Lipase variants and polynucleotides encoding them

Abstract

The present invention relates to lipase variants of a parent lipase having lipase activity, having at least 75% but less than 100% sequence identity to SEQ ID NO: 3, and comprising a substitution at one or more positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 16, 19, 30, 31, 34, 36, 37, 39, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 84, 86, 88, 90, 92, 93, 95, 96, 100, 101, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135, 137, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 181, 182, 183, 189, 190, 192, 194, 196, 202, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 240, 242, 246, 247, 248, 252, 259, 262, 264, 269 of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells comprising these polynucleotides, and methods of using these variants.

Description

[0001] References to sequence lists

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

[0003] This invention relates to lipase variants, polynucleotides encoding these variants, methods for generating these variants, and methods for using these variants. Background of the Invention

[0005] Lipases are important biocatalysts that have been shown to be useful for various applications. Lipases have been used to remove lipid stains and have been added to different compositions. Current cleaning and / or fabric care compositions contain many active ingredients that interfere with the ability of lipases to remove lipid stains. Therefore, there is a need for lipases that can function in the harsh environments of cleaning compositions. Invention Overview

[0007] This invention provides lipase variants with improved properties compared to their parents, particularly lipase variants for cleaning. These variants are achieved by introducing modifications, such as substitutions, into the parental lipase. The improved properties can be one or more selected from: increased thermal stability; increased thermal stability in detergents; increased hydrolytic activity in detergents; and increased stability in detergents. This invention provides lipase variants, including but not limited to those that are particularly useful in various cleaning applications. This invention also provides cleaning methods using the lipase variants of this invention.

[0008] In one embodiment, the present invention relates to a lipase variant of a parental lipase, the variant having lipase activity, having at least 75% but less than 100% sequence identity with SEQ ID NO:3, and having positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 16, 19, 30, 31, 34, 36, 37, 39, 3, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 88, 92, 93, 95, 96, 100, 101, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135 corresponding to SEQ ID NO:3. One or more of the following positions include substitution: 137, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 181, 182, 183, 189, 190, 192, 194, 196, 202, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 240, 242, 246, 247, 248, 252, 259, 262, 264, 269.

[0009] The present invention also relates to polynucleotides encoding these variants; nucleic acid constructs, vectors, and host cells containing these polynucleotides; and methods for generating and using these variants.

[0010] Detailed disclosure of the invention

[0011] definition

[0012] Lipase: The terms "lipase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to an enzyme of class EC3.1.1 as defined by enzyme nomenclature. It can possess lipase activity (triacylglycerol lipase, EC3.1.1.3), keratinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13), and / or wax ester hydrolase activity (EC3.1.1.50). For the purposes of this invention, lipase activity is determined according to the procedure described in the examples. In one aspect, variants of the present invention have at least 20% of the lipase activity of the polypeptide of SEQ ID NO:3, for example at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0013] 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 alteration 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.

[0014] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription of mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. The early initial RNA transcript is a precursor to mRNA, which undergoes a series of processing steps, including splicing, before it becomes mature spliced ​​mRNA.

[0015] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame that begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). A coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0016] Control Sequences: The term "control sequence" refers to the nucleic acid sequence required 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. Such control sequences include, but are not limited to, precursors, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters and transcription and translation termination signals. These control sequences may be provided with 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 polynucleotide encoding the variant.

[0017] Expression: The term “expression” includes any step involved in the production of a variant, including (but not limited to) transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0018] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule that includes a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0019] Fragment: The term "fragment" means a polypeptide in which one or more (e.g., several) amino acids are absent from the amino and / or carboxyl termini of a mature polypeptide; wherein the fragment has lipase activity. In one aspect, the fragment comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of amino acids 1 to 269 of SEQ ID NO:3, but less than 100%.

[0020] Highly stringent conditions: 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 cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0021] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors including the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0022] Improved properties: The term "improved properties" refers to characteristics that are improved relative to the parent of a variant. Such improved properties include, but are not limited to: stability, such as thermal stability, thermal stability in detergents, and stability in detergents; activity, such as hydrolytic activity, hydrolytic activity in detergents; substrate-specific activity; detergency, such as lipid detergency; and washing performance.

[0023] Isolated: The term “isolated” means a substance that does not exist in nature in a form or environment. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to the kind of substance found in nature; 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.

[0024] Low stringency conditions: The term "low stringency 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 cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 50°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0025] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, a mature polypeptide is amino acids 1 to 269 of SEQ ID NO:2, which are identical to amino acids 1 to 269 of SEQ ID NO:3. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide. Therefore, in one aspect, the mature polypeptide may begin with amino acids 1, 2, 3, 4, or 5 of SEQ ID NO:3.

[0026] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide with lipase activity. In one respect, the mature polypeptide coding sequence is nucleotides 292 to 1098 of SEQ ID NO:1.

[0027] Medium-tough conditions: 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 55°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0028] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization for probes 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 frogfish sperm DNA, and 35% formamide for 12 to 24 hours. Vector materials are finally washed three times at 60°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0029] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[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 that is modified to contain segments of nucleic acid in a manner not normally found in nature, or that is synthetic and includes one or more control sequences.

[0031] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is placed in the appropriate position relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0032] Parent or parental lipase: The term "parent" or "parental lipase" refers to a lipase to which a modification is made to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

[0033] Sequence consistency: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0034] For the purposes of this invention, 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) was used to determine sequence consistency between two amino acid sequences. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of the "longest consistency" annotated by Needle (obtained using the -non-simplification option) was used as the percentage consistency and calculated as follows:

[0035] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0036] For the purposes of this invention, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, ibid.) implemented in the Nieder program, such as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later), was used to determine sequence identity between two deoxyribonucleotide sequences. The parameters used were 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 Nieder-annotated "longest consistency" output (obtained using the -non-simplified option) was used as the percentage consistency and calculated as follows:

[0037] (Consistent deoxyribonucleotides x 100) / (Alignment length - total number of vacancies in the alignment)

[0038] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more (e.g., several) nucleotides are missing from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity. In one aspect, the subsequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of nucleotides 292 to 1098 of SEQ ID NO:1.

[0039] Variants: The term "variant" refers to a polypeptide having lipase activity that contains 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 an amino acid adjacent to and immediately following the amino acid occupying a position. These variants of the present invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the lipase activity of SEQ ID NO:3.

[0040] Very High Tough Conditions: The term "very high tough 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 cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 70°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0041] Very low stringency conditions: The term "very low stringency 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 cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 45°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0042] Wild-type lipase: The term "wild-type" lipase refers to a lipase expressed by a naturally occurring microorganism (such as a bacterium, yeast, or filamentous fungus) found in nature.

[0043] Variant Naming Conventions

[0044] For the purposes of this invention, the polypeptide disclosed in SEQ ID NO:3 is used to determine the corresponding amino acid residues in another lipase. The amino acid sequence of the other lipase is aligned with SEQ ID NO:3, and based on this alignment, the Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in SEQ ID NO:3. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0045] The identification of corresponding amino acid residues in another lipase can be determined by using several computer programs that compare multiple peptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Kato et al., 2005, Nucleic Acids Research 33:511-518; Kato, 2007, Bioinformatics 23:372-374; Kato et al., 2009, Methods in Molecular Biology). Biology) 537: 39-64; Kato Kazuto, 2010, Bioinformatics 26: 1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22: 4673-4680).

[0046] When other enzymes are dissimilar to the polypeptide of SEQ ID NO:3, making traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, J.Mol.Biol. 295:613-615), other pairwise sequence comparison algorithms can be applied. 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 is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J.Mol.Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J.Mol.Biol. 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0047] 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 (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures, and implementations of these algorithms can also be used to query structure databases with structures of interest to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0048] In the description of variations of the invention, the following nomenclature is provided for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are used.

[0049] replace. For amino acid substitutions, the following nomenclature is used: original 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 plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) with arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) with phenylalanine (F).

[0050] Missing For amino acid deletions, use the following nomenclature: original amino acid, position, *. Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195"... * +Ser411 * "or "G195 * +S411 * ".

[0051] insertFor amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0052] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0053] <![CDATA[ Parent: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0054] Multiple changes Variants containing multiple changes are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0055] Different changes. When different changes can be introduced at a single position, these changes are separated by commas. 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:

[0056] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

[0057] variants

[0058] This invention relates to a lipase variant of a parental lipase, the variant having lipase activity and having at least 75% but less than 100% sequence identity with SEQ ID NO:3, and at positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 16, 19, 30, 31, 34, 36, 37, 39, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 88, 92, 93, 95, 96, 100, 101, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135, corresponding to SEQ ID NO:3. One or more of the following positions include substitution: 137, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 181, 182, 183, 189, 190, 192, 194, 196, 202, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 240, 242, 246, 247, 248, 252, 259, 262, 264, 269.

[0059] The present invention also relates to variants, wherein the variant is at position 1 (C, F, G, H, I, L, M, P, Q, R, V, W, Y), 2 (A, C, D, M, N, P, Q, R, S, T, V), 3 (A, E, G, K, Q, S, T, V), 4 (A, P, Q, R, S, T), 5 (E, K, S, T, V, Y), 6 (A, H, K, N, P, Q, R, S, V, W), 7 (P), 8 (C, G, H, N, P, Q, R, S, W, Y), 9 (Y), 10 (G, R, S, Y), 11 (G, H, I, L, P, Q, R, T), 12 (A, I, K, L, M, N, R, S, T), 16 (P, R, S, T, W), 19 (C, E, H, L, W, T), 30 (A, F, H, I, L, N, P, S, T, V, W), 31 (A, C, D, E, F, G, H, I, K, P, Q, R, S, V), 34 (A, G, V), 36 (D, E, G, K, Q, R, S, V), 37 (A, E, G, S, V), 39 (F, K, L, N, S), 40 (A, D, E, G, I, L, R, V), 41 (A, D, E, G, H, K, L, P, Q, R, S, V, W), 42 (A, E, G, K, L, M, R, S, T, V), 44 (A, C, E, G, H, K, M, P, Q, R, S, V, W), 51 (A, D, G, H, K, L, M, V, Y), 52 (L, V), 53 (A, D, G, H, I, L, M, P, Q, R, S, V), 54 (A, D, E, G, I, K, L, S, V), 56 (A, P, Q, R, T, V, W), 58 (C, G, I, P, R, S, V, W), 59 (A, D, F, G, H, L, M, P, R, S, T, V), 61 (G, M), 70 (A, E, G, R), 71 (E, K, N, Q, R, W, Y), 72 (A, C, D, G, K, R, S, T, V), 73 (E, G, L, N, Q, R, S, T), 83 (A), 84 (A, E, L, P, W, Y), 86 (A, G, I, K, M, W), 88 (M, Q, R, S), 90 (D, F, G, L, S, T, V, W), 91 (E, F, G, Q, Y), 92 (A, C, D, F, P, S, T, V, W), 93 (A, I, R, V), 95 (C, G, I, Q, R, S), 96 (A, C, D, E, G, N, Q, R, S, W, Y), 98 (A, D, E, G, K, N, R, T, V, W), 100 (A, E, G, K, L, R, S, V, W), 101 (D, H, R, S, V), 102 (H, I, P, R, S), 103 (A, C, D, F, G, L, P, V), 104 (A, C, D, E, K, L, M, P, Q,R,V,W,Y)、106(A,C,G,L,P,Q,R,S,T,W)、109(A,D,E,G,H,L,N,P,Q,R,S,T)、110(C,P,S,V)、112(F,H,Q,R,W)、113(M,W)、116(A,C,D,F,R,S,T)、117(A,D,G,N,P,R,S,V)、119(D,E,F,I,R,S,T,V)、120(A,E,G,H,L,M,S,T,V,Y)、124(C,D,G,H,L,M,Q,R,S,V,W)、125(A,D,E,G,I,K,L,M,Q,R,S,V)、127(A,F,G,H,I,Q,R,S,V,Y)、128(A,E,G,H,M,Q,S,T)、131(A,G,I,M,S,T,V,W)、132(G,S,V)、133(A,E,K,L,M,R,V,W)、134(A,D,E,G,K,R,S,T,V)、135(A,C,G,P,Q,R,T,V,W,Y)、137(A,F,G,P,R)、158(A,C,G,I,L,N,R,S,T,V,W)、159(G,T)、160(A,D,G,I,L,Q,V)、161(C,G,I,K,L,P,Q,R,S)、162(A,D,G,L,M,P,R,S,V,W)、163(A,D,E,H,L,M,Q,V,Y)、165(A,C,G,K,L,M,P,R,V,W)、166(A,C,E,G,L,,P,R,W)、167(C,F,H,I,L,M,R,V)、168(A,C,D,G,R,S,V,W)、170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y)、181(A,C,E,G,L,Q,R,S,V,W)、182(A,C,F,G,I,L,R,S,T,V,W,Y)、183(E,V)、186(A,C,E,G,H,K,L,R,S,T,W)、189(A,C,G,L,M,P,R,S)、190(A,E,G,I,K,N,R,V,W)、192(A,I,L,M,P,R,T,V)、194(A,D,E,F,G,H,L,Q,T,V)、196(A,Q,Y)、201(D,G,L,P)、202(I,M)、204(E,K,L,P,R)、208(E,M,P,R,W)、210(A,F,L,Q)、211(C,E,G,P,S,V,W)、212(D,E,G,H,K,N,P,Q,R,S,T,V,W)、213(E,H,L,M,P,Q,R,W)、220(A,C,D,G,L,P,R,S,T,W), 225(A,L,M,P,Q,S), 226(C,E,F,G,P,R,W,Y), 227(A,C,G,P,R,S,V,W), 228 (A,C,D,G,L,P,R,V,W), 229(A,I,L,N,R,S,T), 230(A,G,H,L,P,Q,R,S,T,V,W), 2 31(A,C,G,H,K,R,S,V,W,Y), 233(A,F,H,L,M,S,T), 236(C,L,P,S,V), 237(F,G,H ,L,M,N,R,V), 238(A,E,F,G,H,M,P,R,S,V,W), 239(F,G,S,Y), 240(A), 241(M,V), 242(A,C,G,I,L,M,P,R,T,V,Y), 243(A,C,E,F,G,M), 246(A,E,K,P,S,T,V), 247( A,E,L,P,T,Y), 248(A,G,P,V), 252(A,D,E,G,N,Q,S,V), 255(C,E,M,Q,R), 259(A, One or more positions of C,D,E,G,I,K,L,M,Q,R,T,V,W,Y), 262 (A,D,E,K,M,R,T,V), 264 (C,G,I,L,M,P,R,S,T), 267 (E,H,K,M,Q,W), and 269 (A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) include substitutions.

[0060] In some aspects, the present invention relates to variants selected from the group consisting of: (a) a polypeptide having at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the polypeptide of SEQ ID NO:3; (b) a polypeptide encoded by a polynucleotide hybridizing with (i) the polypeptide-coding sequence of SEQ ID NO:1 or (ii) the full-length complement of (i) under low-strict, medium-strict, medium-high-strict, high-strict, or very high-strict conditions; and (c) a polypeptide encoded by a polynucleotide that hybridizes with the polypeptide of SEQ ID NO:1 under low-strict, medium-strict, medium-high-strict, high-strict, or very high-strict conditions. The polypeptide coding sequence of SEQ ID NO:3 has at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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% but less than 100% sequence identity; and (d) a fragment of the polypeptide of SEQ ID NO:3, wherein the fragment has lipase activity.

[0061] In some aspects, the present invention relates to variants having at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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%, but less than 100% sequence identity with the parental lipase.

[0062] In some aspects, the present invention relates to variants that have at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, 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%, but less than 100% sequence identity with SEQ ID NO:3.

[0063] In some aspects, the present invention relates to variants, wherein the number of substitutions in the variants of the invention is 1 to 40, for example 1 to 30, 1 to 20, 1 to 10 and 1 to 5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 substitutions.

[0064] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S1. In another aspect, the amino acid at the position corresponding to position S1 is substituted with C, F, G, H, I, L, M, P, Q, R, V, W, or Y. In yet another aspect, the variant comprises or consists of the substitution S1 (C, F, G, H, I, L, M, P, Q, R, V, W, or Y) of SEQ ID NO:3.

[0065] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position I2. In another aspect, the amino acid at the position corresponding to position I2 is substituted with A, C, D, M, N, P, Q, R, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted I2 (A, C, D, M, N, P, Q, R, S, T, V) of SEQ ID NO:3.

[0066] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D3. In another aspect, the amino acid at the position corresponding to position D3 is substituted with A, E, G, K, Q, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted D3 (A, E, G, K, Q, S, T, or V) of SEQ ID NO:3.

[0067] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G4. In another aspect, the amino acid at the position corresponding to position G4 is substituted with A, P, Q, R, S, or T. In yet another aspect, the variant comprises or consists of the substituted G4 (A, P, Q, R, S, T) of SEQ ID NO:3.

[0068] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G5. In another aspect, the amino acid at the position corresponding to position G5 is substituted with E, K, S, T, V, or Y. In yet another aspect, the variant comprises or consists of the substituted G5 (E, K, S, T, V, Y) of SEQ ID NO:3.

[0069] In some aspects, the present invention relates to variants, which include substitutions or constitute the amino acid at the position corresponding to position I6. In another aspect, the amino acid at the position corresponding to position I6 is substituted with A, H, K, N, P, Q, R, S, V, or W. In yet another aspect, the variant comprises or constitutes the substituted I6 (A, H, K, N, P, Q, R, S, V, W) of SEQ ID NO:3.

[0070] In some aspects, the present invention relates to variants, which include substitutions or constitute the amino acid at the position corresponding to position I6. In another aspect, the amino acid at the position corresponding to position I6 is substituted with A, H, K, N, P, Q, R, S, V, or W. In yet another aspect, the variant comprises or constitutes the substituted I6 (A, H, K, N, P, Q, R, S, V, W) of SEQ ID NO:3.

[0071] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position R7. In another aspect, the amino acid at the position corresponding to position R7 is substituted with P. In yet another aspect, the variant comprises or consists of the substituted R7(P) of SEQ ID NO:3.

[0072] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position A8. In another aspect, the amino acid at the position corresponding to position A8 is substituted with C, G, H, N, P, Q, R, S, W, or Y. In yet another aspect, the variant comprises or consists of the substituted A8 (C, G, H, N, P, Q, R, S, W, Y) of SEQ ID NO:3.

[0073] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position A9. In another aspect, the amino acid at the position corresponding to position A9 is substituted with Y. In yet another aspect, the variant comprises or consists of the substitution A8(Y) of SEQ ID NO:3.

[0074] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T10. In another aspect, the amino acid at the position corresponding to position T10 is substituted with G, R, S, Y. In yet another aspect, the variant comprises or consists of the substituted T10 (G, R, S, Y) of SEQ ID NO:3.

[0075] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S11. In another aspect, the amino acid at the position corresponding to position S11 is substituted with G, H, I, L, P, Q, R, or T. In yet another aspect, the variant comprises or consists of the substituted S11 (G, H, I, L, P, Q, R, T) of SEQ ID NO:3.

[0076] In some aspects, the present invention relates to variants comprising or consisting of a substitution corresponding to position Q12. In another aspect, the amino acid corresponding to position Q12 is substituted with A, I, K, L, M, N, R, S, T. In yet another aspect, the variant comprises or consists of the substituted Q12 (A, I, K, L, M, N, R, S, T) of SEQ ID NO:3.

[0077] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E16. In another aspect, the amino acid at the position corresponding to position E16 is substituted with P, R, S, T, or W. In yet another aspect, the variant comprises or consists of the substituted E16 (P, R, S, T, W) of SEQ ID NO:3.

[0078] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Y19. In another aspect, the amino acid at the position corresponding to position Y19 is substituted with C, E, H, L, W, or T. In yet another aspect, the variant comprises or consists of the substituted Y19 (C, E, H, L, W, T) of SEQ ID NO:3.

[0079] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position R30. In another aspect, the amino acid at the position corresponding to position R30 is substituted with A, F, H, I, L, N, P, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substitutions R30 (A, F, H, I, L, N, P, S, T, V, W) of SEQ ID NO:3.

[0080] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T31. In another aspect, the amino acid at the position corresponding to position T31 is substituted with A, C, D, E, F, G, H, I, K, P, Q, R, S, V. In yet another aspect, the variant comprises or consists of the substituted T31 (A, C, D, E, F, G, H, I, K, P, Q, R, S, V) of SEQ ID NO:3.

[0081] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P34. In another aspect, the amino acid at the position corresponding to position P34 is substituted with A, G, or V. In yet another aspect, the variant comprises or consists of the substituted P34 (A, G, V) of SEQ ID NO:3.

[0082] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position A36. In another aspect, the amino acid at the position corresponding to position A36 is substituted with D, E, G, K, Q, R, S, V. In yet another aspect, the variant comprises or consists of the substitution A36 (D, E, G, K, Q, R, S, V) of SEQ ID NO:3.

[0083] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position T37. In another aspect, the amino acid at the position corresponding to position T37 is substituted with A, E, G, S, or V. In yet another aspect, the variant comprises or consists of the substituted T37 (A, E, G, S, V) of SEQ ID NO:3.

[0084] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D39. In another aspect, the amino acid at the position corresponding to position D39 is substituted with F, K, L, N, or S. In yet another aspect, the variant comprises or consists of the substitution D39 (F, K, L, N, S) of SEQ ID NO:3.

[0085] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position C40. In another aspect, the amino acid at the position corresponding to position C40 is substituted with A, D, E, G, I, L, R, or V. In yet another aspect, the variant comprises or consists of the substituted C40 (A, D, E, G, I, L, R, or V) of SEQ ID NO:3.

[0086] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position I41. In another aspect, the amino acid at the position corresponding to position I41 is substituted with A, D, E, G, H, K, L, P, Q, R, S, V, W. In yet another aspect, the variant comprises or consists of the substituted I41 (A, D, E, G, H, K, L, P, Q, R, S, V, W) of SEQ ID NO:3.

[0087] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position H42. In another aspect, the amino acid at the position corresponding to position H42 is substituted with A, E, G, K, L, M, R, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted H42 (A, E, G, K, L, M, R, S, T, or V) of SEQ ID NO:3.

[0088] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D44. In another aspect, the amino acid at the position corresponding to position D44 is substituted with A, C, E, G, H, K, M, P, Q, R, S, V, W. In yet another aspect, the variant comprises or consists of the substitution D44 (A, C, E, G, H, K, M, P, Q, R, S, V, W) of SEQ ID NO:3.

[0089] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position I51. In another aspect, the amino acid at the position corresponding to position I51 is substituted with A, D, G, H, K, L, M, V, or Y. In yet another aspect, the variant comprises or consists of the substituted I51 (A, D, G, H, K, L, M, V, Y) of SEQ ID NO:3.

[0090] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position I52. In another aspect, the amino acid at the position corresponding to position I52 is substituted with L or V. In yet another aspect, the variant comprises or consists of the substituted I52 (L, V) of SEQ ID NO:3.

[0091] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position K53. In another aspect, the amino acid at the position corresponding to position K53 is substituted with A, D, G, H, I, L, M, P, Q, R, S, V. In yet another aspect, the variant comprises or consists of the substituted K53 (A, D, G, H, I, L, M, P, Q, R, S, V) of SEQ ID NO:3.

[0092] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position T54. In another aspect, the amino acid at the position corresponding to position T54 is substituted with A, D, E, G, I, K, L, S, V. In yet another aspect, the variant comprises or consists of the substituted T54 (A, D, E, G, I, K, L, S, V) of SEQ ID NO:3.

[0093] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S56. In another aspect, the amino acid at the position corresponding to position S56 is substituted with A, P, Q, R, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted S56 (A, P, Q, R, T, V, W) of SEQ ID NO:3.

[0094] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L58. In another aspect, the amino acid at the position corresponding to position L58 is substituted with C, G, I, P, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted L58 (C, G, I, P, R, S, V, W) of SEQ ID NO:3.

[0095] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position I59. In another aspect, the amino acid at the position corresponding to position I59 is substituted with A, D, F, G, H, L, M, P, R, S, T, V. In yet another aspect, the variant comprises or consists of the substitution I59 (A, D, F, G, H, L, M, P, R, S, T, V) of SEQ ID NO:3.

[0096] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D61. In another aspect, the amino acid at the position corresponding to position D61 is substituted with G or M. In yet another aspect, the variant comprises or consists of the substituted D61 (G, M) of SEQ ID NO:3.

[0097] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position D70. In another aspect, the amino acid at the position corresponding to position D70 is substituted with A, E, G, or R. In yet another aspect, the variant comprises or consists of the substitutions (A, E, G, R) of SEQ ID NO:3.

[0098] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S71. In another aspect, the amino acid at the position corresponding to position S71 is substituted with E, K, N, Q, R, W, or Y. In yet another aspect, the variant comprises or consists of the substitutions (E, K, N, Q, R, W, Y) of SEQ ID NO:3.

[0099] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E72. In another aspect, the amino acid at the position corresponding to position E72 is substituted with A, C, D, G, K, R, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted E72 (A, C, D, G, K, R, S, T, or V) of SEQ ID NO:3.

[0100] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position K73. In another aspect, the amino acid at the position corresponding to position K73 is substituted with E, G, L, N, Q, R, S, or T. In yet another aspect, the variant comprises or consists of the substituted K73 (E, G, L, N, Q, R, S, T) of SEQ ID NO:3.

[0101] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position S83. In another aspect, the amino acid at the position corresponding to position S83 is substituted with A. In yet another aspect, the variant comprises or consists of the substitution S83(A) of SEQ ID NO:3.

[0102] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S84. In another aspect, the amino acid at the position corresponding to position S84 is substituted with A, E, L, P, W, or Y. In yet another aspect, the variant comprises or consists of the substitutions (A, E, L, P, W, Y) of SEQ ID NO:3.

[0103] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position R86. In another aspect, the amino acid at the position corresponding to position R86 is substituted with A, G, I, K, M, or W. In yet another aspect, the variant comprises or consists of the substituted R86 (A, G, I, K, M, W) of SEQ ID NO:3.

[0104] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position W88. In another aspect, the amino acid at the position corresponding to position W88 is substituted with M, Q, R, or S. In yet another aspect, the variant comprises or consists of the substituted W88 (M, Q, R, S) of SEQ ID NO:3.

[0105] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position A90. In another aspect, the amino acid at the position corresponding to position A90 is substituted with D, F, G, L, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted A90 (D, F, G, L, S, T, V, W) of SEQ ID NO:3.

[0106] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D91. In another aspect, the amino acid at the position corresponding to position D91 is substituted with E, F, G, Q, or Y. In yet another aspect, the variant comprises or consists of the substituted D91 (E, F, G, Q, Y) of SEQ ID NO:3.

[0107] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L92. In another aspect, the amino acid at the position corresponding to position L92 is substituted with A, C, D, F, P, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted L92 (A, C, D, F, P, S, T, V, W) of SEQ ID NO:3.

[0108] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position T93. In another aspect, the amino acid at the position corresponding to position T93 is substituted with A, I, R, or V. In yet another aspect, the variant comprises or consists of the substituted T93 (A, I, R, V) of SEQ ID NO:3.

[0109] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position V95. In another aspect, the amino acid at the position corresponding to position V95 is substituted with C, G, I, Q, R, or S. In yet another aspect, the variant comprises or consists of the substituted V95 (C, G, I, Q, R, S) of SEQ ID NO:3.

[0110] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P96. In another aspect, the amino acid at the position corresponding to position P96 is substituted with A, C, D, E, G, N, Q, R, S, W, or Y. In yet another aspect, the variant comprises or consists of the substituted P96 (A, C, D, E, G, N, Q, R, S, W, Y) of SEQ ID NO:3.

[0111] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S98. In another aspect, the amino acid at the position corresponding to position S98 is substituted with A, D, E, G, K, N, R, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted S98 (A, D, E, G, K, N, R, T, V, W) of SEQ ID NO:3.

[0112] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position P100. In another aspect, the amino acid at the position corresponding to position P100 is substituted with A, E, G, K, L, R, S, V, W. In yet another aspect, the variant comprises or consists of the substituted P100 (A, E, G, K, L, R, S, V, W) of SEQ ID NO:3.

[0113] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position P101. In another aspect, the amino acid at the position corresponding to position P101 is substituted with D, H, R, S, V. In yet another aspect, the variant comprises or consists of substituted P101 (D, H, R, S, V) of SEQ ID NO:3.

[0114] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position V102. In another aspect, the amino acid at the position corresponding to position V102 is substituted with H, I, P, R, or S. In yet another aspect, the variant comprises or consists of the substituted V102 (H, I, P, R, S) of SEQ ID NO:3.

[0115] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S103. In another aspect, the amino acid at the position corresponding to position S103 is substituted with A, C, D, F, G, L, P, or V. In yet another aspect, the variant comprises or consists of the substitutions of S103 (A, C, D, F, G, L, P, V) of SEQ ID NO:3.

[0116] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G104. In another aspect, the amino acid at the position corresponding to position G104 is substituted with A, C, D, E, K, L, M, P, Q, R, V, W, or Y. In yet another aspect, the variant comprises or consists of the substituted G104 (A, C, D, E, K, L, M, P, Q, R, V, W, Y) of SEQ ID NO:3.

[0117] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position K106. In another aspect, the amino acid at the position corresponding to position K106 is substituted with A, C, G, L, P, Q, R, S, T, or W. In yet another aspect, the variant comprises or consists of the substituted K106 (A, C, G, L, P, Q, R, S, T, W) of SEQ ID NO:3.

[0118] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position K109. In another aspect, the amino acid at the position corresponding to position K109 is substituted with A, D, E, G, H, L, N, P, Q, R, S, or T. In yet another aspect, the variant comprises or consists of the substituted K109 (A, D, E, G, H, L, N, P, Q, R, S, T) of SEQ ID NO:3.

[0119] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position G110. In another aspect, the amino acid at the position corresponding to position G110 is substituted with C, P, S, or V. In yet another aspect, the variant comprises or consists of the substituted G110 (C, P, S, V) of SEQ ID NO:3.

[0120] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L112. In another aspect, the amino acid at the position corresponding to position L112 is substituted with F, H, Q, R, or W. In yet another aspect, the variant comprises or consists of the substituted L112 (F, H, Q, R, W) of SEQ ID NO:3.

[0121] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D113. In another aspect, the amino acid at the position corresponding to position D113 is substituted with M or W. In yet another aspect, the variant comprises or consists of the substituted D113 (M, W) of SEQ ID NO:3.

[0122] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G116. In another aspect, the amino acid at the position corresponding to position G116 is substituted with A, C, D, F, R, S, or T. In yet another aspect, the variant comprises or consists of the substituted G116 (A, C, D, F, R, S, T) of SEQ ID NO:3.

[0123] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E117. In another aspect, the amino acid at the position corresponding to position E117 is substituted with A, D, G, N, P, R, S, or V. In yet another aspect, the variant comprises or consists of the substituted E117 (A, D, G, N, P, R, S, V) of SEQ ID NO:3.

[0124] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Q119. In another aspect, the amino acid at the position corresponding to position Q119 is substituted with D, E, F, I, R, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted Q119 (D, E, F, I, R, S, T, or V) of SEQ ID NO:3.

[0125] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position N120. In another aspect, the amino acid at the position corresponding to position N120 is substituted with A, E, G, H, L, M, S, T, V, or Y. In yet another aspect, the variant comprises or consists of the substituted N120 (A, E, G, H, L, M, S, T, V, Y) of SEQ ID NO:3.

[0126] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position A124. In another aspect, the amino acid at the position corresponding to position A124 is substituted with C, D, G, H, L, M, Q, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted A124 (C, D, G, H, L, M, Q, R, S, V, W) of SEQ ID NO:3.

[0127] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T125. In another aspect, the amino acid at the position corresponding to position T125 is substituted with A, D, E, G, I, K, L, M, Q, R, S, V. In yet another aspect, the variant comprises or consists of the substituted T125 (A, D, E, G, I, K, L, M, Q, R, S, V) of SEQ ID NO:3.

[0128] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L127. In another aspect, the amino acid at the position corresponding to position L127 is substituted with A, F, G, H, I, Q, R, S, V, Y. In yet another aspect, the variant comprises or consists of the substituted L127 (A, F, G, H, I, Q, R, S, V, Y) of SEQ ID NO:3.

[0129] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D128. In another aspect, the amino acid at the position corresponding to position D128 is substituted with A, E, G, H, M, Q, S, or T. In yet another aspect, the variant comprises or consists of the substituted D128 (A, E, G, H, M, Q, S, T) of SEQ ID NO:3.

[0130] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position K131. In another aspect, the amino acid at the position corresponding to position K131 is substituted with A, G, I, M, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted K131 (A, G, I, M, S, T, V, W) of SEQ ID NO:3.

[0131] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Q132. In another aspect, the amino acid at the position corresponding to position Q132 is substituted with G, S, or V. In yet another aspect, the variant comprises or consists of the substituted Q132 (G, S, V) of SEQ ID NO:3.

[0132] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Y133. In another aspect, the amino acid at the position corresponding to position Y133 is substituted with A, E, K, L, M, R, V, or W. In yet another aspect, the variant comprises or consists of the substituted Y133 (A, E, K, L, M, R, V, W) of SEQ ID NO:3.

[0133] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P134. In another aspect, the amino acid at the position corresponding to position P134 is substituted with A, D, E, G, K, R, S, T, V. In yet another aspect, the variant comprises or consists of the substituted P134 (A, D, E, G, K, R, S, T, V) of SEQ ID NO:3.

[0134] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S135. In another aspect, the amino acid at the position corresponding to position S135 is substituted with A, C, G, P, Q, R, T, V, W, or Y. In yet another aspect, the variant comprises or consists of the substitutions of SEQ ID NO:3 for S135 (A, C, G, P, Q, R, T, V, W, Y).

[0135] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position K137. In another aspect, the amino acid at the position corresponding to position K137 is substituted with A, F, G, P, or R. In yet another aspect, the variant comprises or consists of the substituted K137 (A, F, G, P, R) of SEQ ID NO:3.

[0136] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Y158. In another aspect, the amino acid at the position corresponding to position Y158 is substituted with A, C, G, I, L, N, R, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted Y158 (A, C, G, I, L, N, R, S, T, V, W) of SEQ ID NO:3.

[0137] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Q159. In another aspect, the amino acid at the position corresponding to position Q159 is substituted with G or T. In yet another aspect, the variant comprises or consists of the substituted Q159 (G, T) of SEQ ID NO:3.

[0138] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position R160. In another aspect, the amino acid at the position corresponding to position R160 is substituted with A, D, G, I, L, Q, or V. In yet another aspect, the variant comprises or consists of the substitutions R160 (A, D, G, I, L, Q, or V) of SEQ ID NO:3.

[0139] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E161. In another aspect, the amino acid at the position corresponding to position E161 is substituted with C, G, I, K, L, P, Q, R, or S. In yet another aspect, the variant comprises or consists of the substituted E161 (C, G, I, K, L, P, Q, R, S) of SEQ ID NO:3.

[0140] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position E162. In another aspect, the amino acid at the position corresponding to position E162 is substituted with A, D, G, L, M, P, R, S, V, W. In yet another aspect, the variant comprises or consists of the substituted E162 (A, D, G, L, M, P, R, S, V, W) of SEQ ID NO:3.

[0141] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G163. In another aspect, the amino acid at the position corresponding to position G163 is substituted with A, D, E, H, L, M, Q, V, or Y. In yet another aspect, the variant comprises or consists of the substituted G163 (A, D, E, H, L, M, Q, V, Y) of SEQ ID NO:3.

[0142] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S165. In another aspect, the amino acid at the position corresponding to position S165 is substituted with A, C, G, K, L, M, P, R, V, or W. In yet another aspect, the variant comprises or consists of the substitutions of SEQ ID NO:3 for S165 (A, C, G, K, L, M, P, R, V, W).

[0143] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S166. In another aspect, the amino acid at the position corresponding to position S166 is substituted with A, C, E, G, L, P, R, or W. In yet another aspect, the variant comprises or consists of the substitutions of S166 (A, C, E, G, L, P, R, W) of SEQ ID NO:3.

[0144] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position S167. In another aspect, the amino acid at the position corresponding to position S167 is substituted with C, F, H, I, L, M, R, or V. In yet another aspect, the variant comprises or consists of the substitution of S167 (C, F, H, I, L, M, R, or V) of SEQ ID NO:3.

[0145] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position N168. In another aspect, the amino acid at the position corresponding to position N168 is substituted with A, C, D, G, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted N168 (A, C, D, G, R, S, V, W) of SEQ ID NO:3.

[0146] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position F170. In another aspect, the amino acid at the position corresponding to position F170 is substituted with A, C, E, G, H, I, K, L, M, Q, R, S, V, W, or Y. In yet another aspect, the variant comprises or consists of the substituted F170 (A, C, E, G, H, I, K, L, M, Q, R, S, V, W, Y) of SEQ ID NO:3.

[0147] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D181. In another aspect, the amino acid at the position corresponding to position D181 is substituted with A, C, E, G, L, Q, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted D181 (A, C, E, G, L, Q, R, S, V, W) of SEQ ID NO:3.

[0148] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P182. In another aspect, the amino acid at the position corresponding to position P182 is substituted with A, C, F, G, I, L, R, S, T, V, W, or Y. In yet another aspect, the variant comprises or consists of the substituted P182 (A, C, F, G, I, L, R, S, T, V, W, Y) of SEQ ID NO:3.

[0149] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position A183. In another aspect, the amino acid at the position corresponding to position A183 is substituted with E or V. In yet another aspect, the variant comprises or consists of the substitution of A183 (E, V) of SEQ ID NO:3.

[0150] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position N186. In another aspect, the amino acid at the position corresponding to position N186 is substituted with A, C, E, G, H, K, L, R, S, T, or W. In yet another aspect, the variant comprises or consists of the substituted N186 (A, C, E, G, H, K, L, R, S, T, W) of SEQ ID NO:3.

[0151] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position V189. In another aspect, the amino acid at the position corresponding to position V189 is substituted with A, C, G, L, M, P, R, or S. In yet another aspect, the variant comprises or consists of the substituted V189 (A, C, G, L, M, P, R, S) of SEQ ID NO:3.

[0152] In some aspects, the present invention relates to variants comprising or consisting of substitutions at positions corresponding to position S190. In another aspect, the amino acid at position S190 is substituted with A, E, G, I, K, N, R, V, or W. In yet another aspect, the variant comprises or consists of the substitutions (A, E, G, I, K, N, R, V, W) of SEQ ID NO:3.

[0153] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G192. In another aspect, the amino acid at the position corresponding to position G192 is substituted with A, I, L, M, P, R, T, V. In yet another aspect, the variant comprises or consists of the substituted G192 (A, I, L, M, P, R, T, V) of SEQ ID NO:3.

[0154] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P194. In another aspect, the amino acid at the position corresponding to position P194 is substituted with A, D, E, F, G, H, L, Q, T, or V. In yet another aspect, the variant comprises or consists of the substituted P194 (A, D, E, F, G, H, L, Q, T, V) of SEQ ID NO:3.

[0155] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position R196. In another aspect, the amino acid at the position corresponding to position R196 is substituted with A, Q, or Y. In yet another aspect, the variant comprises or consists of the substitution of R196 (A, Q, Y) of SEQ ID NO:3.

[0156] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E201. In another aspect, the amino acid at the position corresponding to position E201 is substituted with D, G, L, or P. In yet another aspect, the variant comprises or consists of the substituted E201 (D, G, L, P) of SEQ ID NO:3.

[0157] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position R202. In another aspect, the amino acid at the position corresponding to position R202 is substituted with I or M. In yet another aspect, the variant comprises or consists of the substituted R202 (I, M) of SEQ ID NO:3.

[0158] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position I204. In another aspect, the amino acid at the position corresponding to position I204 is substituted with E, K, L, P, or R. In yet another aspect, the variant comprises or consists of the substituted I204 (E, K, L, P, R) of SEQ ID NO:3.

[0159] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L208. In another aspect, the amino acid at the position corresponding to position L208 is substituted with E, M, P, R, or W. In yet another aspect, the variant comprises or consists of the substituted L208 (E, M, P, R, W) of SEQ ID NO:3.

[0160] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position P210. In another aspect, the amino acid at the position corresponding to position P210 is substituted with A, F, L, or Q. In yet another aspect, the variant comprises or consists of the substituted P210 (A, F, L, Q) of SEQ ID NO:3.

[0161] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position A211. In another aspect, the amino acid at the position corresponding to position A211 is substituted with C, E, G, P, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted A211 (C, E, G, P, S, V, W) of SEQ ID NO:3.

[0162] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position A212. In another aspect, the amino acid at the position corresponding to position A212 is substituted with D, E, G, H, K, N, P, Q, R, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted A212 (D, E, G, H, K, N, P, Q, R, S, T, V, W) of SEQ ID NO:3.

[0163] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position F213. In another aspect, the amino acid at the position corresponding to position F213 is substituted with E, H, L, M, P, Q, R, or W. In yet another aspect, the variant comprises or consists of the substituted F213 (E, H, L, M, P, Q, R, W) of SEQ ID NO:3.

[0164] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position E220. In another aspect, the amino acid at the position corresponding to position E220 is substituted with A, C, D, G, L, P, R, S, T, or W. In yet another aspect, the variant comprises or consists of the substituted E220 (A, C, D, G, L, P, R, S, T, W) of SEQ ID NO:3.

[0165] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T225. In another aspect, the amino acid at the position corresponding to position T225 is substituted with A, L, M, P, Q, or S. In yet another aspect, the variant comprises or consists of the substituted T225 (A, L, M, P, Q, S) of SEQ ID NO:3.

[0166] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D226. In another aspect, the amino acid at the position corresponding to position D226 is substituted with C, E, F, G, P, R, W, or Y. In yet another aspect, the variant comprises or consists of the substituted D226 (C, E, F, G, P, R, W, Y) of SEQ ID NO:3.

[0167] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position N227. In another aspect, the amino acid at the position corresponding to position N227 is substituted with A, C, G, P, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substituted N227 (A, C, G, P, R, S, V, W) of SEQ ID NO:3.

[0168] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S228. In another aspect, the amino acid at the position corresponding to position S228 is substituted with A, C, D, G, L, P, R, V, or W. In yet another aspect, the variant comprises or consists of the substitutions of S228 (A, C, D, G, L, P, R, V, W) of SEQ ID NO:3.

[0169] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position P229. In another aspect, the amino acid at the position corresponding to position P229 is substituted with A, I, L, N, R, S, T. In yet another aspect, the variant comprises or consists of the substituted P229 (A, I, L, N, R, S, T) of SEQ ID NO:3.

[0170] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position E230. In another aspect, the amino acid at the position corresponding to position E230 is substituted with A, G, H, L, P, Q, R, S, T, V, or W. In yet another aspect, the variant comprises or consists of the substituted E230 (A, G, H, L, P, Q, R, S, T, V, W) of SEQ ID NO:3.

[0171] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T231. In another aspect, the amino acid at the position corresponding to position T231 is substituted with A, C, G, H, K, R, S, V, W, or Y. In yet another aspect, the variant comprises or consists of the substituted T231 (A, C, G, H, K, R, S, V, W, Y) of SEQ ID NO:3.

[0172] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position Q233. In another aspect, the amino acid at the position corresponding to position Q233 is substituted with A, F, H, L, M, S, or T. In yet another aspect, the variant comprises or consists of the substituted Q233 (A, F, H, L, M, S, T) of SEQ ID NO:3.

[0173] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position T236. In another aspect, the amino acid at the position corresponding to position T236 is substituted with C, L, P, S, or V. In yet another aspect, the variant comprises or consists of the substituted T236 (C, L, P, S, V) of SEQ ID NO:3.

[0174] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position S237. In another aspect, the amino acid at the position corresponding to position S237 is substituted with F, G, H, L, M, N, R, or V. In yet another aspect, the variant comprises or consists of the substitution S237 (F, G, H, L, M, N, R, or V) of SEQ ID NO:3.

[0175] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D238. In another aspect, the amino acid at the position corresponding to position D238 is substituted with A, E, F, G, H, M, P, R, S, V, or W. In yet another aspect, the variant comprises or consists of the substitution D238 (A, E, F, G, H, M, P, R, S, V, W) of SEQ ID NO:3.

[0176] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position L239. In another aspect, the amino acid at the position corresponding to position L239 is substituted with F, G, S, or Y. In yet another aspect, the variant comprises or consists of the substituted L239 (F, G, S, Y) of SEQ ID NO:3.

[0177] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position E240. In another aspect, the amino acid at the position corresponding to position E240 is substituted with A. In yet another aspect, the variant comprises or consists of the substitution E240(A) of SEQ ID NO:3.

[0178] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position T241. In another aspect, the amino acid at the position corresponding to position T241 is substituted with M or V. In yet another aspect, the variant comprises or consists of the substitution of T241 (M, V) of SEQ ID NO:3.

[0179] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S242. In another aspect, the amino acid at the position corresponding to position S242 is substituted with A, C, G, I, L, M, P, R, T, V, or Y. In yet another aspect, the variant comprises or consists of the substitutions of SEQ ID NO:3 for S242 (A, C, G, I, L, M, P, R, T, V, Y).

[0180] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position D243. In another aspect, the amino acid at the position corresponding to position D243 is substituted with A, C, E, F, G, or M. In yet another aspect, the variant comprises or consists of the substitution of D243 (A, C, E, F, G, M) of SEQ ID NO:3.

[0181] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position N246. In another aspect, the amino acid at the position corresponding to position N246 is substituted with A, E, K, P, S, T, or V. In yet another aspect, the variant comprises or consists of the substituted N246 (A, E, K, P, S, T, or V) of SEQ ID NO:3.

[0182] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position S247. In another aspect, the amino acid at the position corresponding to position S247 is substituted with A, E, L, P, T, or Y. In yet another aspect, the variant comprises or consists of the substitution of S247 (A, E, L, P, T, Y) of SEQ ID NO:3.

[0183] In some aspects, the present invention relates to variants comprising or consisting of a substitution corresponding to position I248. In another aspect, the amino acid corresponding to position I248 is substituted with A, G, P, or V. In yet another aspect, the variant comprises or consists of the substituted I248 (A, G, P, V) of SEQ ID NO:3.

[0184] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T252. In another aspect, the amino acid at the position corresponding to position T252 is substituted with A, D, E, G, N, Q, S, or V. In yet another aspect, the variant comprises or consists of the substituted T252 (A, D, E, G, N, Q, S, V) of SEQ ID NO:3.

[0185] In some aspects, the present invention relates to variants that include substitutions or are composed of the amino acid at the position corresponding to position L255. In another aspect, the amino acid at the position corresponding to position L255 is substituted with C, E, M, Q, or R. In yet another aspect, the variant comprises or is composed of the substituted L255 (C, E, M, Q, R) of SEQ ID NO:3.

[0186] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position S259. In another aspect, the amino acid at the position corresponding to position S259 is substituted with A, C, D, E, G, I, K, L, M, Q, R, T, V, W, or Y. In yet another aspect, the variant comprises or consists of the substitutions of S259 (A, C, D, E, G, I, K, L, M, Q, R, T, V, W, Y) of SEQ ID NO:3.

[0187] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position G262. In another aspect, the amino acid at the position corresponding to position G262 is substituted with A, D, E, K, M, R, T, or V. In yet another aspect, the variant comprises or consists of the substituted G262 (A, D, E, K, M, R, T, V) of SEQ ID NO:3.

[0188] In some aspects, the present invention relates to variants comprising or consisting of a substitution at the position corresponding to position N264. In another aspect, the amino acid at the position corresponding to position N264 is substituted with C, G, I, L, M, P, R, S, T. In yet another aspect, the variant comprises or consists of the substituted N264 (C, G, I, L, M, P, R, S, T) of SEQ ID NO:3.

[0189] In some aspects, the present invention relates to variants that include substitutions at or constitute the position corresponding to position L267. In another aspect, the amino acid at the position corresponding to position L267 is substituted with E, H, K, M, Q, or W. In yet another aspect, the variant comprises or constitutes the substituted L267 (E, H, K, M, Q, W) of SEQ ID NO:3.

[0190] In some aspects, the present invention relates to variants comprising or consisting of substitutions at the position corresponding to position T269. In another aspect, the amino acid at the position corresponding to position T269 is substituted with A, D, E, G, H, K, L, M, N, Q, R, S, V, Y. In yet another aspect, the variant comprises or consists of the substituted T269 (A, D, E, G, H, K, L, M, N, Q, R, S, V, Y) of SEQ ID NO:3.

[0191] In some aspects, the present invention relates to variants wherein an improvement factor (IF) greater than 1.0 is measured, the variant having one or more (e.g., several) improved properties relative to the parent, wherein such improved properties are selected from the group consisting of: thermal stability; thermal stability in detergents; hydrolytic activity in detergents; and stability in detergents.

[0192] In some aspects, the present invention relates to variants wherein the improvement factor (IF) is greater than 1.0 in at least one assay selected from the group consisting of: A thermal stability assay; B thermal stability assay in detergent; C performance screening assay; and D stability assay in detergent.

[0193] In some aspects, the present invention relates to variants in which the improvement factor (IF) is greater than 1.0 in at least two, at least three, or at least four determinations.

[0194] In some aspects, the present invention relates to variants wherein the improvement factor (IF) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.3, 2.4 or 2.5.

[0195] The amino acid positions within the molecule used to create the variant are such that substitution at at least one of these positions results in a variant that, compared to the unchanged molecule, i.e., the parent, exhibits improved characteristics, i.e., IF > 1.0. This improved characteristic can be determined using the assays described in Example 1: A. thermal stability assay; B. thermal stability assay in detergent; C. detergent stability assay; or D. performance screening assay.

[0196] In some aspects, the present invention relates to variants having lipase activity and at positions 1, 2, 3, 4, 5, 6, 11, 12, 16, 30, 31, 37, 39, 40, 42, 44, 51, 53, 54, 58, 59, 71, 72, 73, 88, 92, 93, 95, 96, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 134, 135, 13 One or more (e.g., several) of the positions 7, 158, 160, 162, 163, 165, 166, 167, 168, 170, 181, 182, 190, 194, 196, 202, 210, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 242, 246, 259, 264, and 269 include substitutions, the polypeptide having 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, and having improved thermal stability, i.e., IF > 1.0, compared to the parent. This improved property can be measured using a "thermal stability test" as described in Example 1.

[0197] In some aspects, the present invention relates to variants, wherein the variant has lipase activity and at positions corresponding to SEQ ID NO: 3: 1 (C,F,G,H,I,L,M,P,Q,R,V,W,Y), 2 (C,M,N,P,R,T,V), 3 (A,E,G,K,T,V), 4 (A,P,Q,R,S,T), 5 (E,K,Y), 6 (H,K,P,Q,R), 8 (C,P,R,S,W), 11 (I,L,P,R), 12 (A,I,L,M,R,S,T), 16 (P,R,W), 30 (L,P,S), 31 (F,Q,V), 37 (A), 39 (N), 40 (E,I,L), 41 (A,D,E,G,H,L,P,Q,R,S,V,W), 42 (A,E,G,L,M,R,T,V), 44 (A,C,G,K,M,P,R,V,W), 51 (A,G,K,L,M,V), 53 (A,D,G,H,I,L,M,Q,R,S,V), 54 (A,D,E,G,I,K,L,S,V), 58 (C,G,P,R,W), 59 (A,H,M,R,S,T,V), 71 (K,R), 72 (A,C,D,G,K,R,S,V), 73 (E,G,L,N,Q,R), 84 (A,E,L,P,W,Y), 88 (M,Q,R), 90 (D,G,S,T,V,W), 91 (G,Q,Y), 92 (A,D,F,S,T,V), 93 (A,R,V), 95 (C,G,I,Q,R,S), 96 (C,D,E,G,N,Q,S,W,Y), 98 (A,D,E,G), 102 (H,I,R), 103 (A,C,D,F,G,P), 104 (K,L,R), 106 (L,P,Q,R), 109 (A,E,R,S), 110 (C,V), 112 (F,Q,W), 117 (A,R,S), 119 (E,R,S), 124 (G,L,S,W), 125 (A,M,R,S,V), 127 (A,F,H,R,S), 128 (A,E,Q,T), 131 (G,M,T), 132 (G,V), 134 (A,D,E,G,K,R,S,T,V), 135 (A,G,Q,V,W,Y), 137 (A,P,R), 158 (W), 160 (A,D,G,I,Q,V), 162 (A,G,L,M,P,R,S,V,W), 163 (A,D,E,Q), 165 (A,G,K,R), 166 (A,G,P), 167 (H,I,L), 168 (A,C,G,R,S,V,W), 170 (A,E,G,H,I,K,L,M,R,V,Y), 181 (E,W), 182 (F,I,R,Y), 186 (A,C,E,G,K,R,S), 190 (A,E,G,K,N,R,W), 194(A,D,E,H,L,T), 196(A,Q,Y), 201(D,G,L,P), 202(I,M) ,204(E,K,L,P,R),208(E,M,R,W),210(A),212(D,E,G,H,K,N,P,Q,R,S, T,V,W), 213(E,H,L,M,Q,R,W), 220(A,C,G,L,P,R,S,T), 225(L,M), 226( C,F,G,P,R,W,Y), 227(S), 228(P,R,V), 229(A,L,N,R,S,T), 230(G,H,P, One or more (e.g., several) positions of the following peptides include substitutions: 231(A,C,G,H,K,R,S,V,Y), 233(A,F,H,L,M,S), 236(V), 237(F,G,H,R), 238(A,E,F,G,H,M,P,R,S,V,W), 239(F,Y), 242(A,C,P,T), 243(A,C,F,G,M), 246(P), 255(C,E,M,Q,R), 259(C,E,L,M,R,T,V), 264(C,P,R,S), 267(E), 269(D,E,G,L,M,Q,R,V), the peptide is similar to SEQ. ID NO:3 exhibits 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, and demonstrates improved thermal stability compared to the parent, i.e., IF > 1.0. This improved property can be measured using the "A Thermal Stability Test" as described in Example 1.

[0198] In some aspects, the present invention relates to variants having lipase activity and at positions 1, 2, 3, 4, 5, 6, 7, 10, 11, 16, 19, 30, 31, 36, 42, 44, 51, 52, 54, 56, 58, 59, 70, 71, 72, 73, 88, 92, 93, 96, 100, 101, 104, 106, 109, 110, 112, 119, 124, 125, 127, 128, 131, 133, 134, 135, 158, 160 corresponding to SEQ ID NO:3 or the polypeptide. One or more (e.g., several) positions of SEQ ID NO: 161, 162, 163, 165, 166, 167, 170, 181, 182, 189, 190, 192, 194, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 242, 246, 247, 248, 252, 259, 264, 269 include substitutions, and the polypeptide is similar to SEQ ID NO: 164, 165, 166, 167, 170, 181, 182, 189, 190, 192, 194, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 242, 246, 247, 248, 252, 259, 264, 269. ID NO:3 has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, and exhibits improved thermal stability in detergents, i.e., IF > 1.0, compared to the parent. This improved property can be measured using the "Determination of Thermal Stability in Detergent B" as described in Example 1.

[0199] In some aspects, the present invention relates to variants, where the variants have lipase activity and correspond to the positions of SEQ ID NO: 3 or the polypeptide: 1 (C, F, G, H, I, M, Q, R, V, W, Y), 2 (A, C, D, N, P, Q, R, S, T, V), 3 (E, G, Q, S), 4 (A, P, S, T), 5 (S, T, V, Y), 6 (A, H, K, N, P, Q, R, S, V, W), 7 (P), 8 (C, G, H, N, P, Q, R, S, W, Y), 10 (S, Y), 11 (H, P, Q, R, T), 16 (S, T), 19 (C, E, H, L, W), 30 (A, F, H, L, P, S, T, W), 31 (C, D, E, G, I, K, Q, S, V), 36 (E, G, K, Q), 42 (G, K, L, R, S, V), 44 (A, E, G, H, K, P, Q, S, V), 51 (V), 52 (L, V), 54 (A, I), 56 (Q, T), 58 (C, G, I, P, R, W), 59 (D, F, H, L, T, V), 70 (A, E, G, R), 71 (E, K, N, Q, R, W, Y), 72 (A, T, V), 73 (E, G, N, R, T), 86 (G, I, K, M), 88 (R, S), 90 (F), 91 (F, Y), 92 (A, S, V), 93 (A, I), 96 (A, C, D, E, G, N, Q, R, W, Y), 98 (A, E, G, K, N, T, V, W), 100 (A, E, K, L, R, V, W), 101 (D, H, R, S, V), 103 (A, D, G, L, V), 104 (A, C, D, E, K, L, M, P, Q, R, V, W, Y), 106 (A, C, L, P, T), 109 (A, D, E, G, H, L, Q, R, S, T), 110 (C, S, V), 112 (F, H, Q, R, W), 116 (A, S, T), 119 (D, E, F,, R, S, T, V), 120 (A, E, G, H, L, M, S, T, V, Y), 124 (C, D, G, H, L, M, Q, R, S, V, W), 125 (A, D, G, K, M, R, S, V), 127 (F, G, H, I, R, S, V, Y), 128 (A, G, Q, T), 131 (A, G, I, M, S, T, V, W), 133 (A, E, K, L, R, W), 134 (A, D, E, G, K, R, S, T), 135 (A, C, G, P, R, W, Y), 158 (C, G, I, L, N, R, S, T, V), 160 (V), 161 (C, G, I, L, Q, R, S), 162 (A, D, L, M, P, R, S, V), 163 (A, D, E, H, L, M, Q, V), 165 (C, G, K, L, M, P, R, V,W), 166(A,C,G,P,W), 167(C,F,I,L,M,R,V), 170(A,C), 181(A,G,L,Q,R,S,V,W), 182(A,C,F,G,I,L,R,S ,T,V,W,Y), 186(A,E,G,H,L,R,S,T,W), 189(A,C,G,L,M,P,R,S), 190(A,E,G,I,K,R,V), 192(A,I,L,M,P, R,T,V), 194(E,G,Q), 201(D,G), 204(K,P), 208(M), 210(L), 211(G,P,S,V,W), 212(D,E,G,H,K,N,R,S), 2 13(E,H,L,M,P,R,W), 220(D,G,R,S,W), 225(A,L,P,S), 226(C,E,G,P,W), 227(C,G,P,R,S,V), 228(A,G,L ,R), 229(I,L,N,S), 230(A,G,H,L,P,Q,R,S,T,W), 231(C,G,H,K,R,S), 233(A,S,T), 236(C,L,P,S,V), 23 7(F,L,M,N,R,V), 238(A,E,G,P,R,S,V), 239(S), 241(M,V), 242(A,I,L,M,P,R,T,V,Y), 243(M), 246(A,K One or more (e.g., several) positions of SEQ (S,T), 247 (A,E,L,T,Y), 248 (V), 252 (A,E,G,N,S,V), 255 (C,M,Q), 259 (A,C,D,E,M,Q,T,V,W,Y), 264 (G,I,L,M,P,S,T), 267 (E,H,K,M,Q,W), and 269 (A,E,G,H,K,L,M,N,Q,R,S,V,Y) include substitutions, and the polypeptide is similar to SEQ (S,T). ID NO:3 exhibits 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 and, compared to its parent, demonstrates improved thermal stability in detergents, i.e., IF > 1.0. This improved property can be measured using the "Determination of Thermal Stability in Detergent B" as described in Example 1.

[0200] In some aspects, the present invention relates to variants having lipase activity and at positions 1, 2, 5, 6, 9, 10, 11, 12, 19, 30, 31, 34, 36, 37, 39, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 92, 93, 95, 96, 100, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135, 13 One or more (e.g., several) positions of 7, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 181, 182, 183, 189, 190, 192, 194, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 238, 239, 240, 242, 246, 247, 248, 252, 259, 262, 264 include substitutions, and the polypeptide is similar to SEQ ID NO. ID NO:3 exhibits 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 and, compared to the parent, demonstrates improved hydrolytic activity in detergents, i.e., IF > 1.0. This improved property can be measured using the “C performance screening assay” as described in Example 1.

[0201] In some aspects, the present invention relates to variants, wherein the variants have lipase activity and correspond to the positions of SEQ ID NO: 3 or the polypeptide: 1 (M), 2 (Q, T), 5 (S), 6 (K, N, V), 9 (Y), 10 (G, R, S), 11 (G, H, I, L, P, R), 12 (I, K, L, M, N, R, S, T), 19 (C, T), 30 (A, F, I, L, N, P, T, V), 31 (A, G, H, P, R), 34 (A, G, V), 36 (D, E, G, K, R, S, V), 37 (A, E, G, S, V), 39 (F, K, L, S), 40 (A, E, G, I, L, V), 41 (A, H, K, L, P, R, V), 42 (A, R, V), 44 (G, P), 51 (A, D, G, H, V, Y), 52 (L, V), 53 (G, H, L, P, Q, R), 54 (K, V), 56 (A, P, Q, R, T, V, W), 58 (C, S, V), 59 (D, G, P, R, V), 61 (G, M), 70 (A, G), 71 (N, W), 72 (A, G, K, R, T), 73 (E, G, Q, R, S), 83 (A), 86 (A, G, W), 90 (F, L), 91 (E), 92 (C, P, V, W), 93 (A, V), 95 (G, R), 96 (C, G, S, Y), 98 (A, D, R, V), 100 (A, G, R, S, W), 102 (P, R, S), 103 (A, G, L, P, V), 104 (L, M, R), 106 (A, G, S, W), 109 (L, N, P, R), 110 (P, S), 112 (Q, R), 113 (M, W), 116 (C, D, F, R), 117 (A, D, G, N, P, R, V), 119 (E, I, R, S, T), 120 (M), 124 (L, M, Q, R, S, V), 125 (A, D, E, G, I, L, M, Q, R, S, V), 127 (Q, V), 128 (E, Q, T), 131 (A, V), 132 (S), 133 (E, M, V, W), 134 (A, D, G, K, R), 135 (G, R, T, Y), 158 (C, G, I, N, R, S), 159 (G, T), 160 (A, L, Q, V), 161 (C,, K, L, P, Q, R), 162 (A, G, P, S, W), 163 (E, L, Q, Y), 165 (A, L, R, V, W), 166 (A, C, E, G, L, R), 167 (H), 168 (A, D), 170 (C), 181 (C, E), 182 (A, C, G, I, L, R, S, T, V, W, Y), 183 (E, V), 186 (G, S), 189 (G, L, P, R), 190 (A, E, G, K, R,V), 192(P,T), 194(A,F,G,L,V), 201(G,L), 208(P,R,W), 210(F,Q), 211(C,E,G,S,V,W), 212(D,G,H,P,Q,S,T), 213(M,Q,R), 220(A,C,D,G,P,S), 225(L,Q), 226(C,E,G,R,W), 227(A,C,G,W), 228(A,C,D,G,R,V,W), 229(L), 230(G,P,R,S), 231(A,G,H,R One or more (e.g., several) positions of SEQ ID NO(A,G,H,V), 238(A,G,H,V), 239(G,S), 240(A), 241(V), 242(A,G,P,R,T,V), 243(A,E,M), 246(A,E,P,S,V), 247(L,P), 248(A,G,P), 252(A,D,G,N,Q,S,V), 259(A,G,I,K,L,M,Q,R,V,Y), 262(A,D,E,K,M,R,T,V), and 264(I,P,R) include substitutions, and the polypeptide is similar to SEQ ID NO(A,G,H,V). ID NO:3 exhibits 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 and, compared to the parent, demonstrates improved hydrolytic activity in detergents, i.e., IF > 1.0. This improved property can be measured using the “C performance screening assay” as described in Example 1.

[0202] In some aspects, the present invention relates to variants having lipase activity and comprising substitutions at one or more (e.g., several) positions corresponding to SEQ ID NO:3 or the polypeptide, wherein the polypeptide is similar to SEQ ID NO:3. NO:3 exhibits 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 and, compared to its parent, improved stability in detergents, i.e., IF > 1.0. This improved property can be measured using the "Stability Determination in Detergent D" as described in Example 1.

[0203] In some aspects, the present invention relates to variants having lipase activity and at positions corresponding to SEQ ID NO:3 or the polypeptide: 2(A,D,Q,S), 3(E,K), 4(A,P,Q,R,S), 5(K,T,Y), 6(K,P), 7(P), 10(S), 16(T), 30(L), 31(E), 36(R), 42(K,L,R), 44(A,G,H,K,S), 54(L,V), 71(W,Y), 72(K), 92(F,S) ,T), 93(A,R,V), 96(A,C,Q,R,W), 98(E), 104(C,M,P), 106(T), 109(R,S), 112(H,R,W), 1 19(D,R), 127(S), 131(A,G,I,M,S,T,V,W), 133(A,E,W), 135(C,P,W), 158(I,S), 161(S), 162(G,L,P,R,S,V), 163(A,D,E,M), 165(G), 166(A,C,G,W), 167(R,V), 182(I,T), 192(I ,L,M,R,T,V), 201(G,L), 213(E,L), 220(W), 226(C,G), 227(G,P,R,S), 228(L,R), 229(R, One or more (e.g., several) positions of SEQ ID NO:3 include substitutions, the polypeptide having 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, and having improved stability in detergents, i.e., IF > 1.0, compared to the parent. This improved property can be measured using the "Stability Determination in D Detergent" as described in Example 1.

[0204] These variants may further include one or more additional substitutions in one or more (e.g., several) other locations.

[0205] These amino acid changes can be minute in nature, 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 amino-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 tracts, antigenic epitopes, or binding domains.

[0206] 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.

[0207] Alternatively, amino acid alterations have the property of changing the physicochemical properties of peptides. For example, amino acid alterations can improve the thermal stability of peptides, change substrate specificity, and alter the optimal pH, etc.

[0208] For example, these variants may further include substitutions at one or more locations corresponding to: positions A8T, Y28LC, I41T, C43S, D48E, K50E, Y60AC, D61NKRAVLSTC, R80C, S84C, I86C, N87C, A90C, D91NKRAVLST, F94LTK, S98P, S103R, D113NKRAVLST, S114C, G116V, N120KD, K131F, G156D, N18 6Y, E201QKRAVLST, I204VATSG, V205IL, L208VATSG, F213VATSG, H217N, D226NKRAVLST, T236A, T2 41S, D243NKRAVLSTH, V254IATSG, L255VATSG, D256NKRAVLST, L258VATSG, Y260WCGV, L267VATSG.

[0209] Essential amino acids in peptides can be identified using procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the lipase activity of the resulting mutant molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, Journal of Biochemistry 271:4699-4708. 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. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Essential amino acids can also be identified by comparison with related peptides.

[0210] In one embodiment, the variant exhibits improved thermal stability compared to the parent enzyme. This thermal stability can be determined using assay A as described in Example 1.

[0211] In one embodiment, this variant exhibits improved thermal stability in detergents compared to the parent enzyme. This improved stability can be determined using assay B as described in Example 1.

[0212] In one embodiment, this variant exhibits improved hydrolytic activity in detergents compared to the parent enzyme. This improved stability in detergents can be determined using assay C as described in Example 1.

[0213] In one embodiment, this variant exhibits improved stability in detergents compared to the parent enzyme. This improved stability in detergents can be determined using assay D as described in Example 1.

[0214] Parental lipase

[0215] The parental lipase may be (a) a polypeptide having at least 75% sequence identity with SEQ ID NO:3; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the coding sequence of the mature polypeptide of SEQ ID NO:1, (ii) the full-length complement of (i) or (ii); or (c) a polypeptide encoded by a polynucleotide that has at least 75% sequence identity with the coding sequence of the mature polypeptide of SEQ ID NO:1.

[0216] In one respect, the parent has at least 75%, for example, 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 having lipase activity. In another respect, the amino acid sequence of the parent differs from that of SEQ ID NO:3 by up to 40 amino acids, for example, 1, 3, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0217] 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 another aspect, the parent comprises or consists of SEQ ID NO:3. In another aspect, the parent comprises or consists of amino acids 1 to 269 of SEQ ID NO:3.

[0218] On the other hand, the parent is a fragment of the polypeptide of SEQ ID NO:3. On the other hand, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids 1 to 269 of SEQ ID NO:3, but less than 100%.

[0219] In another embodiment, the parent is an allele variant of SEQ ID NO:3.

[0220] On the other hand, the parent is encoded by a polynucleotide that hybridizes with (i) the mature polypeptide coding sequence of SEQ ID NO:1 and (ii) the full-length complement of (i) under very low stringency, low stringency, medium stringency, medium-high stringency, high stringency, or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, New York).

[0221] The polynucleotide or a subsequence thereof of SEQ ID NO:1 and the polypeptide or a fragment thereof of SEQ ID NO:2 can be used to design nucleic acid probes to identify and clone a parental DNA encoding a strain from different genera or species according to methods well known in the art. Specifically, such probes can be hybridized with the genomic DNA or cDNA of the cell 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, for example at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probes are labeled (e.g., with...). 32 P, 3 H, 35 This invention covers probes containing biotin (or avidin) to detect corresponding genes.

[0222] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened for DNA that hybridizes to and encodes the parental DNA using 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 and immobilized on nitrocellulose or other suitable vector materials. The vector material is used for DNA blotting to identify clones or DNA that hybridize to SEQ ID NO:1 or its subsequences.

[0223] For the purposes of this invention, hybridization indicates that a polynucleotide hybridizes with a labeled nucleic acid probe under very low to very high stringency conditions, the probe corresponding to (i) SEQ ID NO:1; (ii) the mature polypeptide coding sequence of SEQ ID NO:1; (iii) its full-length complement; or (iv) its subsequence. Molecules hybridizing with the nucleic acid probe under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0224] In one respect, the nucleic acid probe is the coding sequence of the mature polypeptide of SEQ ID NO:1. In another respect, the nucleic acid probe is nucleotides 292 to 1098 of SEQ ID NO:1. In another respect, the nucleic acid probe is a polynucleotide encoding a polypeptide of SEQ ID NO:2, its mature polypeptide, or a fragment thereof. In yet another respect, the nucleic acid probe is SEQ ID NO:1.

[0225] In another embodiment, the parent is encoded by a polynucleotide having at least 60%, such as 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 the mature polypeptide coding sequence of SEQ ID NO:1.

[0226] 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.

[0227] 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 into the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking coding sequences of the peptides such that they are in a frame and that the expression of the fusion peptide is under the control of one or more promoters and terminators. Fusion peptides can also be constructed using intron peptide techniques, wherein the fusion peptide is generated post-translational (Cooper et al., 1993, Journal of the European Society for Molecular Biology 12:2575-2583; Dawson, 1994, Science 266:776-779).

[0228] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, *Journal of Industrial Microbiology and Biotechnology*, 3:568-576; Svetina et al., 2000, *Journal of Biotechnology*, 76:245-251; Rasmussen-Wilson et al., 1997, *Applied and Environmental Microbiology*, 63:3488-3493; Ward et al., 2008. (ard et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.)

[0229] 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 should 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.

[0230] The parent can be a filamentous fungus lipase, such as those in the genera *Cladosporium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Briestrogenus*, *Botryospaeria*, *Pseudomonas*, *Trichoderma*, *Aureospora*, *Ceraviridae*, *Cyclophora*, *Cryptococcus*, *Cryptococcus*, *Diplosporium*, *Auricularia*, *Filibasidium*, *Fusarium*, *Gibberellinus*, *Pterygodium*, *Pyrophyllus*, *Phyllostachys*, *Lentinula*, and *Leptozoa*. spaeria), Magnaporthe, Melanocarpus, Meripius, Mucor, Hymenopterus, Neomycetes, Alternaria, Penicillium, Penicillium, Leptotrichum, Ruminocyticum, Poitrasia, Pseudomonas, Pseudomonas, Rhizopus, Schizophyllum, Columnar spores, Basilaria, Thermophilus, Clostridium, Trichoderma, Trichoderma, Verticillium, Cladosporium, or Anthracnose lipase.

[0231] On the other hand, the parent species are *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, and *Chrysosporium queenslandense*. The fungi listed are: queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, and Fusarium sulfide. Fusarium sulphureum, Fusarium torulosum, Fusarium pseudofilariaetrichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thievora achromatica, Thievora albomyces, Thievora albopilosa, Thievora australisinsis, Thievora foetida Thievora fimeti, Thievora microspora, Thievora ovispora, Thievora peruviana, Thievora setosa, Thievora spededonium, Thievora subthermophila, Thievora terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

[0232] On the other hand, the parent is a Rhizopus miltiorrhiza lipase, such as the lipase of SEQ ID NO:2 or its mature polypeptide identical to SEQ ID NO:3.

[0233] It should be understood that, for the foregoing species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0234] 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 Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the U.S. Agricultural Research Service.

[0235] 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 habitat 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 techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0236] Preparation of variants

[0237] The present invention also relates to a method for obtaining lipase variants and / or fragments thereof, the method comprising placing the lipase at positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 16, 19, 30, 31, 34, 36, 37, 39, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 88, 92, 93, 95, 96, 100, 101, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135, 137, 158, 159, 160, 161 corresponding to SEQ ID NO:3. The substitution of one or more of the following positions is introduced into the parental lipase, wherein the variant and / or fragment has lipase activity; and the variant and / or fragment is recovered.

[0238] In some aspects, the present invention relates to methods for obtaining lipase variants or fragments thereof, the method comprising substituting at positions corresponding to positions 1 (C,F,G,H,I,L,M,P,Q,R,V,W,Y), 2 (A,C,D,M,N,P,Q,R,S,T,V), 3 (A,E,G,K,Q,S,T,V), 4 (A,P,Q,R,S,T), 5 (E,K,S,T,V,Y), 6 (A,H,K,N,P,Q,R,S,V,W), 7 (P), 8 (C,G,H,N,P,Q,R,S,W,Y), 9 (Y), 10 (G,R,S,Y), 11 (G,H,I,L,P,Q,R,T), 12 (A,I,K,L,M,N,R,S,T), 16 (P,R,S,T,W), 19 (C,E,H,L,W,T), 30 (A,F,H,I,L,N,P,S,T,V,W), 31 (A,C,D,E,F,G,H,I,K,P,Q,R,S,V), 34 (A,G,V), 36 (D,E,G,K,Q,R,S,V), 37 (A,E,G,S,V), 39 (F,K,L,N,S), 40 (A,D,E,G,I,L,R,V), 41 (A,D,E,G,H,K,L,P,Q,R,S,V,W), 42 (A,E,G,K,L,M,R,S,T,V), 44 (A,C,E,G,H,K,M,P,Q,R,S,V,W), 51 (A,D,G,H,K,L,M,V,Y), 52 (L,V), 53 (A,D,G,H,I,L,M,P,Q,R,S,V), 54 (A,D,E,G,I,K,L,S,V), 56 (A,P,Q,R,T,V,W), 58 (C,G,I,P,R,S,V,W), 59 (A,D,F,G,H,L,M,P,R,S,T,V), 61 (G,M), 70 (A,E,G,R), 71 (E,K,N,Q,R,W,Y), 72 (A,C,D,G,K,R,S,T,V), 73 (E,G,L,N,Q,R,S,T), 83 (A), 84 (A,E,L,P,W,Y), 86 (A,G,I,K,M,W), 88 (M,Q,R,S), 90 (D,F,G,L,S,T,V,W), 91 (E,F,G,Q,Y), 92 (A,C,D,F,P,S,T,V,W), 93 (A,I,R,V), 95 (C,G,I,Q,R,S), 96 (A,C,D,E,G,N,Q,R,S,W,Y), 98 (A,D,E,G,K,N,R,T,V,W), 100 (A,E,G,K,L,R,S,V,W), 101 (D,H,R,S,V), 102 (H,I,P,R,S), 103 (A,C,D,F,G,L,P,V)、104(A,C,D,E,K,L,M,P,Q,R,V,W,Y)、106(A,C,G,L,P,Q,R,S,T,W)、109(A,D,E,G,H,L,N,P,Q,R,S,T)、110(C,P,S,V)、112(F,H,Q,R,W)、113(M,W)、116(A,C,D,F,R,S,T)、117(A,D,G,N,P,R,S,V)、119(D,E,F,I,R,S,T,V)、120(A,E,G,H,L,M,S,T,V,Y)、124(C,D,G,H,L,M,Q,R,S,V,W)、125(A,D,E,G,I,K,L,M,Q,R,S,V)、127(A,F,G,H,I,Q,R,S,V,Y)、128(A,E,G,H,M,Q,S,T)、131(A,G,I,M,S,T,V,W)、132(G,S,V)、133(A,E,K,L,M,R,V,W)、134(A,D,E,G,K,R,S,T,V)、135(A,C,G,P,Q,R,T,V,W,Y)、137(A,F,G,P,R)、158(A,C,G,I,L,N,R,S,T,V,W)、159(G,T)、160(A,D,G,I,L,Q,V)、161(C,G,I,K,L,P,Q,R,S)、162(A,D,G,L,M,P,R,S,V,W)、163(A,D,E,H,L,M,Q,V,Y)、165(A,C,G,K,L,M,P,R,V,W)、166(A,C,E,G,L,,P,R,W)、167(C,F,H,I,L,M,R,V)、168(A,C,D,G,R,S,V,W)、170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y)、181(A,C,E,G,L,Q,R,S,V,W)、182(A,C,F,G,I,L,R,S,T,V,W,Y)、183(E,V)、186(A,C,E,G,H,K,L,R,S,T,W)、189(A,C,G,L,M,P,R,S)、190(A,E,G,I,K,N,R,V,W)、192(A,I,L,M,P,R,T,V)、194(A,D,E,F,G,H,L,Q,T,V)、196(A,Q,Y)、201(D,G,L,P)、202(I,M)、204(E,K,L,P,R)、208(E,M,P,R,W)、210(A,F,L,Q)、211(C,E,G,P,S,V,W)、212(D,E,G,H,K,N,P,Q,R,S,T,V,W)、213(E,H,L,M,P,Q,R,W), 220(A,C,D,G,L,P,R,S,T,W), 225(A,L,M,P,Q,S), 226(C,E,F,G,P,R,W,Y), 227(A,C ,G,P,R,S,V,W), 228(A,C,D,G,L,P,R,V,W), 229(A,I,L,N,R,S,T), 230(A,G,H,L,P,Q,R,S ,T,V,W), 231(A,C,G,H,K,R,S,V,W,Y), 233(A,F,H,L,M,S,T), 236(C,L,P,S,V), 237(F,G, H,L,M,N,R,V), 238(A,E,F,G,H,M,P,R,S,V,W), 239(F,G,S,Y), 240(A), 241(M,V), 242(A,C ,G,I,L,M,P,R,T,V,Y), 243(A,C,E,F,G,M), 246(A,E,K,P,S,T,V), 247(A,E,L,P,T,Y), 24 8(A,G,P,V), 252(A,D,E,G,N,Q,S,V), 255(C,E,M,Q,R), 259(A,C,D,E,G,I,K,L,M,Q,R,T,V The substitution of one or more positions of 262(A,D,E,K,M,R,T,V), 264(C,G,I,L,M,P,R,S,T), 267(E,H,K,M,Q,W), and 269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) is introduced into the parental lipase, wherein the variant and / or fragment possesses lipase activity; and the variant and / or fragment is recovered.

[0239] 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.

[0240] 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.

[0241] 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 mutated oligonucleotide into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0242] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0243] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0244] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques involving the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0245] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or truncation, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0246] The activity of cloned, mutagenic peptides expressed by host cells can be detected by combining mutagenesis / reorganization methods with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0247] 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 involves combining the process of synthesizing polynucleotide fragments with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenesis primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0248] Polynucleotides

[0249] The present invention also relates to polynucleotides encoding variants of the invention.

[0250] Nucleic acid constructs

[0251] 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.

[0252] 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 before its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0253] 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.

[0254] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). (crobiology) 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, see above. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0255] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei endodextranase I, Trichoderma reesei endodextranase II, Trichoderma reesei endodextranase III, Trichoderma reesei endodextranase IV, Trichoderma reesei endodextranase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei Trichoderma β-xylosidase, and the NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-restrictive examples include modified promoters derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and its mutant promoters, truncated promoters, and heterozygous promoters.

[0256] In yeast hosts, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8:423-488, describe other useful promoters in yeast host cells.

[0257] 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.

[0258] Preferred terminators for bacterial host cells were obtained from the genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0259] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0260] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above by Romanos et al., 1992.

[0261] The control sequence can also be a stable region of mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0262] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0263] This control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the polynucleotide encoding that variant. Any leader that functions in the host cell can be used.

[0264] Preferred precursors for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0265] Precursors suitable for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0266] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of the variant-coding sequence and recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0267] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0268] The polyadenylated sequences useful for yeast host cells are described in Guo and Sherman, 1995, Molecular Cellular Biology, 15:5983-5990.

[0269] 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 include 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 may simply replace the native signal peptide coding sequence to increase 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.

[0270] Effective signal peptide coding sequences for bacterial host cells are obtained from the following genes: maltose amylase produced by Bacillus NCIB 11837, subtilisin protease from Bacillus licheniformis, β-lactamase from Bacillus licheniformis, α-amylase from Bacillus thermophilus, neutral proteases (nprT, nprS, nprM) from Bacillus thermophilus, and prsA from Bacillus subtilis. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0271] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus pubescens lipase, and Aspergillus oryzae aspartic protease.

[0272] Signal peptides useful to yeast host cells are obtained from genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Romanos et al., 1992, see above, describe the coding sequences of other useful signal peptides.

[0273] 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. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Mucor mannii aspartic protease, and Saccharomyces cerevisiae α-factor.

[0274] 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.

[0275] It is also desirable to add regulatory sequences that modulate the expression of the variant relative 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 the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0276] expression carrier

[0277] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0278] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that facilitates recombinant DNA procedures and induces the expression of polynucleotides. 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.

[0279] 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 from the genome to be introduced into the host cell), or transposons can be used.

[0280] 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.

[0281] 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). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate-amylase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in *Aspergillus* cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces hygroscopicus*.

[0282] 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.

[0283] 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 integrative 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 integrative elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrative 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.

[0284] 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 replicator" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0285] 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.

[0286] Examples of replication origins used in yeast host cells are 2-micron replication origins ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6.

[0287] Examples of origins of replication used in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acid Research 15:9163-9175; WO 00 / 24883). The isolation of the AMA1 gene and the construction of plasmids or vectors containing this gene can be performed according to the methods disclosed in WO 00 / 24883.

[0288] 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 the 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.

[0289] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0290] host cells

[0291] 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.

[0292] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0293] 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.

[0294] 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.

[0295] 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.

[0296] The bacterial host cell can also be any Streptomyces cell, including but not limited to chromogenic Streptomyces, insecticidal Streptomyces, blue Streptomyces, gray Streptomyces, and pale blue Streptomyces cells.

[0297] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168:111-115), and competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubnau and David Dubnau). Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into *E. coli* cells via protoplast transformation (see, e.g., Hanahan, 1983, Journal of Molecular Biology 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, Journal of Microbiological Methods, 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol., 71:51-57).DNA can be introduced into Streptococcus cells via the following methods: native competent cells (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0298] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0299] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, along with Oomycota and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0300] The host cell of this fungus can be a yeast cell. As used herein, "yeast" includes *Entomophycetes* (Endosporales), *Basidiomycetes*, and yeasts belonging to the class Deuteromycetes (Bacillus). Since the classification of yeast may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium, No. 9, 1980).

[0301] Yeast host cells can be cells from the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Yarrowia*, such as *Kluyveromyces lactis*, *Kluyveromyces*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces davidiana*, *Saccharomyces douglas*, *Kluyveromyces*, *Nordiya*, *Ovoyces*, or *Yarrowia lipolytica*.

[0302] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the phylum Eumycota and subphyla of Oomycetes (as defined by Hawksworth et al., 1995, see above). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal extension, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentation.

[0303] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Magnaporthe*, *Mucor*, *Hydrophyllus*, *Neurophyllus*, *Penicillium*, *Penicillium*, *Phlebia*, *Ruminocyticum*, *Pleurotus*, *Schizophyllum*, *Basilella*, *Thermophila*, *Fusarium*, *Trametes*, or *Trichoderma*.

[0304] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium lucknowense*, and *Chrysosporium foetida*. merdarium), rent spores, Queensland golden spores (Chrysosporium queenslandicum), tropical golden spores, brown golden spores (Chrysosporium zonatum), gray-capped coprinus (Coprinus cinereus), hairy-skinned spores (Coriolushirsutus), rod-shaped spores (Fusarium), cereal spores (Fusarium), kuweiss spores (Fusarium), large-knife spores (Fusarium), grass spores (Fusarium), red spores (Fusarium), heterospores (Fusarium), albinofuss spores (Fusarium), acuminata spores (Fusarium), multibranched spores (Fusarium), pink spores (Fusarium), elderberry spores (Fusarium), skin-colored spores (Fusarium), pseudo-clastic spores (Fusarium), sulfur spores (Fusarium), round spores (Fusarium), pseudo-filamentous spores (Fusarium), patchy spores (Fusarium), specific humic molds (Fusarium), soft-haired humic molds (Fusarium), rice black molds (Mucor), thermophilic filamentous molds (Fusarium), rough spores (Nephrolepis), purpuric molds (Penicillium), Phanerochaete chrysosporium (Phanerochaete chrysosporium), radiata (Phlebia radiata), Pleurotus eryngii (Pleurotus) eryngii), terrestrial clostridium, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0305] Fungal cells can be transformed through processes involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1920.

[0306] Production methods

[0307] The present invention also relates to a method for generating a variant, the method comprising: (a) culturing a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.

[0308] These host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. The culture occurs using procedures known in the art in a suitable nutrient medium 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.

[0309] The variant can be detected using methods known in the art that are specific to these variants. 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, an enzyme assay can be used to determine the activity of the variant, such as the assay described in the following examples.

[0310] This 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.

[0311] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0312] In an alternative, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0313] Composition

[0314] In one aspect, the present invention relates to a composition comprising a lipase variant of a parental lipase having lipase activity, having at least 75% but less than 100% sequence identity with SEQ ID NO:3, and at positions 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 16, 19, 30, 31, 34, 36, 37, 39, 40, 42, 44, 51, 52, 53, 54, 56, 58, 59, 70, 71, 72, 73, 83, 88, 92, 93, 95, 96, 100, 101, 102, 104, 106, 109, 110, 112, 117, 119, 124, 125, 127, 128, 131, 132, 133, 134, 135 corresponding to SEQ ID NO:3. One or more of the following positions include substitution: 137, 158, 159, 160, 161, 162, 163, 165, 166, 167, 168, 170, 181, 182, 183, 189, 190, 192, 194, 196, 202, 210, 211, 212, 220, 225, 227, 228, 229, 230, 231, 233, 237, 238, 239, 240, 242, 246, 247, 248, 252, 259, 262, 264, 269.

[0315] In another aspect, the present invention relates to a composition comprising a lipase variant of a parental lipase, the variant having lipase activity, having at least 75% but less than 100% sequence identity with SEQ ID NO:3, and corresponding to SEQ ID NO:3 positions 1(C,F,G,H,I,L,M,P,Q,R,V,W,Y), 2(A,C,D,M,N,P,Q,R,S,T,V), 3(A,E,G,K,Q,S,T,V), 4(A,P,Q,R,S,T), 5(E,K,S,T,V,Y), 6(A ,H,K,N,P,Q,R,S,V,W), 7(P), 8(C,G,H,N,P,Q,R,S,W,Y), 9(Y), 10(G,R,S,Y), 11(G,H,I,L,P,Q,R,T), 12(A,I,K,L,M,N,R,S,T), 16(P,R, S,T,W), 19(C,E,H,L,W,T), 30(A,F,H,I,L,N,P,S,T,V,W), 31(A,C,D,E,F,G,H,I,K,P,Q,R,S,V), 34(A,G,V), 36(D,E,G,K,Q,R,S,V), 37( A,E,G,S,V), 39(F,K,L,N,S), 40(A,D,E,G,I,L,R,V), 41(A,D,E,G,H,K,L,P,Q,R,S,V,W), 42(A,E,G,K,L,M,R,S,T,V), 44(A,C,E,G,H,K, M,P,Q,R,S,V,W), 51(A,D,G,H,K,L,M,V,Y), 52(L,V), 53(A,D,G,H,I,L,M,P,Q,R,S,V), 54(A,D,E,G,I,K,L,S,V), 56(A,P,Q,R,T,V,W), 5 8(C,G,I,P,R,S,V,W), 59(A,D,F,G,H,L,M,P,R,S,T,V), 61(G,M), 70(A,E,G,R), 71(E,K,N,Q,R,W,Y), 72(A,C,D,G,K,R,S,T,V), 73(E,G, L,N,Q,R,S,T), 83(A), 84(A,E,L,P,W,Y), 86(A,G,I,K,M,W), 88(M,Q,R,S), 90(D,F,G,L,S,T,V,W), 91(E,F,G,Q,Y), 92(A,C,D,F,P,S,T, V,W), 93(A,I,R,V), 95(C,G,I,Q,R,S), 96(A,C,D,E,G,N,Q,R,S,W,Y), 98(A,D,E,G,K,N,R,T,V,W), 100(A,E,G,K,L,R,S,V,W), 101(D,H,R,S,V)、102(H,I,P,R,S)、103(A,C,D,F,G,L,P,V)、104(A,C,D,E,K,L,M,P,Q,R,V,W,Y)、106(A,C,G,L,P,Q,R,S,T,W)、109(A,D,E,G,H,L,N,P,Q,R,S,T)、110(C,P,S,V)、112(F,H,Q,R,W)、113(M,W)、116(A,C,D,F,R,S,T)、117(A,D,G,N,P,R,S,V)、119(D,E,F,I,R,S,T,V)、120(A,E,G,H,L,M,S,T,V,Y)、124(C,D,G,H,L,M,Q,R,S,V,W)、125(A,D,E,G,I,K,L,M,Q,R,S,V)、127(A,F,G,H,I,Q,R,S,V,Y)、128(A,E,G,H,M,Q,S,T)、131(A,G,I,M,S,T,V,W)、132(G,S,V)、133(A,E,K,L,M,R,V,W)、134(A,D,E,G,K,R,S,T,V)、135(A,C,G,P,Q,R,T,V,W,Y)、137(A,F,G,P,R)、158(A,C,G,I,L,N,R,S,T,V,W)、159(G,T)、160(A,D,G,I,L,Q,V)、161(C,G,I,K,L,P,Q,R,S)、162(A,D,G,L,M,P,R,S,V,W)、163(A,D,E,H,L,M,Q,V,Y)、165(A,C,G,K,L,M,P,R,V,W)、166(A,C,E,G,L,,P,R,W)、167(C,F,H,I,L,M,R,V)、168(A,C,D,G,R,S,V,W)、170(A,C,E,G,H,I,K,L,M,Q,R,S,V,W,Y)、181(A,C,E,G,L,Q,R,S,V,W)、182(A,C,F,G,I,L,R,S,T,V,W,Y)、183(E,V)、186(A,C,E,G,H,K,L,R,S,T,W)、189(A,C,G,L,M,P,R,S)、190(A,E,G,I,K,N,R,V,W)、192(A,I,L,M,P,R,T,V)、194(A,D,E,F,G,H,L,Q,T,V)、196(A,Q,Y)、201(D,G,L,P)、202(I,M)、204(E,K,L,P,R)、208(E,M,P,R,W)、210(A,F,L,Q)、211(C,E,G,P,S,V,W)、112(D,E,G,H,K,N,P,Q,R,S,T,V,W), 213(E,H,L,M,P,Q,R,W), 220(A,C,D,G,L,P,R,S,T,W), 225( A,L,M,P,Q,S), 226(C,E,F,G,P,R,W,Y), 227(A,C,G,P,R,S,V,W), 228(A,C,D,G,L,P,R,V,W ), 229(A,I,L,N,R,S,T), 230(A,G,H,L,P,Q,R,S,T,V,W), 231(A,C,G,H,K,R,S,V,W,Y), 23 3(A,F,H,L,M,S,T), 236(C,L,P,S,V), 237(F,G,H,L,M,N,R,V), 238(A,E,F,G,H,M,P,R,S,V ,W), 239(F,G,S,Y), 240(A), 241(M,V), 242(A,C,G,I,L,M,P,R,T,V,Y), 243(A,C,E,F,G,M ), 246(A,E,K,P,S,T,V), 247(A,E,L,P,T,Y), 248(A,G,P,V), 252(A,D,E,G,N,Q,S,V), 255( One or more positions of C,E,M,Q,R), 259(A,C,D,E,G,I,K,L,M,Q,R,T,V,W,Y), 262(A,D,E,K,M,R,T,V), 264(C,G,I,L,M,P,R,S,T), 267(E,H,K,M,Q,W), and 269(A,D,E,G,H,K,L,M,N,Q,R,S,V,Y) include substitutions.

[0316] The non-limiting list of compositional components set forth below is suitable for use in these compositions, and the methods herein may be suitably incorporated into certain embodiments of the invention, for example, to aid or enhance cleaning performance, for treating substrates to be cleaned, or to enhance the aesthetic appeal of the composition when used in conjunction with fragrances, colorants, dyes, or the like. The level of any such component incorporated into any composition is in addition to any materials previously referenced for incorporation. The precise properties of these additional components and their incorporation levels will depend on the physical form of the composition and the nature of the cleaning operation in which the composition will be used. Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as a component may include additional functionality as will be understood by one of ordinary skill.

[0317] Unless otherwise stated, amounts expressed as percentages are by weight (wt%) of the composition. Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes and enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymeric dispersants, clay removers / anti-redeposition agents, brighteners, foam inhibitors, dyes, tinting dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble additives, processing aids, solvents, and / or pigments. Suitable examples and levels of use of other such components, in addition to the disclosures below, are found in US 5576282, US 6306812, and US 6326348, which are hereby incorporated by reference.

[0318] Therefore, in some embodiments, the present invention does not contain one or more of the following auxiliary materials: surfactants, soaps, detergent builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymer dispersants, clay removal / anti-redeposition agents, brightening agents, foam inhibitors, dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble additives, processing aids, solvents, and / or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

[0319] surfactants - The compositions according to the invention may include a surfactant or surfactant system, wherein the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, facultative zwitterionic surfactants, semipolar nonionic surfactants, and mixtures thereof. When present, the surfactant is typically present at levels ranging from 0.1 wt% to 60 wt%, from 0.2 wt% to 40 wt%, from 0.5 wt% to 30 wt%, from 1 wt% to 50 wt%, from 1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1 wt% to 20 wt%, from 3 wt% to 10 wt%, from 3 wt% to 5 wt%, from 5 wt% to 40 wt%, from 5 wt% to 30 wt%, from 5 wt% to 15 wt%, from 3 wt% to 20 wt%, from 3 wt% to 10 wt%, from 8 wt% to 12 wt%, from 10 wt% to 12 wt%, or from 20 wt% to 25 wt%.

[0320] Suitable anionic detergency surfactants include sulfate and sulfonate detergency surfactants.

[0321] Suitable sulfonate detergency surfactants include alkylbenzene sulfonates, which in one aspect are C 10-13Alkylbenzene sulfonates. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonation of commercially available linear alkylbenzenes (LABs). Suitable LABs include low-carbon 2-phenyl LABs, such as... or Other suitable LABs include high-carbon 2-phenyl LABs, such as... Suitable anionic detergency surfactants are alkylbenzene sulfonates obtained via DETAL catalytic processes, but other synthetic routes (such as HF) may also be suitable. In one approach, magnesium salts of LAS are used.

[0322] Suitable sulfate detergency surfactants include alkyl sulfates, in one respect, C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfates.

[0323] Another suitable sulfate detergency surfactant is alkylalkoxylated sulfate, which is alkylethoxylated sulfate on one hand and C on the other. 8-18 Alkyl alkoxylated sulfates, on the other hand, are C 8-18 Alkyl ethoxylated sulfates, typically alkyl alkoxylated sulfates have an average degree of alkoxylation from 0.5 to 20 or from 0.5 to 10, and are typically C10-2 ... 8-18 Alkyl ethoxylated sulfates having an average degree of ethoxylation of 0.5 to 10, 0.5 to 7, 0.5 to 5, or 0.5 to 3.

[0324] Alkyl sulfates, alkylalkoxylated sulfates, and alkylbenzene sulfonates can be linear or branched, substituted or unsubstituted.

[0325] The detergency surfactant can be a medium-chain branched detergency surfactant, on one hand a medium-chain branched anionic detergency surfactant, on the other hand a medium-chain branched alkyl sulfate and / or medium-chain branched alkylbenzene sulfonate, such as a medium-chain branched alkyl sulfate. On one hand, the medium chain branch is C 1-4 Alkyl groups, typically methyl and / or ethyl.

[0326] Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES, 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.

[0327] Suitable nonionic detergency surfactants are selected from the following group, which consists of the following items: C8-C 18 Alkyl ethoxylates, such as C6-C 12 Alkylphenol alkoxylates, wherein the alkoxyl unit may be an ethyleneoxy unit, an propyleneoxy unit, or a mixture thereof; C 12 -C 18 Alcohols and C6-C 12 Condensations of alkylphenols with ethylene oxide / propylene oxide block polymers, such as Pranic. C 14 -C 22 Medium-chain branched alcohols; C 14 -C 22 Medium-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation from 1 to 30; alkyl polysaccharides, in one respect as alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

[0328] Suitable nonionic detergency surfactants include alkyl polysaccharides and / or alkyl alkoxylated alcohols.

[0329] On the one hand, nonionic detergency surfactants include alkylalkoxylated alcohols, and on the other hand, they are C 8-18 Alkyl alkoxylated alcohols, such as C 8-18 Alkyl ethoxylated alcohols, wherein the alkyl alkoxylated alcohols may have an average degree of alkoxylation ranging from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C 8-18Alkyl ethoxylated alcohols have an average degree of ethoxylation ranging from 1 to 10, from 1 to 7, more commonly from 1 to 5, or from 3 to 7. Alkyl alkoxylated alcohols can be straight-chain or branched, and substituted or unsubstituted. Suitable nonionic surfactants include...

[0330] 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.

[0331] Suitable cationic detergency surfactants include alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds, and mixtures thereof.

[0332] Suitable cationic detergency surfactants are quaternary ammonium compounds having the following general formula: (R)(R1)(R2)(R3)N + X - Where R is a straight or branched, substituted or unsubstituted C 6-18 The alkyl or alkenyl moiety, R1 and R2 are independently selected from the methyl or ethyl moiety, R3 is a hydroxy, hydroxymethyl, or hydroxyethyl moiety, and X is an anion that provides charge neutrality; suitable anions include halides, such as chlorides; sulfates; and sulfonates. Suitable cationic detergency surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. A highly suitable cationic detergency surfactant is mono-C... 8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C 10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

[0333] 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, ester quaternary ammonium compounds, and combinations thereof.

[0334] Suitable amphoteric / zombietic surfactants include amine oxides and betaines (such as alkyldimethyl betaine, sulfobetaine), or combinations thereof. The amine-neutralized anionic surfactants of the present invention—anionic surfactants and associated anionic cosurfactants—may be present in acidic form, and said acidic form may be neutralized to form surfactant salts desired for use in the detergent compositions of the present invention. Typical neutralizing agents include metal counterionic bases, such as hydroxides, like NaOH or KOH. Further preferred agents for neutralizing the anionic surfactants of the present invention and associated anionic surfactants or cosurfactants in their acidic form include ammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other straight-chain or branched alkanolamines known in the art; for example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization can proceed to a complete or partial extent. For example, a portion of an anionic surfactant mixture can be neutralized by sodium or potassium, and a portion of an anionic surfactant mixture can be neutralized by amines or alkanolamines.

[0335] Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyl dimethylamine oxides.

[0336] A surfactant system comprising one or more anionic surfactants and one or more other nonionic surfactants, and optionally a mixture with other surfactants such as cationic surfactants, is preferred. A preferred weight ratio of anionic to nonionic surfactants is at least 2:1, or at least 1:1 to 1:10.

[0337] soap - The compositions herein may contain soap. Without being theoretically limited, it is desirable to include soap because it partially acts as a surfactant and partially as a detergent builder, and can be used to suppress foaming; furthermore, it can advantageously interact with a variety of cationic compounds in the composition to enhance the softness of textile fabrics treated with the compositions of the present invention. Any soap known in the art for use in laundry detergents may be used. In one embodiment, these compositions contain from 0 wt% to 20 wt%, from 0.5 wt% to 20 wt%, from 4 wt% to 10 wt%, or from 4 wt% to 7 wt% soap.

[0338] Examples of soaps useful here include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps with different chain lengths and degrees of substitution. One such mixture is top-coated palm kernel fatty acid.

[0339] In one embodiment, the soap is selected from free fatty acids. Suitable fatty acids are saturated and / or unsaturated and can be obtained from natural sources such as plant or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tallow oil, castor oil, tallow and fish oil, fats and oils, and mixtures thereof), or synthesized (e.g., via the oxidation of petroleum or via the Fisher-Tropsch process of hydrogenating carbon monoxide).

[0340] Examples of suitable saturated fatty acids for use in the compositions of the present invention include decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and benzyl acid. Suitable unsaturated fatty acids include palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and ricinoleic acid. Examples of preferred fatty acids are saturated Cn fatty acids, saturated Ci2-Ci4 fatty acids, and saturated or unsaturated Cn to Ci8 fatty acids, and mixtures thereof.

[0341] When present, the weight ratio of the fabric-softening cationic auxiliary surfactant to the fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, and most preferably about 1:1.

[0342] The levels of soap and non-soap anionic surfactants mentioned herein are percentages by weight of the detergent composition specified on an acidic basis. However, as is generally understood in the art, in practice, sodium, potassium, or alkanol ammonium bases such as sodium hydroxide or monoethanolamine are used to neutralize the anionic surfactants and soap.

[0343] Water-soluble- The compositions of the present invention may include one or more co-hydrophilic solvents. A co-hydrophilic solvent is a compound that dissolves a hydrophobic compound (or conversely, a polar substance 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 self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12:121-128. Co-hydrophilic solvents do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and lipid formation of micelles, thin layers, or other well-defined intermediate phases occur. Many co-hydrophilic solvents, on the contrary, exhibit a continuous aggregation process, in which the size of the aggregates increases with increasing concentration. However, many water-soluble 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). Water-soluble solvents are classically used across industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble solvents in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0344] Detergents may contain 0 to 10 wt%, such as 0 to 5 wt%, 0.5 wt% to 5 wt%, or 3 wt% to 5 wt% 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 hydroxynaphthenate, sodium hydroxynaphthenate, sodium ethylhexylsulfonate, and combinations thereof.

[0345] builder - The compositions of the present invention may include one or more detergent builders, co-builders, detergent builder systems, or mixtures thereof. When a detergent builder is used, the cleaning composition will typically include 0 to 65 wt%, at least 1 wt%, 2 wt% to 60 wt%, or 5 wt% to 10 wt% of the detergent builder. In dishwashing cleaning compositions, the level of detergent builder is typically 40 wt% to 65 wt% or 50 wt% to 65 wt%. The composition may be substantially free of detergent builders; substantially free of zeolite and / or phosphates, meaning "no intentionally added". Typical zeolite detergent builders include zeolite A, zeolite P, and zeolite MAP. Typical phosphate detergent builders are sodium tripolyphosphate.

[0346] 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 detergents may be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hearst Corporation), ethanolamines such as 2-aminoethanol (MEA), iminodiethanol (DEA), and 2,2',2'-triazine (TEA), and carboxymethyl inulin (CMI), and combinations thereof.

[0347] Cleaning compositions 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 phosphonates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-dimethylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), and N-(2-sulfoethyl)aspartic acid (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), anthranilic acid-N,N-diacetic acid (ANDA), p-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), aminoethanesulfonic acid-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations thereof and salts thereof. Further exemplary builders and / or co-builders are described in, for example, WO 09 / 102854, US 5977053.

[0348] Chelating agents and crystal growth inhibitors- The composition herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, ionic, and / or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), disodium hydroxide of 1,2-dihydroxybenzene-3,5-disulfonic acid, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin, and 2-phosphinocarboxybutane-1,2,4-tricarboxylic acid ( AM) and its derivatives. Typically, the composition may include chelating agents or crystal growth inhibitors from 0.005 wt% to 15 wt% or from 3.0 wt% to 10 wt%.

[0349] bleaching components Suitable bleaching components for incorporation into the methods and compositions of the present invention include one or a mixture of more than one bleaching component. Suitable bleaching components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, and mixtures thereof. Typically, when using bleaching components, the compositions of the present invention may include from 0 to 30 wt%, from 0.00001 wt% to 90 wt%, from 0.0001 wt% to 50 wt%, from 0.001 wt% to 25 wt%, or from 1 wt% to 20 wt%. Examples of suitable bleaching components include:

[0350] (1) Preformed peracid: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of preformed peroxyacids or their salts, typically peroxycarboxylic acids or their salts, or persulfate or their salts.

[0351] The pre-formed peroxyacid or its salt is preferably a peroxycarboxylic acid or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0352]

[0353] Where: R 14 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 14 The functional group can be straight-chain or branched, substituted or unsubstituted; and Y is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 14 Is it a linear or branched, substituted or unsubstituted C? 6-9Alkyl group. Preferably, the peroxy acid or its salt is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and their salts, or any combination thereof. Particularly preferred peroxy acids are phthalimide-peroxy-alkanoic acids, especially ε-phthalimide-peroxyhexanoic acid (PAP). Preferably, the peroxy acid or its salt has a melting point in the range of 30°C to 60°C.

[0354] The pre-formed peroxyacid or its salt can also be persulfate or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0355]

[0356] Where: R 15 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 15 The group can be straight-chain or branched, substituted or unsubstituted; and Z is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 15 Is it a linear or branched, substituted or unsubstituted C? 6-9 Alkyl groups. Preferably, such bleaching components may be present in the compositions of the present invention in an amount from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

[0357] (2) Hydrogen peroxide sources include, for example, inorganic hydrogen peroxide salts, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, sodium salts of persilicates, and mixtures thereof. In one aspect of the invention, inorganic hydrogen peroxide salts are, for example, those selected from the group consisting of perborates, sodium salts of percarbonates, and mixtures thereof. When used, inorganic hydrogen peroxide salts are typically present in amounts from 0.05 wt% to 40 wt% or 1 wt% to 30 wt% of the total composition and are typically incorporated into such compositions as crystalline solids that can be coated. Suitable coatings include: inorganic salts, such as alkali metal silicates, carbonates, or borates, or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils, or fatty soaps. Preferably, such bleaching components may be present in the compositions of the invention in amounts from 0.01 wt% to 50 wt% or 0.1 wt% to 20 wt%.

[0358] (3) The term bleaching activator herein refers to a compound that reacts with hydrogen peroxide to form a peracid via a perhydrolysis reaction. The peracid formed in this manner constitutes an activated bleaching agent. Suitable bleaching activators to be used herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable bleaching activators are those having R-(C=O)-L, wherein R is an alkyl group (preferably branched), having from 6 to 14 carbon atoms or from 8 to 12 carbon atoms when the bleaching activator is hydrophobic, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleaching activator is hydrophilic; and is an L leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives—especially benzenesulfonates. Suitable bleaching activators include dodecyloxybenzenesulfonate, decyloxybenzenesulfonate, decyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecyloxy)benzene-1-sulfonate (LOBS), 4-(decyloxy)benzene-1-sulfonate, 4-(decyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A family of bleaching activators disclosed in EP 624154, and particularly preferred in that family, is triethyl acetyl citrate (ATC). ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly. Furthermore, triethyl acetylacetic acid (ATC) and triacetin exhibit good hydrolytic stability in the product during storage and are effective bleaching activators. Finally, ATC is versatile because the citrate released during the hydrolysis reaction can act as a detergent builder. Alternatively, the bleaching system may include, for example, peroxy acids of the form of amides, imides, or sulfones. The bleaching system may also include peracids, such as 6-(phthalimide)peroxyhexanoic acid (PAP). Suitable bleaching activators are also disclosed in WO 98 / 17767. Although any suitable bleaching activator may be used, in one aspect of the invention, the subject cleaning composition may include NOBS, TAED, or mixtures thereof. When present, peracids and / or bleaching activators are typically present in the composition in amounts of 0.1 wt% to 60 wt%, 0.5 wt% to 40 wt%, or 0.6 wt% to 10 wt%, depending on the fabric and home care composition. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors. Preferably, such bleaching components may be present in the compositions of the present invention in an amount of 0.01 wt% to 50 wt% or 0.1 wt% to 20 wt%.

[0359] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0360] (4) Diacyl peroxides - Preferred diacyl peroxide bleaching types include those selected from diacyl peroxides having the following general formula: R 1 -C(O)-OO-(O)CR 2 , where R 1 Represents a C6-C 18 Alkyl groups, preferably comprising a straight chain having at least 5 carbon atoms and optionally containing one or more substituents (e.g., -N). + (CH3)3, -COOH or -CN) and / or one or more interruptions inserted between adjacent carbon atoms of an alkyl group (e.g., -CONH- or -CH=CH-) at C6-C 12 Alkyl groups, and R 2 This represents an aliphatic group compatible with the peroxide moiety, such that R 1 and R 2 Together they contain a total of 8 to 30 carbon atoms. In a preferred aspect, R 1 and R 2 It is a straight-chain, unsubstituted C6-C 12 Alkyl chain. Most preferably, R 1 and R 2 They are the same. Diacyl peroxide (where R) 1 and R 2 All are C6-C 12 Alkyl groups are particularly preferred. Preferably, at least one of the R groups (R1 or R2) does not contain a branched or drooping ring at the α-position, or preferably does not contain a branched or drooping ring at either the α- or β-position, or most preferably does not contain a branched or drooping ring at either the α-, β-, or γ-position. In a further preferred embodiment, the DAP may be asymmetric, such that the hydrolysis of the R1 acyl group is preferably rapid to produce a superacid, but the hydrolysis of the R2 acyl group is slow.

[0361] Tetraacyl peroxide bleaching agents are preferably selected from tetraacyl peroxides having the following general formula: R 3 -C(O)-OO-C(O)-(CH2)nC(O)-OO-C(O)-R 3 , where R 3 Indicates C1-C9 alkyl, or C 3- The C7 group, and n represents an integer from 2 to 12 or from 4 to 10 (inclusive of the end value).

[0362] Preferably, the diacyl and / or tetraacyl peroxide bleaching agents are present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm of detergent solution by weight. In a preferred embodiment, these bleaching agents are present in an amount sufficient to provide detergent solution from 0.5 ppm to 300 ppm, or from 30 ppm to 150 ppm by weight.

[0363] Preferably, the bleaching component includes bleaching catalysts (5 and 6).

[0364] (5) Preferably, organic (non-metallic) bleaching catalysts are included, comprising bleaching catalysts capable of accepting oxygen atoms from peroxy acids and / or their salts and transferring those oxygen atoms to an oxidizable substrate. Suitable bleaching catalysts include, but are not limited to: imine-onium cations and polyions; imine-onium zwitterions; modified amines; modified amine oxides; N-sulfonylimides; N-phosphonylimides; N-acylimides; thiadiazole dioxides; perfluoroimides; cyclic glycoketones and mixtures thereof.

[0365] Suitable imine-onium cations and polyions include, but are not limited to, N-methyl-3,4-dihydroisoquinolineonium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (e.g., compound 4, page 433); N-methyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5360569 (e.g., column 11, example 1); and n-octyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5360568 (e.g., column 10, example 3).

[0366] Suitable imine-onium zwitterions include, but are not limited to, N-(3-sulfopropyl)-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5576282 (e.g., column 31, Example II); N-[2-(sulfonoxy)dodecyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5817614 (e.g., column 32, Example V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in WO 05 / 047264 (e.g., page 18, Example 8); and 2-[3-[(2-butyloctyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt.

[0367] Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinoline alcohol, which can be prepared according to the method described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxygen transfer catalysts include, but are not limited to, sodium 1-hydroxy-N-oxy-N-[2-(sulfonoxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0368] Suitable N-sulfonylimide oxygen transfer catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide prepared according to the procedure described in the Journal of Organic Chemistry (1990), 55(4), 1254-61.

[0369] Suitable N-phosphonylimide oxygen transfer catalysts include, but are not limited to, [R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-p-phenyl-p-(2,4,6-trimethylphenyl)phosphonamide, which can be prepared according to the method described in the Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70.

[0370] Suitable N-acylimide oxygen transfer catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, which can be manufactured according to the procedure described in the Polish Journal of Chemistry (2003), 77(5), 577-590.

[0371] Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, which may be manufactured according to the procedure described in US 5753599 (Column 9, Example 2).

[0372] Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butyrylimide fluoride, which can be prepared according to the method described in Tetrahedron Letters (1994), 35(34), 6329-30.

[0373] Suitable cyclic glycoketone oxygen transfer catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexanedione-2,6-pyranose, as prepared in US 6649085 (column 12, Example 1).

[0374] Preferably, the bleaching catalyst comprises imine-onium and / or carbonyl functional groups, and is generally capable of forming oxaziridinium and / or diethylene oxide functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises oxaziridinium functional groups and / or is capable of forming oxaziridinium functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises cyclic imine-onium functional groups, preferably wherein the cyclic portion has a ring size of five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleaching catalyst comprises arylimine-onium functional groups, preferably bicyclic arylimine functional groups, and more preferably 3,4-dihydroisoquinoline-onium functional groups. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary peroxyimine cation functional group when accepting an oxygen atom, particularly when accepting an oxygen atom from a peroxy acid and / or its salt. On the other hand, the detergent composition comprises a logP having a value not greater than 0, not greater than -0.5, not greater than -1.0, not greater than -1.5, not greater than -2.0, not greater than -2.5, not greater than -3.0, or not greater than -3.5. o / w The bleaching component. The following describes in more detail the components used to determine logP. o / w The method.

[0375] Typically, bleaching agents can produce X values ​​ranging from 0.01 to 0.30, from 0.05 to 0.25, or from 0.10 to 0.20. SO The types of bleaching are described in more detail below for determining X. SO The method. For example, bleaching components with an isoquinolineonium structure can produce bleaching species with a peroxyimine cation structure. In this example, X SO It is X, a type of peroxide imine cation bleaching. SO .

[0376] Preferably, the bleaching catalyst has a chemical structure corresponding to the following chemical formula:

[0377]

[0378] Where: n and m independently range from 0 to 4, preferably both n and m are 0; each R 1Independently selected, either substituted or unsubstituted, groups selected from the group consisting of: hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocyclic, fused heterocyclic, nitro, halogen, cyano, sulfonate, alkoxy, ketone, carboxyl, and alkoxycarbonyl; and any two R groups in the connected positions 1 Substituents can merge to form fused aryl, fused carbocyclic, or fused heterocyclic groups; each R 2 Independently selected, either substituted or unsubstituted, groups independently selected from the group consisting of: hydrogen, hydroxyl, alkyl, cycloalkyl, alkylaryl, aryl, aralkyl, alkylene, heterocyclic, alkoxy, arylcarbonyl, carboxyalkyl, and amide groups; any R 2 It can be used with any other R 2 They are combined to form part of a common ring; any harmonic R 2 They can merge to form a carbonyl group; and any two R groups can be combined to form a carbonyl group. 2 They can merge to form a substituted or unsubstituted fused unsaturated portion; R 3 It is C1 to C 20 Substituted or unsubstituted alkyl groups; R 4 Is it hydrogen or Q? t -A portion, where: Q is a branched or unbranched alkene, t = 0 or 1, and A is an anionic group selected from the group consisting of the following: OSO3 - SO3 - CO2 - OCO2 - OPO3 2- OPO3H - and OPO2 - ;R 5 Is it hydrogen or -CR? 11 R 12 -YG b -Y c -[(CR 9 R 10 ) y -O] k -R 8 Part, wherein: each Y is independently selected from the following group, which consists of: O, S, NH, or NR. 8 And each R 8 Independently selected from the group consisting of alkyl, aryl, and heteroaryl groups, wherein the moiety is substituted or unsubstituted, and wherein the substituted or unsubstituted moiety has less than 21 carbons; each G is independently selected from the group consisting of CO, SO2, SO, PO, and PO2; R 9 and R 10 Independently selected from the group consisting of: H and C1-C4 alkyl groups; R11 and R 12 Independently selected from the group consisting of: H and alkyl groups, or those that can combine to form a carbonyl group when placed together; b = 0 or 1; c can = 0 or 1, but if b = 0, c must = 0; y is an integer from 1 to 6; k is an integer from 0 to 20; R 6 It is an H, or alkyl, aryl, or heteroaryl moiety; said moiety is substituted or unsubstituted; and X, if present, is a suitable charge-balancing counterion, preferably when R 4 When it is hydrogen, X exists. Suitable X includes, but is not limited to: chlorides, bromides, sulfates, methoxysulfates, sulfonates, p-toluenesulfonates, borotetrafluorides, and phosphates.

[0379] In one embodiment of the present invention, the bleaching catalyst has a structure corresponding to the following general formula:

[0380]

[0381] Where R 13 It is a branched alkyl group (including branched carbon atoms) containing three to 24 carbon atoms or a straight-chain alkyl group containing one to 24 carbon atoms; preferably, R 13 It is a branched alkyl group containing eight to 18 carbon atoms or a straight-chain alkyl group containing eight to eighteen carbon atoms; preferably, R 13 Selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isothidecyl, and isopentetranyl; preferably, R 13 Selected from the group consisting of the following: 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, isotridecyl and isopentadecanyl.

[0382] Preferably, in addition to the bleaching catalyst, particularly an organic bleaching catalyst, the bleaching component also includes a peracid source. The peracid source may be selected from (a) pre-formed peracid; (b) percarbonate, perborate, or percarbonate (hydrogen peroxide source), preferably in combination with a bleaching activator; and (c) perhydrolase and ester, used to form the peracid in situ in the presence of water during textile or hard surface treatment steps.

[0383] When present, based on the composition, the peracid and / or bleaching activator are typically present in the composition in amounts from 0.1 wt% to 60 wt%, from 0.5 wt% to 40 wt%, or from 0.6 wt% to 10 wt%. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors.

[0384] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

[0385] (6) Metal-containing bleaching catalysts – the bleaching component can be provided by a catalytic metal complex. One type of metal-containing bleaching catalyst is a catalytic system comprising a transition metal cation (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation) having defined bleaching catalytic activity, an auxiliary metal cation (e.g., zinc or aluminum cation) having little or no bleaching catalytic activity, and an insulator having defined stability constants for both the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts. Such catalysts are disclosed in US 4430243. Preferred catalysts are described in WO 09 / 839406, US 6218351, and WO 00 / 012667. Transition metal catalysts or ligands thus serving as cross-bridged multidentate N-donor ligands are particularly preferred.

[0386] If desired, the composition herein can be catalyzed using manganese compounds. Such compounds and the levels of use are well known in the art and include, for example, manganese-based catalysts disclosed in US 5576282.

[0387] Useful cobalt bleaching catalysts are known and described, for example, in US 5597936 and US 5595967. Such cobalt catalysts can be readily prepared by known procedures, such as those taught in US 5597936 and US 5595967.

[0388] The compositions herein may also suitably include transition metal complexes of ligands, such as bispidone (US 7501389) and / or multicyclic rigid ligands – abbreviated as “MRL”. As a practical problem and not for limitation, the compositions and methods herein may be adapted to provide approximately at least one part per hundred million of active MRL species in an aqueous washing medium, and will typically provide MRL levels from 0.005 ppm to 25 ppm, from 0.05 ppm to 10 ppm, or from 0.1 ppm to 5 ppm in the washing solution.

[0389] Suitable transition metals for readily available transition metal bleaching catalysts include, for example, manganese, iron, and chromium. Suitable MRLs include 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitable transition metal MRLs can be readily prepared by known procedures, such as those described in US 6225464 and WO 00 / 32601.

[0390] (7) Photobleaching agents – Suitable photobleaching agents include, for example, sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, zeatin dyes, and mixtures thereof. Preferred bleaching components used in these compositions of the invention include a hydrogen peroxide source, a bleaching activator, and / or an organic peroxyacid, optionally generated in situ by a reaction of the hydrogen peroxide source and the bleaching activator in combination with a bleaching catalyst. Preferred bleaching components include a bleaching catalyst, preferably an organic bleaching catalyst as described above.

[0391] The preferred bleaching component is a bleaching catalyst, especially an organic bleaching catalyst.

[0392] Exemplary bleaching systems are also described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242.

[0393] Fabric colorant - The composition may include a fabric colorant. Suitable fabric colorants include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes belonging to the following colorimetric index (CI) classifications: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet, and basic red, or mixtures thereof.

[0394] On the other hand, suitable small molecule dyes include those selected from the group consisting of the following: colorimetric index (Society of Dyers and Colorists, Bradford, UK) number: Direct Violet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, Acid Violet 50. Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75, Basic Blue 159 and mixtures thereof. On the other hand, suitable small molecule dyes include those selected from the group consisting of: colorimetric index (Institute of Dyers and Colorists, Bradford, UK) numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, Acid Red 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, Acid Blue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51, and mixtures thereof. On the other hand, suitable small molecule dyes include those selected from the group consisting of: colorimetric index (Institute of Dyers and Colorists, Bradford, UK) numbers Acid Violet 17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113, and mixtures thereof.

[0395] Suitable polymer dyes include polymer dyes selected from the group consisting of polymers containing conjugated chromogens (dye-polymer conjugates) and polymers copolymerized with chromogens into the polymer backbone, and mixtures thereof.

[0396] On the other hand, suitable polymer dyes include polymer dyes selected from the group consisting of: Substantial fabric colorants under the name Milliken are dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of: polymers comprising portions selected from the group consisting of hydroxyl moieties, primary amine moieties, secondary amine moieties, thiols, and mixtures thereof. In another aspect, suitable polymer dyes include polymer dyes selected from the group consisting of: Purple CT, carboxymethyl cellulose (CMC) conjugated with reactive blue, reactive purple or reactive red dyes, such as CMC conjugated with CI reactive blue 19 (sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC), alkoxylated triphenylmethane polymer colorants, alkoxylated thiophene polymer colorants, and mixtures thereof.

[0397] Preferred tinting dyes include whitening agents found in WO 08 / 87497. These whitening agents can be characterized by the following structure (I):

[0398]

[0399] R1 and R2 can be independently selected from:

[0400] a)[(CH2CR'HO) x (CH2CR"HO) y H]

[0401] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

[0402] b) R1 = alkyl, aryl, or arylalkyl, and R2 = [(CH2CR'HO) x (CH2CR"HO) y H]

[0403] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

[0404] c) R1 = [CH2CH2(OR3)CH2OR4], and R2 = [CH2CH2(OR3)CH2OR4]

[0405] R3 is selected from the following group, which consists of the following items: H, (CH2CH2O). z H, and mixtures thereof; and wherein z = 0 to 10;

[0406] R4 is selected from the following group, which consists of the following items: (C1-C 16 )alkyl, aryl, and mixtures thereof; and

[0407] d) Wherein R1 and R2 may be independently selected from styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, tert-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl hexadecyl ether, followed by an addition of 1 to 10 epoxide units.

[0408] The preferred whitening agent of the present invention can be characterized by the following structure (II):

[0409]

[0410] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

[0411] Another preferred whitening agent of the present invention can be characterized by the following structure (III):

[0412]

[0413] Typically, this includes mixtures having a total of 5 EO groups. Suitable preferred molecules are those in structure I that have the side groups described in “part a” above.

[0414] Table A:

[0415] R1 R2 R’ R” x y R’ R” x y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c = b H H 1 1 H H 2 1 d = a H H 0 1 H H 3 1

[0416] Other whitening agents used include those described in US 2008 / 34511 (Unilever). The preferred agent is "Violet 13".

[0417] Suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of at least one cationic / basic dye and green clay, and mixtures thereof. On the other hand, suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of cationic / basic dyes and clay, wherein the cationic / basic dye is selected from the group consisting of: CI Basic Yellow 1 to 108, CI Basic Orange 1 to 69, CI Basic Red 1 to 118, CI Basic Violet 1 to 51, CI Basic Blue 1 to 164, CI Basic Green 1 to 14, CI Basic Brown 1 to 23, CI Basic Black 1 to 11, and the clay is selected from the group consisting of: montmorillonite clay, hydrophoretic clay, soapstone clay, and mixtures thereof. On the other hand, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: montmorillonite Basic Blue B7C.I.42595 conjugate, montmorillonite Basic Blue B9C.I.52015 conjugate, montmorillonite Basic Violet V3C.I.42555 conjugate, montmorillonite Basic Green G1C.I.42040 conjugate, montmorillonite Basic Red R1C.I.45160 conjugate, montmorillonite CI Basic Black 2 conjugate, sphagnum alexandrite Basic Blue B7C.I.42595 conjugate, and sphagnum alexandrite Basic Blue B9C.I.52015 conjugate. 5. Conjugates, including: sphagnum basic violet V3C.I.42555 conjugate, sphagnum basic green G1C.I.42040 conjugate, sphagnum basic red R1C.I.45160 conjugate, sphagnum basic black CI 2 conjugate, saponite basic blue B7C.I.42595 conjugate, saponite basic blue B9C.I.52015 conjugate, saponite basic violet V3C.I.42555 conjugate, saponite basic green G1C.I.42040 conjugate, saponite basic red R1C.I.45160 conjugate, saponite CI 2 conjugate, and mixtures thereof.

[0418] Suitable pigments include pigments selected from the group consisting of: flavonoids, indanone, chlorinated indanone containing 1 to 4 chlorine atoms, pinanthrone, dichloropinanthrone, monobromodichloropinanthrone, dibromodichloropinanthrone, tetrabromopinanthrone, dinaphthyl-3,4,9,10-tetracarboxylic acid diimide (wherein the imide group may be unsubstituted or substituted with C1-C3-alkyl or phenyl or heterocyclic groups, and wherein the phenyl and heterocyclic groups may additionally have substituents that do not impart solubility in water), anthraquinone pyrimidine carboxylic acid amide, anthrone violet, isoanthrone violet, dioxazine pigments, copper phthalocyanine per molecule that may contain up to 2 chlorine atoms, polychlorinated copper phthalocyanine or polybrominated chloro-copper phthalocyanine per molecule containing up to 14 bromine atoms, and mixtures thereof.

[0419] On the other hand, suitable pigments include those selected from the group consisting of ultramarine (CI Pigment Blue 29), ultramarine violet (CI Pigment Violet 15), and mixtures thereof.

[0420] The above-mentioned fabric toners can be used in combination (any mixture of fabric toners can be used). Suitable toners are described in more detail in US 7208459. The preferred level of dye in the compositions of the present invention is 0.00001 wt% to 0.5 wt%, or 0.0001 wt% to 0.25 wt%. The concentration of dye used in water for treatment and / or cleaning steps is preferably from 1 ppb to 5 ppm, 10 ppb to 5 ppm, or 20 ppb to 5 ppm. In preferred compositions, the concentration of surfactant will be from 0.2 to 3 g / L.

[0421] Encapsulation - The composition may comprise an encapsulated compound. In one aspect, the encapsulated compound comprises a core and a shell having an inner surface and an outer surface, the shell encapsulating the core.

[0422] In one aspect of the encapsulated compound, the core may comprise a material selected from the group consisting of: fragrances; brighteners; dyes; insect repellents; silicones; waxes; flavorings; vitamins; fabric softeners; skin care agents, in one aspect, paraffin wax; enzymes; antibacterial agents; bleaching agents; sensates; and mixtures thereof; and the shell may comprise a material selected from the group consisting of: polyethylene; polyamides; polyvinyl alcohol, optionally including other comonomers; polystyrene; polyisoprene; polycarbonate; polyester; polyacrylate; amino plastics, in one aspect, the amino plastics may include polyurea, polyurethane and / or polyurea polyurethane, in one aspect, the polyurea may include polyoxymethylene urea and / or melamine formaldehyde; polyolefins; polysaccharides, in one aspect, the polysaccharides may include alginate and / or chitosan; gelatin; shellac; epoxy resins; water-insoluble inorganic vinyl polymers; silicones; and mixtures thereof.

[0423] In one aspect of the encapsulated compound, the core may include a fragrance.

[0424] In one aspect of the encapsulated compound, the shell may comprise melamine formaldehyde and / or cross-linked melamine formaldehyde.

[0425] On one hand, it is disclosed that suitable encapsulation compounds may include a core material and a shell, the shell at least partially surrounding the core material. 85% or 90% of the encapsulation compound may have a tensile strength from 0.2 MPa to 10 MPa, from 0.4 MPa to 5 MPa, from 0.6 MPa to 3.5 MPa, or from 0.7 MPa to 3 MPa; and have a beneficial reagent leakage of 0% to 30%, 0% to 20%, or 0% to 5%.

[0426] In one respect, 85% or 90% of the encapsulated material may have a particle size ranging from 1 to 80 micrometers, from 5 to 60 micrometers, from 10 to 50 micrometers, or from 15 to 40 micrometers.

[0427] In one respect, 85% or 90% of the encapsulated material may have a particle wall thickness of 30 to 250 nm, 80 to 180 nm, or 100 to 160 nm.

[0428] In one aspect, the core material of the encapsulated compound may include materials selected from the group consisting of: flavoring ingredients and / or optionally materials selected from the group consisting of: vegetable oils, including undiluted and / or blended vegetable oils (including castor oil, coconut oil, cottonseed oil, grape oil, rapeseed oil, soybean oil, corn oil, palm oil, flaxseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil, and mixtures thereof); vegetable oil esters, esters, This includes dibutyl adipate, dibutyl phthalate, benzyl butyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl phosphate, and mixtures thereof; straight-chain or branched hydrocarbons, including those with boiling points above about 80°C; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, including monoisopropyl biphenyls, alkyl naphthalenes (including dipropyl naphthalene), gasoline (including kerosene), mineral oils, and mixtures thereof; aromatic solvents, including benzene, toluene, and mixtures thereof; silicone oils; and mixtures thereof.

[0429] In one aspect, the wall material of the encapsulation may include a suitable resin comprising a reaction product of an aldehyde and an amine, with suitable aldehydes including formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycourea, and mixtures thereof. Suitable melamines include hydroxymethyl melamine, methylated hydroxymethyl melamine, iminomelamine, and mixtures thereof. Suitable ureas include dihydroxymethyl urea, methylated dihydroxymethyl urea, urea-resorcinol, and mixtures thereof.

[0430] On one hand, before, during, or after the addition of the encapsulation compound to the composition, a suitable formaldehyde scavenger may be used with and / or added to such composition, for example, in a capsule slurry. Suitable capsules may be made according to the following teachings of US 2008 / 0305982; and / or US 2009 / 0247449.

[0431] In a preferred aspect, the composition may further comprise a deposition acid, preferably composed of a group consisting of cationic or nonionic polymers. Suitable polymers include cationic starch, cationic hydroxyethyl cellulose, polyethylene formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene terephthalate, and polymers comprising dimethylaminoethyl methacrylate and optionally having one or more monomers selected from the group consisting of acrylic acid and acrylamide.

[0432] spices- In one aspect, the composition comprises a fragrance containing one or more fragrance raw materials selected from the group consisting of: 1,1'-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol, monoacetic acid ester; (3-methylbutoxy)acetic acid, 2-propenyl ester; β-methylcyclododecaneethanol; 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol; oxetane-hexadecyl-2-one; α-methyl-benzyl alcohol acetate; trans-3-ethoxy-1,1,5-trimethylcyclohexane; 4-(1,1-dimethylethyl)cyclohexanol acetate; dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; β-methylphenylpropanal; β-methyl-3-(1-methylethyl)phenylpropanal; 4-phenyl-2-butanone; 2-methylbutyric acid, ethyl ester; benzaldehyde; 2-methylbutyric acid, 1-methylethyl ester; dihydro-5-pentyl-2(3H)furanone; (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecaaldehyde; undecanoal; 2-ethyl-α,α-dimethylphenylpropanal; decanal; α,α-dimethylphenylethanol acetate; 2-( 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoic acid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one; 2-pentylcyclopentanone; 3-oxo-2-pentylcyclopentaneacetic acid, methyl ester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde; 2-heptylcyclopentanone; 1-(4-methylphenyl)ethyl ketone; (3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one; (3E)-4-(2,6,6-trimethyl-3-cyclohexen-1-yl)-3-buten-2-one; (2H-1-benzopyran-2-one); phenethyl alcohol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde; 10-undecenal; propionic acid, phenyl methyl ester; β-methylphenylpentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene; α,α-dimethylphenethyl alcohol; (2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid, phenyl methyl ester; cyclohexanepropionic acid, 2-propenyl ester; hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene; 1,5-dimethyl-bicyclo[3.2].1] Oct-8-one oxime; 4-(4-hydroxy-4-methylpentyl)-3-cyclohexen-1-carboxaldehyde; 3-buten-2-ol; 2-[[[2,4(or 3,5)-dimethyl-3-cyclohexenyl-1-yl]methylene]amino]benzoic acid, methyl ester; 8-cyclohexadecyl-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol; 2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-octadien-1-ol; 2-hydroxy-benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenoic acid; 4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; acetic acid, (2-methylbutoxy)-, 2-propenyl ester; benzoic acid, 2-hydroxy-, 3-methylbutyl ester; 2-buten-1-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; cyclopentanecarboxylic acid, 2-hexyl-3-oxo- methyl esters; phenylpropanal, 4-ethyl-α,α-dimethyl-; 3-cyclohexene-1-carboxaldehyde, 3-(4-hydroxy-4-methylpentyl)-; acetone, 1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanolchamomile-5-yl)-, [3R-(3.α.,3a.β.,7.β.,8a.α.)]-; undecaldehyde, 2-methyl-2H-pyran-2-one, 6-butyltetrahydro-; phenylpropanal, 4-(1,1-dimethylacetone) α-methyl-; 2(3H)-furanone, 5-heptyldihydro-; benzoic acid, 2-[(7-hydroxy-3,7-dimethyl-octyl)amino]-, methyl; benzoic acid, 2-hydroxy-, phenylmethyl ester; naphthalene, 2-methoxy-; 2-cyclopenten-1-one, 2-hexyl-; 2(3H)-furanone, 5-hexyldihydro-; oxacyclopropanecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester; 2-oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; phenylpentanol, γ-.-Methyl-; 3-octanol, 3,7-dimethyl-; 3,7-dimethyl-2,6-octadienone; 3,7-dimethyl-6-octen-1-ol; terpineol acetate; 2-methyl-6-methylene-7-octen-2-ol, dihydro derivative; 3a,4,5,6,7,7a-hexahydro-4,7-methanol-1H-inden-6-phenol propionate; 3-methyl-2-buten-1-ol acetate; (Z)-3-hexen-1-ol acetate; 2-ethyl-4 -(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol; 4-(octahydro-4,7-methanol-5H-indenyne-5-yl)-butanal; 3-2,4-dimethyl-cyclohexen-1-carboxaldehyde; 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)-acetone; 2-hydroxybenzoic acid, methyl ester; 2-hydroxybenzoic acid, hexyl ester; 2-phenoxy-ethanol; 2-hydroxy-benzene Formic acid, pentyl ester; 2,3-heptanedione; 2-hexen-1-ol; 6-octen-2-ol, 2,6-dimethyl-; daemonone (α, β, γ or δ or mixtures thereof), 4,7-methanol-1H-inden-6-phenol, 3a,4,5,6,7,7a-hexahydro-, acetate; 9-undecenal; 8-undecenal; heterocyclic citral; acetone, 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl) -; 3-cyclohexene-1-carboxaldehyde, 3,5-dimethyl-; 3-cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-octadien-3-ol, 3,7-dimethyl-; 1,6-octadien-3-ol, 3,7-dimethyl-, acetate; lily aldehyde (pt-Bucinal); and cyclopentanone, 2-[2-(4-methyl-3-cyclohexene-1-yl)propyl]- and 1-methyl-4-(1-methylvinyl)cyclohexene and mixtures thereof.

[0433] In one aspect, the composition may include encapsulated fragrance particles containing a water-soluble hydroxy compound or melamine-formaldehyde or modified polyvinyl alcohol. In another aspect, the encapsulation comprises (a) at least partially water-soluble solid matrix containing one or more water-soluble hydroxy compounds, preferably starch; and (b) fragrance oil encapsulated by the solid matrix.

[0434] On the other hand, the flavoring can be pre-complexed with a polyamine (preferably polyethyleneimine) to form a Schiff base.

[0435] polymer- The composition may include one or more polymers. Examples include carboxymethyl cellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazolium), polycarboxylate (such as polyacrylate), maleic acid / acrylic acid copolymer, and lauryl methacrylate / acrylic acid copolymer.

[0436] The composition may include one or more amphiphilic cleaning polymers, such as compounds having the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + -(CH3)-bis((C2H5O)(C2H4O)n), where n = from 20 to 30 and x = from 3 to 8, or its sulfonated or sulfonated variants.

[0437] The composition may include amphiphilic alkoxylated grease-removing polymers that have a balanced hydrophilic and hydrophobic property, enabling them to remove grease particles from fabrics and surfaces. Specific embodiments of the amphiphilic alkoxylated grease-removing polymers of the present invention include a core structure and a plurality of alkoxylated groups attached to that core structure. These may include alkoxylated polyalkylenimines, preferably having an inner ethylene oxide block and an outer propylene oxide block.

[0438] Here, alkoxylated polycarboxylate esters (such as those prepared from polyacrylates) can be used to provide additional grease-removing properties. Such materials are described in WO 91 / 08281 and PCT 90 / 01815. Chemically, these materials comprise polyacrylates having one ethoxylated side chain per 7-8 acrylate units. The side chain has the chemical formula -(CH2CH2O). m (CH2) n CH3, where m is 2-3 and n is 6-12. Side chains are esters attached to the polyacrylate "main chain" to provide a "comb-like" polymer-type structure. Molecular weights can vary, but are typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylic acid esters can be included in the compositions herein from 0.05 wt% to 10 wt%.

[0439] The isoprene-derived surfactants of the present invention, and mixtures thereof formed with other auxiliary surfactants and adjuvants, are particularly suitable for use with amphiphilic graft copolymers, preferably comprising (i) a polyethylene glycol backbone; and (ii) and at least one dangling portion selected from polyvinyl acetate, polyvinyl alcohol, and mixtures thereof. A preferred amphiphilic graft copolymer is Sokalan HP22 supplied by BASF. Suitable polymers include random graft copolymers, preferably polyvinyl acetate-grafted polyethylene oxide copolymers, having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000, and the weight ratio of polyethylene oxide to polyvinyl acetate is 40:60, with no more than one grafting point per 50 ethylene oxide units.

[0440] Carboxylic acid ester polymers - The compositions of the present invention further include one or more carboxylate polymers, such as maleate / acrylate random copolymers or polyacrylate homopolymers. In one aspect, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of from 4,000 Da to 9,000 Da or from 6,000 Da to 9,000 Da.

[0441] Fouling release polymers - The compositions of the present invention may further comprise one or more dirt-releasing polymers having a structure as defined by one of the following structures (I), (II) or (III):

[0442] (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d

[0443] (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e

[0444] (III)-[(OCHR 5 -CHR 6 ) c -OR 7 ] f

[0445] in:

[0446] a, b, and c are from 1 to 200;

[0447] d, e, and f are from 1 to 50;

[0448] Ar is a 1,4-substituted phenylene;

[0449] sAr is a 1,3-substituted phenylene with SO3Me substituted at the 5-position;

[0450] Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, mono-, di-, tri-, or tetraalkylammonium, wherein the alkyl group is C1-C. 18 Alkyl or C2-C 10 Hydroxyalkyl groups, or mixtures thereof;

[0451] R 1 R 2 R 3 R 4 R 5 and R 6 Independently selected from H or C1-C 18 n- or iso-alkyl; and

[0452] R 7 Is it a straight or branched C1-C? 18 Alkyl groups, or straight-chain or branched C2-C 30 Alkenyl, or cycloalkyl having 5 to 9 carbon atoms, or C8-C 30 Aryl, or C6-C 30 Arylalkyl.

[0453] Suitable detergency polymers are polyester detergency polymers, such as Repel-o-tex polymers, including Repel-o-tex, SF-2, and SRP6, supplied by Rhodia. Other suitable detergency polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, supplied by Clariant. Other suitable detergency polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0454] Cellulose polymers The compositions of the present invention further include one or more cellulose polymers, including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, and alkylcarboxyalkyl cellulose. In one aspect, the cellulose polymer is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof. In one aspect, the carboxymethyl cellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 and a molecular weight from 100,000 Da to 300,000 Da.

[0455] enzymes- The composition may include one or more additional enzymes that provide cleaning properties and / or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, xanthan gumase, ligninase, amylopectinase, tanninase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, and amylase, or mixtures thereof. A typical combination is an enzyme mixture that may contain, for example, protease and lipase together with amylase. When present in the composition, the aforementioned additional enzymes may be present at a level of enzyme protein from 0.00001 wt% to 2 wt%, from 0.0001 wt% to 1 wt%, or from 0.001 wt% to 0.5 wt% by weight of the composition.

[0456] 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.

[0457] Cellulase Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered variants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Cladosporium*, such as the fungal cellulases produced by *Pyrophyllus*, *Thermophyllus*, and *Fusarium* disclosed in US 4435307, US 5648263, US 5691178, US5776757, and WO 89 / 09259.

[0458] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0531315, US 5457046, US5686593, US 5763254, WO 95 / 24471, WO 98 / 12307, and WO 99 / 001544.

[0459] Other cellulases are endoglucanases having a sequence that is at least 97% identical to the amino acid sequence at positions 1 to 773 of SEQ ID NO:2 of WO 02 / 099091, or a family 44 xyloglucanase having a sequence that is at least 60% identical to the amino acid sequence at positions 40-559 of SEQ ID NO:2 of WO 01 / 062903.

[0460] Commercially available cellulases include Celluzyme TM and Carezyme TM (Novozymes A / S), Carezyme Premium) TM (Novozymes), Celluclean TM (Novozymes), Celluclean Classic TM (Novozymes), Cellusoft TM (Novozymes), Whitezyme TM (Novozymes), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM (Kao Corporation).

[0461] protease Suitable 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 or protein-engineered variants. It can be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases can be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from family M4 or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0462] The term "subtilisinase" 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. Subtilisins can be classified into six subfamilies: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanoxanthipeptidase family, the Kexin family, and the Pyrolysin family.

[0463] Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus lentus*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and *Lentus*, *Novo*, *Carlsberg*, *Bacillus licheniformis*, *BPN'*, *309*, *147*, and *168* as described in WO 89 / 06279, and *PD138* as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., from porcine or bovine sources) 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).

[0464] Further preferred proteases are alkaline proteases from Bacillus tarda DSM 5483 (as described in, for example, WO95 / 23221), and their variants (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0465] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0466] Examples of useful proteases are described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO Variants in 11 / 036264, especially those with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, use BPN' numbers. More preferably, these Bacillus subtilis enzyme variants may include the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

[0467] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm Durazym Tm , Ultra Ultra Ultra Ultra and (Novozymes), those sold under the following product names: Preferenz Tm Purafect Purafect Purafect Effectenz Tm , as well as (Danisco / DuPont), Axapem TM (Gist-Brocases N.V.), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0468] Lipase and keratinase Suitable lipases and keratins include those of bacterial or fungal origin. This 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 EP258068 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* (EP 331376), *Pseudomonas* strain SD705 (WO 95 / 06720 & WO 96 / 27002), *Pseudomonas wisconsinensis* (WO 96 / 12012); and GDSL-type *Streptomyces* lipases (WO... 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).

[0469] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO94 / 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.

[0470] Preferred commercially available lipase products include Lipolase TM Lipex™; Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0471] 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).

[0472] amylase- Suitable amylases that can be used with the enzymes / variants / enzyme blends of the present invention can be α-amylases or glucosylamylases and can be of bacterial or fungal origin. This includes chemically modified or protein-engineered variants. Amylases include, for example, α-amylases obtained from the genus *Bacillus*, such as α-amylases of specific strains of *Bacillus licheniformis* described in more detail in GB 1296839.

[0473] Suitable amylases include the amylase having SEQ ID NO:2 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO:3. Preferred variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, 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.

[0474] Suitable amylases include the amylase having SEQ ID NO:6 in WO 02 / 010355 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193.

[0475] Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* in SEQ ID NO:6 of WO 06 / 066594 and residues 36-483 of the *Bacillus licheniformis* α-amylase in SEQ ID NO:4 of WO 06 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those having substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4 is those having the following substitutions:

[0476] M197T;

[0477] H156Y+A181T+N190F+A209V+Q264S; or

[0478] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0479] Suitable alternative amylases are those having SEQ ID NO:6 in WO 99 / 019467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182 or positions H183 and G184.

[0480] Other amylases that can be used are those of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 with WO 96 / 023873, or variants thereof having 90% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. SEQ ID 2 of WO 96 / 023873 is used for numbering. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants are those selected from two positions of 181, 182, 183, and 184, such as 181 and 182, 182 and 183, or positions 183 and 184 having deletions. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 are those having deletions at positions 183 and 184 and having substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0481] 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.

[0482] 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. A more preferred variant of SEQ ID NO:2 is one 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 variant of SEQ ID NO:2 is one having the following substitutions:

[0483] N128C+K178L+T182G+Y305R+G475K;

[0484] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0485] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0486] 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.

[0487] Other suitable amylases are α-amylases having SEQ ID NO:12 in WO 01 / 66712 or variants having at least 90% sequence identity with SEQ ID NO:12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO:12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions for R118K, N195F, R320K, and R458K, as well as variants having 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 being variants having substitutions at all of these positions.

[0488] Other examples are, for instance, those amylase variants described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0489] 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).

[0490] Peroxidase / oxidase- The suitable peroxidase according to the present invention is a peroxidase included in enzyme classification EC1.11.1.7 as stated by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment thereof exhibiting peroxidase activity.

[0491] Suitable peroxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered variants. Examples of useful peroxidases include peroxidases from the genus *Coprinus*, such as *C. cinerea* (EP 179486), and its variants, such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257.

[0492] Peroxidases also include halogenated peroxidases, such as chloride peroxidase, bromoperoxidase, and compounds exhibiting chloride or bromoperoxidase activity. Halogenated peroxidases are classified according to their specificity for halide ions. Chloride peroxidase (EC1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

[0493] In one embodiment, the halogenated peroxidase of the present invention is a chloride peroxidase. Preferably, the halogenated peroxidase is a vanadium halide peroxidase, i.e., a vanadate-containing halogenated peroxidase. In a preferred method of the present invention, the vanadate-containing halogenated peroxidase is combined with a chloride ion source.

[0494] Halogenated peroxidases have been isolated from many different fungi, particularly from the dematiaceous hyphomycete fungal group, such as Caldariomyces (e.g., Coal Caldariomyces fumago), Alternaria, Curvularia (e.g., Curvularia verruculosa and Curvularia inaequalis), Helicobacter endolymphaeus, Helicobacter spp., and Botrytis.

[0495] Halogenated peroxidases have also been isolated from bacteria such as Pseudomonas (e.g., P. pyrrocinia) and Streptomyces (e.g., Streptomyces aureofaciens).

[0496] In a preferred embodiment, the halogenated peroxidase may be derived from the genus Curvularia, particularly Curvularia verruculosa and Curvularia anisotropis, such as Curvularia anisotropis CBS102.42 as described in WO 95 / 27046 or Curvularia verruculosa CBS 147.63 or Curvularia verruculosa CBS 444.70 as described in WO 97 / 04102; or it may be derived from Drechslera hartlebii as described in WO 01 / 79459, Dendryphiella salina as described in WO 01 / 79458, Phaeochrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium sp. as described in WO 01 / 79460.

[0497] The oxidases according to the invention specifically include any laccase or fragment thereof exhibiting laccase activity, or a compound exhibiting similar activity, as encompassed by enzyme classification EC 1.10.3.2, such as catechol oxidase (EC 1.10.3.1), o-aminophenol oxidase (EC 1.10.3.4), or bilirubin oxidase (EC 1.3.3.5).

[0498] Preferred laccases are microbial enzymes. These enzymes can originate from plants, bacteria, or fungi (including filamentous fungi and yeast).

[0499] Suitable examples of fungi include laccases derived from strains of the following: *Aspergillus*, *Neurospora* (e.g., *Neurospora crassa*), *Strigera*, *Botrytis*, *Collybia*, *Fomes*, *Lentinula*, *Pleurotus*, *Coralus* (e.g., *Coralus longifolius* and *Coralus discolor*), *Rhizoctonia* (e.g., *Rhizoctonia solani*), *Coprinus* (e.g., *Coprinus comatus*, *Coprinus friesii*, and *Coprinus spp.*). *Psathyrella* (e.g., *P. condelleana*), *P. papilionaceus* (e.g., *P. papilionaceus*), *Schytalidium* (e.g., *S. thermophilum*), *P. pinsitus*, *P. radiata* (e.g., *P. radiata*) (WO 92 / 01046), or *C. hirsutus* (e.g., *C. hirsutus*) (JP 2238885).

[0500] Suitable examples from bacteria include laccases that may originate from strains of the genus Bacillus.

[0501] The preferred laccases are those derived from the genera *Coprinus* or *Desmodium*; particularly those derived from *Coprinus gracilistylus*, as disclosed in WO 97 / 08325; or those derived from *Desmodium thermophilum*, as disclosed in WO 95 / 33836.

[0502] Other preferred enzymes include those listed in the trademark name. The pectin lyase sold below, and the trademark name The mannanase sold below (Novozymes), and (Danisco / DuPont).

[0503] The one or more detergent enzymes can be included in the detergent composition by adding a single additive containing one or more enzymes, or by adding a combination additive containing all of these enzymes. The detergent additives of the present invention, i.e., single additives or combination additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-dust granules; liquids, especially stabilized liquids; or slurries.

[0504] Non-dust particles can be manufactured, for example, as disclosed in US 4106991 and US 4661452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols containing 12 to 20 carbon atoms and having 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP238216.

[0505] Dye transfer inhibitors - The 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 composition, the dye transfer inhibitor may be present at levels from 0.0001 wt% to 10 wt%, from 0.01 wt% to 5 wt%, or from 0.1 wt% to 3 wt%.

[0506] Brightening agent - The compositions of the present invention may also contain additional components that can color the cleaned article, such as fluorescent brighteners.

[0507] The composition may include CI fluorescent brightener 260 in an α-crystalline form having the following structure:

[0508]

[0509] On one hand, the brightener is a cold water-soluble brightener, such as CI fluorescent brightener 260 in α-crystalline form. On the other hand, the brightener is primarily in α-crystalline form, meaning that typically at least 50 wt%, at least 75 wt%, at least 90 wt%, at least 99 wt%, or even substantially all of CI fluorescent brightener 260 is in α-crystalline form.

[0510] Brightening agents are typically in the form of microparticles with a weighted average primary particle size ranging from 3 to 30 micrometers, from 3 to 20 micrometers, or from 3 to 10 micrometers.

[0511] The composition may include CI fluorescent brightener 260 in β-crystalline form, and the weight ratio of (i) CI fluorescent brightener 260 in α-crystalline form to (ii) CI fluorescent brightener 260 in β-crystalline form may be at least 0.1 or at least 0.6. BE 680847 relates to a method for obtaining CI fluorescent brightener 260 in α-crystalline form.

[0512] Commercial optical brighteners that can be used in this invention can be classified into several subgroups, which include, but are not necessarily limited to, stilbene, pyrazoline, coumarin, carboxylic acids, methinecyanine, dibenzothiophene-5,5-dioxide, azoles, derivatives of 5- and 6-membered heterocyclic rings, and other hybrids. Examples of such brighteners are disclosed in “Production and Application of Fluorescent Brighteners,” M. Zahradnik, John Wiley & Sons, New York (1982). Specific, non-limiting examples of optical brighteners that can be used in the compositions of this invention are those identified in US 4790856 and US3646015.

[0513] Other suitable brightening agents have the following structure:

[0514]

[0515] Suitable levels of fluorescent brighteners range from lower levels of 0.01 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt% to higher levels of 0.5 wt% or 0.75 wt%.

[0516] On one hand, the brightening agent can be loaded onto the clay to form particles. Silicates – The compositions of the present invention may also contain silicates, such as sodium silicate or potassium silicate. The composition may include silicates from 0 wt% to less than 10 wt%, up to 9 wt%, or up to 8 wt%, or up to 7 wt%, or up to 6 wt%, or up to 5 wt%, or up to 4 wt%, or up to 3 wt%, or even up to 2 wt%, and from more than 0 wt%, or from 0.5 wt%, or from 1 wt%. A suitable silicate is sodium silicate.

[0517] dispersant - The compositions of the present invention may further include a dispersant. 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.

[0518] Enzyme stabilizers - The enzymes used in the composition can be stabilized by various techniques. The enzymes used herein can be stabilized in the presence of a water-soluble source of calcium and / or magnesium ions. Examples of conventional stabilizers are, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, boric acid or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, and the composition can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708. In the case of aqueous compositions containing proteases, reversible protease inhibitors, such as boron compounds comprising borates, 4-formylphenylboronic acid, phenylboronic acid and their derivatives, or compounds such as calcium formate, sodium formate and 1,2-propanediol, can be added to further improve stability.

[0519] solvent Suitable solvents include water and other solvents, such as lipophilic fluids. Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerol derivatives (e.g., glycerol ethers), perfluorinated amines, perfluorinated and hydrofluoroether solvents, low-volatility nonfluorinated organic solvents, glycol solvents, other environmentally friendly solvents, and mixtures thereof.

[0520] Structurer / Thickener- Structured liquids can be internally structured, whereby the structure is formed by primary components (e.g., surfactant materials), and / or externally structured by providing a three-dimensional matrix structure through the use of secondary components (e.g., polymers, clays, and / or silicate materials). The composition may include 0.01 wt% to 5 wt%, or 0.1 wt% to 2.0 wt% of a structuring agent. The structuring agent is typically selected from the group consisting of: diglycerides and triglycerides, ethylene glycol stearate diester, microcrystalline cellulose, cellulose-based materials, microfibrillated cellulose, hydrophobically modified basic expandable emulsions (e.g., Polygel W30 (3VSigma)), biopolymers, xanthan gum, glucan gum, and mixtures thereof. Suitable structuring agents include hydrogenated castor oil and its non-ethoxylated derivatives. Suitable structuring agents are disclosed in US6855680. Such structuring agents have a thread-like structuring system with a range of aspect ratios. Other suitable structuring agents and methods for their preparation are described in WO 10 / 034736.

[0521] regulator - The compositions of the present invention may include high-melting-point fatty acid compounds. Useful high-melting-point fatty acid compounds herein have a melting point of 25°C or higher and are selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof. Such compounds having low melting points are not intended to be included in this section. Non-limiting examples of high-melting-point compounds are found in the International Dictionary of Cosmetic Ingredients, 5th Edition, 1993, and the CTFA Handbook of Cosmetic Ingredients, 2nd Edition, 1992.

[0522] In view of providing improved conditioning benefits (such as a smooth, soft feel during application to wet hair and a moisturizing feel on dry hair), high melting point fatty compounds are included in the composition at levels ranging from 0.1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1.5 wt% to 16 wt%, and from 1.5 wt% to 8 wt%.

[0523] The compositions of the present invention may comprise cationic polymers. The concentration of the cationic polymer in the composition typically ranges from 0.05 wt% to 3 wt%, from 0.075 wt% to 2.0 wt%, or from 0.1 wt% to 1.0 wt%. Suitable cationic polymers will have a cationic charge density of at least 0.5 meq / gm, at least 0.9 meq / gm, at least 1.2 meq / gm, at least 1.5 meq / gm, or less than 7 meq / gm, and less than 5 meq / gm at the pH at which the composition is intended to be used, in a pH range generally from pH 3 to pH 9, or between pH 4 and pH 8. Here, the “cationic charge density” of the polymer refers to the ratio of the number of positive charges on the polymer to the molecular weight of the polymer. Such suitable cationic polymers will have an average molecular weight generally between 10,000 and 10 million, between 50,000 and 5 million, or between 100,000 and 3 million.

[0524] Suitable cationic polymers for use in the compositions of the present invention comprise a cationic nitrogen-containing moiety, such as a quaternary ammonium or a cationic protonated amino moiety. Any anionic counterion may be used in association with the cationic polymer, provided that the polymer remains dissolved in water, in the composition, or in the condensed phase of the composition, and provided that the counterion is physically and chemically compatible with the main component of the composition or otherwise does not unduly impair the properties, stability, or aesthetics of the composition. Non-limiting examples of such counterions include halides (e.g., chlorides, fluorides, bromides, iodides), sulfates, and methyl sulfates.

[0525] Non-limiting examples of such polymers are described in the CTFA Dictionary of Cosmetic Ingredients, Third Edition, by Estrin, Crosley, and Haynes (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, 1982).

[0526] Other suitable cationic polymers for use in this composition include polysaccharide polymers, cationic guar gum derivatives, tetravalent nitrogen-containing cellulose ethers, synthetic polymers, and copolymers of etherified cellulose, guar gum, and starch. When used, the cationic polymer herein is soluble in the composition or in a soluble complex condensed phase formed from the cationic polymer described above and anionic, amphoteric, and / or facultative zwitterionic surfactant components. The complex condensate of the cationic polymer may also form with other charged materials in the composition. Suitable cationic polymers are described in US 3962418; US 3958581; and US 2007 / 0207109.

[0527] The compositions of the present invention may include nonionic polymers as modifiers. Polyalkylene glycols having a molecular weight greater than 1000 are useful here. Those having the following general formulas are useful:

[0528]

[0529] Where R 95 The following group is selected, which consists of: H, methyl groups, and mixtures thereof. Modifiers, and in particular silicones, may be included in the composition. Modifiers used in the compositions of the present invention typically include water-insoluble, water-dispersible, non-volatile liquids that form emulsion liquid particles. Suitable modifiers for use in this composition are those generally characterized as: silicones (e.g., silicone oils, cationic silicones, silicone gels, high-refractive silicones, and silicone resins), organic modifier oils (e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinations thereof, or those modifiers that otherwise form liquid dispersion particles in an aqueous surfactant matrix herein. Such modifiers should be physically and chemically compatible with the main components of the composition and should not otherwise unduly impair the stability, aesthetics, or performance of the composition.

[0530] The concentration of the modifier in the composition should be sufficient to provide the desired moderating benefits. This concentration can vary depending on the modifier, the desired moderating performance, the average size of the modifier particles, the type and concentration of other components, and other similar factors.

[0531] The concentration range of silicone modifiers is typically from 0.01 wt% to 10 wt%. Non-limiting examples of suitable silicone modifiers and optional suspending agents for silicones are described in U.S. Republication Patent Nos. 34,584; US 5104646; US5106609; US 4152416; US 2826551; US ​​3964500; US 4364837; US 6607717; US 6482969; US5807956; US 5981681; US ​​6207782; US 7465439; US 7041767; US 7217777; US 2007 / 0286837 A1; US ​​2005 / 0048549 A1; US ​​2007 / 0041929 A1; GB 849433; DE All references are cited in 10036533; Chemistry and Technology of Silicones, New York: Academic Press (1968); GE Silicone Rubber Product Data Sheets SE 30, SE 33, SE54 and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and Encyclopedia of Polymer Science and Engineering, Volume 15, 2nd Edition, pp. 204-308, John Wiley & Sons, Inc. (1989).

[0532] The compositions of the present invention may further comprise from 0.05 wt% to 3 wt% of at least one organic conditioning oil as a conditioning agent, alone or in combination with other conditioning agents such as silicone (described herein). Suitable conditioning oils include hydrocarbon oils, polyolefins, and fatty esters. Also suitable for use in the compositions herein are the conditioning agents described in US 5674478 and US 5750122 or in US 4529586; US 4507280; US 4663158; US 4197865; US 4217914; US 4381919; and US 4422853.

[0533] Hygiene and Odor -The compositions of the present invention may further include zinc ricinoleate, thymol, quaternary ammonium salts (e.g., zinc ricinoleate, thymol, quaternary ammonium salts). ), polyethyleneimine (e.g., from BASF) ) and its zinc complexes, silver and silver compounds (especially those designed to slowly release Ag) +One or more of the following: (or those nano-silver dispersions).

[0534] Probiotics These compositions may include prebiotics, such as those described in WO 09 / 043709.

[0535] foaming agent - If high foaming is desired, use a foaming agent (e.g., C). 10 -C 16 Alkylamide or C 10 -C 14 Alkyl sulfates can typically be incorporated into the composition at levels ranging from 1 wt% to 10 wt%. 10 -C 14 Monoethanol and diethanolamides illustrate typical categories of such foaming agents. These foaming agents are also advantageous when used with high levels of foaming adjuvants and surfactants (e.g., the aforementioned amine oxides, betaine, and sulfobetaine). If desired, water-soluble magnesium and / or calcium salts (e.g., MgCl2, MgSO4, CaCl2, CaSO4, etc.) can typically be added at levels from 0.1 wt% to 2 wt% to provide additional foam and enhance grease removal performance.

[0536] Foam inhibitors - Compounds for reducing or inhibiting foam formation may be incorporated into the compositions of the present invention. Foam inhibition may be particularly important in so-called “high-concentration cleaning processes” as described in US 4489455 and US 4489574, and in front-loading-style washing machines. A wide variety of materials can be used as foam inhibitors, and foam inhibitors are well known to those skilled in the art. See, for example, Kirk Othmer Encyclopedia of Chemical Technology, 3rd Edition, Volume 7, pp. 430-447 (John Willie & Sons, 1979). Examples of foam inhibitors include monocarboxylic fatty acids and their soluble salts, high molecular weight hydrocarbons such as paraffin, fatty acid esters (e.g., fatty acid triglycerides), fatty acid esters of monovalent alcohols, aliphatic C 18 -C 40Ketones (e.g., stearone), N-alkylated aminotriazines, preferably wax hydrocarbons having a melting point below about 100°C, silicone foam inhibitors, and secondary alcohols. Foam inhibitors are described in US 2954347; US 4265779; US 4265779; US3455839; US 3933672; US 4652392; US 4978471; US ​​4983316; US 5288431; US ​​4639489; US4749740; US 4798679; US 4075118; EP 89307851.9; EP 150872; and DOS 2,124,526.

[0537] For any detergent composition intended for use in an automatic washing machine, foam should not form to the extent that it overflows the washing machine. When used, the foam inhibitor is preferably present in a "foam-inhibiting amount." "Foam-inhibiting amount" refers to the amount of this foam control agent that the formulator of the composition can choose to adequately control foam to result in a low-foaming laundry detergent for use in an automatic washing machine.

[0538] The compositions herein will typically include 0 to 10 wt% of a foam inhibitor. When used as a foam inhibitor, monocarboxylic fatty acids and their salts will typically be present in amounts up to 5 wt%. Preferably, a fatty monocarboxylic acid ester foam inhibitor is used in amounts from 0.5 wt% to 3 wt%. Silicone foam inhibitors are typically used in amounts up to 2.0 wt%, although higher amounts may be used. Monostearyl phosphate foam inhibitors are typically used in amounts ranging from 0.1 wt% to 2 wt%. Hydrocarbon foam inhibitors are typically used in amounts ranging from 0.01 wt% to 5 wt%, although higher levels may be used. Alcohol foam inhibitors are typically used in amounts from 0.2 wt% to 3 wt%.

[0539] The compositions herein can exhibit cleaning activity over a wide pH range. In some embodiments, these compositions have cleaning activity from pH 4 to pH 11.5. In other embodiments, these compositions are active from pH 6 to pH 11, from pH 7 to pH 11, from pH 8 to pH 11, from pH 9 to pH 11, or from pH 10 to pH 11.5.

[0540] The compositions herein can exhibit cleaning activity over a wide temperature range (e.g., from 10°C or lower to 90°C). Preferably, this temperature will be below 50°C, 40°C, or even 30°C. In some embodiments, the optimal temperature range for these compositions is from 10°C to 20°C, from 15°C to 25°C, from 15°C to 30°C, from 20°C to 30°C, from 25°C to 35°C, from 30°C to 40°C, from 35°C to 45°C, or from 40°C to 50°C.

[0541] Forms of the composition

[0542] The compositions described herein are advantageously used in applications such as laundry, hard surface cleaning, dishwashing, and cosmetic applications (e.g., dentures, teeth, hair, and skin). The compositions of the invention are specifically solid or liquid cleaning and / or treatment compositions. In one aspect, the invention relates to a composition selected from the group consisting of: regular, compressed, or concentrated liquids; gels; ointments; soap bars; regular or compressed powders; granular solids; homogeneous or multilayer tablets having two or more layers (same or different phases); bags having one or more chambers; single or multiple chamber unit dosage forms; or any combination thereof.

[0543] The composition is designed to physically separate the components from each other in multiple compartments (e.g., like a water-soluble pouch) or different layers of a tablet. This avoids negative storage interactions between the components. Furthermore, the different dissolution profiles of each compartment in the washing solution can also cause delayed dissolution of selected components.

[0544] The bag can be configured as a single or multiple chambers. It can have any form, shape, and material suitable for containing 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 containing 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, 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, such as those sold by MonoSol LLC, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may differ in composition from chambers comprising solid components (US 2009 / 0011970 A1).

[0545] Lipase granules

[0546] The lipase variants contained in the water-soluble membrane of the present invention can be present as lipase particles. These lipase particles may even contain one or more additional enzymes, as described below.

[0547] Lipase particles are any form of lipase variant in solid particulate form. Lipase particles can be lipase crystals, lipase precipitates, spray-dried or lyophilized lipase, or any form of granular lipase, as a powder or suspension in a liquid. Typically, the particle size of lipase particles, measured as equivalent spherical diameter (average particle size based on volume), is less than 2 mm, preferably less than 1 mm, less than 0.5 mm, less than 0.25 mm, or less than 0.1 mm; and greater than 0.05 μm, preferably greater than 0.1 μm, greater than 0.5 μm, greater than 1 μm, greater than 5 μm, or greater than 10 μm.

[0548] In a preferred embodiment, the particle size of the lipase particles ranges from 0.5 μm to 100 μm.

[0549] The lipase particles contain at least 1% w / w lipase protein, preferably at least 5% w / w lipase protein, at least 10% w / w lipase protein, at least 20% w / w lipase protein, at least 30% w / w lipase protein, at least 40% w / w lipase protein, at least 50% w / w lipase protein, at least 60% w / w lipase protein, at least 70% w / w lipase protein, at least 80% w / w lipase protein, or at least 90% w / w lipase protein.

[0550] In a preferred embodiment, the lipase particles are lipase crystals, or the lipase protein is in a crystal form.

[0551] Enzyme crystallization can be carried out in a variety of ways as known in the art (e.g., as described in WO 91 / 09943 or WO94 / 22903).

[0552] Lipases can be formulated in lipase particles as known in the art for use in solid enzyme formulations, such as formulations for reducing dust, improving stability, and / or altering enzyme release rates. Lipase particles can also be formulated in a matrix or coated with reagents that inhibit the dissolution of the enzyme particles in PVOH / membrane solutions used to prepare water-soluble membranes.

[0553] The lipase molecules on the surface of lipase particles can also be cross-linked, such as CLEC (cross-linked enzyme crystals) or CLEA (cross-linked enzyme aggregates).

[0554] Water-soluble membrane

[0555] Water-soluble films, optional components therein, and methods for preparing them are well known in the art. In one type of embodiment, the water-soluble film comprises PVOH. PVOH is a synthetic resin typically prepared by alcoholysis (commonly referred to as hydrolysis or saponification) of polyvinyl acetate. Fully hydrolyzed PVOH, in which almost all acetate groups have been converted to alcohol groups, is a highly crystalline polymer with strong hydrogen bonds that dissolves only in hot water (above about 140°F (60°C)). If a sufficient number of acetate groups are allowed to remain after the hydrolysis of polyvinyl acetate, then the PVOH polymer is called partially hydrolyzed, with weaker hydrogen bonds and lower crystallinity, and is soluble in cold water (below about 50°F (10°C)). An intermediate cold / hot water-soluble film may comprise, for example, intermediately partially hydrolyzed PVOH (e.g., having a degree of hydrolysis of about 94% to about 98%), and is readily soluble only in warm water (e.g., rapidly dissolving at temperatures of about 40°C and above). Both fully and partially hydrolyzed PVOH types are commonly referred to as PVOH homopolymers, although the partially hydrolyzed type is technically a vinyl alcohol-vinyl acetate copolymer.

[0556] The degree of hydrolysis of PVOH contained in the water-soluble membrane disclosed herein can be from about 75% to about 99%. When the degree of hydrolysis decreases, the membrane made from the resin will have reduced mechanical strength but will dissolve more quickly at temperatures below about 20°C. When the degree of hydrolysis increases, the membrane made from the resin will tend to have higher mechanical strength and its thermoformability will tend to decrease. The degree of hydrolysis of PVOH can be selected such that the water solubility of the resin is temperature-dependent, and thus the solubility of the membrane made from the resin, compatibility reagents, and other components is also affected. In one type of embodiment, the membrane is cold-water soluble. A cold-water soluble membrane (soluble in water at temperatures below 10°C) may include PVOH with a degree of hydrolysis in the range of about 75% to about 90%, or in the range of about 80% to about 90%, or in the range of about 85% to about 90%. In another type of embodiment, the membrane is hot-water soluble. A hot-water soluble membrane (soluble in water at temperatures of at least about 60°C) may include PVOH with a degree of hydrolysis of at least about 98%.

[0557] In addition to or as an alternative to PVOH, other film-forming resins used may include, but are not limited to, modified polyvinyl alcohol, polyacrylates, water-soluble acrylate copolymers, polyacrylates, polyacrylamide, polyvinylpyrrolidone, pullulan, water-soluble natural polymers including but not limited to guar gum, xanthan gum, carrageenan, and starch, and water-soluble polymer derivatives including but not limited to ethoxylated starch and hydroxypropylated starch, poly(acrylamido-2-methylpropanesulfonate), polymethyl maleate, copolymers thereof, and combinations thereof. In one embodiment, the film-forming resin is a terpolymer composed of vinyl alcohol, vinyl acetate, and acrylamide-2-methylpropanesulfonate. Surprisingly, water-soluble films based on the terpolymer of vinyl alcohol, vinyl acetate, and acrylamide-2-methylpropanesulfonate have shown high percentages of enzyme recovery.

[0558] The water-soluble resin can be included in the water-soluble membrane in any suitable amount, for example, in the range of about 35 wt% to about 90 wt%. The preferred weight ratio of the amount of the water-soluble resin to the combined amount of all enzymes, enzyme stabilizers and auxiliary additives can be any suitable ratio, for example, in the range of about 0.5 to about 5, or about 1 to about 3, or about 1 to about 2.

[0559] The water-soluble resins (including, but not limited to, PVOH resins) used in the membranes described herein can be characterized by any viscosity suitable for the desired membrane properties, optionally in the range of about 5.0 to about 30.0 cP, or about 10.0 cP to about 25 cP. The viscosity of the PVOH resin is determined by measuring the freshly prepared solution using a Brookfield LV viscometer with a UL adapter, as described in Annex E of British Standard EN ISO 15023-2:2006, Brookfield Test Method. It is international practice to describe the viscosity of a 4% aqueous polyvinyl alcohol solution at 20°C. All PVOH viscosities specified herein in cP should be understood to refer to the viscosity of a 4% aqueous polyvinyl alcohol solution at 20°C, unless otherwise stated.

[0560] It is well known in the art that the viscosity of a PVOH resin is related to its weight-average molecular weight. Related, and this viscosity is often used as Therefore, the weight-average molecular weight of the water-soluble resin may optionally be in the range of about 35,000 to about 190,000, or about 80,000 to about 160,000. The molecular weight of the resin only needs to be sufficient for it to be molded into a plastic film using suitable techniques.

[0561] The water-soluble film disclosed herein may include, for example, other optional additive components in an amount suitable for its intended purpose, including but not limited to, plasticizers, surfactants, defoamers, film-forming agents, anti-blocking agents, internal release agents, anti-yellowing agents, and other functional components.

[0562] Water is considered a very effective plasticizer for PVOH and other polymers; however, its volatility limits its effectiveness because polymer films need to have at least some tolerance (robustness) to a variety of environmental conditions, including low and high relative humidity. Glycerin is much less volatile than water and has been well established as an effective plasticizer for PVOH and other polymers. If the levels used in film formulations are too high, glycerin or other such liquid plasticizers can themselves cause surface “sweating” and greasiness. This can lead to problems in films such as giving consumers an unacceptable tactile experience and, if not mitigated in some way (e.g., surface dusting), can even cause the film to become stuck on rollers or in stacks of sheets. This can be characterized as overplasticization. However, if too little plasticizer is added to the film, the film may lack sufficient stretch and flexibility for many end uses, such as when being converted to end-use types, such as bags.

[0563] Plasticizers used in the water-soluble membranes disclosed herein include, but are not limited to, sorbitol, glycerol, diglycerol, propylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol (up to 400 MW), 2-methyl-1,3-propanediol, lactic acid, glyceryl monoacetate, triacetyl ester, triethyl citrate, 1,3-butanediol, trimethylolpropane (TMP), polyether triols, and combinations thereof. As mentioned above, polyols are generally used as plasticizers. The less plasticizer used, the more brittle the membrane may become, while the more plasticizer used, the more likely the membrane may lose tensile strength. Plasticizers may be included in the water-soluble membrane in amounts ranging from, for example, from about 25 phr to about 50 phr, or from about 30 phr to about 45 phr, or from about 32 phr to about 42 phr.

[0564] Surfactants used in water-soluble membranes are well known in the art. Optionally, surfactants are included to assist in the dispersion of the resin solution during casting. Suitable surfactants for the water-soluble membranes disclosed herein include, but are not limited to, dialkyl sulfosuccinates, lactated fatty acid esters of glycerol and propylene glycol, fatty acid lactyl esters, sodium alkyl sulfate, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkyl polyoxyethylene ethers, lecithin, acetylated fatty acid esters of glycerol and propylene glycol, sodium lauryl sulfate, acetylated fatty acid esters, myristyl dimethylamine oxide, trimethyl tallow alkyl ammonium chloride, quaternary ammonium compounds, their salts, and combinations thereof. Thus, surfactants may be included in the water-soluble membrane in amounts, for example, less than about 2 phr, such as less than about 1 phr, or less than about 0.5 phr.

[0565] One type of auxiliary ingredient to be considered is a defoamer. Defoamers can help coalesce foam bubbles. Suitable defoamers for use in water-soluble films according to this disclosure include, but are not limited to, hydrophobic silica, such as fine-grained silica or calcined silica, including Foam. Defoamers (available from Emerald Performance Materials), including Foam 327, Foam UVD, Foam 163. Foam 269. Foam 338, Foam 290, Foam 332, Foam 349. Foam 550 and Foam 339, these are proprietary non-mineral oil defoamers. In embodiments, the defoamer may be used in amounts of 0.5 phr or less, such as 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr. Preferably, significant amounts of silica will be avoided to prevent stress whitening.

[0566] Methods for preparing water-soluble articles (including films) include casting, blow molding, extrusion, or blow extrusion, as known in the art. One class of considered embodiments is characterized by the formation of water-soluble films described herein by casting, for example by applying the mixture to a surface by mixing the components described herein with water to produce an aqueous mixture (e.g., a solution having optionally dispersed solids), and drying to remove the water to produce a film. Similarly, other compositions can be formed by drying the mixture while confining it to a desired shape.

[0567] In one considered embodiment, the water-soluble film is formed by casting a water-soluble mixture, wherein the water-soluble mixture is prepared according to the following steps:

[0568] (a) Provide a mixture of water-soluble resin, water, and any optional additives (excluding plasticizers);

[0569] (b) Boil the mixture for 30 minutes;

[0570] (c) Degas the mixture in an oven at a temperature of at least 40°C; optionally in the range of 40°C to 70°C, for example about 65°C;

[0571] (d) Add one or more enzymes, plasticizers, and additional water to the mixture at a temperature of 65°C or lower; and

[0572] (e) Stir the mixture without vortexing until the mixture appears substantially uniform in color and consistency; optionally for a period ranging from 30 to 90 minutes, optionally for at least 1 hour; and

[0573] (f) Pour the mixture quickly after the mixing period (e.g., within 4 hours, 2 hours, or 1 hour).

[0574] If enzymes are added to a mixture too early (e.g., along with auxiliary additives or resins), enzyme activity may decrease. Without intending to be bound by any specific theory, it is believed that boiling a mixture containing enzymes causes enzyme denaturation, and prolonged storage in solution also leads to decreased enzyme activity.

[0575] In one embodiment, the water-soluble membrane according to this disclosure is maintained at high enzyme activity by rapid drying under moderate to mild conditions. As used herein, rapid drying means a drying time of less than 24 hours, optionally less than 12 hours, optionally less than 8 hours, optionally less than 2 hours, optionally less than 1 hour, optionally less than 45 minutes, optionally less than 30 minutes, optionally less than 20 minutes, optionally less than 10 minutes, for example, in the range of about 6 minutes to about 10 minutes or 8 minutes. As used herein, moderate to mild conditions mean a drying temperature below 170°F (77°C), optionally in the range of about 150°F to about 170°F (about 66°C to about 77°C), for example, 165°F (74°C). As the drying temperature increases, the enzyme tends to denature more quickly, while as the drying temperature decreases, the drying time increases, thereby exposing the enzyme to solution for an extended period of time.

[0576] This membrane is used to produce a package containing a composition, such as a laundry or dishwashing composition, thereby forming a bag. The membrane described herein can also be used to prepare a package having two or more compartments, made from the same membrane or a membrane combined with other polymeric materials. Additional membranes can be obtained, for example, by casting, blow molding, extrusion, or blow extrusion of the same or different polymeric materials, as known in the art. In one type of embodiment, polymers, copolymers, or derivatives thereof suitable for use as additional membranes are selected from polyvinyl alcohol, polyvinylpyrrolidone, polyepoxides, polyacrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetate, polycarboxylic acids and salts, polyamino acids or peptides, polyamides, polyacrylamide, maleic acid / acrylic acid copolymers, polysaccharides (including starch and gelatin), and natural gums (e.g., xanthan gum and carrageenan). For example, the polymer may be selected from polyacrylate and water-soluble acrylate copolymers, methylcellulose, sodium carboxymethylcellulose, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylate and combinations thereof, or from polyvinyl alcohol, polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC) and combinations thereof.

[0577] The bags and / or packages disclosed herein include at least one sealed compartment. Therefore, these bags may include a single compartment or multiple compartments. These bags may have enzyme-containing and enzyme-free areas. In embodiments including multiple compartments, each compartment may contain the same and / or different compositions. Furthermore, these compositions may take any suitable form, including but not limited to liquids, solids, and combinations thereof (e.g., solids suspended in a liquid). In some embodiments, these bags include first, second, and third compartments, wherein each compartment contains a different first, second, and third composition, respectively. In some embodiments, as described in EP 2258820, these compositions may be visually different.

[0578] The compartments of a multi-compartment bag and / or pack may have one or more identical or different sizes and / or volumes. The compartments of the multi-compartment bag of the present invention may be separate or combined in any suitable manner. In some embodiments, the second and / or third and / or subsequent compartments are stacked on top of the first compartment. In one embodiment, the third compartment may be stacked on top of the second compartment, which in turn is stacked on top of the first compartment in a sandwich configuration. Alternatively, the second and third compartments may be stacked on top of the first compartment. However, it is also contemplated that the first, second, and optionally third and subsequent compartments may be attached to each other in a side-by-side relationship. These compartments may be packaged in a string, each compartment being separable individually by perforated lines. Thus, each compartment can be individually torn off by the end user from the remainder of the string.

[0579] In some embodiments, a multi-compartment bag and / or package comprises three compartments, consisting of a large first compartment and two smaller compartments. The smaller second and third compartments are stacked on top of the large first compartment. The dimensions and geometry of these compartments are chosen to make this arrangement feasible. The geometries of the compartments may be the same or different. In some embodiments, the second and optionally third compartments each have a different geometry and shape compared to the first compartment. In these embodiments, the second and optionally third compartments are arranged on top of the first compartment in a design. This design may be decorative, didactic, or illustrative, for example, to illustrate a concept or guide, and / or to indicate the origin of the product. In some embodiments, the first compartment is the largest compartment, having two large, perimeter-sealed surfaces, while the second compartment is smaller, covering less than about 75%, or less than about 50%, of the surface area of ​​one surface of the first compartment. In embodiments where a third compartment exists, the above structure may be the same, but the second and third compartments cover less than about 60%, or less than about 50%, or less than about 45% of the surface area of ​​one side of the first compartment.

[0580] The bags and / or pouches disclosed herein may include one or more different films. For example, in a single-compartment embodiment, the pouch may be made of a wall folded onto itself and sealed at the edges, or alternatively, of two walls sealed together at the edges. In a multi-compartment embodiment, the pouch may be made of one or more films such that any given pouch compartment may include walls made of a single film or multiple films with different compositions. In one embodiment, a multi-compartment bag includes at least three walls: an outer upper wall; an outer lower wall; and a partition wall. The outer upper wall and the outer lower wall are generally opposite each other and form the exterior of the bag. The partition wall is inside the bag and is secured to these generally opposite outer walls along a sealing line. The partition wall divides the interior of the multi-compartment bag into at least one first compartment and one second compartment. In one type of embodiment, the partition wall may be the only membrane containing the enzyme, thereby minimizing consumer exposure to these enzymes.

[0581] Bags and pouches can be made using any suitable equipment and methods. For example, individual compartment bags can be made using vertical filling, horizontal filling, or drum filling techniques known in the art. Such processes can be continuous or intermittent. The membrane can be wetted and / or heated to improve its ductility. The method may also involve using a vacuum to draw the membrane into a suitable mold. The vacuum drawing the membrane into the mold can be applied for about 0.2 to about 5 seconds, or about 0.3 to about 3 seconds, or about 0.5 to about 1.5 seconds, once the membrane is on the horizontal portion of the surface. This vacuum can be such that it provides a pressure, for example, in the range of 10 mbar to 1000 mbar, or in the range of 100 mbar to 600 mbar.

[0582] These molds (in which bags can be made) can have any shape, length, width, and depth, depending on the dimensions required for these bags. These molds can also differ from one another in size and shape, if desired. For example, the final bag volume can be from about 5 ml to about 300 ml, or from about 10 to 150 ml, or from about 20 to about 100 ml, and the size of these molds can be adjusted accordingly.

[0583] In one embodiment, the package includes a first and a second sealed compartment. Typically, the second compartment is stacked on top of the first sealed compartment, such that the second sealed compartment and the first sealed compartment share a partition wall inside the bag.

[0584] In one embodiment, the package including a first and a second compartment further includes a third sealed compartment. Typically, the third sealed compartment is superimposed on the first sealed compartment such that the third sealed compartment and the first sealed compartment share a partition wall inside the bag.

[0585] In different embodiments, the first composition and the second composition are selected from one of the following combinations: liquid, liquid; liquid and powder; powder, powder; and powder and liquid.

[0586] In different embodiments, the first, second and third compositions are selected from one of the following combinations: solid, liquid, liquid and liquid, liquid, liquid.

[0587] In one embodiment, the single compartment or multiple sealed compartments contain a composition. The multiple compartments may each contain the same or different compositions. The composition is selected from a liquid, a solid, or a combination thereof.

[0588] In this method, heat can be applied to the film, commonly referred to as thermoforming. Heat can be applied by any suitable method. For example, the film can be directly heated before or once it is supplied to a surface by placing it under a heating element or by hot air. Alternatively, for example, it can be indirectly heated by heating the surface or by applying a hot article to the film. Infrared light can be used to heat the film. The film can be heated to at least 50°C, for example, about 50°C to about 150°C, about 50°C to about 120°C, about 60°C to about 130°C, about 70°C to about 120°C, or about 60°C to about 90°C.

[0589] Alternatively, the membrane can be wetted by any suitable means, for example, by spraying a wetting agent (including water, a solution of the membrane composition, a plasticizer for the membrane composition, or any combination thereof) directly onto the membrane before or once it is supplied to a surface, or indirectly by wetting the surface or by applying a wetting article to the membrane.

[0590] Once the film has been heated and / or wetted, it can be drawn into a suitable mold, preferably using a vacuum. For example, the film can be thermoformed at a stretch ratio of at least about 1.5, and optionally up to a stretch ratio of 2. The molding film can be filled using any suitable means. In some embodiments, the most preferred method will depend on the product form and the required filling speed. In some embodiments, the molding film is filled using an in-line filling technique. The filled open bags are then closed using any suitable method with a second film to form a pouch. This can be done while in a horizontal position and in continuous, uniform motion. The closure can be achieved by continuously supplying a second film (preferably a water-soluble film) above and over these open bags, and then preferably sealing the first and second films together, typically in the area between the molds and thus between the bags.

[0591] Any suitable sealing package and / or its individual compartments can be used. Non-limiting examples of such means include heat sealing, solvent welding, solvent sealing, or liquid sealing, and combinations thereof. The water-soluble package and / or its individual compartments can be heat-sealed at a temperature of at least 200°F (93°C), for example, in the range of about 220°F (about 105°C) to about 290°F (about 145°C), or about 230°F (about 110°C) to about 280°F (about 140°C). Typically, only the area to be sealed is treated with heat or solvent. Typically, heat or solvent can be applied to the sealing material by any method, and typically only to the area to be sealed. If solvent sealing, liquid sealing, or welding is used, it is preferable to also apply heat. Preferred liquid sealing or solvent sealing / welding methods involve selectively applying a solvent to the area between molds or to the sealing material, by spraying or printing it onto these areas, and then applying pressure to these areas to form a seal. For example, sealing rollers and belts as described above can be used (optionally, heat is also provided).

[0592] The formed bag can then be cut using a cutting device. Any known method can be used to perform the cutting. Preferably, the cutting can be performed continuously, and preferably at a constant speed and preferably when in a horizontal position. The cutting device can be, for example, a sharp object, a hot object, or a laser, whereby, in the latter case, the hot object or laser 'burns' through the film / sealable area.

[0593] The different compartments of a multi-compartment bag can be made together in a side-by-side pattern, wherein the resulting one-piece bag can be separated by cutting or can remain together. Alternatively, the compartments can be made separately.

[0594] In some embodiments, the bag may be manufactured according to a method including the following steps:

[0595] a) Forming a first compartment (as described above);

[0596] b) Forming notches in some or all of the closed compartments formed in step (a) to create a second molded compartment superimposed on the first compartment;

[0597] c) The second compartment is filled and sealed by means of a third membrane;

[0598] d) Seal the first, second, and third membranes; and

[0599] e) Cut these membranes to create a multi-compartment bag.

[0600] The notch formed in step (b) can be achieved by applying a vacuum to the compartment prepared in step (a).

[0601] In some embodiments, the second and / or third compartments may be made in separate steps and then combined with the first compartment, as described in EP 2088187 or WO 2009 / 152031.

[0602] In other embodiments, the bag can be made according to a method including the following steps:

[0603] a) Optionally using heat and / or vacuum, a first compartment is formed on a first forming machine using a first membrane;

[0604] b) Fill the first compartment with the first composition;

[0605] c) On a second molding machine, heat and vacuum are optionally used to deform the second film to form a second and optionally a third molded compartment;

[0606] d) Fill the second and optionally third compartment;

[0607] e) Seal the second and optionally third compartments using a third membrane;

[0608] f) Place the sealed second and optionally third compartments onto the first compartment;

[0609] g) Seal the first, second, and optionally third compartments; and

[0610] h) Cut these membranes to create a multi-compartment bag.

[0611] The suitability of the above methods can be selected based on the first and second molding machines. In some embodiments, the first molding machine is preferably a horizontal molding machine, and the second molding machine is preferably a rotary drum molding machine, preferably located above the first molding machine.

[0612] It should be understood that, by using appropriate feed stations, it is possible to manufacture multi-compartment bags incorporating a variety of different or unique compositions and / or different or unique liquid, gel, or paste compositions.

[0613] Method for manufacturing the composition

[0614] The compositions of the present invention can be formulated in any suitable form and can be prepared by any method of choice of the formulation party, non-limiting examples of which are described in the applicant’s examples and in US 4990280; US20030087791 A1; US ​​20030087790 A1; US ​​20050003983 A1; US ​​20040048764 A1; US4762636; US 6291412; US 20050227891 A1; EP 1070115 A2; US 5879584; US 5691297; US5574005; US 5569645; US 5565422; US 5516448; US 5489392; US 5486303, all of which are incorporated herein by application. The compositions of the present invention, or compositions prepared according to the present invention, comprise cleaning and / or treatment compositions, including but not limited to compositions for treating fabrics, hard surfaces, and any other surfaces in the fields of fabric and home care, including: air care (including air fresheners and odor delivery systems), car care, dishwashing, fabric conditioning (including softening and / or freshening), laundry detergents, laundry and rinsing additives and / or care, hard surface cleaning and / or treatments (including floor and toilet cleaners), general-purpose or “heavy-duty” detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, gel, or paste form, especially so-called heavy-duty liquid types; liquid fine-fabric detergents; hand or light-duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including various tablet, granule, liquid, and rinsing aid types for use in homes and public institutions: car or carpet shampoos, bathroom cleaners (including toilet cleaners); and cleaning aids, such as bleaching additives and “stain-stick” or pretreatment types, matrix-loaded compositions (e.g., sheets with added desiccants). Preferred are compositions and methods for cleaning and / or treating textiles and / or hard surfaces (most preferably textiles). The compositions are preferably used in the pretreatment step or the main washing step of the washing process (most preferably for the textile washing step).

[0615] As used herein, the term "fabric and / or hard surface cleaning and / or treatment composition" is a subset of cleaning and treatment compositions, which, unless otherwise indicated, includes general-purpose or "heavy-duty" detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, glue, or paste form, especially so-called heavy-duty liquid types; liquid fine fabric detergents; hand or light-duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including various tablet, granular, liquid, and rinsing aid types for use in homes and public institutions; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners (including toilet cleaners); fabric conditioning compositions (including softeners and / or fresheners), which may be in liquid, solid, and / or desiccant sheet form; and matrix-loaded compositions (e.g., desiccant sheets) together with cleaning aids, such as bleach additives and "stain remover sticks" or pretreatment types. All applicable such compositions may be in standard, concentrated, or even highly concentrated forms, even to the point that such compositions may be non-aqueous in some respects.

[0616] How to use

[0617] This invention includes a method for cleaning any surface (including treating textiles or hard or other surfaces) in the field of fabrics and / or home care. In one aspect of the invention, the method includes a step of contacting the surface to be treated in a pretreatment step or a main washing step of the washing process (most preferably for use in textile washing steps or alternatively for use in dishwashing (including both manual and automatic / mechanical dishwashing)). In one embodiment of the invention, a lipase variant and other components are sequentially added to the method for cleaning and / or treating the surface. Alternatively, the lipase variant and other components are added simultaneously.

[0618] As used herein, washing includes, but is not limited to, scrubbing and mechanical agitation. Washing can be performed using foam compositions (as described in WO 08 / 101958) and / or by applying alternating pressure (pressure / vacuum) as an additional or alternative method to scrubbing and mechanical agitation. Drying such surfaces or fabrics can be accomplished by any of the common means employed in domestic or industrial environments. The cleaning compositions of the present invention are ideally suited for use in laundry and dishwashing applications. Therefore, the present invention includes methods for cleaning objects (including, but not limited to, fabrics, tableware, knives, and kitchen utensils). The method includes the step of contacting the object to be cleaned with the cleaning composition, which includes at least one embodiment of the applicant's cleaning composition, cleaning additives, or mixtures thereof. Fabrics can include most fabrics capable of being washed under normal consumer or public institution use conditions. The solution can have a pH from 8 to 10.5. The composition can be used in the solution at concentrations from 500 ppm to 15,000 ppm. The water temperature range is typically from 5°C to 90°C. The water-to-fabric ratio is typically from 1:1 to 30:1.

[0619] In one aspect, the present invention relates to a method for producing a composition using a polypeptide having at least 75% similarity to SEQ ID NO:3. In another aspect, the present invention relates to the use of the composition for cleaning objects.

[0620] In one aspect, the present invention relates to a method of producing a composition, the method comprising adding the variant. In another aspect, the present invention relates to a method of producing the composition, the method comprising adding a polypeptide having at least 75% similarity to SEQ ID NO:3, and a surfactant. In another aspect, the present invention relates to a method for cleaning a surface, the method comprising contacting lipid stains present on the surface to be cleaned with the cleaning composition. In another aspect, the present invention relates to a method for hydrolyzing stains and / or lipids present on a surface, the method comprising contacting the stains and / or dirt with a cleaning composition. In another aspect, the present invention relates to a method for hydrolyzing a lipase substrate, the method comprising contacting said substrate with the variant.

[0621] The scope of the invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to exemplify several aspects of the invention. Any equivalent aspects are contemplated to be within the scope of the invention. In fact, various modifications of the invention, other than those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such embodiments are also intended to fall within the scope of the appended claims. In case of conflict, the disclosure including the definition shall prevail.

[0622] Example

[0623] Example 1: Measurement Protocol

[0624] A 6 mM stock solution of 4-nitrophenyl palmitate (pNP-palmitic acid) was prepared by dissolving 226.5 mg of 4-nitrophenyl palmitate (Sigma catalog number N2752) in 100 mL of anhydrous ethanol (Merck catalog number 1.00983.0511).

[0625] A 625 μM pNP-palmitic acid working solution was prepared by adding 10 mL of the stock substrate solution to 90 mL of assay buffer (50 mL 1 M TRIS pH 8.0 (Sigma catalog number T6066); 1.35 g solid deoxycholic acid / sodium deoxycholic acid monohydrate (Sigma catalog number D5670); 0.7 g AOS (BIO-TERGE AS-40); up to 500 mL Milli Q water).

[0626] Store the substrate and working solution in the dark at 4°C until use.

[0627] Table 1: Standard Detergent Composition

[0628] Element Amount (wt%) Linear alkylbenzene sulfonic acid (LAS) (97%) 5.00 Ether sulfate (AEOS) (70.5%) 10.00 Sodium alkyl sulfate (AS) (90%) 4.50 Cocoa fatty acids (>99%) 1.00 AEO; alcohol ethoxide with 7 mol EO (approximately 100%) 5.00 MEA, monoethanolamine (99.5%) 0.30 MPG (>98%) 3.00 EtOH, propan-2-ol (90%) 1.35 DTPA, diethylenetriaminepentaacetic acid, sodium pentachloride (40%) 0.10 Sodium citrate (100%) 4.00 Sodium formate (>95%) 1.00 NaOH, granules (>99%) 0.66 Add water until 100

[0629] A: Thermal stability determination

[0630] Thermal stability was determined by measuring the enzyme activity observed in the culture supernatant of the variant or wild-type control exposed to 50°C.

[0631] At room temperature (control) or 50°C, 10 μL of supernatant sample was heat-treated in a 96-well Abgene PCR plate (Thermo Scientific, catalog number...

Claims

1. A lipase variant of a parental lipase derived from *Rhizomucor miehei*, wherein the parental lipase has at least 99% sequence identity with SEQ ID NO: 3, and wherein the variant has lipase activity and contains S1C, S1F, S1G, S1H, S1I, S1L, S1M, S1P, S1Q, S1R, S1V, S1W or S1Y substitutions corresponding to position 1 of SEQ ID NO: 3, wherein the variant has improved thermal stability relative to the parent, measured as an improvement factor (IF) greater than 1.1, wherein the improvement factor (IF) is determined using thermal stability assay A.

2. The variant of claim 1, wherein the improvement factor (IF) is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.3, 2.4 or 2.

5.

3. A method for hydrolyzing a lipase substrate, the method comprising contacting the substrate with a variant as described in any one of claims 1-2.

4. A composition comprising any variant of claims 1-2.

5. A method for producing the composition of claim 4, the method comprising adding a variant as described in any one of claims 1-2.

6. A cleaning method comprising bringing a surface or article into contact with a variant according to any one of claims 1-2.

7. A polynucleotide encoding a variant as described in any one of claims 1-2.

8. A nucleic acid construct comprising the polynucleotide as described in claim 7.

9. An expression vector comprising the polynucleotide as described in claim 7.

10. A host cell comprising the polynucleotide as described in claim 7.

11. A method for producing a lipase variant, the method comprising: a. Culture the host cells as described in claim 10 under conditions suitable for expressing the variant; and b. Recycle the variant.

12. A method for obtaining a lipase variant, the method comprising introducing a substitution of S1C, S1F, S1G, S1H, S1I, S1L, S1M, S1P, S1Q, S1R, S1V, S1W or S1Y into a parental lipase at position 1 corresponding to SEQ ID NO: 3, wherein the parental lipase is derived from Rhizopus miltiorrhiza and has at least 99% sequence identity with SEQ ID NO: 3, wherein the variant has lipase activity; and recovering the variant.

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