatty acids and / or synthetic soaps. Laundry soap bars have a physical form that is solid at room temperature, rather than liquid, gel, or powder. The term solid is defined as a physical form that does not change significantly over time, i.e., if a solid object (e.g., a laundry soap bar) is placed in a container, the solid object will not change to fill the container containing the solid object. The bar is typically in bar form but can be a solid in other solid shapes, such as round or oval.

[0538] The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hyposulfate adducts or hemiacetal adducts), boric acid, borates, borax and / or phenylboronic acid derivatives such as 4-carboxylic acid, one or more soaps or synthetic surfactants, polyols such as glycerol, pH-controlling compounds such as fatty acids, citric acid, acetic acid and / or formic acid, and / or salts of monovalent cations and organic anions, wherein the monovalent cation may be, for example, Na+. + K + or NH4 + Furthermore, the organic anion can be, for example, a formate, acetate, citrate, or lactate, and therefore the salt of the monovalent cation and the organic anion can be, for example, sodium formate.

[0539] Washing soap bars may also contain complexing agents such as EDTA and HEDP, fragrances and / or different types of fillers, surfactants such as anionic synthetic surfactants, builders, polymerized soil releasers, detergent chelators, stabilizers, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, defoamers, structural agents, binders, leachants, bleach activators, clay detergents, anti-redeposition agents, polymeric dispersants, brighteners, fabric softeners, fragrances and / or other compounds known in the art.

[0540] Laundry soap bars can be processed in conventional laundry soap bar manufacturing equipment, such as, but not limited to, mixers, pressing machines (e.g., two-stage vacuum pressing machines), extruders, cutters, logo-stamper machines, cooling tunnels, and packaging machines. This invention is not limited to preparing laundry soap bars by any single method. Premixes can be added to the soap at different stages of the process. For example, a premix comprising soap, enzymes, optionally one or more other enzymes, protease inhibitors, and salts of monovalent cations and organic anions can be prepared and then the mixture can be pressed into bars. Enzymes, such as protease inhibitors in liquid form, and optionally other enzymes can be added simultaneously. In addition to the mixing and pressing steps, the process may further include grinding, extrusion, cutting, molding, cooling, and / or packaging steps.

[0541] Granular Detergent Formulation

[0542] As described in WO 09 / 092699, EP 1705241, EP 1382668, WO 07 / 001262, US 6472364, WO04 / 074419 or WO 09 / 102854, granular detergents can be formulated. Other useful detergent formulations are described in the following: WO 09 / 124162, WO 09 / 124163, WO 09 / 117340, WO 09 / 117341, WO 09 / 117342, WO09 / 072069, WO 09 / 063355, WO 09 / 132870, WO 09 / 121757, WO 09 / 112296, WO 09 / 112298, WO09 / 103822, WO 09 / 087033, WO 09 / 050026, WO 09 / 047125, WO 09 / 047126, WO 09 / 047127, WO09 / 047128, WO 09 / 021784, WO 09 / 010375、WO 09 / 000605、WO 09 / 122125、WO 09 / 095645、WO09 / 040544、WO 09 / 040545、WO 09 / 024780、WO 09 / 004295、WO 09 / 004294、WO 09 / 121725, WO09 / 115391, WO 09 / 115392, WO 09 / 074398, WO 09 / 074403, WO 09 / 068501, WO 09 / 065770, WO09 / 021813, WO 09 / 030632 and WO 09 / 015951.

[0543] WO 2011025615、WO 2011016958、WO 2011005803、WO 2011005623、WO2011005730、WO 2011005844、WO 2011005904、WO 2011005630、WO 2011005830、WO2011005912、WO 2011005905、WO 2011005910、WO 2011005813、WO 2010135238、WO2010120863、WO 2010108002、WHERE 2010111365, WO 2010108000, WO 2010107635, WO2010090915, WO 2010033976, WO 2010033746, WO 2010033747, WO 2010033897, WO2010033979, WO 2010030540, WO 2010030541, WO 2010030539, WO 2010024467, WO2010024469, WO 2010024470, WO 2010025161, WO 2010014395、WO 2010044905、

[0544] WO 2010145887, WO 2010142503, WO 2010122051, WO 2010102861, WO2010099997, WO 2010084039, WO 2010076292, WO 2010069742, WO 2010069718, WO2010069957, WO 2010057784, WO 2010054986, WO 2010018043, WO 2010003783, WO2010003792

[0545] WO 2011023716, WO 2010142539, WO 2010118959, WO 2010115813, WO2010105942, WO 2010105961, WO 2010105962, WO 2010094356, WO 2010084203, WO2010078979, WO 2010072456, WO 2010069905, WO 2010076165, WO 2010072603, WO2010066486, WO 2010066631, WO 2010066632, WO 2010063689、WO 2010060821, WO2010049187, WO 2010031607, WO 2010000636.

[0546] use

[0547] The Bacillus subtilis enzyme variants or combinations thereof according to the invention can be used in the washing of textiles and fabrics (e.g., household and industrial garment washing).

[0548] The Bacillus subtilis enzyme variants or combinations thereof according to the invention can also be used in methods for cleaning hard surfaces such as floors, tables, walls, roofs, etc., as well as for cleaning hard objects such as car (car wash) and tableware (dishwasher).

[0549] The detergent composition may be formulated as, for example, a hand or machine washing detergent composition, including a laundry detergent additive composition suitable for pretreating stained 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 washing dishwashing operations.

[0550] The polypeptides of this invention can be added to detergent additives.

[0551] The cleaning process or textile care process can be, for example, a laundry process, a dishwashing process, or the cleaning of hard surfaces (such as bathroom tiles, floors, countertops, drains, sinks, and basins). The laundry process can be, for example, household laundry, but it can also be industrial laundry. A method for washing fabrics and / or garments, wherein the method includes treating the fabric with a washing solution containing a detergent composition to which at least one variant of the Bacillus subtilis enzyme of the present invention may be added. For example, the cleaning process or textile care process can be carried out during machine washing or manual washing. The washing solution can be, for example, an aqueous washing solution containing a detergent composition.

[0552] In recent years, there has been a growing interest in replacing detergent components, stemming from the use of renewable biological components such as enzymes and peptides to replace petrochemical products without compromising washing performance. When detergent compositions incorporate new enzymes with altered activity or alternative and / or improved properties compared to commonly used detergent enzymes (such as proteases), lipases and amylases are needed to achieve similar or improved washing performance compared to conventional detergent compositions.

[0553] This invention further relates to the use of the Bacillus subtilis enzyme variant of the invention in the removal of protein stains. Protein stains may be stains such as food stains, including baby food, sebum, cocoa, eggs, blood, milk, ink, grass, or combinations thereof.

[0554] 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 washed 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 wash water, for example, by removing metal ions from the liquid.

[0555] Compositions including the Bacillus subtilis enzyme variants of the present invention may include one or more detergent components, such as surfactants, water-soluble solvents, builders, co-builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric toners, fabric conditioning agents, foaming agents, defoaming agents, dispersants, dye transfer inhibitors, fluorescent brighteners, fragrances, optical brighteners, bactericides, fungicides, dirt suspending agents, dirt-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0556] The composition may include the Bacillus subtilis enzyme variant of the present invention and one or more other enzymes selected from the group consisting of: proteases, amylases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases, or any mixture thereof.

[0557] The composition may include the Bacillus subtilis enzyme variant of the present invention, one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases or any mixture thereof, and one or more detergent components, such as surfactants, water-soluble solvents, builders, co-builders, chelators or chelating agents, bleaching systems or bleaching components, polymers, fabric toners, fabric conditioning agents, foaming agents, defoaming agents, dispersants, dye transfer inhibitors, fluorescent whitening agents, fragrances, optical brighteners, bactericides, fungicides, soil suspending agents, soil-releasing polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.

[0558] Washing method

[0559] A cleaning method includes the step of contacting the object with a detergent composition comprising a Bacillus subtilis enzyme variant of the present invention, under conditions suitable for cleaning the object.

[0560] A method for removing stains from fabrics or tableware may include contacting the fabric or tableware with a composition comprising a Bacillus subtilis enzyme variant of the present invention, under conditions suitable for cleaning the object.

[0561] Compositions and methods for treating fabrics (e.g., desizing textiles) may use one or more of the Bacillus subtilis enzyme variants of the present invention. These variants can be used in any fabric treatment method well known in the art (see, for example, US 6,077,316). For example, in one aspect, the feel and appearance of a fabric are improved by contacting the fabric with a protease in a solution. In another aspect, the fabric is treated with the solution under pressure.

[0562] The detergent composition is suitable for use in laundry and hard surface applications (including dishwashing). Such methods involve contacting the fabric / dishware to be cleaned with a solution comprising the detergent composition. Fabrics may include any fabric capable of being washed under normal consumer use conditions. Dishware may include any tableware, such as earthenware, tableware, ceramics, plastics (e.g., melamine), metals, porcelain, glass, and acrylics. The solution preferably has a pH from about 5.5 to about 11.5. The composition can 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 ratio of water to fabric typically ranges from about 1:1 to about 30:1.

[0563] One or more enzymes in the detergent composition can be stabilized using conventional stabilizers and protease inhibitors, such as polyols (e.g., propylene glycol or glycerol), sugars or sugar alcohols, various salts (e.g., NaCl, KCl), lactic acid, formic acid, boric acid, or boric acid derivatives (e.g., aromatic borate esters, or phenyl borate derivatives (e.g., 4-formylphenyl borate)), or peptide aldehydes (e.g., dipeptide aldehydes, tripeptide aldehydes, or tetrapeptide aldehydes or aldehyde analogs) (or having the form B1-B0-R, where R is H, CH3, CX3, CHX2, or CH2X (X = halogen), B0 is a single amino acid residue (preferably having an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two, or three), optionally including an N-terminal protecting group, or as described in WO 09118375, WO The composition may be formulated as described in, for example, WO 92 / 19709, WO 92 / 19708, and US 6,472,364, using a protein-type protease inhibitor such as RASI, BASI, WASI (a bifunctional α-amylase / subtilisin inhibitor for rice, barley, and wheat), or CI2 or SSI. In some embodiments, the enzymes utilized herein are stabilized by a water-soluble source of zinc(II), calcium(II), and / or magnesium(II) ions present in the finished composition providing such ions for these enzymes, along with other metal ions (e.g., barium(II), scandium(II), iron(II), manganese(II), aluminum(III), tin(II), cobalt(II), copper(II), nickel(II), and vanadium(IV)).

[0564] These detergent compositions are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 11.5, or, in alternative embodiments, even from about 6.0 to about 10.5. In some preferred embodiments, particulate or liquid laundry products are formulated to have a pH of from about 6 to about 8. Techniques for controlling the pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.

[0565] The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0566] Example

[0567] Materials and Methods

[0568] Automated Mechanical Stress Measurement (AMSA) for Clothing Washing

[0569] To evaluate washing performance in clothing, washing experiments were conducted using an Automated Mechanical Stress Measurement (AMSA). AMSA allows for the examination of the washing performance of large quantities of small-volume enzyme detergent solutions. The AMSA plate has numerous slits and caps for the test solution, with the caps forcefully pressing against the wash sample (the textile to be washed) at all slit openings. During the washing period, the plate, test solution, textile, and caps vibrate violently, thereby bringing the test solution into contact with the textile and applying mechanical stress in a regular, periodic oscillating manner. For further description, see WO 02 / 42740, particularly the "Special method embodiments" paragraph on pages 23-24.

[0570] Washing performance is measured as the brightness of the color of the washed textiles. Brightness can also be expressed as the intensity of light reflected from the sample when illuminated with white light. When a sample is soiled, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of reflected light can be used to measure washing performance.

[0571] Using a professional flatbed scanner (Kodak iQsmart, Midtager 29, DK-2605) (Denmark) Color measurements are performed using a scanner that captures images of the washed textiles.

[0572] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by summing the RGB values ​​as vectors and then considering the length of the resulting vector.

[0573]

[0574] Table 1: Composition of Standard Detergents and Test Materials

[0575] The standard detergent and test materials are as follows:

[0576]

[0577] Tergo-O-Tometer (TOM)

[0578] The Tergo-O-tometer (TOM) is a medium-scale standard washing system that can be used to simultaneously test 12 different washing conditions. Essentially, a TOM is a large, temperature-controlled water bath containing up to 12 open metal beakers submerged within it. Each beaker constitutes a small top-loaded washing machine, and during the experiment, each of them contains a solution of a specific detergent / enzyme system and its performance is tested on both soiled and unsoiled fabrics. Mechanical stress is achieved by rotating agitator arms that agitate the liquid (1L) within each beaker. Because the TOM beakers are lidless, it is possible to retrieve samples during a TOM experiment and analyze information online during washing. Factors such as the ballast-to-dirt ratio and the fabric-to-wash ratio can vary in a TOM experiment. Therefore, TOM provides a link between small-scale experiments (such as AMSA and microwashing) and the more time-consuming full-scale experiments in a full-scale washing machine.

[0579] After washing and rinsing, lay the small fabric samples flat and allow them to air dry overnight at room temperature. Evaluate all washes the day after washing. Evaluate the light reflectance of the small fabric samples using a Macbeth Color Eye 7000 reflectance spectrophotometer with a large aperture. Measurements are taken under UV-free incident light conditions, and reflectance is extracted at 460 nm. Measurements are performed on both unwashed and washed small fabric samples. Place the test fabric sample to be measured on top of another small fabric sample of the same type and color (paired samples).

[0580] The reflectance of a single fabric sample is calculated by subtracting the reflectance of an un-enzyme-washed (blank) fabric sample from the reflectance of a group of fabric samples washed with enzymes.

[0581] The effect of the protease variant on each stain was calculated by taking measurements from small samples of fabric washed with enzymes and subtracting measurements from small samples of fabric not washed with enzymes.

[0582] The performance of the new protease variant was compared with the reference performance (REF) of SEQ ID NO:3 by calculating the relative performance (RP) as follows:

[0583] RP = (R 蛋白酶变体 -R空白 ) / (R REF -R 空白 )

[0584] Conventional molecular biology methods:

[0585] Unless otherwise noted, DNA manipulation and transformation are performed using standard molecular biology methods (Sambrook et al. (1989); Ausubel et al. (1995); Harwood and Cutting (1990)).

[0586] Protease activity assay:

[0587] 1) Assay for Suc-AAPF-pNA activity:

[0588] 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, which is yellow in color and can therefore be measured spectrophotometrically at a wavelength of 405 nm. The Suc-AAPF-PNA substrate is prepared by Bachem (catalog number L1400, dissolved in DMSO).

[0589] The protease sample to be analyzed was diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay was performed by transferring 60 μl of the diluted enzyme sample to a 96-well microtiter plate and adding 140 μl of substrate working solution (0.72 mg / mL in 100 mM Tris, pH 8.6). The solution was mixed at room temperature and the absorbance was measured at OD 405 nm over 5 minutes at 20-second intervals.

[0590] Under a given set of conditions, the slope of the time-dependent absorption curve (absorbance per minute) is directly proportional to the specific activity of the protease in question (activity / mg enzyme). The protease sample should be diluted to a level where the slope is linear.

[0591] Accelerated storage stability determination

[0592] The storage stability of the protease variant in liquid detergent was assessed using an accelerated assay after incubation at elevated temperatures for up to 24 hours.

[0593] Based on absorbance at 280 nm and theoretical extinction coefficient, all purified protease samples were diluted to concentrations of 0.2 and 0.1 mg / mL using 0.01% Triton X-100. For each variant, two wells with a high protease concentration and two wells with a low concentration were included. For reference, SEQ ID NO:3 was included on each microtiter plate. Using a magnetic rod (on a Zephyr stage (Caliper LifeSciences), 30 μl of protease sample was mixed with 270 μl of detergent (CNS EDTA pH 9) in the wells of a microtiter plate (Nunc U96PP 0.5 mL). Then, 20 μl of this mixture was transferred to another microtiter plate (Nunc U96PP 0.5 mL with a magnetic rod) and mixed with 150 μl of 100 mM Tris (pH 8.6) (on a Zephyr stage for at least 5 min). 30 μl of this dilution was transferred to Nunc F 96-MTP, and after adding 70 μl of substrate solution, the initial activity of the non-stressed sample was determined by measuring absorbance at 405 nm every 20 seconds for 5 min (on SpectraMax Plus). After sealing, the detergent plate was incubated in an Eppendorf thermostat (without shaking) at the appropriate temperature (47 °C for CNS, pH 9 for EDTA). After incubation for 1–4 and 20–25 hours, 20 μl of sample was drawn, and the residual activity of the stressed sample was measured as with the initial non-stressed activation.

[0594] The decrease in activity during incubation with detergent was assumed to be exponential. The half-life (T1 / 2) was determined from linear regression of Log(activity) against incubation time (0, 1–4, and 20–25 h), and the half-life improvement factor (T1 / 2IF) was calculated as the half-life of the protease variant relative to the half-life referenced in SEQ ID NO:3.

[0595] Detergent

[0596]

[0597]

[0598] Example 1: Variant preparation and expression

[0599] Expression cassettes were introduced into Bacillus subtilis by utilizing the natural competence of the organism to transform suitable expression cassettes.

[0600] DNA manipulation, such as introducing mutations and constructing expression cassettes into Bacillus subtilis, was performed via PCR (e.g., Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press), and all DNA manipulations were reproducible by those skilled in the art. Recombinant Bacillus subtilis constructs encoding Bacillus subtilis enzyme variants were inoculated into shake flasks containing nutrient-rich medium (e.g., PS-1: 100 g / L sucrose (Denisco catalog 109-0429), 40 g / L soybean hulls (soybean meal), 10 g / L Na₂HPO₄·12H₂O (Merck catalog 6579), 0.1 ml / L Replace-Dowfax 63N10 (Dow Chemicals)). Incubation was typically carried out for 4 days at 30°C with shaking at 220 rpm.

[0601] Example 2: Fermentation of Variants

[0602] Fermentation can be carried out using methods well-known in the art or as follows. A strain of *Bacillus subtilis* with the relevant expression plasmid is streaked onto an LB agar plate and grown overnight at 37°C. The colonies are then transferred to 100 ml of PS-1 medium in a 500 ml shake flask. Cells and other undissolved material are removed from the fermentation broth by centrifugation at 4500 rpm for 20–25 minutes. The supernatant is then filtered to obtain a clear solution.

[0603] Example 3: Purification of Variants

[0604] The culture medium was centrifuged (26000x g, 20 min), and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove any remaining Bacillus host cells. The pH of the 0.2 μm filtrate was adjusted to pH 8 with 3 M Tris base, and the pH-adjusted filtrate was applied to a MEP Hypercel column (from Pall Corporation) equilibrated in 20 mM Tris / HCl and 1 mM CaCl2 (pH 8.0). After washing the column with equilibration buffer, it was gradually eluted with 20 mM CH3COOH / NaOH and 1 mM CaCl2 (pH 4.5). The fractions from the column were analyzed for protease activity (using the Suc-AAPF-pNA assay at pH 9), and the peak fractions were combined. The pH of the pooled fraction from the MEP Hypercel column was adjusted to pH 6 using 20% ​​(v / v) CH3COOH or 3M Tris base, and the pH-adjusted fraction was diluted with deionized water to the same conductivity as 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). The diluted fraction was applied to an SP-agarose FF column (from GE Healthcare) equilibrated in 20 mM MES / NaOH, 2 mM CaCl2 (pH 6.0). After washing the column with equilibration buffer, the protease was eluted with a linear NaCl gradient (0-->0.5 M) in the same buffer for five column volumes. The fractions from the column were analyzed for protease activity (using the Suc-AAPF-pNA assay at pH 9), and the activity fractions were analyzed by SDS-PAGE. The fractions (which showed only one band on a Coomassie-stained SDS-PAGE gel) were combined into a purified formulation and used for further experiments.

[0605] Example 4: Stability of variants of the invention

[0606] The variants of the invention were generated and purified as described in Examples 1-3, and their stability in liquid detergents was tested at 47°C using the stability tests disclosed above, and the half-life was calculated. The reference polypeptide is a subtilisin having SEQ ID NO:3.

[0607] Table 2: Stability of variants of SEQ ID NO:3. The first column indicates the substitutions in SEQ ID NO:3. The second column indicates the half-life observed in experiments, and in the third column, the data are presented relative to SEQ ID NO:3. The last column indicates the standard deviation.

[0608]

[0609] Under these conditions, all variants exhibit equivalent, improved, or significantly improved stability in liquid detergents.

[0610] Example 5 Residual Activity

[0611] The stability of the variants of the invention in liquid detergents was tested using the stability test disclosed above at a temperature of 45°C, and the half-life and residual activity were calculated after 19 hours. The parent polypeptide is a subtilisin having SEQ ID NO:3.

[0612] Table 3: Stability of variants of SEQ ID NO:3. The first column indicates substitutions compared to SEQ ID NO:3. The second column indicates the calculated residual activity after 19 hours, and the last column indicates the residual activity with standard deviation:

[0613]

[0614]

[0615] The data show that these variants have increased stability under the test conditions and also have increased residual activity after 19 hours compared to the parental subtilis enzyme with SEQ ID NO:3.

[0616] Example 6

[0617] The washing performance of the variants in the detergent was determined using the following standard soil samples:

[0618] A: Egg yolk on cotton fabric: Product number 10EG, available from: W-Testgewebe GmbH, Christenfeld 10, 41379, Brüggen, Germany

[0619] B: Blood on the cotton fabric: Product number CS01, available from: CFT (Test Materials Center) BV, Vlaardingen, Netherlands.

[0620] C: Egg on cotton fabric: Product number C37, available from: CFT (Test Materials Center) BV, Vlaardingen, Netherlands.

[0621] D: Blood on the cotton cloth: Product number 111, obtained from the following: Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal Materials and Testing Agency, Testmaterials], St. Gallen, Switzerland.

[0622] The following stain ERs are all available from CFT (Center for Testing Materials) BV, Vlaardingen, Netherlands:

[0623] E: Cocoa on the cotton fabric: Product number CH-09

[0624] F: Egg on cotton cloth: Product number C38

[0625] G: Chocolate on cotton cloth - Milk: Product No. C03

[0626] H: Cocoa & Oatmeal: Product No. CS-54

[0627] I: Chocolate-Milk on Polyester / Cotton Fabric: Product No. PC-3-009

[0628] J: Cocoa cooked with milk on cotton cloth: Product No. C-H019

[0629] K: Meal replacement mixed beverage on cotton cloth: Product No. C-H165

[0630] L: Chocolate pudding on cotton cloth: Product No. C-H118

[0631] M: Chocolate Pudding on Cotton Fabric: Product No. C-H172

[0632] N: Meat Pate Vallette: Product No. KC-H 171

[0633] O: Chocolate pudding on cotton cloth: Product No. KC-H 172

[0634] P: Aged chocolate ice cream on cotton fabric: Product No. CS-68

[0635] Q: Chocolate pudding on cotton cloth: Product number CS-69

[0636] R: Aged egg yolk carbon black: Product No. CS-38

[0637] S: Egg on cotton fabric: Product number WFK 10N available from W-Test Fabrics GmbH, Christenfeld 10, 41379 Brüggen, Germany

[0638] T: Cocoa on cotton fabric: Product number EMPA 112, from Available at Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal Materials and Testing Agency, Testmaterials], St. Gallen, Switzerland.

[0639] U: Blood on cotton cloth - Milk / Ink: Product No. C05

[0640] Peanut oil pigment / ink on cotton fabric: Product No. C10

[0641] W: Straw clippings on cotton cloth: Product number 164, from Available at Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal materials and testing agency, Testmaterials], St. Gallen, Switzerland.

[0642] X: Cocoa cooked with milk: Product No. C-H010

[0643] Y: Blood / Milk / Ink, Product No. EMPA 117, from Available at Material-und Prüfanstalt (EMPA) Testmaterialien AG [Federal Materials and Testing Agency, Testmaterials], St. Gallen, Switzerland.

[0644] Z: Chocolate milk and ash, product number CFT C03, available from CFT (Test Materials Centre) BV, Vlaardingen, Netherlands:

[0645] Use a liquid detergent with the following composition as the base formulation (all values ​​are by weight percentage): 0% to 0.5% xanthan gum, 0.2% to 0.4% defoamer, 0.2% to 8% triethanolamine, 1% to 7% glycerin, 0.3% to 3% ethanol, 0% to 12% FAEOS (fatty alcohol ether sulfate), 1% to 28% nonionic surfactant, 0.5% to 4% boric acid, 0.5% to 6% sodium citrate (dihydrate), 1% to 6% baking soda, 0% to 16% coconut fatty acid, 0.5% to 6% HEDP (1-hydroxyethane-(1,1-diphosphonic acid)), 0% to 0.4% PVP (polyvinylpyrrolidone), 0% to 0.05% optical brightener, 0% to 0.001% pigment, and the remainder being deionized water.

[0646] Based on this basic formulation, different detergents were prepared by adding the corresponding proteases as indicated in Table 4. The reference is a protease having the amino acid sequence in SEQ ID NO. 3, which has shown good washing performance (especially in liquid detergents). The same amount of protease was added based on the total protein content (5 mg / L of detergent liquid).

[0647] The liquid detergent dosage ratio is 4.7 g / L of washing liquid, and the washing program is carried out for 60 minutes at temperatures of 20°C and 40°C, with the water having a hardness between 15.5° and 16.5° (German hardness).

[0648] Whiteness (i.e., the whitening effect on dirt) is determined by photometric measurement as an indicator of washing performance. A Minolta CM508d spectrophotometer was used, which was calibrated beforehand using a whiteness standard with provided units.

[0649] The results obtained are the difference between the remission units obtained using the detergent and those obtained using a detergent containing a reference protease. Therefore, a positive value indicates the improved washing performance of the variant in the detergent. It is clear from Tables 4a (results at 40°C) and 4b (results at 20°C) that the variant according to the invention exhibits improved washing performance.

[0650] Tables 4a and 4b: Washing performance of protease variants having the same amino acid sequence as SEQ ID NO:3 (except for substitutions) against stains as shown at 40°C, according to the table below; reference is the protease according to SEQ ID NO:3.

[0651] a)

[0652]

[0653] b)

[0654] Protease variants U G V T W S163G 0,9 1,5 1,8 nd 0,9 G61D 1,5 1,7 nd nd nd S156D 1,6 2,1 0,5 nd nd H120D 0,8 1,6 2,4 nd nd G195E V199M 1,5 0,7 1,1 nd nd A228V N261D 0,5 1,3 1,6 nd nd V244T 1,3 1,1 2,7 nd nd T58L nd 2,0 1,1 0,9 0,4 S3T V4I N261D 1,5 1,7 0,4 nd nd A194P G195E V199M V205I 0,8 nd nd 1,1 nd H120D A228V 2,1 1,9 nd nd nd H120D N261D nd nd 0,8 1,2 nd N76D A228V N261D nd 0,5 nd 1,8 nd

[0655] Tables 4c-e: Washing performance of protease variants having the same amino acid sequence as SEQ ID NO:3 (except for substitutions) at 20°C for stains as shown, according to the table below; reference is the protease according to SEQ ID NO:3.

[0656] c)

[0657]

[0658] d)

[0659]

[0660]

[0661] e)

[0662]

[0663] f)

[0664]

[0665]

[0666] Example 7: Washing performance of lipase variants

[0667] The following table lists the washing performance of protease variants having the same amino acid sequence as SEQ ID NO:3 (except for substitutions) against the stains shown; reference is the protease according to SEQ ID NO:3. The washing performance was evaluated using an automated mechanical stress assay (AMSA) during laundry washing, where the washing performance of numerous small-volume enzyme detergent solutions can be examined. An AMSA plate has numerous tanks for the test solution and a lid that forcefully presses the textile to be washed against the tank openings. During washing, the plate, test solution, textile, and lid are violently vibrated to bring the test solution into contact with the textile and apply mechanical stress in a regular, periodic oscillating manner. For further description, see WO 02 / 42740, particularly the “Specific Method Examples” paragraph on pages 23-24.

[0668] The clothing washing experiment was conducted under the experimental conditions specified in Table 5.

[0669] Table 5

[0670] Detergent dosage 2.0g / L Test solution volume 160 μL (20 μL enzyme + 140 μL detergent) pH 8.4 Washing time 20 minutes temperature 20℃ water hardness 12°dH

[0671] The standard detergents and test materials are listed in Table 1:

[0672] Table 6: Composition of Standard Detergents and Test Materials

[0673]

[0674] The test material was obtained from the Center for Testmaterials (BV), 3133KT Vlaardien, Netherlands.

[0675] By using CaCl2, MgCl2 and NaHCO3 (Ca 2+ :Mg 2+ Add a 2:1:4.5 ratio to the test system to adjust the water hardness to 12°dH. After washing, rinse the textiles with tap water and dry.

[0676] Washability is measured as the brightness of the color of the washed textiles. Brightness can also be expressed as the intensity of light reflected from the sample when illuminated with white light. When the sample is soiled, the intensity of the reflected light is lower than that of a clean sample. Therefore, the intensity of the reflected light can be used to measure washability.

[0677] Using a Kodak iQsmart flatbed scanner (Kodak Midtager 29, DK-2605) (In Denmark) Color measurements are performed using a scanner that captures images of the textiles being washed.

[0678] To extract light intensity values ​​from a scanned image, the 24-bit pixel values ​​from the image are converted into red, green, and blue (RGB) values. The intensity value (Int) can be calculated by adding the RGB values ​​as vectors and then considering the length of the resulting vector:

[0679]

[0680] The results are shown in Table 7. The results are given as relative performance at an enzyme concentration of 30 nM on three different small cloth samples compared to SEQ ID NO:3.

[0681] Table 7: AMSA relative performance of variants compared to SEQ ID NO:3.

[0682]

[0683]

[0684] Table 8 shows the relative performance of the enzyme at a concentration of 30 nM on two different small cloth samples compared to SEQ ID NO:3.

[0685]

[0686]

[0687] The results showed that the stabilized variant of SEQ ID NO 3 exhibited equivalent or improved washing performance compared to that of SEQ ID NO 3.

[0688] Example 8: Results of washing measurements using Terg-O-tometer (TOM)

[0689] Conduct TOM washing experiments under the following specified experimental conditions:

[0690]

[0691]

[0692] Table 10 shows the relative performance of the Bacillus subtilis enzyme variant with the mutation shown compared to SEQ ID NO:3.

[0693]

[0694]

[0695] Example 9: Washing performance of protease variants

[0696] Washing was performed under various conditions, including AMSA washing with a low concentration of protease (30 nM), AMSA washing with a high concentration of protease (300 nM), TOM washing, and full-scale washing (FSW), as shown in Table 15. The detergents disclosed in Table 1 and PC-03 (chocolate-milk / ink on cotton / polyester) test material were used for washing. The results were compared with the performance of the reference protease containing SEQ ID NO:3 and the performance shown in Table 11 below, where the result for the reference protease was set to 1.00.

[0697] Table 11

[0698]

[0699] sequence list <110> Friis, Esben Peter <120> Bacillus subtilis enzyme variants <130> 12755-WO-PCT <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 275 <212> PRT <213> Bacillus amyloliquefaciens <400> 1 Ala Gln Ser Val Pro Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Val Ala Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser Ala Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val Val Val Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser Ser Ser Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 Val Gly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190 Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210 215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn 225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr Thr Thr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala 260 265 270 Ala Ala Gln 275 <210> 2 <211> 269 <212> PRT <213> Bacillus lentus <400> 2 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Asp Gly Arg Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265 <210> 3 <211> 269 <212> PRT <213> Artificial <220> <223> BLAP R101E (Numbered according to SEQ ID NO.1) <400> 3 Ala Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 Gly Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 Asp Gly Glu Gly Ala Ile Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110 Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120 125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130 135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Ser Ser Ile Ser 145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Gly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 260 265

Claims

1. A Bacillus subtilis enzyme variant having a glutamic acid residue (E) at position 101, and a substitution of S156D+L262E based on SEQ ID NO:3 or a substitution of S156D+S163G+L262E based on SEQ ID NO:3, wherein these positions correspond to the positions in SEQ ID NO:

1.

2. The subtilisin variant of claim 1, wherein the subtilisin further has improved washing performance compared to the subtilisin having SEQ ID NO:3, when determined according to the AMSA and / or TOM methods described herein.

3. The subtilisin variant of claim 1, wherein, when measured according to the accelerated storage stability assay described herein, the subtilisin has improved stability in the liquid detergent composition compared to the subtilisin having SEQ ID NO:

3.

4. A nucleotide sequence encoding a variant according to any one of claims 1-3.

5. An expression vector comprising the nucleotide sequence as described in claim 4.

6. A recombinant host cell comprising the nucleotide sequence of claim 4 or the expression vector of claim 5.

7. A method for producing a variant according to any one of claims 1-3, the method comprising: a. Providing the recombinant host cell as described in claim 6; b. Culture the recombinant host cells under conditions that induce the expression of this variant; and c. Separate the variant.

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