Lipase variants and microcapsule compositions comprising such lipase variants

Abstract

To provide lipase with improved wash performance, reduced odor-generation and / or improved storage stability / longer shelf life / increased thermostability.SOLUTION: The present invention relates to variants of a parent lipase which has lipase activity and comprises one or more substitutions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T using SEQ ID NO: 2 for numbering. The present invention also relates to compositions or microcapsule compositions comprising a lipase variant of the invention and to liquid products comprising a microcapsule composition of the invention as well as the use of the microcapsule composition for stabilizing lipase variants of the invention.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. [Background technology]

[0002] The present invention relates to lipase variants, polynucleotides encoding said variants, and methods for making said variants. The present invention also relates to compositions and microcapsule compositions comprising the lipase variants of the invention, and liquid products comprising the microcapsule compositions of the invention.

[0003] Lipases are important biocatalysts that have been shown to be useful for a variety of applications. Wild-type Thermomyces lanuginosus lipase (synonymous with Humicola lanuginosa), sold under the trade name LIPOLASE™, and its variants are commercially available as active ingredients in cleaning compositions to remove lipid soils by hydrolyzing triglycerides to produce fatty acids. Summary of the Invention [Problem to be solved by the invention]

[0004] Detergent, cleaning and / or fabric care compositions contain active ingredients that interfere with the ability of lipases to remove lipid soils. Many known Thermomyces lanuginosus lipase variants with good cleaning performance form odor-producing short-chain fatty acids during the wash and / or have short storage stability.

[0005] Therefore, there is still a need and desire for lipases with improved cleaning performance, reduced odor production and / or improved storage stability / longer shelf life / enhanced thermal stability. [Means for solving the problem]

[0006] The present invention relates to variants of a parent lipase that have lipase activity and have at least 60% but less than 100% sequence identity with SEQ ID NO:2 and contain one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R, N233R, T244E, and P256T.

[0007] The present invention further relates to compositions comprising the lipase variants of the present invention and their use for hydrolyzing lipid substrates. Furthermore, the present invention relates to polynucleotides encoding the variants of the present invention, nucleic acid constructs, vectors, and host cells comprising the polynucleotides.

[0008] In one aspect, the present invention relates to a microcapsule composition, wherein the membrane of the microcapsule is produced by cross-linking a hyperbranched polyamine having a molecular weight of more than 1 kDa, and wherein the microcapsule comprises a lipase variant of the present invention.

[0009] In a further aspect, the present invention relates to a microcapsule composition comprising a lipase variant of the present invention encapsulated within a compartment formed by a membrane, wherein the membrane is produced by crosslinking (a) a hyperbranched polyamine having a molecular weight of more than 800 Da and (b) an aliphatic or aromatic amine having a molecular weight of less than 300 Da; and wherein the weight ratio of (a) / (b) is in the range of 0.1 to 1000.

[0010] Finally, the present invention relates to liquid products comprising the microcapsule compositions of the present invention.

[0011] definition Lipase: The terms "lipase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to enzymes belonging to class EC 3.1.1 as defined by enzyme nomenclature. They may have lipase activity (triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13), and / or wax-ester hydrolase activity (EC 3.1.1.50). For purposes of the present invention, lipase activity is determined according to the procedures described in the Examples. In one aspect, a variant of the invention has at least 20%, such as 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% of the lipase activity of the polypeptide of SEQ ID NO:2.

[0012] Allelic variant: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation and can result in polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide having an altered amino acid sequence. An allelic variant of a polypeptide is one encoded by an allelic variant of a gene.

[0013] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The original primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as a spliced ​​mature mRNA.

[0014] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be DNA, cDNA, synthetic DNA, or a combination thereof.

[0015] Control sequences: The term "control sequences" refers to all nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from different genes) to the polynucleotide encoding the variant, or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide.

[0016] 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.

[0017] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that contains a polynucleotide that encodes a variant and is operably linked to regulatory nucleotides that effect expression.

[0018] Fragment: The term "fragment" refers to a polypeptide lacking one or more (e.g., several) amino acids from the amino and / or carboxyl terminus of the polypeptide; wherein the fragment has lipase activity. In one aspect, the fragment has 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%, and at least 95%, but less than 100%, of the number of amino acids 1-269 of SEQ ID NO:2.

[0019] High stringency conditions: The term "high stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, using 200 micrograms / ml fragmented and modified salmon sperm DNA in 5xSSPE, 0.3% SDS, and 50% formamide at 42°C, followed by prehybridization and hybridization for 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 65°C.

[0020] Host cell: The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the invention. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell by mutations that occur during replication.

[0021] Improved properties: The term "improved properties" refers to improved characteristics associated with the variant compared to the parent lipase. Such improved properties include, but are not limited to, detergent stability, stability in detergents in the presence of proteases, protease stability, chemical stability, oxidative stability, pH stability, stability under storage conditions, and thermal stability.

[0022] Isolated: The term "isolated" refers to a substance in a form or environment that is not found in nature. Non-limiting examples of isolated substances include: (1) any non-naturally occurring substance; (2) any enzyme, mutant, nucleic acid, protein, peptide, or cofactor that is at least partially removed from one or more or all of the naturally occurring components with which it is naturally associated, including, but not limited to: (3) any substance that has been artificially modified relative to a naturally occurring substance; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of the gene encoding the substance; use of a stronger promoter than that naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

[0023] Low stringency conditions: The term "low stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, using prehybridization and hybridization in 5xSSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, and 25% formamide at 42°C for 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 50°C.

[0024] 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 embodiment, the mature polypeptide is amino acids 1-269 of SEQ ID NO: 2. It is known in the art that a host cell may produce a mixture of two or more different mature polypeptides (i.e., having different C-terminal and / or N-terminal amino acids) expressed by the same polynucleotide.

[0025] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having lipase activity. In one embodiment, the mature polypeptide coding sequence is nucleotides 1 to 807 of SEQ ID NO:1.

[0026] Medium stringency conditions: The term "medium stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, including prehybridization and hybridization in 5xSSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, and 35% formamide at 42°C for 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 55°C.

[0027] Medium-high stringency conditions: The term "medium-high stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, including prehybridization and hybridization in 5xSSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, 35% formamide at 42°C, followed by 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 60°C.

[0028] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0029] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, which is single-stranded or double-stranded, isolated from a native gene or modified to contain segments of nucleic acid that are not naturally occurring in nature or that have been synthesized, and which includes one or more regulatory sequences.

[0030] Operably linked: The term "operably linked" refers to a configuration in which a control sequence is positioned relative to a coding sequence of a polynucleotide so that the control sequence effects expression of the coding sequence.

[0031] Parent or Parent Lipase: The term "parent" or "parent lipase" refers to a lipase that has been modified to result in an enzyme variant of the invention. The parent lipase may be a naturally occurring (wild-type) polypeptide or a variant thereof.

[0032] Sequence identity: The relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity."

[0033] For purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity, calculated as follows: (equivalent residues × 100) / (length of alignment − total number of gaps in alignment)

[0034] For the purposes of the present invention, sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably the Needle program in version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment − total number of gaps in alignment)

[0035] Stability: The stability of the lipase variants of the present invention may be expressed as the residual activity or performance of the lipase during or after exposure to various test conditions (stress conditions), such as storage in a detergent composition at different temperatures, at different pHs, in the presence of different ingredients such as proteases, chemicals, and / or oxidizing agents, or during use in a cleaning process. The stability of the lipase variant may be measured relative to the known activity or performance of a parent lipase, such as the lipase set forth in SEQ ID NO: 2, or alternatively, relative to the known activity or performance of the lipase variant when initially added to a detergent composition that has optionally been stored at low temperature or frozen, or relative to the lipase variant that has been stored at low temperature or frozen (non-stress conditions).

[0036] Subsequence: The term "subsequence" refers to a polypeptide lacking one or more (e.g., several) nucleotides from the 5' and / or 3' end of 1-807 of SEQ ID NO:1; wherein the subsequence encodes a fragment that has lipase activity. In one aspect, a subsequence of the invention has 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 of the mature polypeptide-encoding sequence.

[0037] Variant: The term "variant" refers to a polypeptide having lipase activity that contains a modification, i.e., a substitution, insertion, and / or deletion, at one or more (e.g., several) positions. A substitution refers to replacing an amino acid occupying a position with a different amino acid; a deletion refers to removing an amino acid occupying a position; and an insertion refers to adding an amino acid adjacent to and immediately after the amino acid occupying a position. Variants of the invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lipase activity of the polypeptide of SEQ ID NO:2.

[0038] Ultra-high stringency conditions: The term "ultra-high stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, using 200 micrograms / ml fragmented and modified salmon sperm DNA in 5xSSPE, 0.3% SDS, and 50% formamide at 42°C, followed by prehybridization and hybridization for 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 70°C.

[0039] Ultra-low stringency conditions: The term "ultra-low stringency conditions" refers to standard Southern blotting procedures for probes at least 100 nucleotides in length, using 200 micrograms / ml fragmented and modified salmon sperm DNA in 5xSSPE, 0.3% SDS, and 25% formamide at 42°C, followed by prehybridization and hybridization for 12-24 hours. The carrier material is finally washed three times for 15 minutes each with 2xSSC, 0.2% SDS at 45°C.

[0040] Wild-type lyase: The term "wild-type" lipase refers to a lipase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus, as found in nature.

[0041] Conventions for naming variants For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another lipase. The amino acid sequence of the other lipase is aligned with SEQ ID NO: 2, and based on the alignment, the number of the amino acid position corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453), preferably as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), version 5.0.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0042] Identification of the corresponding amino acid residues in the other lipase can be performed using, but is not limited to, MUSCLE (multiple sequence comparison by log-expectation; 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; Katoh et al., 2005, Nucleic Acids Research 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680) using ClustalW, using their respective default parameters.

[0043] Other pairwise sequence comparison algorithms can be used when other enzymes are divergent from the polypeptide of SEQ ID NO:2 and traditional sequence-based comparisons cannot detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615). Greater sensitivity in sequence-based searches can be achieved by searching databases using search programs that utilize probabilistic representations (profiles) of polypeptide families. For example, the PSI-BLAST program generates 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 when a polypeptide family or superfamily has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure predictions, structural alignment profiles, and solvation potential) as input to neural networks that predict structural folds for query sequences. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.

[0044] For proteins of known structure, several tools and resources are available for searching and generating structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and these alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747). In addition, implementations of these algorithms can be used to search structural databases with structures of interest to find potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0045] In describing the variants of the present invention, the following nomenclature is adopted for ease of reference: accepted IUPAC one-letter or three-letter amino acid abbreviations are used.

[0046] Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, and replacement amino acid. Thus, a substitution of threonine with alanine at position 226 is named "Thr226Ala" or "T226A." Multiple mutations are separated by a symbol ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent a substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0047] Deletion. In amino acid deletion, the original amino acid, position, * The following nomenclature is used for the deletion of glycine at position 195: * " or "G195 * Multiple deletions are separated by a symbol ("+"), e.g., "Gly195* +Ser411 * " or "G195 * +S411 * " are cited as examples.

[0048] Insertions. For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, an insertion of lysine after glycine at position 195 would be named "Gly195GlyLys" or "G195GK." Multiple amino acid insertions are named as follows: [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, an insertion of lysine and alanine after glycine at position 195 would be designated as "Gly195GlyLysAla" or "G195GKA."

[0049] In such cases, the inserted amino acid residue is numbered by adding a lower case letter to the position number of the amino acid residue preceding the inserted amino acid residue. Thus, in the above example, the sequence would be:

[0050] [Table 1]

[0051] Multiple Alterations. Variants containing multiple alterations are separated by a symbol ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively.

[0052] Different modifications. When different modifications can be introduced at a position, the different modifications are separated by a comma, for example, "Arg170Tyr,Glu" or "R170Y,E" represents a substitution of arginine with tyrosine or glutamic acid at position 170. Thus, "Tyr167Gly,Ala + Arg170Gly,Ala" represents the following variant: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala" Refers to... DETAILED DESCRIPTION OF THE INVENTION

[0053] Disclosed are variants of a parent lipase that have lipase activity, for example variants having at least 60% but less than 100% sequence identity with SEQ ID NO: 2 from Thermomyces lanuginosus.

[0054] Mutants The present invention provides variants of a parent lipase that have lipase activity, wherein the variant has at least 60% but less than 100% sequence identity to SEQ ID NO:2 and includes one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R, N233R, T244E, and P256T.

[0055] Preferably, the lipase variants have at least 60% but less than 100% sequence identity with SEQ ID NO: 2 and contain one or more substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T. Preferably, the variants further contain one or both substitutions at positions corresponding to T231R and / or N233R.

[0056] A preferred mutant contains a substitution at a position corresponding to G23S. A preferred mutant contains a substitution at a position corresponding to D27N. A preferred mutant contains a substitution at a position corresponding to A401. A preferred mutant contains a substitution at a position corresponding to F51I,L. A preferred mutant contains a substitution at a position corresponding to E56R. A preferred mutant contains a substitution at a position corresponding to D57N. A preferred mutant contains a substitution at a position corresponding to V60E,K. A preferred mutant contains a substitution at a position corresponding to K98I. A preferred mutant contains a substitution at a position corresponding to N101D. A preferred mutant contains a substitution at a position corresponding to R118F. A preferred mutant contains a substitution at a position corresponding to G163S. A preferred mutant contains a substitution at a position corresponding to Y220F. A preferred mutant contains a substitution at a position corresponding to T244E. A preferred mutant contains a substitution at a position corresponding to P256T.

[0057] Preferred mutants contain one or more substitutions at positions corresponding to F51I,L, E56R and / or R118F.

[0058] In preferred embodiments, the variants of the invention comprise any one of the following sets of substitutions:

[0059] [Table 2]

[0060] In one embodiment, the variant comprises a substitution at a position corresponding to T231R+N233R, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

[0061] In a preferred embodiment, the variant comprises substitutions corresponding to E56R+T231R+N233R, as well as one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

[0062] In a preferred embodiment, the variant comprises a substitution at a position corresponding to R118F+T231R+N233R, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

[0063] In a more preferred embodiment, the variant comprises a substitution at a position corresponding to E56R+R118F+T231R+N233R, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

[0064] In an even more preferred embodiment, the variant comprises a substitution at a position corresponding to E56R+R118F+T231R+N233R+P256T, as well as one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0065] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+R118F+T231R+N233R, as well as one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E and P256T.

[0066] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0067] In another more preferred embodiment, the variant comprises substitutions at positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R, as well as one or more (e.g., several) substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

[0068] In an even more preferred embodiment, the variant comprises a substitution at a position corresponding to D27N+F51I,L+E56R+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0069] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R, as well as one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E and P256T.

[0070] In an even more preferred embodiment, the variant comprises a substitution at a position corresponding to D27N+F51I,L+E56R+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0071] In an even more preferred embodiment, the variant comprises a substitution at a position corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0072] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+D57N+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0073] In a further preferred embodiment, the variant comprises a substitution at a position corresponding to F51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E. In an even more preferred embodiment, the variant comprises a substitution at a position corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, V60EK, N101D, Y220F, and T244E.

[0074] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+T244E+P256T, as well as one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D and Y220F.

[0075] In a further preferred embodiment, the variant comprises substitutions at positions corresponding to F51I, L+E56R+D57N+V60E, K+K98I+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, N101D, G163S, Y220F, and T244E.

[0076] In a further preferred embodiment, the variant comprises a substitution at a position corresponding to F51I,L+E56R+D57N+V60E,K+K98I+N101D+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, G163S, Y220F, and T244E. In a further preferred embodiment, the variant comprises a substitution at a position corresponding to F51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E.

[0077] A particularly preferred embodiment is the following set of substitutions: R118F+T231R+N233R+P256T; A40I+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R; E56R+D57N+R118F+T231R+N233R; E56R+V60K+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R; D57N+E56R+R118F+T231R+N233R; K98I+E56R+R118F+T231R+N233R; G163S+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R; A40I+D57N+E56R+R118F+T231R+N233R; A40I+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+D57N+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K98I+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K98I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G163S+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+F51L+D57N+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+F51L+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+F51L+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+F51L+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+D57N+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+D57N+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+D57N+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+K98I+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> A40I+K98I+E56R+R118F+T231R+N233R+P256T; A40I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+E56R+R118F+T231R+N233R; F51L+D57N+G163S+E56R+R118F+T231R+N233R; F51L+D57N+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R; F51L+K98I+E56R+R118F+T231R+N233R+P256T; F51L+G163S+E56R+R118F+T231R+N233R+P256T; D57N+K98I+G163S+E56R+R118F+T231R+N233R; D57N+K98I+E56R+R118F+T231R+N233R+P256T; D57N+G163S+E56R+R118F+T231R+N233R+P256T; K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R+P256T; D27N+A40I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; D27N+E56R+R118F+T231R+N233R+P256T; A40I+F51I+E56R+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R+T244E; A40I+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+P256T; E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; G23S+E56R+R118F+T231R+N233R+T244E+P256T; G23S+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+E56R+V60E+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R; D27N+A40I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; D27N+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E; A40I+F51I+E56R+R118F+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E: G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; F51I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R; K98I+E56R+R118F+T231R+N233R; Y220F+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+K98I+E56R+R118F+T231R+N233R; G23S+Y220F+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R; D27N+K98I+E56R+R118F+T231R+N233R; D27N+Y220F+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; D27N+E56R+R118F+T231R+N233R+P256T; F51I+K98I+E56R+R118F+T231R+N233R; F51I+Y220F+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+P256T; K98I+Y220F+E56R+R118F+T231R+N233R; K98I+E56R+R118F+T231R+N233R+T244E; K98I+E56R+R118F+T231R+N233R+P256T; Y220F+E56R+R118F+T231R+N233R+T244E; Y220F+E56R+R118F+T231R+N233R+P256T; E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+K98I+E56R+R118F+T231R+N233R; G23S+D27N+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+E56R+R118F+T231R+N233R; G23S+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+K98I+E56R+R118F+T231R+N233R+T244E; G23S+K98I+E56R+R118F+T231R+N233R+P256T; G23S+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R; D27N+F51I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R; D27N+K98I+E56R+R118F+T231R+N233R+T244E; D27N+K98I+E56R+R118F+T231R+N233R+P256T; D27N+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R; F51I+K98I+E56R+R118F+T231R+N233R+T244E; F51I+K98I+E56R+R118F+T231R+N233R+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; K98I+Y220F+E56R+R118F+T231R+N233R+T244E; K98I+Y220F+E56R+R118F+T231R+N233R+P256T; K98I+E56R+R118F+T231R+N233R+T244E+P256T; Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+K98I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T and variants containing a substitution at a position corresponding to one of:

[0078] The lipase variants of the present invention have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the parent lipase.

[0079] In preferred embodiments, the variants of the invention have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:2.

[0080] A variant of the present invention may have 1 to 40, 1 to 30, 1 to 20, such as 1 to 12, for example 1 to 11, for example 1 to 10, such as 1 to 9, for example 1 to 8, such as 1 to 7, for example 1 to 6, such as 1 to 5, for example 1 to 4, for example 1 to 3, or such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions.

[0081] The variants of the present invention may have one or more of the following properties compared to the parent lipase: improved cleaning performance, reduced odor generation, improved storage stability, longer shelf life and / or enhanced thermal stability.

[0082] The lipase variants of the present invention may further comprise one or more additional substitutions at one or more (eg, several) other positions.

[0083] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small extensions of the amino or carboxyl termini, e.g., amino-terminal methionine residues; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, e.g., a poly-histidine tract, an antigenic epitope, or a binding domain.

[0084] Examples of conservative substitutions include those in the following groups: 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 activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions 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.

[0085] Alternatively, the amino acid changes may be of a nature such that the physicochemical properties of the polypeptide are modified, e.g., the amino acid changes may improve the thermostability of the polypeptide, alter its substrate specificity, or change its pH optimum.

[0086] Essential amino acids in a polypeptide can be identified according to 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, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues that are critical for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of the enzyme or other biological interactions can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutations of putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides.

[0087] A variant may consist of or comprise 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% by number of amino acids of SEQ ID NO:2.

[0088] Parent Lipase The parent lipase is a) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO:2; b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, under medium stringency conditions, under medium-high stringency conditions, under high stringency conditions, or under very high stringency conditions to (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i); c) a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; and d) a fragment of the polypeptide of SEQ ID NO: 2 may be selected from the group consisting of:

[0089] In one aspect of the invention, the parent lipase has lipase activity and has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:2.

[0090] In one aspect, the parent amino acid sequence differs from the polypeptide of SEQ ID NO: 2 by at most 40 amino acids, for example 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 amino acids.

[0091] In another embodiment, the parent comprises or consists of the amino acid sequence of SEQ ID NO:2.

[0092] In another embodiment, the parent is a fragment of the polypeptide of SEQ ID NO:2 that 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 of SEQ ID NO:2.

[0093] In another embodiment, the parent is an allelic variant of the polypeptide of SEQ ID NO:2.

[0094] In another aspect, the parent lipase is encoded by a polynucleotide that hybridizes to (i) the polypeptide coding sequence of SEQ ID NO: 1, (ii) the full-length complement of (i) under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0095] The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, and the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding the parental gene from strains of different genera or species, according to methods well known in the art. In particular, such probes can be used to hybridize with genomic DNA or cDNA of a cell of interest and identify and isolate the corresponding gene therein, according to standard Southern blotting procedures. Such probes can be significantly shorter than the entire sequence, but should be at least 15, e.g., 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, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled (e.g., with 32 P, 3 H, 35 S, biotin, or avidin) for detecting the corresponding gene, and such probes are encompassed by the present invention.

[0096] Genomic DNA or cDNA libraries prepared from such other strains may be screened for DNA that hybridizes with the above-described probes and encodes the parent. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA may be transferred and immobilized on nitrocellulose or other suitable support material. The support material is used in a Southern blot to identify clones or DNA that hybridize to SEQ ID NO:1 or a subsequence thereof.

[0097] For purposes of this invention, hybridization refers to the hybridization of a polynucleotide under very low to very high stringency conditions to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the polypeptide coding sequence of SEQ ID NO: 1; (iii) a full-length complement thereof; or (iv) a subsequence thereof. A molecule of interest to which the nucleic acid probe hybridizes under these conditions can be detected, for example, using X-ray film or any other detection means known in the art.

[0098] In one embodiment, the nucleic acid probe is the polypeptide coding sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid probe is 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 nucleotides of SEQ ID NO: 1. In another embodiment, the nucleic acid probe is a polynucleotide encoding the polypeptide of SEQ ID NO: 2; or a polypeptide thereof; or a fragment thereof. In another embodiment, the nucleic acid probe is SEQ ID NO: 1.

[0099] In another embodiment, the parent is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide-encoding sequence of SEQ ID NO:1.

[0100] The polypeptides may be hybrid polypeptides in which a region of one polypeptide is fused at the N-terminus or C-terminus to a region of another polypeptide.

[0101] The parent lipase may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides may also be constructed using intein technology, such that the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0102] The fusion polypeptide may further comprise a cleavage site between the two polypeptides, which is cleaved upon secretion of the fusion protein, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those described in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward 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. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0103] The parent lipase may be obtained from a microorganism of any genus. For purposes of the present invention, the term "obtained from," when used herein in reference to a given source, shall mean that the parent encoded by the polynucleotide is produced by the source or by the strain into which the polynucleotide from the source is inserted. In one aspect, the parent is secreted extracellularly.

[0104] The parent may be a bacterial lipase, for example a Gram-positive lipase such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase. The lipase may be a gram-positive bacterial polypeptide, or a gram-negative bacterial polypeptide such as Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma lipase.

[0105] In one embodiment, the parent is Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis lipase.

[0106] In another embodiment, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus lipase.

[0107] In another embodiment, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans lipase.

[0108] In another embodiment, the parent is a Thermobifida alba or Thermobifida fusca (formerly known as Thermomonaspora fusca) lipase.

[0109] The parent may be a fungal lipase. For example, the parent may be a yeast lipase, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipase, such as an Acremonium, Agaricus, Alternaria, Aspergillus ), Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria ria), Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocal Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,The lipase may be derived from Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

[0110] In another embodiment, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis lipase.

[0111] In another embodiment, the parent is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophilasubthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

[0112] In another embodiment, the parent is a Thermomyces lanuginosus lipase, such as, in particular, the lipase of SEQ ID NO:2.

[0113] It will be understood that the present invention encompasses both perfect and imperfect forms of the above species, as well as other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the equivalence of appropriate equivalents.

[0114] Strains of these species are readily publicly accessible at several culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0115] The parent lipase may be identified and obtained from other sources, including microorganisms isolated from nature (e.g., pollutants, compost, water, etc.), or DNA samples may be obtained directly from natural materials (e.g., pollutants, compost, water, etc.) using the probes described above. Techniques for isolating DNA directly from microorganisms and natural habitats are well known in the art. Polynucleotides encoding the parent may then be obtained by similarly screening genomic DNA or cDNA libraries of the other microorganism or mixed DNA samples. Once the polynucleotides encoding the parent have been detected using the probes, the polynucleotides may be isolated or cloned using techniques known to those skilled in the art (see, e.g., Sambrook et al., 1989 (supra)).

[0116] Preparation of mutants The present invention also relates to a method for obtaining a lipase variant of the present invention, comprising: (a) introducing substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, K98I, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T; (b) selecting variants that have lipase activity and, relative to the parent lipase, have one of the desired properties listed above; and (c) recovering the variants.

[0117] Variants can be prepared using any mutagenesis method known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, and the like.

[0118] Site-directed mutagenesis is a technique in which one or more (eg, several) mutations are introduced at one or more defined sites in a polynucleotide encoding a parent lipase.

[0119] Site-directed mutagenesis can be achieved in vitro by PCR using oligonucleotide primers bearing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves restriction enzyme cleavage at a site within a plasmid containing a polynucleotide encoding the parent lipase, followed by ligation of an oligonucleotide bearing the mutation into the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to be ligated together. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0120] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, e.g., 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).

[0121] Any site-directed mutagenesis method can be used in the present invention. Many commercial kits are available that can be used to prepare mutants.

[0122] Synthetic gene construction involves the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using several techniques, such as the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques in which oligonucleotides are synthesized and assembled on a photoprogrammable microfluidic chip.

[0123] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by associated screening methods, such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, 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; U.S. Pat. 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).

[0124] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using methods standard in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0125] Semisynthetic gene construction is achieved by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semisynthetic construction is typified by a process that utilizes synthesized polynucleotide fragments in combination with PCR technology. Thus, defined regions of a gene may be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, while other regions may be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.

[0126] Polynucleotides The present invention also relates to isolated polynucleotides encoding the lipase variants of the invention. In certain aspects, the invention relates to nucleic acid constructs comprising the polynucleotides of the invention. In certain aspects, the invention relates to expression vectors comprising the polynucleotides of the invention. In certain aspects, the invention relates to host cells comprising the polynucleotides of the invention. In certain aspects, the invention relates to methods for producing lipase variants, comprising: (a) culturing the host cells of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

[0127] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions appropriate to the control sequences.

[0128] Polynucleotides may be manipulated in a variety of ways to result in expression of variants. Manipulation of a polynucleotide prior to its insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0129] The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide. The promoter has transcriptional control sequences that mediate expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular polypeptides that are either homologous or heterologous to the host cell.

[0130] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the invention in bacterial host cells include the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus licheniformis penicillinase gene (penP), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus subtilis levansucrase gene (sacB), the Bacillus subtilis xylA and xylB genes, the Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13:97-107), the E. coli lac operon, the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase genes (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in "Useful Proteins from Recombinant Bacteria" by Gilbert et al., 1980, Scientific American 242:74-94; and Sambrook et al., 1989 (supra). Examples of tandem promoters are disclosed in WO 99 / 43835.

[0131] Examples of promoters suitable for directing transcription of the nucleic acid constructs of the invention in filamentous fungal host cells include those encoding Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum f. sp. oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reeseiPromoters obtained from the genes for Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, and the NA2-tpi promoter (a modified promoter from the Aspergillus neutral alpha-amylase gene in which the non-translated leader has been replaced by the non-translated leader from the Aspergillus triose phosphate isomerase gene; a non-limiting example is the Aspergillus niger neutral alpha-amylase gene in which the non-translated leader has been replaced by the non-translated leader from Aspergillus nidulans or Aspergillus oryzae) in which the untranslated leader from the triosephosphate isomerase gene has been replaced by the untranslated leader from the gene); and truncated mutants, as well as hybrid promoters thereof.

[0132] In yeast hosts, useful promoters can be obtained from genes encoding Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described in Romanos et al., 1992, Yeast 8:423-488.

[0133] The control sequence may also be a transcription terminator recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used.

[0134] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0135] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0136] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992 (supra).

[0137] The regulatory sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene that enhances expression of the gene.

[0138] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0139] The control sequence may also be a non-translated leader region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0140] Preferred leaders in filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0141] Suitable leaders for yeast host cells are obtained from the genes in Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0142] The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of the variant coding sequence, which, when transcribed, is recognized by the host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0143] Preferred polyadenylation sequences in filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0144] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0145] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of the variant and directs the variant into the secretory pathway of the cell. The 5' end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the variant. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required if the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0146] Useful signal peptide coding sequences for bacterial host cells include those obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides have been described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0147] Effective signal peptide coding sequences for filamentous fungal host cells are those obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0148] Useful signal peptides in yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992 (supra).

[0149] The regulatory sequence may also be a propeptide coding sequence encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes an enzyme precursor). Propolypeptides are generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences may be obtained from genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0150] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the variant, and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0151] It may be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, and the Aspergillus oryzae glucoamylase promoter may be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein gene, which is amplified using heavy metals. In these cases, the polynucleotide encoding the variant would be operably linked to the regulatory sequence.

[0152] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcriptional and translational stop signals. Various nucleotides and control sequences may be ligated together to create a recombinant expression vector, which may contain one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding the variant at such site. Alternatively, a polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. When creating an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0153] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of a polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0154] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, and its replication is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for ensuring autonomous replication. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, together carrying the entire DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0155] Vectors preferably carry one or more selectable markers which permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0156] Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenylyltransferase), and trpC (anthranilate synthase), and their equivalents. In Aspergillus cells, the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus are preferred.

[0157] The vector preferably has elements that allow the vector to be integrated into the genome of a host cell or to replicate autonomously within the cell independent of the genome.

[0158] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may have additional polynucleotides to direct integration into the host cell genome by homologous recombination at a precise location in the chromosome. To increase the likelihood of integration at a precise location, the integration element must contain a sufficient number of nucleic acids, e.g., 100-10,000 base pairs, 400-10,000 base pairs, and 800-10,000 base pairs, with a high degree of sequence identity to the corresponding target sequence to increase the probability of homologous recombination. The integration element may be any sequence that is homologous to the target sequence in the host cell genome. Furthermore, the integration element may be a non-coding or coding polynucleotide. Alternatively, the vector may be integrated into the host cell genome by non-homologous recombination.

[0159] In autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that allows a plasmid or vector to replicate in vivo.

[0160] Examples of bacterial replication origins are the replication origins of plasmids pBR322, pUC19, pACYC177, and pACYC184, which enable replication in E. coli, and the replication origins of pUB110, pE194, pTA1060, and pAMβ1, which enable replication in Bacillus.

[0161] Examples of origins of replication that can be used in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0162] Examples of replication origins useful in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acids Res. 15:9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and construction of a plasmid or vector containing the gene can be carried out according to the methods disclosed in WO 00 / 24883.

[0163] To increase the production of variants, more than one copy of a polynucleotide of the invention may be inserted into a host cell. An increase in polynucleotide copy number may 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, where cells carrying an amplified copy of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected by culturing the cells in the presence of the appropriate selectable agent.

[0164] The procedures used to ligate the above-described elements to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

[0165] host cell The present invention also relates to recombinant host cells comprising a polynucleotide encoding a variant of the invention operably linked to one or more control sequences that direct the production of the variant. A construct or vector comprising the polynucleotide is introduced into a host cell, whereby the construct or vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector, as described above. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of host cell will depend largely on the gene encoding the variant and its source.

[0166] The host cell may be any cell useful in the recombinant production of the variant, e.g., prokaryotic or eukaryotic.

[0167] Prokaryotic host cells can be any Gram-positive or Gram-negative bacterium, including, but not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0168] Bacterial host cells include, but are not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus The cell may be any Bacillus cell, including Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0169] The bacterial host cell may also be any Streptococcus cell, including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0170] A bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0171] Introduction of DNA into Bacillus cells may be by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81:823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56:209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). Introduction of DNA into E. coli cells may be by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). Introduction of DNA into Streptomyces cells may be by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98:6289-6294). Introduction of DNA into Pseudomonas cells may be by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64:391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71:51-57).Introduction of DNA into Streptococcus cells may be by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0172] The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0173] The host cell may be a fungal cell. "Fungi," as used herein, includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all vegetative spore-forming 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).

[0174] The fungal host cell may be a yeast cell. "Yeast," as used herein, includes ascosporogenous yeasts (Endomycetales), basidiomycete yeasts, and yeasts belonging to the Fungi Imperfecti (Blastomycetes). Because the classification of yeasts may change in the future, for purposes of the present invention, yeasts will be defined as set forth in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0175] Yeast host cells include Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norvensis, and the like. norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cells.

[0176] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts, such as Saccharomyces cerevisiae, is by budding of a unicellular thallus, and carbon catabolism may be fermentative.

[0177] Filamentous fungal host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, and Myceliophthora. , Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cells.

[0178] For example, filamentous fungal host cells include Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilbescens, and the like. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, white rot fungi (Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolorversicolor), Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0179] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable methods for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, 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 may be transformed using the methods described by Becker and Guarente, in Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0180] Generation method The present invention also relates to a method for producing a lipase variant of the invention, comprising: (a) culturing a host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

[0181] The host cells are cultured in a nutrient medium suitable for the production of the variant using methods known in the art. For example, the cells may be cultured in shake flask cultures or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors conducted in a suitable medium and under conditions that allow for the expression and / or isolation of the variant. Culturing is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using methods known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in the catalog of the American 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.

[0182] Mutants may be detected using methods known in the art that are specific for the variant. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, enzyme assays may be used to measure the activity of the variants as described in the Examples.

[0183] The variant may be recovered using methods known in the art. For example, the variant may be recovered from the nutrient medium by conventional methods including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0184] The variants may be purified by a variety of methods known in the art, including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, isoelectric focusing, and size exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction to obtain a substantially pure variant (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989).

[0185] In an alternative embodiment, the variants are not recovered, but rather the host cells of the invention that express the variants are used as a source of the variants.

[0186] composition The present invention also includes compositions comprising the lipase variants of the present invention.

[0187] In certain aspects, the present invention relates to compositions comprising variants of a parent lipase having lipase activity and having at least 60% but less than 100% sequence identity to SEQ ID NO:2 and comprising one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, K98I, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T.

[0188] In certain embodiments, the composition includes a substitution at a position corresponding to R118F+T231R+N233R, as well as one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, K98I, G163S, Y220F, T244E, and P256T.

[0189] In certain embodiments, the variants have improved cleaning performance, reduced odor production and / or improved storage stability / longer shelf life / enhanced heat stability.

[0190] The non-limiting list of composition ingredients set forth herein below are suitable for use in the present compositions and methods herein may desirably be incorporated into certain embodiments of the present invention, for example, to aid or enhance cleaning performance for purposes of treating the substrate being cleaned, or to modify the aesthetics of the composition, as in the case of fragrances, colorants, dyes, etc. The level of such optional ingredients incorporated into any composition is in addition to any materials previously listed for incorporation. The exact nature of these additional ingredients and their level of incorporation will depend on the physical form of the composition and the nature of the cleaning operation for which it is used. The ingredients below are categorized under general headings according to specific functions, but as will be recognized by those skilled in the art, this should not be construed as limiting, as ingredients may include additional functions.

[0191] Unless otherwise indicated, percentage amounts are based on the weight of the composition (wt%).Suitable ingredient materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removers / anti-redeposition agents, brighteners, suds suppressors, dyes, tinting dyes, perfumes, perfume delivery systems, structural softeners, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments.In addition to the disclosure below, suitable examples and use levels of other such ingredients can be found in U.S. Patent Nos. 5,576,282, 6,306,812, and 6,326,348, which are incorporated herein by reference.

[0192] Thus, in certain embodiments, the present invention does not contain one or more of the following adjunct materials: surfactants, soaps, builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removers / anti-redeposition agents, brighteners, suds suppressors, dyes, fragrances, fragrance delivery systems, resilience agents, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments. However, if one or more ingredients are present, one or more of the ingredients as detailed below may be present.

[0193] Surfactant—The compositions according to the present invention may comprise a surfactant or surfactant system, which may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. When present, the surfactant is typically present at a level of 0.1 to 60%, 0.2 to 40%, 0.5 to 30%, 1 to 50%, 1 to 40%, 1 to 30%, 1 to 20%, 3 to 10%, 3 to 5%, 5 to 40%, 5 to 30%, 5 to 15%, 3 to 20%, 3 to 10%, 8 to 12%, 10 to 12%, 20 to 25%, or 25 to 60% by weight.

[0194] Suitable anionic cleansing surfactants include sulfate and sulfonate cleansing surfactants.

[0195] Suitable sulfonate cleaning surfactants include alkyl benzene sulfonates, in one aspect C 10~13Suitable alkylbenzene sulfonates (LAS) include alkylbenzene sulfonates. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonating commercially available linear alkylbenzenes (LAB); suitable LABs include lower 2-phenyl LABs such as Isochem® or Petrelab®, while other suitable LABs include higher 2-phenyl LABs such as Hyblene®. Suitable anionic detersive surfactants are alkylbenzene sulfonates obtained by the DETAL catalysis process, although other synthetic routes, such as HF, may also be suitable. In one embodiment, magnesium salts of LAS are used.

[0196] Suitable sulfate cleansing surfactants include alkyl sulfates, in one aspect C 8~18 Alkyl sulfate or mainly C 12 Contains alkyl sulfates.

[0197] Other suitable sulfate cleansing surfactants are alkyl alkoxylated sulfates, in one aspect alkyl ethoxylated sulfates, in one aspect C 8~18 Alkyl alkoxylated sulfates, in another embodiment C 8~18 alkyl ethoxylated sulfates, typically the alkyl alkoxylated sulfates have an average degree of alkoxylation of 0.5 to 20 or 0.5 to 10, typically the alkyl alkoxylated sulfates have an average degree of ethoxylation of 0.5 to 10, 0.5 to 7, 0.5 to 5, or 0.5 to 3 C 8~18 It is an alkyl ethoxylated sulfate.

[0198] The alkyl sulfates, alkyl alkoxylated sulfates, and alkyl benzene sulfonates may be linear or branched, substituted or unsubstituted.

[0199] The detersive surfactant may be a mid-chain branched detersive surfactant, in one aspect a mid-chain branched anionic detersive surfactant, in one aspect a mid-chain branched alkyl sulfate, and / or a mid-chain branched alkyl benzene sulfonate, e.g., a mid-chain branched alkyl sulfate. In one aspect, the mid-chain branched alkyl benzene sulfonate is selected from the group consisting of C 1~4 The alkyl groups are typically methyl and / or ethyl groups.

[0200] Non-limiting examples of anionic surfactants include sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkane sulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkenesulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates, and the like. sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfofatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonates (MES), alkyl- or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, di- and monoesters of sulfo-succinic acid or soaps, and combinations thereof.

[0201] Suitable non-ionic cleansing surfactants are selected from the group consisting of: C8-C9 such as NEODOL® 18 Alkyl ethoxylates; C6-C6 alkyl alkoxylate units, which may be ethyleneoxy units, propyleneoxy units, or mixtures thereof. 12Alkylphenol alkoxylates; C with ethylene oxide / propylene oxide block polymers, such as Pluronic® 12 ~C 18 Alcohols and C6-C 12 Alkylphenol condensate; C 14 ~C 22 Mid-chain branched alcohols; typically C with an average degree of alkoxylation of 1 to 30 14 ~C 22 mid-chain branched alkyl alkoxylates; alkyl polysaccharides, and in one aspect, alkyl polyglycosides; polyhydroxy fatty acid amides; ether-capped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

[0202] Suitable non-ionic detersive surfactants include alkyl polyglucosides and / or alkyl alkoxylated alcohols.

[0203] In one aspect, the nonionic detersive surfactant includes an alkyl alkoxylated alcohol, in one aspect, C 8~18 Alkyl alkoxylated alcohols, such as C 8~18 Included are alkyl ethoxylated alcohols, which can have an average degree of alkoxylation of 1 to 50, 1 to 30, 1 to 20, or 1 to 10. In one aspect, the alkyl alkoxylated alcohols are C alkoxylated alcohols having an average degree of ethoxylation of 1 to 10, 1 to 7, or even 1 to 5 or 3 to 7. 8~18 The alkyl ethoxylated alcohol may be linear or branched, and may be substituted or unsubstituted. Suitable nonionic surfactants include Lutensol®.

[0204] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFAs), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (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 (glucamides, GA, or fatty acid glucamides, FAGA), and products available under the tradenames SPAN and TWEEN®, and combinations thereof.

[0205] Suitable cationic detersive surfactants include alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl tertiary sulfonium compounds, and mixtures thereof.

[0206] Suitable cationic detersive surfactants are quaternary ammonium compounds having the general formula: (R)(R1)(R2)(R3)N + X - , wherein R is a linear or branched, substituted or unsubstituted C 6~18 R1 and R2 are independently selected from methyl or ethyl moieties; R3 is a hydroxyl, hydroxymethyl, or hydroxyethyl moiety; and X is an anion that provides charge neutrality; suitable anions include halides, such as chloride; sulfate; and sulfonate. Suitable cationic detersive surfactants include mono C 6~18 Alkyl monohydroxyethyl dimethyl quaternary ammonium chloride. A highly suitable cationic detersive surfactant is mono C 8~10 Alkylmonohydroxyethyldimethyl quaternary ammonium chloride, mono C10~12 Alkyl monohydroxyethyl dimethyl quaternary ammonium chloride, and mono C 10 Alkyl monohydroxyethyl dimethyl quaternary ammonium chloride.

[0207] Non-limiting examples of cationic surfactants include alkyl dimethylethanolamine quats (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyl distearyl ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.

[0208] Suitable zwitterionic surfactants include amine oxides and betaines, such as alkyldimethylbetaines, sulfobetaines, or combinations thereof. Amine-neutralized anionic surfactants—The anionic surfactants and auxiliary anionic co-surfactants of the present invention may exist in acid form, which can be neutralized to form the surfactant salts desired for use in the detergent compositions. Typical neutralizing agents include metal counterion bases such as hydroxides, e.g., NaOH or KOH. Further preferred agents for neutralizing the anionic surfactants and auxiliary anionic surfactants or co-surfactants of the present invention in acid form include ammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other linear or branched alkanolamines known in the art, such as 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Neutralization with an amine can be carried out to a total or partial extent, for example, a portion of the anionic surfactant mixture can be neutralized with sodium or potassium and a portion of the anionic surfactant mixture can be neutralized with an amine or alkanolamine.

[0209] Non-limiting examples of semi-polar surfactants include amine oxides (AOs), such as alkyldimethylamine oxides.

[0210] Surfactant systems comprising a mixture of one or more anionic surfactants and, optionally, one or more nonionic surfactants with additional surfactants such as cationic surfactants may be preferred. The preferred weight ratio of anionic surfactant to nonionic surfactant is at least 2:1, or at least 1:1 to 1:10.

[0211] In one embodiment, the surfactant system can include a mixture of isoprenoid surfactants represented by Formula A and Formula B below: [ka] wherein Y is CH or absent, and Z can be selected such that the resulting surfactant is selected from the following surfactants: alkyl carboxylate surfactants, alkyl polyalkoxy surfactants, alkyl anionic polyalkoxy sulfate surfactants, alkyl glycerol ester sulfonate surfactants, alkyl dimethylamine oxide surfactants, alkyl polyhydroxy-based surfactants, alkyl phosphate ester surfactants, alkyl glycerol sulfonate surfactants, alkyl polygluconate surfactants, alkyl polyphosphate ester surfactants, alkyl phosphonate surfactants, alkyl polyglycoside surfactants, alkyl monoglycoside surfactants, alkyl diglycoside surfactants, alkyl sulfosuccinate surfactants, alkyl disulfate surfactants, alkyl disulfonate surfactants, alkyl sulfosuccinamate surfactants, alkyl glucamide surfactants, alkyl taurinate surfactants, alkyl sarcosinate surfactants, alkyl glycinate surfactants, alkyl isethionate surfactants, alkyl dialkanol surfactants. Amido surfactants, alkyl monoalkanolamide surfactants, alkyl monoalkanolamide sulfate surfactants, alkyl diglycolamide surfactants, alkyl diglycolamide sulfate surfactants, alkyl glycerol ester surfactants, alkyl glycerol ester sulfate surfactants, alkyl glycerol ether surfactants, alkyl glycerol ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglycerol ether surfactants, alkyl polyglycerol ether sulfate surfactants, alkyl sorbitan ester surfactants, alkyl ammonioalkanesulfonate surfactants, alkylamidopropyl betaine surfactants, alkyl aryl quat-based surfactants, alkyl monohydroxyalkyl-di-alkylated quat-based surfactants, alkyl dihydroxyalkyl monoalkyl quat-based surfactants, alkylated quat surfactants, alkyl trimethylammonium quat-based surfactants, alkyl polyhydroxyalkyloxypropyl quat-based surfactants,alkyl glycerol ester quat surfactants, alkyl glycol amine quat surfactants, alkyl monomethyl dihydroxyethyl quaternary ammonium surfactants, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactants, alkyl trimethyl ammonium surfactants, alkyl imidazoline-based surfactants, alkene-2-yl succinate surfactants, alkyl a-sulfonated carboxylic acid surfactants, alkyl a-sulfonated carboxylic acid alkyl ester surfactants, alpha olefin sulfonate surfactants, alkyl phenol ethoxylate surfactants, alkyl benzene sulfonate surfactants, alkyl sulfobetaine surfactants, alkyl hydroxysulfobetaine surfactants, alkyl ammoniocarboxylate betaine surfactants, alkyl sucrose ester surfactants, alkyl alkanolamide surfactants, alkyl di(polyoxyethylene) monoalkyl ammonium surfactants, alkyl mono(polyoxyethylene) dialkyl ammonium surfactants, alkyl benzyl dimethyl ammonium surfactants, alkyl amino propionate surfactants, alkyl amido propyl dimethyl amine surfactants, or mixtures thereof; and when Z is a charged moiety, Z is charge balanced by a suitable metal or organic counterion. Suitable counterions include metal counterions, amines, or alkanolamines, such as C1-C6 alkanolammonium. More specifically, suitable counterions include Na+, Ca+, Li+, K+, Mg+, such as monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, 1-amino-3-propanol, or mixtures thereof. In one embodiment, the composition contains 5% to 97% of one or more non-isoprenoid surfactants and one or more auxiliary cleaning additives, and the weight ratio of the surfactant of formula A to the surfactant of formula B is 50:50 to 95:5.

[0212] Soap—The compositions herein can contain soap. Without being bound by theory, it may be desirable to include soap, which acts partly as a surfactant and partly as a builder, may be useful for suds suppression, and may also interact favorably with the various cationic compounds of the composition to enhance the softness of fabrics rubbed with the compositions of the present invention. Any soap known in the art for use in laundry detergents can be utilized. In one embodiment, the composition contains 0% to 20%, 0.5% to 20%, 4% to 10%, or 4% to 7% by weight of soap.

[0213] Examples of soaps useful herein include oleic acid soap, palmitic acid soap, palm kernel fatty acid soap, and mixtures thereof.Typical soaps are in the form of a mixture of fatty acid soaps with various chain lengths and degrees of substitution.One such mixture is topped palm kernel fatty acid.

[0214] In one embodiment, the soap is selected from free fatty acids. Suitable fatty acids can be saturated and / or unsaturated and can be obtained from natural sources such as vegetable or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil, beef and fish oils, fats and oils, and mixtures thereof), or can be prepared synthetically (e.g., via oxidation of petroleum or by hydrogenation of carbon monoxide by the Fisher-Tropsch method).

[0215] Examples of suitable saturated fatty acids for use in the compositions of the present invention include captic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. Suitable unsaturated fatty acid species include palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and ricinoleic acid. Examples of preferred fatty acids are saturated Cn fatty acids, saturated C1-C4 fatty acids, and saturated or unsaturated Cn-C8 fatty acids, and mixtures thereof.

[0216] When present, the weight ratio of the fabric softening cationic co-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.

[0217] The soap and non-soap anionic surfactant levels herein are weight percent of the detergent composition specified based on the acid form, although, as is commonly understood in the art, anionic surfactants and soaps are in practice neutralized using sodium, potassium, or alkanolammonium bases such as sodium hydroxide or monoethanolamine.

[0218] Hydrotrope—The compositions of the present invention can include one or more hydrotropes. Hydrotropes are compounds that solubilize hydrophobic compounds in aqueous solutions (or, conversely, polar substances in non-polar environments). Typically, hydrotropes possess both hydrophilic and hydrophobic properties (so-called amphiphilicity, as known from surfactants); however, the molecular structure of hydrotropes generally does not promote spontaneous self-aggregation (see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128). Hydrotropes do not exhibit a critical concentration at which self-aggregation occurs, as observed for surfactants and lipids that form micellar, lamellar, or other well-defined mesophases. Instead, many hydrotropes exhibit a continuous aggregation process in which the size of the aggregates increases with increasing concentration. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing polar and non-polar substances, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are traditionally used in industries ranging from pharmaceuticals, personal care, and food to technical applications. The use of hydrotropes in detergent compositions allows for more concentrated surfactant formulations without undesirable phenomena such as phase separation or high viscosity (as occurs in the process of compacting liquid detergents by removing water).

[0219] The detergent can contain 0 to 10% by weight of a hydrotrope, e.g., 0 to 5% by weight, 0.5 to 5% by weight, or 3 to 5% by weight. Any hydrotrope known in the art for use in detergents can be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0220] Builders—The compositions of the present invention can include one or more builders, co-builders, builder systems, or mixtures thereof. When builders are used, cleaning compositions will typically include 0-65 wt.%, at least 1 wt.%, 2-60 wt.%, or 5-10 wt.% builder. In dishwashing compositions, builder levels are typically 40-65 wt.% or 50-65 wt.%. The compositions may be substantially free of builders; by substantially free, it is meant that zeolite and / or phosphate are "not intentionally added." Typical zeolite builders include zeolite A, zeolite P, and zeolite MAP. A typical phosphate builder is sodium tripolyphosphate.

[0221] The builder and / or co-builder may be, in particular, a chelating agent that forms a water-soluble complex with Ca and Mg. Any builder and / or co-builder known in the art for use in detergents can be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol (DEA), and 2,2',2"-nitrilotriethanol (TEA), and carboxymethyl inulin (CMI), and combinations thereof.

[0222] The cleaning composition can contain a co-builder alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of co-builders include polyacrylate homopolymers or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl- or alkenyl succinic acids.Further specific examples include 2,2',2"-nitrilotriacetic 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-diylbis(phosphonic acid) (HEDP), ethylenediaminetetrakis(methylene)tetrakis(phosphonic acid) ) (EDTMPA), diethylenetriaminepentakis(methylene)pentakis(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2- Sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), 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), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfome Examples of suitable builders and / or co-builders include methyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylidenediamine triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and U.S. Pat. No. 5,977,053.

[0223] Chelating Agents and Crystal Growth Inhibitors - The compositions herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, iron, and / or manganese chelating agents, and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin, and 2-phosphonobutane 1,2,4-tricarboxylic acid (Bayhibit® AM), and derivatives thereof. Typically, the composition can contain 0.005 to 15% by weight or 3.0 to 10% by weight of a chelating agent or crystal growth inhibitor.

[0224] Bleaching Component—Bleaching components suitable for incorporation into the methods and compositions of the present invention include one bleaching component or a mixture of two or more bleaching components. Suitable bleaching components include bleach catalysts, photobleaches, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, and mixtures thereof. Generally, when a bleaching component is used, the compositions of the present invention can include 0-30 wt.%, 0.00001-90 wt.%, 0.0001-50 wt.%, 0.001-25 wt.%, or 1-20 wt.%. Suitable bleaching components include: (1) Preformed Peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of preformed peracids or salts thereof, typically either peroxycarboxylic acids or salts thereof, or peroxysulfonic acids or salts thereof.

[0225] The preformed peracid or salt thereof is preferably a peroxycarboxylic acid or salt thereof, which typically has a chemical structure corresponding to the following formula: [ka] and In the formula, R 14 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; R 14 The group may be linear or branched, substituted or unsubstituted; Y is any suitable counterion that achieves charge neutrality, preferably Y is selected from hydrogen, sodium or potassium. 14 is a linear or branched, substituted or unsubstituted C 6~9 The peracid or its salt is preferably selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, any salt thereof, or any combination thereof. Particularly preferred peracids are phthalimido-peroxy-alkanoic acids, especially ε-phthalimido-peroxy-hexanoic acid (PAP). Preferably, the peracid or its salt has a melting point in the range of 30°C to 60°C.

[0226] The preformed peracid or salt thereof may be a peroxysulfonic acid or salt thereof, which typically has a chemical structure corresponding to the following formula: [ka] and In the formula, R 15 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; R 15 The group may be linear or branched, substituted or unsubstituted; Z is any suitable counterion that achieves charge neutrality, preferably Z is selected from hydrogen, sodium or potassium. 15 is a linear or branched, substituted or unsubstituted C 6~9Preferably, such bleaching components may be present in the compositions of the present invention in an amount of 0.01 to 50% by weight, or 0.1 to 20% by weight.

[0227] (2) Hydrogen peroxide sources include inorganic peroxide hydrate salts, including alkali metal salts such as sodium salts of perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, persilicate, and mixtures thereof. In one embodiment of the present invention, inorganic peroxide hydrate salts, such as those selected from the group consisting of perborates, percarbonates, and mixtures thereof, are used. When used, inorganic peroxide hydrate salts are present in an amount of 0.05 to 40% by weight, or 1 to 30% by weight, of the total composition. They are typically incorporated into the composition as a crystalline solid, which may 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 dispersible polymers, waxes, oils, or fatty acid soaps. Preferably, such bleaching components are present in the compositions of the present invention in an amount of 0.01 to 50% by weight, or 0.1 to 20% by weight.

[0228] (3) The term bleach activator, as used herein, refers to a compound that reacts with hydrogen peroxide to form a peracid via perhydrolysis. The peracid thus formed constitutes the activated bleach. Suitable bleach activators for use herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable bleach activators have the formula R-(C=O)-L, where R is an optionally branched alkyl group having 6 to 14 carbon atoms or 8 to 12 carbon atoms if the bleach activator is hydrophobic, or less than 6 carbon atoms or less than 4 carbon atoms if the bleach activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups include benzoic acid and its derivatives, especially benzenesulfonate. Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A family of bleach activators is disclosed in EP 624154, and a particularly preferred member of that family is acetyl triethyl citrate (ATC). Short-chain triglycerides such as ATC or triacetin have the advantage of being environmentally friendly. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in products during storage and are also effective bleach activators. Finally, ATC is multifunctional because the citrate released in the perhydrolysis reaction can function as a builder. Alternatively, the bleaching system can include, for example, amide, imide, or sulfone-type peroxyacids. The bleaching system can also include peracids such as 6-(phthaloylimido)percaproic acid (PAP).Suitable bleach activators are also disclosed in WO 98 / 17767. While any suitable bleach activator can be used, in one aspect of the present invention, the subject cleaning compositions can include NOBS, TAED, or mixtures thereof. When present, peracids and / or bleach activators are generally present in the compositions in amounts of 0.1 to 60%, 0.5 to 40%, or 0.6 to 10% by weight, based on the fabric and home care composition. One or more hydrophobic peracids or precursors thereof can be used in combination with one or more hydrophilic peracids or precursors thereof. Preferably, such bleaching components are present in the compositions of the present invention in amounts of 0.01 to 50%, or 0.1 to 20% by weight.

[0229] The amount of hydrogen peroxide source and peracid or bleach activator can be selected to provide a molar ratio of available oxygen (from the peroxide source) to peracid of from 1:1 to 35:1, or even from 2:1 to 10:1.

[0230] (4) Diacyl Peroxides—Preferred diacyl peroxide bleaching species include those having the general formula: 1 -C(O)-OO-(O)CR 2 (In the formula, R 1 contains a linear chain of at least 5 carbon atoms and optionally contains one or more substituents (e.g., -N + C6-C alkyl groups containing one or more interrupting moieties (e.g., -CONH- or -CH=CH-) inserted between adjacent carbon atoms of the alkyl group (e.g., -(CH3)3, -COOH, or -CN) and / or alkyl groups. 18 Alkyl, preferably C6-C 12 R represents an alkyl group; 2 indicates an aliphatic group that is compatible with the peroxide moiety, so that R 1 and R 2 In one preferred embodiment, R 1 and R 2 is a straight chain unsubstituted C6-C 12 It is an alkyl chain. Most preferably, R 1 and R2 are identical. R 1 and R 2 Particularly preferred are diacyl peroxides in which both R groups (R1 or R2) are C6-C12 alkyl groups. Preferably, at least one of the R groups (R1 or R2), and most preferably only one, does not contain a branch or pendant ring at the alpha position, or preferably at either the alpha or beta position, or most preferably at either the alpha, beta, or gamma position. In a further preferred embodiment, the DAP can be asymmetric, preferably such that hydrolysis of the R1 acyl group is rapid to produce peracid, while hydrolysis of the R2 acyl group is slow.

[0231] The tetraacyl peroxide bleaching species preferably has the general formula: R 3 -C(O)-OO-C(O)-(CH2)nC(O)-OO-C(O)-R 3 (In the formula, R 3 represents a C1 to C9 alkyl group or a C3 to C7 alkyl group; and n represents an integer of 2 to 12 or 4 to 10 (inclusive).

[0232] Preferably, the diacyl and / or tetraacyl peroxide bleaching species is present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm by weight of the wash liquor. In preferred embodiments, the bleaching species is present in an amount sufficient to provide from 0.5 to 300 ppm, from 30 to 150 ppm by weight of the wash liquor.

[0233] Preferably, the bleaching component comprises bleach catalysts (5 and 6).

[0234] (5) Preferred organic (non-metallic) bleach catalysts include those capable of accepting an oxygen atom from a peracid and / or its salt and transferring the oxygen atom to an oxidizable substrate. Suitable bleach catalysts include, but are not limited to, iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphonylimines; N-acylimines; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones, and mixtures thereof.

[0235] Suitable iminium cations and polyions include, but are not limited to, N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (e.g., compound 4, p. 433); N-methyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in U.S. Pat. No. 5,360,569 (e.g., Column 11, Example 1); and N-octyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in U.S. Pat. No. 5,360,568 (e.g., Column 10, Ex. 3).

[0236] Suitable iminium zwitterions include, but are not limited to, the following: N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, an inner salt prepared as described in U.S. Pat. No. 5,576,282 (e.g., Column 31, Ex. II); N-[2-(sulfoxy)dodecyl]-3,4-dihydroisoquinolinium, an inner salt prepared as described in U.S. Pat. No. 5,817,614 (e.g., Column 32, Ex. V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, an inner salt prepared as described in WO 05 / 047264 (e.g., page 18, Ex. 8); and 2-[3-[(2-butyloctyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, an inner salt.

[0237] Suitable modified amine oxygen transport catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can be prepared according to the method described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxygen transport catalysts include, but are not limited to, sodium 1-hydroxy-N-oxy-N-[2-(sulfoxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0238] Suitable N-sulfonylimine oxygen transport catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, prepared according to the method described in Journal of Organic Chemistry (1990), 55(4), 1254-61.

[0239] Suitable N-phosphonylimine oxygen transport catalysts include, but are not limited to, "R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-P-phenyl-P-(2,4,6-trimethylphenyl)-phosphinic acid amide, prepared according to the method described in Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70.

[0240] Suitable N-acylimine oxygen transport catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, prepared according to the method described in Polish Journal of Chemistry (2003), 77(5), 577-590.

[0241] Suitable thiadiazole dioxide oxygen transport catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, prepared according to the method described in U.S. Pat. No. 5,753,599 (see Column 9, Ex. 2).

[0242] Suitable perfluoroimine oxygen transport catalysts include, but are not limited to, 2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butanimidoyl fluoride, which can be prepared according to the method described in Tetrahedron Letters (1994), 35(34), 6329-30.

[0243] Suitable cyclic sugar ketone oxygen transport catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiulo-2,6-pyranose, prepared according to the method described in U.S. Pat. No. 6,649,085 (Column 12, Ex. 1).

[0244] Preferably, the bleach catalyst comprises an iminium and / or carbonyl functional group, typically capable of forming an oxaziridium and / or dioxirane functional group upon acceptance of an oxygen atom, particularly from a peracid and / or its salt. Preferably, the bleach catalyst comprises an oxaziridium functional group and / or capable of forming an oxaziridium functional group upon acceptance of an oxygen atom, particularly from a peracid and / or its salt. Preferably, the bleaching component comprises a cyclic iminium functional group, preferably the cyclic moiety having a ring size of 5 to 8 atoms (including the nitrogen atom), preferably 6 atoms. Preferably, the bleach catalyst comprises an aryliminium functional group, preferably a bicyclic aryliminium functional group, preferably a 3,4-dihydroisoquinolinium functional group. Typically, the imine functional group is a quaternary imine functional group, typically capable of forming a quaternary oxaziridinium functional group upon acceptance of an oxygen atom, particularly from a peracid and / or its salt. In another embodiment, the detergent composition has a logP o / w , logP less than or equal to -0.5 o / w , logP less than or equal to -1.0 o / w , logP less than or equal to -1.5 o / w , logP less than or equal to -2.0 o / w , logP less than or equal to -2.5 o / w , logP less than or equal to -3.0 o / w , or logP less than -3.5 o / w Contains a bleaching component having a logP o / w The method for determining is described in detail below.

[0245] Typically, the bleaching component has an X of 0.01 to 0.30, 0.05 to 0.25, or 0.10 to 0.20. SO A bleached species having X can be produced. SO Methods for determining X are described in detail below. For example, a bleaching component having an isoquinolinium structure can produce a bleaching species having an oxaziridium structure. In this example, X SO is that of the oxaziridium bleaching species.

[0246] Preferably, the bleach catalyst has the following formula: [ka] and having a chemical structure corresponding to In the formula, n and m are independently 0 to 4, and preferably, both n and m are 0; 1 are independently selected from substituted or unsubstituted radicals selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocycle, fused heterocycle, nitro, halo, cyano, sulfonato, alkoxy, keto, carboxyl, and carboalkoxy radicals; and any two adjacent R 1 The substituents may be joined to form a fused aryl, fused carbocyclic, or fused heterocyclic ring; each R 2 are independently selected from substituted or unsubstituted radicals selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylene, heterocycle, alkoxy, arylcarbonyl, carboxyalkyl, and amido; 2 is any other R 2 may be bonded to form part of a common ring; any geminal R 2 may combine to form a carbonyl; and any two R 2 may be linked to form a substituted or unsubstituted fused unsaturated moiety; R 3 is C1~C 20 is a substituted or unsubstituted alkyl; R 4 is hydrogen or the moiety Q t -A, where Q is branched or unbranched alkylene, t=0 or 1, and A is OSO3 - , SO3 - , CO2 - , OCO2 - , OPO3 2- , OPO3H - and OPO2 - an anionic group selected from the group consisting of: R 5 is hydrogen or partial -CR 11 R 12 -YG b -Yc -[(CR 9 R 10 ) y -O] k -R 8 wherein each Y is independently O, S, NH, or NR 8 each R 6 is independently selected from the group consisting of alkyl, aryl, and heteroaryl, and said moieties are substituted or unsubstituted, and whether substituted or unsubstituted, said moieties have less than 21 carbons; each G is independently selected from the group consisting of CO, SO, SO, PO, and PO; R 9 and R 10 is independently selected from the group consisting of H and C1-C4 alkyl; R 11 and R 12 are independently selected from the group consisting of H and alkyl, or may combine to form a carbonyl when taken together; b=0 or 1; c=0 or 1, provided that when b=0, c=0; y is an integer from 1 to 6; k is an integer from 0 to 20; R 6 is H, or an alkyl, aryl, or heteroaryl moiety; said moiety may be substituted or unsubstituted; X, if present, is a suitable charge-balancing counterion; R 4 is hydrogen, X is preferably present, and suitable X include, but are not limited to, chloride, bromide, sulfate, methosulfate, sulfonate, p-toluenesulfonate, boron tetrafluoride, and phosphate.

[0247] In one embodiment of the present invention, the bleach catalyst has the following general formula: [ka] has a chemical structure corresponding to In the formula, R 13 is a branched alkyl group containing 3 to 24 carbon atoms (including the branched carbon atom) and a linear alkyl group containing 1 to 24 carbon atoms; preferably, R 13is a branched alkyl group containing 8 to 18 carbon atoms or a linear alkyl group containing 8 to 18 carbon atoms; preferably, R 13 is 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and iso-pentadecyl; preferably, R 13 is selected from the group consisting of 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

[0248] Preferably, the bleaching composition comprises, in addition to the bleaching catalyst, a peracid source, especially an organic bleaching catalyst. The peracid source may be selected from: (a) preformed peracid, (b) percarbonate, perborate or persulfate salts (perhydrogen persulfate sources), preferably in combination with a bleach activator; and (c) perhydrolase enzymes and esters that form peracid in situ in the presence of water during the textile or hard surface treatment step.

[0249] When present, the peracid and / or bleach activator is generally present in an amount of 0.1 to 60%, 0.5 to 40%, or 0.6 to 10% by weight of the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof.

[0250] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source):peracid is from 1:1 to 35:1, alternatively from 2:1 to 10:1.

[0251] (6) Metal-Containing Bleach Catalyst—The catalyst component may be provided by a catalytic metal complex. One type of metal-containing bleach catalyst is a catalyst system containing a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation; an auxiliary metal cation with little or no bleach catalytic activity, such as zinc or aluminum cation; and a sequestrate with a defined stability constant for the catalyst and auxiliary metal cation, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphate), and their water-soluble salts. Such catalysts are disclosed in U.S. Pat. No. 4,430,243. Preferred catalysts are described in WO 09 / 839406, U.S. Pat. No. 6,218,351, and WO 00 / 012667. Particularly preferred are transition metal catalysts or bridging multidentate N-donor ligands.

[0252] If desired, the compositions herein can be catalyzed using manganese compounds, such compounds and use levels being known to those skilled in the art, for example, the manganese-based catalysts disclosed in U.S. Patent No. 5,576,282.

[0253] Cobalt bleach catalysts useful in the present invention are known and are described, for example, in U.S. Patent Nos. 5,597,936 and 5,595,967. Such cobalt catalysts are readily prepared by known methods, such as those taught in U.S. Patent Nos. 5,597,936 and 5,595,967.

[0254] The compositions of the present invention suitably comprise transition metal complexes of ligands such as bispidones (U.S. Pat. No. 7,501,389) and / or macropolycyclic rigid ligands (abbreviated "MRLs"). In practice, and without limitation, the compositions and methods of the present invention can be adjusted to provide at least around 1 part per billion of active MRL species in the aqueous cleaning medium, typically 0.005 to 25 ppm, 0.05 to 10 ppm, or even 0.1 to 5 ppm MRL in the cleaning solution.

[0255] Suitable transition metals in the transition metal bleach catalysts of the present invention include, for example, manganese, iron, and chromium. A suitable MRL includes 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitable transition metal MRLs are readily prepared by known procedures, such as those taught in U.S. Pat. No. 6,225,464 and WO 00 / 32601.

[0256] (7) Photobleaches—Suitable photobleaches include, for example, sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines, xanthene dyes, and mixtures thereof. Preferred bleaching components for use in the compositions of the present invention include a hydrogen peroxide source, a bleach activator, and / or an organic peracid (optionally generated in situ by reaction of the hydrogen peroxide source with the bleach activator), in combination with a bleach catalyst. Preferred bleaching components include a bleach catalyst, preferably an organic bleach catalyst, as described above.

[0257] Particularly preferred bleaching components are bleach catalysts, especially organic bleach catalysts.

[0258] Exemplary bleaching systems are also described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, and WO 2007 / 087242.

[0259] Fabric hueing agent—The composition can include a fabric hueing agent. Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling under the Color Index (CI) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof.

[0260] In another embodiment, suitable small molecule dyes include 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 and a small molecule dye selected from the group consisting of 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, and Basic Blue 159, and mixtures thereof. In another embodiment, suitable small molecule dyes include small molecule dyes selected from the group consisting of 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 Color Index (Society of Dyers and Colorists, Bradford, UK) numbers, and mixtures thereof.In another embodiment, suitable small molecule dyes include small molecule dyes selected from the group consisting of Color Index (Society 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, or mixtures thereof.

[0261] Suitable polymeric dyes include polymeric dyes selected from the group consisting of polymers containing conjugated chromogens (dye-polymer conjugates) and polymers with chromogens copolymerized into the polymer backbone, and mixtures thereof.

[0262] In another embodiment, suitable polymeric dyes include those polymeric dyes selected from the group consisting of fabric colorants sold under the name Liquitint® (Milliken), dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of polymers comprising a moiety selected from the group consisting of a hydroxyl moiety, a primary amine moiety, a secondary amine moiety, a thiol moiety, and mixtures thereof. In yet another embodiment, suitable polymeric dyes include those polymeric dyes selected from the group consisting of Liquitint® Violet CT, carboxymethyl cellulose (CMC) conjugated with a reactive blue, reactive violet, or reactive red dye, such as CMC conjugated with CI Reactive Blue 19 sold by Megazyme, Wicklow, Ireland under the trade name AZO-CM-CELLULOSE, product code S-ACMC, alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophene polymeric colorants, and mixtures thereof.

[0263] Preferred hueing dyes include the brighteners found in WO 08 / 87497. These brighteners can be characterized by the following structure (I): [ka] wherein R1 and R2 can be independently selected from the following: a) [(CH2CR'HO) x (CH2CR”HO) y H] In the formula, R' is H, CH, CHO(CHCHO) z H, and mixtures thereof; R" is selected from the group consisting of H, CH2O(CH2CH2O) z H, and mixtures thereof; x+y≦5; y≧1; and z=0-5; b) R1 = alkyl, aryl, or arylalkyl and R2 = [(CH2CR'HO) x (CH2CR”HO) y H] In the formula, R' is H, CH, CHO(CHCHO) z H, and mixtures thereof; R" is selected from the group consisting of H, CH2O(CH2CH2O) z H, and mixtures thereof; x+y≦10; y≧1; and z=0-5; c) R1 = [CH2CH2(OR3)CH2OR4] and R2 = [CH2CH2(OR3)CH2OR4] Wherein R3 is H, (CH2CH2O) z H, and mixtures thereof; and z=0-10; R4 is (C1~C 16 ) selected from the group consisting of alkyl groups, aryl groups, and mixtures thereof; and d) R1 and R2 can be independently selected from amino addition products of styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, t-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl hexadecyl ether with 1 to 10 alkylene oxide units.

[0264] Preferred brighteners of the present invention can be characterized by the following structure (II): [ka] In the formula, R' is H, CH, CHO(CHCHO) z H, and mixtures thereof; R" is selected from the group consisting of H, CH2O(CH2CH2O) z H, and mixtures thereof; x+y≦5; y≧1; and z=0-5.

[0265] Further preferred brighteners of the present invention can be characterized by the following structure (III): [ka] Typically, it comprises a mixture having a total of 5 EO groups. Suitable preferred molecules are those belonging to structure I in "part a" above, having the following pendant groups:

[0266] [Table 3]

[0267] Further useful brighteners include those described in US Patent Application Publication No. 2008 / 34511 (Unilever). A preferred brightener is "Violet 13."

[0268] Suitable dye clay conjugates include those selected from the group consisting of at least one cationic / basic dye and a smectite clay, as well as mixtures thereof. In another embodiment, suitable dye clay conjugates include those selected from the group consisting of one cationic / basic dye selected from the group consisting of CI Basic Yellow 1-108, CI Basic Orange 1-69, CI Basic Red 1-118, CI Basic Violet 1-51, CI Basic Blue 1-164, CI Basic Green 1-14, CI Basic Brown 1-23, and CI Basic Black 1-11, and a clay selected from the group consisting of montmorillonite clay, hectorite clay, and saponite clay, as well as mixtures thereof.In yet another embodiment, suitable dye clay conjugates include Montmorillonite Basic Blue B7 CI42595 conjugate, Montmorillonite Basic Blue B9 CI52015 conjugate, Montmorillonite Basic Violet V3 CI42555 conjugate, Montmorillonite Basic Green G1 CI42040 conjugate, Montmorillonite Basic Red R1 CI45160 conjugate, Montmorillonite CI Basic Black 2 conjugate, Hectorite Basic Blue B7 CI42595 conjugate, Hectorite Basic Blue B9 CI52015 conjugate, Hectorite Basic Violet V3 CI42555 conjugate, Hectorite Basic Green G1 CI42040 conjugate, Hectorite Basic Red R1 CI45160 conjugate, Hectorite CI Basic Black 2 conjugate, Saponite Basic Blue B7 CI 42595 conjugate, Saponite Basic Blue B9 CI 52015 conjugate, Saponite Basic Violet V3 CI 42555 conjugate, Saponite Basic Green G1 CI 42040 conjugate, Saponite Basic Red R1 CI 45160 conjugate, Saponite CI Basic Black 2 conjugate, and mixtures thereof.

[0269] Suitable pigments include pigments selected from the group consisting of flavanthrone, indanthrone, chlorinated indanthrones having 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide (the imide group may be unsubstituted or substituted with C1-C3 alkyl or phenyl or heterocyclic groups, and the phenyl and heterocyclic groups may carry further substituents that do not impart water solubility), anthrapyrimidinecarboxylic acid amide, violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyanine (which may contain up to two chlorine atoms per molecule), polychloro-copper phthalocyanine or polybromochloro-copper phthalocyanine (containing up to 14 bromine atoms per molecule), and mixtures thereof.

[0270] In another embodiment, suitable pigments include pigments selected from the group consisting of Ultramarine Blue (CI Pigment Blue 29), Ultramarine Violet (CI Pigment Violet 15), and mixtures thereof.

[0271] The aforementioned fabric hueing agents can be used in combination (any mixture of fabric hueing agents can be used). Suitable hueing agents are described in detail in U.S. Pat. No. 7,208,459. The preferred level of dye in the compositions of the present invention is 0.00001 to 0.5% by weight, or 0.0001 to 0.25% by weight. The preferred dye concentration in water for the treatment and / or washing steps is 1 ppb to 5 ppm, 10 ppb to 5 ppm, or 20 ppb to 5 ppm. In preferred compositions, the surfactant concentration is 0.2 to 3 g / L.

[0272] Capsules - The composition can comprise a capsule. In one embodiment, the capsule comprises a core, a shell having an inner surface and an outer surface, the shell encapsulating the core.

[0273] In one embodiment of the capsule, the core can comprise a material selected from the group consisting of fragrances; whitening agents; dyes; insect repellents; silicones; waxes; flavorings; vitamins; fabric softeners; skin care agents, and in one embodiment, paraffin; enzymes; antibacterial agents; bleaching agents; sensation enhancers; and mixtures thereof; the shell can comprise a material selected from the group consisting of polyethylene; polyamides; polyvinyl alcohol, optionally containing other comonomers; polystyrene; polyisoprene; polycarbonates; polyesters; polyacrylates; aminoplasts, and in one embodiment, the aminoplasts can comprise polyureas, polyurethanes, and / or polyureaurethanes, and in one embodiment, the polyureas can comprise polyoxymethylene urea and / or melamine formaldehyde; polyolefins; polysaccharides, and in one embodiment, the polysaccharides can comprise alginates and / or chitosan; gelatin; shellac; epoxy resins; vinyl polymers; water-insoluble inorganics; silicones; and mixtures thereof.

[0274] In one embodiment of the capsule, the core may include a flavoring.

[0275] In one embodiment of the capsule, the shell can comprise melamine formaldehyde and / or crosslinked melamine formaldehyde.

[0276] In one aspect, suitable capsules are disclosed that can include a core material and a shell, the shell at least partially surrounding the core, wherein at least 75%, 85%, or 90% of the capsules can have a breaking strength of 0.2-10 MPa, 0.4-5 MPa, 0.6-3.5 MPa, or 0.7-3 MPa; and a leakage of benefit agent of 0-30%, 0-20%, or 0-5%.

[0277] In one embodiment, at least 75%, 85%, or 90% of the capsules may have a particle size of 1-80 microns, 5-60 microns, 10-50 microns, or 15-40 microns.

[0278] In one embodiment, at least 75%, 85%, or 90% of the capsules can have a particle wall thickness of 30 to 250 nm, 80 to 180 nm, or 100 to 160 nm.

[0279] In one embodiment, the capsule core material is comprised of a material selected from the group consisting of perfume raw materials and / or optionally vegetable oils, such as pure and / or blended vegetable oils including castor oil, coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil, and mixtures thereof; esters of vegetable oils, dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl methyl ester, methyl ... straight or branched chain hydrocarbons including straight or branched chain hydrocarbons having a boiling point above about 80° C.; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls including monoisopropyl biphenyl, alkylated naphthalenes including dipropyl naphthalene, mineral spirits including kerosene, mineral oils, and mixtures thereof; aromatic solvents including benzene, toluene, and mixtures thereof; silicone oils; and mixtures thereof.

[0280] In one embodiment, the capsule wall material may comprise a suitable resin comprising a reaction product of an aldehyde and an amine, where suitable aldehydes include formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril, and mixtures thereof. Suitable melamines include methylolmelamine, methylated methylolmelamine, iminomelamine, and mixtures thereof. Suitable ureas include dimethylolurea, methylated dimethylolurea, urea-resorcinol, and mixtures thereof.

[0281] In one embodiment, a suitable formaldehyde scavenger can be used with the capsules, for example, in a capsule slurry, and / or can be added to the composition before, during, or after the capsules are added to the composition.Suitable capsules can be made according to the teachings of U.S. Patent Application Publication No. 2008 / 0305982; and / or U.S. Patent Application Publication No. 2009 / 0247449.

[0282] In a preferred embodiment, the composition may also contain a deposition aid, preferably from the group comprising cationic polymers or non-ionic polymers. Suitable polymers include cationic starch, cationic hydroxyethyl cellulose, polyvinyl formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene terephthalate, and polymers containing dimethylaminoethyl methacrylate, optionally with one or more monomers selected from the group comprising acrylic acid and acrylamide.

[0283] Perfume - In one embodiment, the composition comprises a perfume comprising one or more perfume raw materials selected from the group consisting of: 1,1'-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol, monoacetate; (3-methylbutoxy)acetic acid, 2-propenyl ester; beta-methylcyclododecaneethanol; 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol; oxacyclohexadecane- 2-one;alpha-methyl-benzenemethanol acetate;trans-3-ethoxy-1,1,5-trimethylcyclohexane;4-(1,1-dimethylethyl)cyclohexanol acetate;dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan;beta-methylbenzenepropanal;beta-methyl-3-(1-methylethyl)benzenepropanal;4-phenyl-2-butanone;2-Methylbutanoic acid, ethyl ester;benzaldehyde;2-Methylbutanoic acid, 1-methylethyl ester;dihydro-5-pentyl 2-(Phenylmethylene)octanal;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;Dodecanal;Undecanal;2-Ethyl-alpha,alpha-dimethylbenzenepropanal;Decanal;Alpha,alpha-Dimethylbenzeneethanol acetate;2-(Phenylmethylene)octanal;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 1-Then-1-one;2-Pentylcyclopentanone;3-Oxo-2-pentylcyclopentaneacetic acid, methyl ester;4-Hydroxy-3-methoxybenzaldehyde;3-Ethoxy-4-oxybenzaldehyde;2-Heptylcyclopentanone;1-(4-Methylphenyl)ethanone;(3E)-4-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-3-buten-2-one;(3E)-4-(2,6,6-Trimethyl-2-cyclohexen-1-yl)-3-buten-2-one;Benzeneethanol;2H-1-Benzopyran-2-one;4-Methoxybenzaldehyde;10-Undecenal;Propanoic acid, phenylmethyl ester;beta-Methylbenzenepentanol;1,1-Diethoxy-3,7-dimethyl-2,6-octadiene;alpha,alpha-Dimethylbenzeneethanol;(2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one;Acetic acid, phenylmethyl ester;Cyclohexanepropanoic acid, 2-propenyl ester;Hexanoic acid, 2-propenyl ester;1,2-Dimethoxy-4-(2-propenyl)benzene; 1,5-Dimethyl-bicyclo[3.2.1]octan-8-one oxime;4-(4-Hydroxy-4-methylpentyl)-3-cyclohexene-1-carboxaldehyde;3-Buten-2-ol;2-[[[2,4(or 3,5)-Dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid, methyl ester;8-Cyclohexadecen-1-one;Methyl ionone;2,6-Dimethyl-7-octen-2-ol;2-Methoxy-4-(2-propenyl)phenol;(2E)-3,7-Dimethyl-2,6-octadiene-1- ol;2-Hydroxybenzoic acid, (3Z)-3-hexenyl ester;2-Tridecenenitrile;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-hexenyl Xyl-3-oxo-, methyl ester; Benzenepropanal, 4-ethyl-.alpha.,.alpha.-dimethyl-; 3-Cyclohexene-1-carboxaldehyde (3-(4-hydroxy-4-methylpentyl)-); Ethanone, 1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-(methanoazulen-5-yl)-, [3R-(3.alpha.,3a.beta.7.beta.,8a.alpha.)]-; Undecanal, 2-methyl-2H-pyran-2-one, 6-butyltetrahydro-;Benzenepropanal, 4-(1,1-dimethylethyl)-.alpha.-methyl-;2(3H)-Furanone, 5-heptyldihydro-;Benzoic acid, 2-[(7-hydroxy-3,7-dimethyloctylidene)amino]-, methyl;Benzoic acid, 2-hydroxy-, phenylmethyl ester;Naphthalene, 2-methoxy-;2-Cyclopenten-1-one, 2-hexyl-;2(3H)-Furanone, 5-hexyldihydro-;Oxirancarboxylic acid, 3-methyl-3-phenyl-, ethyl ester;2-Oxabicyclo[2.2.2]octane, 1,3,3 -Trimethyl-;Benzenepentanol, gamma-methyl-;3-Octanol, 3,7-dimethyl-;3,7-Dimethyl-2,6-octadienenitrile;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-methano-1H-inden-6-ol propanoate;3-Methyl-2-buten-1-ol acetate;(Z)-3-Hexene-1-ol acetate;2-Ethyl-4-(2,2, 3-Trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol;4-(Octahydro-4,7-methano-5H-inden-5-ylidene)-butanal;3-2,4-Dimethyl-cyclohexene-1-carboxaldehyde;1-(1,2,3,4,5,6,7,8-Octahydro-2,3,8,8-tetramethyl-2-naphthalenyl-ethanone;2-Hydroxybenzoic acid, methyl ester;2-Hydroxybenzoic acid, hexyl ester;2-Phenoxyethanol;2-Hydroxybenzoic acid, pentyl ester;2,3-Heptane Dione; 2-Hexene-1-ol; 6-Octen-2-ol, 2,6-dimethyl; Damascone (alpha, beta, gamma, or delta, or mixtures thereof), 4,7-methano-1H-inden-6-ol, 3a,4,5,6,7,7a-hexahydro-, acetate; 9-Undecenal; 8-Undecenal; Isocyclocitral; Ethanone, 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthalenyl)-; 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; lysine (pt-bucinal), and cyclopentanone, 2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and 1-methyl-4-(1-methylethenyl)cyclohexene, and mixtures thereof;

[0284] In one embodiment, the composition can include encapsulated perfume particles comprising either a water-soluble hydroxyl compound or melamine-formaldehyde or modified polyvinyl alcohol. In one embodiment, the capsules comprise (a) an at least partially water-soluble solid matrix, preferably starch, comprising one or more water-soluble hydroxyl compounds; and (b) a perfume oil encapsulated by the solid matrix.

[0285] In a further aspect, the perfume may be pre-complexed with a polyamine, preferably polyethyleneimine, to form a Schiff base.

[0286] Polymers—The composition can include one or more polymers, examples of which include carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0287] 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=20-30 and x=3-8.

[0288] The compositions can include amphiphilic alkoxylated grease-cleaning polymers with balanced hydrophilic and hydrophobic properties to remove grease particles from fabrics and surfaces. Particular embodiments of the amphiphilic alkoxylated grease-cleaning polymers of the present invention include a core structure and a plurality of alkoxylate groups attached to the core structure. These can include alkoxylated polyalkyleneimines, preferably having an inner polyethylene oxide block and an outer polypropylene oxide block.

[0289] Alkoxylated polycarboxylates, such as those prepared from polyacrylates, are useful herein to provide additional grease removal performance. Such materials are described in WO 91 / 08281 and PCT 90 / 01815. Chemically, these materials comprise polyacrylates with one ethoxy side chain per 7-8 acrylate units. The side chains have the formula -(CH2CHO) m (CH2) n CH3 (where m is 2-3 and n is 6-12). The side chains are ester-linked to the polyacrylate "backbone" to give a "comb" polymer type structure. Molecular weights can vary but are typically in the range of 2000-50,000. Such alkoxylated polycarboxylates may be included herein at 0.05%-10% by weight of the compositions.

[0290] The isoprenoid-derived surfactants of the present invention, as well as their mixtures with other cosurfactants and other adjunct ingredients, are particularly suitable for use with amphiphilic graft copolymers, preferably those comprising (i) a polyethylene glycol backbone and (ii) at least one pendant moiety 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 polymers having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000, the weight ratio of polyethylene oxide to polyvinyl acetate is 40 to 60, and there is a maximum of one grafting point per 50 ethylene oxide units.

[0291] Carboxylate Polymers—The compositions of the present invention can also include one or more carboxylate polymers, such as a maleate / acrylate random copolymer or a polyacrylate homopolymer. In one embodiment, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of 4,000 to 9,000 Da or 6,000 to 9,000 Da.

[0292] Contaminant-Releasing Polymers—The compositions of the present invention may also include one or more contaminant-releasing polymers having a structure defined by one of the following structures (I), (II), or (III): (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e (III)-[(OCHR 5 -CHR 6 )c -OR 7 ] f During the ceremony, a, b, and c are 1 to 200; d, e, and f are 1 to 50; Ar is 1,4-substituted phenylene; sAr is 1,3-substituted phenylene substituted at the 5-position with SO3Me; Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, alkyl group C1-C 18 Alkyl or C2-C 10 hydroxyalkyl, or mixtures thereof, mono-, di-, tri-, or tetraalkylammonium; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently H or C1 to C 18 n- or iso-alkyl; and R 7 is straight chain or branched C1-C 18 Alkyl or straight or branched C2-C 30 Alkenyl, or cycloalkyl having 5 to 9 carbon atoms, or C8 to C 30 Aryl group or C6-C 30 It is an arylalkyl group.

[0293] Suitable contaminant release polymers are polyester contaminant release polymers such as Repel-o-tex polymers, including Repel-o-tex, SF-2, and SRP6 supplied by Rhodia. Other suitable contaminant release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325 supplied by Clariant. Other suitable contaminant release polymers include Marloquest polymers, such as Marloquest SL supplied by Sasol.

[0294] Cellulose Polymers—The compositions of the present invention can also include one or more cellulose polymers, including those selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, and alkyl carboxyalkyl celluloses. In one embodiment, the cellulose polymer is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, methylhydroxyethyl cellulose, methylcarboxymethyl cellulose, and mixtures thereof. In one embodiment, the carboxymethyl cellulose has a degree of carboxymethyl substitution of 0.5 to 0.9 and a molecular weight of 100,000 to 300,000 Da.

[0295] Enzyme--The composition can contain one or more enzymes that provide cleaning performance and / or fabric care benefits.Examples of suitable enzymes include, but are not limited to, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, mannanase, pectate lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase, or mixtures thereof.A typical combination is an enzyme cocktail, which can include, for example, a protease and lipase combined with amylase. When present in the composition, the aforementioned additional enzymes may be present at a level of 0.00001 to 2%, 0.0001 to 1%, or 0.001 to 0.5% by weight of enzyme protein, based on the weight of the composition.

[0296] In general, the properties of the enzyme selected should be compatible with the detergent selected (i.e., pH optimum, compatibility with other enzymatic and non-enzymatic components, etc.), and the enzyme should be present in an effective amount.

[0297] In one aspect, preferred enzymes include cellulases. Suitable cellulases include those of bacterial or fungal origin, including mutants obtained by chemical modification or protein engineering. Suitable cellulases include cellulases derived from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced by Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and WO 89 / 09259.

[0298] Particularly suitable cellulases are alkaline or neutral cellulases that provide color care benefits. Examples of such cellulases include those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples include cellulase variants such as those described in WO 94 / 07998, EP 0531315, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK98 / 00299.

[0299] Commercially available cellulases include Celluzyme™ and Carezyme™ (Novozymes A / S), Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0300] In one embodiment, preferred enzymes may include proteases. Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. Mutants obtained by chemical modification or protein engineering are included. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease may be, for example, an S1 family protease, such as trypsin, or an S8 family protease, such as subtilisin. The metalloprotease may be, for example, thermolysin from family M4, or other metalloproteases, such as those from the M5, M7, or M8 families.

[0301] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be classified into six categories: the subtilisin family, thermitase family, proteinase K family, lantibiotic peptidase family, kexin family, and pyrrolysin family.

[0302] Examples of subtilases include those from the genus Bacillus, such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus, and Bacillus gibsonii, as described in U.S. Pat. No. 7,262,042 and WO 09 / 021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, as described in WO 89 / 06279. licheniformis), subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168, and protease PD138 described in (WO 93 / 18140). Other useful proteases may include those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases include trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270, WO 94 / 25583, and WO 05 / 040372, and the chymotrypsin proteases from Cellumonas described in WO 05 / 052161 and WO 05 / 052146.

[0303] Further preferred proteases are the alkaline protease from Bacillus lentus DSM 5483, e.g. as described in WO 95 / 23221, and variants thereof as described in WO 92 / 21760, WO 95 / 23221, EP 1921147, and EP 1921148.

[0304] Examples of metalloproteases include the neutral metalloproteases described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0305] Examples of useful proteases include those 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, and the like. and variants described in WO 11 / 036264, in particular variants having substitutions at one or more of 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 and 274, using the BPN' numbering. More preferably, the subtilase variant may comprise 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, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

[0306] Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Blaze®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, and Esperase®, all available as Ultra® or Evity® (Novozymes A / S), and those sold under the trade names Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™, Purafect Examples include those sold under the names MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco / DuPont), Axapem™ (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of U.S. Pat. No. 5,352,604) and variants thereof (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0307] In one embodiment, preferred enzymes include amylases. Suitable amylases may be alpha-amylases or glucoamylases, and may be of bacterial or fungal origin. These include mutants obtained by chemical modification or protein engineering. Examples of amylases include alpha-amylases obtained from specific strains of Bacillus, such as Bacillus licheniformis, as described in detail in GB Patent No. 1296839.

[0308] Suitable amylases include the amylase having SEQ ID NO: 3 in WO 95 / 10603, or a variant thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are set out in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of 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.

[0309] A different suitable amylase is the amylase having SEQ ID NO: 6 in WO 02 / 010355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 has positions 181 and 182 deleted and position 193 substituted.

[0310] Another suitable amylase is a hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens set forth in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the B. licheniformis alpha-amylase set forth in SEQ ID NO:4 of WO 2006 / 066594, or a variant thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those having substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 have the following substitutions: M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0311] A further suitable amylase is the amylase having SEQ ID NO: 6 in WO 99 / 019467 or a variant thereof having 90% sequence identity to 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.

[0312] Further amylases that can be used are those having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 of WO 96 / 023873, or variants thereof having 90% sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants are those with deletions at positions 181 and 182, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:7 are those having a deletion at positions 183 and 184 and a substitution at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0313] Other amylases that can be used are amylases having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those with substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0314] A further suitable amylase is the amylase having SEQ ID NO: 2 of WO 09 / 061380, or a variant thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those with C-terminal truncations and / or substitutions, deletions, or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 are those that have 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 have deletions at positions R180 and / or S181, or T182 and / or G183. Most preferred amylase variants of SEQ ID NO:2 are those that have the following substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K Here, these variants are C-terminally truncated and optionally further comprise a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0315] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01 / 66712, or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions of 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 a deletion of D183 and G184 and the substitutions R118K, N195F, R320K, and R458K, as well as variants further having substitutions at one or more positions selected from the following group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with variants further having substitutions at all of these positions being most preferred.

[0316] Other examples include amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078, and WO 2013 / 001087.

[0317] Commercially available amylases include Duramyl™, Termamyl™, Termamyl Ultra™, Fungamyl™, Ban™, Stainzyme™, Stainzyme Plus™, Amplify®, Supramyl™, Natalase™, Liquozyme X and BAN™ (manufactured by Novozymes A / S), KEMZYM® AT 9000 Biozym Biotech Trading GmbH Wehlistrasse 27b A-1200 Wien Austria, and Rapidase™, Purastar™ / Effectenz™, Powerase, Preferenz S100, Preferenx S110, ENZYSIZE®, OPTISIZE HT PLUS®, and PURASTAR OXAM® (Danisco / DuPont), and KAM® (Kao).

[0318] Suitable lipases and cutinases include those of bacterial or fungal origin. These include mutant enzymes obtained by chemical modification or protein engineering. Examples include lipases from Thermomyces, such as lipases from T. lanuginosus (formerly Humicola lanuginosus), as described in EP 258068 and EP 305216. Lipases from strains of the genus Pseudomonas (some of which have now been renamed to Burkholderia), such as P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (P.Lipases from Streptomyces wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), cutinase from Magnaporthe grisea (WO 10 / 107560), cutinase from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipase from Thermobifida fusca (WO 11 / 084412, WO 13 / 033318), Geobacillus stearothermophilus Examples of lipases that may be used include lipases from Bacillus stearothermophilus (WO 11 / 084417), Bacillus subtilis (WO 11 / 084599), and lipases from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0319] Other examples include lipase variants such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0320] Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A / S), Lumafast (formerly from Genencor) and Lipomax (formerly from Gist-Brocades).

[0321] Further examples are lipases, sometimes called acyltransferases or perhydrolases, such as acyltransferases homologous to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782), perhydrolases from the CE7 family (WO 09 / 67279), and mutants of M. smegmatis perhydrolase, in particular the S54V mutant used in the product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0322] In one aspect, other preferred enzymes include microbial endoglucanases exhibiting endo-beta-1,4-glucanase activity (EC 3.2.1.4), including bacterial polypeptides endogenous to members of the genus Bacillus having a sequence at least 90%, 94%, 97%, or 99% identical to amino acid sequence SEQ ID NO:2 in U.S. Patent No. 7,141,403, and mixtures thereof. Suitable endoglucanases are sold under the trade names Celluclean® and Whitezyme® (Novozymes).

[0323] Other preferred enzymes include pectate lyases sold under the trade names Pectawash®, Pectaway®, and Xpect®, and mannanases sold under the trade names Mannaway® (Novozymes) and Purabrite® (Danisco / DuPont).

[0324] Detergent enzymes can be included in detergent compositions by adding a separate additive containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additives of the present invention, either separate or combined, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-dusting granules, liquids, especially stabilized liquids, or slurries.

[0325] Non-dusting granules can be prepared, for example, as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452, and optionally coated by methods known in the art. Examples of wax coating materials include poly(ethylene oxide) products (polyethylene glycols, PEGs) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol moiety contains 12 to 20 carbon atoms and 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized-bed techniques are given in British Patent No. 1,483,591. Liquid enzyme preparations can be stabilized, for example, by adding polyols such as propylene glycol, sugars or sugar alcohols, lactic acid, or boric acid, according to established methods. The protected enzyme can be prepared by the method disclosed in EP 238216.

[0326] Dye Transfer Inhibitors—The compositions of the present invention can also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidone, and polyvinylimidazole, and mixtures thereof. When present in the composition, the dye transfer inhibitors can be present at levels of 0.0001% to 10%, 0.01% to 5%, or 0.1% to 3% by weight.

[0327] Brighteners - The compositions of the present invention may also contain additional ingredients that can impart color to the items being washed, such as optical brighteners.

[0328] The composition can include CI Optical Brightener 260 in alpha crystalline form, having the following structure: [ka]

[0329] In one embodiment, the brightener is a cold water soluble brightener, such as the alpha crystalline form of CI Optical Brightener 260. In one embodiment, the brightener is predominantly in the alpha crystalline form, which typically means that at least 50%, at least 75%, at least 90%, at least 99%, or even substantially all of the CI Optical Brightener 260 by weight is in the alpha crystalline form.

[0330] The whitening agent is typically in micronized particle form having a weight average primary particle size of from 3 to 30 micrometers, from 3 to 20 micrometers, or from 3 to 10 micrometers.

[0331] The composition can include the beta crystalline form of CI Optical Brightener 260 and can have a weight ratio of (i) the alpha crystalline form of CI Optical Brightener 260 to (ii) the beta crystalline form of CI Optical Brightener of at least 0.1, or at least 0.6. BE680847 relates to a method for producing the alpha crystalline form of CI Optical Brightener 260.

[0332] Commercially available fluorescent whitening agents that may be useful in the present invention can be divided into subgroups, including, but not limited to, stilbenes, pyrazolines, coumarins, carboxylic acids, methine cyanines, dibenzothiophene-5,5-dioxide, azoles, derivatives of five- and six-membered heterocycles, and various other agents. Examples of such whitening agents are disclosed in "The Production and Application of Fluorescent Brightening Agents," M. Zahradnik, Publisher, John Wiley & Sons, New York (1982). Specific, non-limiting examples of fluorescent whitening agents useful in the present compositions are those identified in U.S. Pat. Nos. 4,790,856 and 3,646,015.

[0333] Further suitable brighteners have the following structure: [ka]

[0334] Suitable optical brightener levels include lower levels of 0.01%, 0.05%, 0.1%, or 0.2% by weight to upper levels of 0.5% or 0.75% by weight.

[0335] In one embodiment, the whitening agent can be loaded onto the clay to form particles.Silicate—The compositions of the present invention can also contain a silicate, such as sodium silicate or potassium silicate.The composition can contain 0% to less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or even 2% by weight of silicate, and can include from 0%, 0.5%, or 1% by weight of silicate as described above.A preferred silicate is sodium silicate.

[0336] Dispersants - The compositions of the present invention can also contain dispersants. Suitable water-soluble organic materials include homopolymeric or copolymeric acids or salts thereof, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0337] Enzyme Stabilizers—Enzymes used in the compositions can be stabilized by various techniques. The enzymes used herein can be stabilized by the presence of a water-soluble source of calcium and / or magnesium ions. Examples of common stabilizers include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, peptide aldehydes, lactic acid, boric acid, or boric acid derivatives, such as aromatic boric acid esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid. The compositions can be formulated as described, for example, in WO 92 / 19709 and WO 92 / 19708. In the case of aqueous compositions containing proteases, reversible protease inhibitors, such as boron compounds including 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. Peptide aldehydes have the formula B2-B1-B0-R, where R is hydrogen, CH3, CX 3、

[0023] The peptide aldehyde may be CHX2 or CH2X, where X is a halogen atom; B0 is a phenylalanine residue having an OH substituent at the p-position and / or m-position; B1 is a single amino acid residue; and B2 consists of one or more amino acid residues, optionally including an N-terminal protecting group. Preferred peptide aldehydes include, but are not limited to, Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H, or Ac-WLVY-H, where Z is benzyloxycarbonyl and Ac is acetyl.

[0338] Solvents—Suitable solvents include water and other solvents such as lipophilic liquids. Examples of suitable lipophilic liquids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerin derivatives such as glycerin ethers, perfluorinated amines, perfluorinated and hydrofluoroether solvents, low volatility non-fluorinated organic solvents, diol solvents, other environmentally friendly solvents, and mixtures thereof.

[0339] Structurants / Thickeners—Structured liquids can be internally structured, whereby the structure is formed by a primary component (e.g., surfactant material), and / or externally structured by using secondary components (e.g., polymers, clays, and / or silicate materials) to provide a three-dimensional matrix structure. The composition can contain 0.01-5 wt. % or 0.1-2.0 wt. % of a structurant. The structurant is typically selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulosic materials, microfiber cellulose, hydrophobically modified alkali-swellable emulsions such as Polygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, and mixtures thereof. Suitable structurants include hydrogenated castor oil and its non-ethoxylated derivatives. Suitable structurants are disclosed in U.S. Pat. No. 6,855,680. Such structurants have a thread-like structure with a range of aspect ratios. Other suitable structurants and methods for making them are described in WO 10 / 034736.

[0340] Conditioning agent--The composition of the present invention can comprise a high-melting-point fatty compound.The high-melting-point fatty compound useful herein has a melting point of 25°C or higher and is selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof.Such compounds with low melting points are not intended to be included in this section.Non-limiting examples of high-melting-point compounds can be found in the International Cosmetic Ingredient Dictionary, 5th Edition, 1993, and the CTFA Cosmetic Ingredient Handbook, 2nd Edition, 1992.

[0341] The high melting point fatty compounds are included in the composition at levels of 0.1 to 40% by weight, 1 to 30% by weight, 1.5 to 16% by weight, 1.5 to 8% by weight, in view of providing benefits indicative of improved conditioning, such as a slippery feel when applied to wet hair, softness and moisturized feel on dry hair.

[0342] The compositions of the present invention may contain a cationic polymer. The concentration of the cationic polymer in the composition is typically in the range of 0.05 to 3 wt. %, 0.075 to 2.0 wt. %, or 0.1 to 1.0 wt. Suitable cationic polymers 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 intended pH of the composition (which generally ranges from pH 3 to pH 9, or from pH 4 to pH 8). As used herein, the "cationic charge density" of a polymer refers to the ratio of the number of positive charges on the polymer to the molecular weight of the polymer. The average molecular weight of such suitable cationic polymers is generally between 10,000 and 10 million, between 50,000 and 5 million, or between 100,000 and 3 million.

[0343] Suitable cationic polymers for use in the compositions of the present invention contain cationic nitrogen-containing moieties, such as quaternary ammonium moieties or cationic protonated amino moieties. Any anionic counterion can be used with the cationic polymer, as long as the polymer is soluble in water, in the composition, or in the coacervate phase of the composition, and as long as the counterion is physically and chemically compatible with the essential components of the composition or does not unduly impair the performance, stability, or aesthetics of the composition. Non-limiting examples of such counterions include halides (e.g., chloride, fluoride, bromide, iodide), sulfate, and methyl sulfate.

[0344] Non-limiting examples of such polymers are described in the CTFA Cosmetic Ingredient Dictionary, 3rd Edition, edited by Estrin, Crosley, and Haynes (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, DC (1982)).

[0345] Other suitable cationic polymers for use in the composition include polysaccharide polymers, cationic guar gum derivatives, quaternary nitrogen-containing cellulose ethers, synthetic polymers, copolymers of etherified cellulose, guar, and starch. When used, the cationic polymers herein are either soluble in the composition or soluble in a complex coacervate phase formed in the composition by the cationic polymer and the anionic, zwitterionic, and / or amphoteric surfactant components described hereinabove. Complex coacervates of cationic polymers can also be formed with other charged substances in the composition. Suitable cationic polymers are described in U.S. Patent Nos. 3,962,418, 3,958,581, and U.S. Patent Application Publication No. 2007 / 0207109.

[0346] The compositions of the present invention can include a nonionic polymer as a conditioning agent. Polyalkylene glycols having a molecular weight greater than 1000 are useful herein. Those having the following general formula are useful: [ka] In the formula, R 95is selected from the group consisting of H, methyl, and mixtures thereof. Conditioning agents, particularly silicones, can be included in the composition. Conditioning agents useful in the compositions of the present invention typically comprise water-insoluble, water-dispersible, non-volatile liquids that form emulsified liquid particles. Conditioning agents suitable for use in the compositions are generally those characterized as silicones (e.g., silicone oils, cationic silicones, silicone gums, high refractive index silicones, and silicone resins), organic conditioning oils (e.g., hydrocarbon oils, polyolefins, and fatty acid esters), or combinations thereof, or conditioning agents that otherwise form liquid dispersed particles in the aqueous surfactant matrix herein. Such conditioning agents should be physically and chemically compatible with the essential components of the composition and should not otherwise unduly impair the stability, aesthetics, or performance of the composition.

[0347] The concentration of conditioning agent in the composition should be sufficient to provide the desired conditioning benefit, and such concentration may vary depending on the conditioning agent, the desired conditioning performance, the average size of the conditioning agent particles, the type and concentration of other ingredients, and other similar factors.

[0348] The concentration of the silicone conditioning agent is typically in the range of 0.01 to 10% by weight. Non-limiting examples of suitable silicone conditioning agents, and optional suspending agents for silicones, are described in U.S. Reissue Patent Nos. 34,584; 5,104,646; 5,106,609; 4,152,416; 2,826,551; 3,964,500; 4,364,837; 6,607,717; 6,482,969; 5,807,956; and 5,981,616. No. 81; U.S. Pat. No. 6,207,782; U.S. Pat. No. 7,465,439; U.S. Pat. No. 7,041,767; U.S. Pat. No. 7,217,777; U.S. Patent Application Publication No. 2007 / 0286837A1; U.S. Patent Application Publication No. 2005 / 0048549A1; U.S. Patent Application Publication No. 2007 / 0041929A1; British Patent No. 849,433; German Patent No. 10,036,533 (all of which are incorporated herein by reference); Chemistry and Technology of Silicones, New York: Academic Press (1968); General Electric Silicone Rubber Product Data Sheets SE 30, SE 33, SE 54 and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and Encyclopedia of Polymer Science and Engineering, vol. 15, 2nd edition, pp 204-308, John Wiley & Sons, Inc. (1989).

[0349] The compositions of the present invention may also contain 0.05 to 3 wt. % of at least one organic conditioning oil as a conditioning agent, either alone or in combination with other conditioning agents, such as silicones (as described herein). Suitable conditioning oils include hydrocarbon oils, polyolefins, and fatty acid esters. Also suitable for use in the compositions herein are conditioning agents described in U.S. Patent Nos. 5,674,478 and 4,529,586, or in U.S. Patent Nos. 5,750,122; 4,507,280; 4,663,158; 4,197,865; 4,217,914; 4,381,919; and 4,422,853.

[0350] Hygiene and Malodor - The compositions of the present invention also contain zinc ricinoleate, thymol, quaternary ammonium salts such as Bardac®, polyethyleneimine (such as Lupasol® from BASF and its zinc complexes), silver and silver compounds, especially Ag + The composition may include one or more of those designed to slow-release or nanosilver dispersions.

[0351] Probiotics - The composition may include probiotics such as those described in WO 09 / 043709.

[0352] Foam booster - if high foaming is desired, C 10 ~C 16 Alkanolamide or C 10 ~C 14 Foam boosters such as alkyl sulfates may be incorporated into the composition, typically at levels of 1 to 10% by weight. 10 ~C 14Monoethanol and diethanolamides are typical examples of such foam boosters. It is also beneficial to use such foam boosters in conjunction with the high-sudsing co-surfactants, such as the amine oxides, betaines, and sultaines mentioned above. If desired, water-soluble magnesium and / or calcium salts, such as MgCl, MgSO, CaCl, and CaSO, can be added, typically at levels of 0.1 to 2% by weight, to provide additional foaming and enhance grease removal performance.

[0353] Suds suppressors—compounds that reduce or inhibit foam formation can be incorporated into the compositions of the present invention. Suds suppression can be particularly important in the so-called "high concentration wash process" described in U.S. Pat. Nos. 4,489,455 and 4,489,574, as well as in front-loading washing machines. A wide variety of substances can be used as suds suppressors, and suds suppressors are well known to those skilled in the art. See, for example, Kirk Othmer Encyclopedia of Chemical Technology, 3rd Edition, Vol. 7, pp. 430-447 (John Wiley & Sons, Inc., 1979). Examples of suds suppressors include monocarboxylic fatty acids and soluble salts thereof, high molecular weight hydrocarbons such as paraffin, fatty acid esters (e.g., fatty acid triglycerides), fatty acid esters of monohydric alcohols, and fatty acid C 18 ~C 40Examples of suitable foam suppressors include ketones (e.g., stearone), N-alkylated aminotriazines, waxy hydrocarbons preferably having a melting point below about 100° C., silicone foam suppressors, and secondary alcohols. Foam suppressors are described in U.S. Pat. No. 2,954,347; U.S. Pat. No. 4,265,779; U.S. Pat. No. 4,265,779; U.S. Pat. No. 3,455,839; U.S. Pat. No. 3,933,672; U.S. Pat. No. 4,652,392; U.S. Pat. No. 4,978,471; U.S. Pat. No. 4,983,316; U.S. Pat. No. 5,288,431; U.S. Pat. No. 4,639,489; U.S. Pat. No. 4,749,740; U.S. Pat. No. 4,798,679; U.S. Pat. No. 4,075,118; EP 89307851.9; EP 150872; and DOS 2,124,526.

[0354] For any detergent composition used in an automatic washing machine, suds should not be formed to the extent that they overflow the washing machine. If used, the suds suppressor is preferably present in a "suds suppressing amount." By "suds suppressing amount," it is meant that the formulator of the composition can select the amount of this suds control agent that will sufficiently control suds to result in a low-sudsing laundry detergent for use in an automatic washing machine.

[0355] The compositions herein will generally contain 0-10% by weight of foam suppressor. Monocarboxylic fatty acids and salts thereof, when used as foam suppressors, will typically be present in amounts up to 5% by weight. Preferably, 0.5-3% by weight of an aliphatic monocarboxylate foam suppressor is used. Silicone foam suppressors are typically used in amounts up to 2.0% by weight, although increased amounts can be used. Monostearyl phosphate foam suppressors are generally used in amounts ranging from 0.1-2% by weight. Hydrocarbon foam suppressors are typically used in amounts ranging from 0.01-5.0% by weight, although increased amounts can be used. Alcohol soap foam suppressors are typically used in amounts ranging from 0.2-3% by weight.

[0356] The compositions herein may have cleaning activity over a wide range of pH. In certain embodiments, the compositions have cleaning activity at pH 4 to pH 11.5. In other embodiments, the compositions are active at pH 6 to pH 11, pH 7 to pH 11, pH 8 to pH 11, pH 9 to pH 11, or pH 10 to pH 11.5.

[0357] The compositions herein may have cleaning activity over a wide range of temperatures, for example, from below 10°C to 90°C. Preferably, the temperature is below 50°C or below 40°C, or even below 30°C. In certain embodiments, the optimum temperature range for the composition is 10°C to 20°C, 15°C to 25°C, 15°C to 30°C, 20°C to 30°C, 25°C to 35°C, 30°C to 40°C, 35°C to 45°C, or 40°C to 50°C.

[0358] Form of composition The compositions described herein are advantageously used, for example, in laundry applications, hard surface cleaning, dishwashing applications, and cosmetic applications such as dentures, teeth, hair, and skin.The compositions of the present invention are particularly solid or liquid cleaning compositions and / or treatment compositions.In one aspect, the present invention relates to a composition whose form is selected from the following: regular, compressed, or concentrated liquid; gel; paste; soap bar; regular or compressed powder; granular solid; homogeneous or multi-layered tablet having two or more layers (same or different phases); pouch with one or more compartments; single or multi-compartment unit dose form; or any combination thereof.

[0359] Depending on the composition's form, the components can be physically separated from each other, for example, in compartments such as a water-soluble pouch or in different layers of a tablet, thereby avoiding negative storage interactions between the components. Different dissolution profiles of each compartment can also delay the dissolution of selected components in the cleaning solution.

[0360] The pouch can be configured as a single or multiple compartments. The pouch can be of any form, shape, and material suitable for retaining the composition without releasing it from the pouch, for example, prior to contact with water. The pouch is made from a water-soluble film that encases an internal volume. The internal volume can be divided into the pouch compartments. Preferred films are polymeric materials, preferably polymers that can be formed into films or sheets. Preferred polymers, copolymers, or derivatives thereof are selected polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer, e.g., PVA, in the film is at least about 60%. Preferred average molecular weights are typically about 20,000 to about 150,000. The film may also be a blended composition comprising a hydrolyzable, water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under the trade name M8630, sold by MonoSol LLC, Indiana, USA) plus a plasticizer such as glycerol, ethylene glycerol, propylene glycol, sorbitol, and mixtures thereof. The pouch may contain a solid laundry cleaning composition or component and / or a liquid laundry cleaning composition or component separated by a water-soluble film. The compartment for the liquid component may be of a different composition than the compartment containing the solid (U.S. Patent Application Publication No. 2009 / 0011970 A1).

[0361] Water-soluble film—The composition of the present invention can also be encapsulated in a water-soluble film. Preferred film materials are preferably polymeric materials. As known in the art, film materials can be obtained, for example, by casting, blow molding, extrusion, or blow extrusion of polymeric materials. Preferred polymers, copolymers, or derivatives thereof suitable for use as pouch materials are selected from polyvinyl alcohol, polyvinylpyrrolidone, polyalkylene oxides, acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetate, polycarboxylic acids and salts, polyamino acids or peptides, polyamides, polyacrylamides, maleic acid / acrylic acid copolymers, polysaccharides including starch and gelatin, and natural gums such as xanthan and carragum. More preferred polymers are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, sodium carboxymethylcellulose, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, and polymethacrylates; most preferably, polyvinyl alcohol, polyvinyl alcohol copolymers, and hydroxypropylmethylcellulose (HPMC), and combinations thereof. Preferably, the level of polymer, e.g., PVA polymer, in the pouch material is at least 60% by weight. The polymer can have any weight-average molecular weight, but preferably is about 1,000 to 1,000,000, about 10,000 to 300,000, or about 20,000 to 150,000. Mixtures of polymers can also be used as pouch materials.

[0362] Of course, different film materials and / or films of different thicknesses can be used to make the compartments of the present invention. An advantage of choosing different films is that the resulting compartments may exhibit different solubility or release characteristics.

[0363] Preferred film materials are PVA films known under MonoSol product numbers M8630, M8900, H8779, and those described in U.S. Pat. Nos. 6,166,117 and 6,787,512, as well as PVA films with comparable solubility and deformability properties.

[0364] The film materials herein may also contain one or more additive components. For example, it may be beneficial to add plasticizers such as glycerol, ethylene glycol, diethylene glycol, propylene glycol, sorbitol, and mixtures thereof. Other additives include functional detergent additives delivered to the wash water, such as organic polymer dispersants.

[0365] Methods of Making the Composition The compositions of the present invention can be formulated into any suitable form and prepared by any method selected by the formulator, non-limiting examples of which are set forth in Applicant's Examples and U.S. Pat. Nos. 4,990,280; 20030087791A1; 20030087790A1; 20050003983A1; 20040048764A1; 4,762,636; 6,291,412; 20050227891; No. 5,489,392; and U.S. Pat. No. 5,486,303, all of which are incorporated herein by reference. The compositions of the present invention, or compositions prepared in accordance with the present invention, may be used in a variety of applications, including, but not limited to, compositions for treating fabrics, hard surfaces, and any other surface in the fabric and home care area (e.g., air care, including air fresheners and scent delivery systems, automotive care, dishwashing, fabric conditioning (including softening and / or freshening), laundry washing, wash and rinse additions and / or care, hard surface cleaning and / or treatment, including floor and toilet bowl cleaners), all-purpose detergents or "heavy duty" detergents in granular or powder form, especially for washing. These include cleaning detergents; all-purpose detergents in liquid, gel, or paste form, especially so-called heavy-duty liquid types; liquid detergents for fine fabrics; hand dishwashing detergents or light-duty dishwashing detergents, especially high-foaming types; dishwashing detergents for machine washing, including various tablet, granule, liquid, and rinse aid types for home and commercial use; bathroom cleaners, including car or carpet shampoos and toilet bowl cleaners; and cleaning and / or treating compositions, including cleaning aids such as bleach additives, and "soil-on" or pre-treatment substrate-loaded compositions such as dryer sheets. Compositions and methods for cleaning and / or treating textiles and / or hard surfaces, most preferably textiles, are preferred.The composition is preferably a composition for use in the pre-treatment step or main wash step of a washing process, most preferably a composition for use in the textile washing step.

[0366] As used herein, the term "fabric and / or hard surface cleaning and / or treatment compositions", unless otherwise indicated, is a subset of cleaning and / or treatment compositions, including all-purpose detergents or "heavy-duty" detergents in granular or powder form, especially cleaning detergents; all-purpose detergents in liquid, gel, or paste form, especially so-called heavy-duty liquid types; liquid detergents for fine fabrics; hand dishwashing detergents or light-duty dishwashing detergents, especially high-foaming types; dishwashing detergents for machine washing, including various tablet, granular, liquid, and rinse aid types for domestic and commercial use; bathroom cleaners, including liquid cleaning and disinfecting agents, car or carpet shampoos, toilet bowl cleaners; fabric conditioning compositions, including softening and / or freshening, which may be in liquid, solid, and / or dryer sheet form; and cleaning aids, such as bleach additives, and "soil-on" or pre-treatment substrate-loaded compositions, such as dryer sheets. All such compositions, where applicable, can be in standard, concentrated, or in certain embodiments, highly concentrated forms that can be non-aqueous.

[0367] How to use The present invention includes a method for cleaning any surface, including treating textiles or hard surfaces, or other surfaces in the fabric and / or home care sector. It is contemplated that such cleaning may be on a small scale, such as for a family household, or on a larger scale, such as in industrial and professional settings. In one aspect of the invention, the method comprises contacting a surface to be treated in a pre-treatment or main washing step of the washing process, most preferably for use in textile washing processes or dishwashing, including both manual and automatic / machine dishwashing. In one embodiment of the invention, the lipase variant and other ingredients are added sequentially or simultaneously during the method for cleaning and / or treating a surface.

[0368] As used herein, cleaning includes, but is not limited to, scrubbing and mechanical agitation. Cleaning can be performed using a foam composition as described in WO 08 / 101958 and / or by alternating application of pressure (pressure / vacuum) in addition to or instead of scrubbing and mechanical agitation. Drying of such surfaces or fabrics can be accomplished by any one of the common means used in either domestic or industrial settings. The cleaning compositions of the present invention are ideally suited for use in laundry and dishwashing applications. Accordingly, the present invention includes a method for cleaning objects, including, but not limited to, fabrics, dishes, cutlery, and kitchen utensils. The method comprises contacting the object to be cleaned with a cleaning composition comprising at least one embodiment of Applicant's cleaning composition, cleaning additive, or mixtures thereof. The fabric can include almost any fabric that can be laundered under normal consumer or institutional use conditions. The pH of the solution can be between 8 and 10.5. The composition can be used at a solution concentration of 500 to 15,000 ppm. The water temperature is typically in the range of 5° C. to 90° C. The water to fabric ratio is typically 1:1 to 30:1.

[0369] In one aspect, the invention relates to a method of using a polypeptide having at least 60% identity to SEQ ID NO: 2 for the manufacture of a composition. In one aspect, the invention relates to the use of a composition for cleansing a subject.

[0370] In one aspect, the invention relates to a method for making a composition, comprising adding a polypeptide having at least 60% identity to SEQ ID NO: 2 and a surfactant. In one aspect, the invention relates to a method for cleaning a surface, comprising contacting lipid soils present on the surface to be cleaned with a cleaning composition. In one aspect, the invention relates to a method for hydrolyzing lipids present on soils and / or soils on a surface, comprising contacting the soils and / or soils with a cleaning composition. In one aspect, the invention relates to the use of the composition in the hydrolysis of carboxylic acid esters. In one aspect, the invention relates to the use of the composition in the hydrolysis, synthesis or transesterification of esters. In one aspect, the invention relates to the use of the composition in the production of a stable formulation.

[0371] plant The present invention also relates to plants, e.g., transgenic plants, plant parts, or plant cells, comprising the polynucleotides of the present invention for expression and production of recoverable amounts of the variants. The variants may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the variants may be used to improve one or more food qualities, e.g., to improve nutritional value, palatability, and rheological properties, or to destroy anti-nutritional factors.

[0372] The transgenic plants can be dicotyledons (double-leafed) or monocotyledons (single-cotyledonous). Examples of monocotyledonous plants are grasses such as pasture grasses (poa), forage grasses such as festuca, lolium, temperate grasses such as agrostis, and cereals such as wheat, oats, rye, barley, rice, sorghum, and maize (corn).

[0373] Examples of dicotyledonous plants are tobacco, legumes such as lupine, potato, sugar beet, pea, bean and soybean, and Brassicaceae plants (family Brassicaceae) such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana.

[0374] Examples of plant parts are stems, calluses, leaves, roots, fruits, seeds, and tubers, as well as the tissues that comprise these parts, such as the epidermis, mesophyll, parenchyma, vascular tissue, and meristem. Specific plant cell compartments, such as the chloroplast, apoplast, mitochondria, vacuoles, peroxisomes, and cytoplasm, are also considered to be plant parts. Furthermore, any plant cell, regardless of tissue origin, is considered to be a plant part. Similarly, plant parts, such as specific tissues and cells isolated to facilitate utilization of the present invention, are also considered to be plant parts, such as embryos, endosperms, aleurone, and seed coats.

[0375] Further, progeny of such plants, plant parts, and plant cells are included within the scope of the present invention.

[0376] Transgenic plants or plant cells expressing the variants may be constructed according to methods known in the art. Briefly, the plant or plant cell is constructed by incorporating one or more expression constructs encoding the variants into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

[0377] An expression construct is conveniently a nucleic acid construct comprising a polynucleotide encoding a variant operably linked to appropriate control sequences required for expression of the polynucleotide in a plant or plant part of choice. In addition, the expression construct may contain a selectable marker useful for identifying plant cells into which the expression construct has been incorporated, as well as DNA sequences necessary for the introduction of the construct into the plant of interest (the latter depending on the DNA introduction method to be used).

[0378] The selection of regulatory sequences, e.g., promoter and terminator sequences, and optionally signal or transit sequences, is determined, for example, based on when, where, and how expression of the variant is desired. For example, expression of the gene encoding the variant may be constitutive or inducible, or may be developmental, stage, or tissue specific, and the gene product may be targeted to specific tissues or plant parts, such as seeds or leaves. Regulatory sequences are described, for example, by Tague et al., 1988, Plant Physiology 86:506.

[0379] For constitutive expression, the 35S-CaMV, maize ubiquitin 1, or rice actin 1 promoters may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18:675-689; Zhang et al., 1991, Plant Cell 3:1155-1165). Organ-specific promoters include, for example, promoters from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or promoters from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), seed-specific promoters such as glutelin, prolamin, globulin, or albumin promoters from rice (Wu et al., 1998, Plant Cell Physiol. 39:885-889), legumin B4 and the Vicia faba promoter from an unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152:708-711), promoters from seed oil body proteins (Chen et al., 1998, Plant Cell Physiol. 39:935-941), the storage protein napA promoter from Brassica napus, or any other seed-specific promoter known in the art, such as those described in WO 91 / 14772.Furthermore, the promoter may be a leaf-specific promoter, such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol. 102:991-1000), the Chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26:85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound-inducible promoter, such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22:573-588). Similarly, promoters may be induced by abiotic treatments such as temperature, drought, or salinity changes, or by exogenously applied substances that activate the promoter, such as ethanol, estrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

[0380] To achieve higher expression of the mutant in plants, a promoter enhancer element may also be used. For example, the promoter enhancer element may be an intron placed between the promoter and the polynucleotide encoding the mutant. For example, Xu et al., 1993 (supra) disclose the use of the first intron of the rice actin 1 gene to enhance expression.

[0381] The selectable marker gene and any other portions of the expression construct may be selected from those available in the art.

[0382] Nucleic acid constructs are integrated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, viral-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244:1293; Potrykus, 1990, Bio / Technology 8:535; Shimamoto et al., 1989, Nature 338:274).

[0383] Agrobacterium tumefaciens-mediated gene transfer is a method for generating transgenic dicotyledonous plants (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:15-38) and for transforming monocotyledonous plants, although other transformation methods may be used for these plants. A method for generating transgenic monocotyledonous plants is biolistic bombardment (microscopic gold or tungsten particles coated with transforming DNA) of embryonic callus or developing embryos (Christou, 1992, Plant J. 2:275-281; ​​Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio / Technology 10:667-674). An alternative method for transforming monocotyledonous plants is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mol. Biol. 21:415-428. Additional transformation methods include those described in U.S. Patent Nos. 6,395,966 and 7,151,204, both of which are incorporated herein by reference in their entireties.

[0384] After transformation, transformants that have incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art. Transformation methods are often designed to allow for the selective removal of the selection gene, either during regeneration or by subsequent generation, for example, by co-transformation of two separate T-DNA constructs or by using site-specific excision with a recombinase specific for the selection gene.

[0385] In addition to direct transformation of a particular plant genotype with a construct of the present invention, transgenic plants may be generated by crossing a plant containing the construct with a second plant lacking the construct. For example, a construct encoding a mutant can be introduced into a particular plant variety by crossing, without the need to have previously directly transformed a plant of the given variety. Thus, the present invention encompasses not only plants directly regenerated from cells transformed according to the present invention, but also the progeny of such plants. As used herein, progeny may refer to any generation of descendants of a parent plant prepared according to the present invention. Such progeny may contain a DNA construct prepared according to the present invention. Crossing results in the introduction of a transgene into a plant line by cross-pollinating the starting line with a donor plant line. Non-limiting examples of such steps are described in U.S. Patent No. 7,151,204.

[0386] Plants may be created through a process of backcross conversion, for example, plants include backcross converted genotypes, lines, inbreds, or plants referred to as hybrids.

[0387] Genetic markers may be used to aid in the introgression of one or more transgenes of the present invention from one genetic background to another. Marker-assisted selection offers an advantage over conventional breeding in that it can be used to avoid errors caused by phenotypic variation. Furthermore, genetic markers may provide data regarding the relative abundance of elite germplasm in each progeny of a particular cross. For example, when a plant with a desired trait that has an otherwise non-agronomically desirable genetic background is crossed with an elite parent, genetic markers may be used to select progeny that not only have the trait of interest but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

[0388] The present invention also relates to a method for producing a mutant of the present invention, comprising: (a) culturing a transgenic plant or plant cell containing a polynucleotide encoding the mutant under conditions conducive to the production of the mutant; and (b) recovering the mutant.

[0389] Microcapsule compositions comprising the lipase variants of the present invention In this aspect, the invention relates to a microcapsule composition in which the membrane of the microcapsule is produced by cross-linking a hyperbranched polyamine having a molecular weight of more than 1 kDa, wherein the microcapsule comprises a lipase variant of the invention.

[0390] The membranes formed by cross-linking hyperbranched polyamines are capable of separating enzymes from (anionic) surfactants in detergents, which are known to be detrimental to enzyme stability.

[0391] A critical parameter when using encapsulated enzymes in detergents, such as for laundry or dishwashing applications, is the ability to release the enzyme immediately upon dilution of the detergent with water. The microcapsules of the present invention have excellent properties in this regard and are capable of releasing the entire encapsulated enzyme within one minute.

[0392] The microcapsules do not require the presence of a core polymer to be capable of releasing the enzyme upon dilution with water. Furthermore, the present invention does not require the enzyme to be in a precipitated form within the core of the microcapsules to control its release over time, as described in WO 97 / 24177.

[0393] When the lipase variants of the present invention, and optionally other enzymes, are encapsulated in microcapsules with semipermeable membranes and have a higher water activity inside these capsules (before adding them to the liquid detergent) than in the liquid detergent, the capsules undergo (partial) collapse (water seeps out) when added to the detergent, thus leaving a more concentrated, more viscous enzyme-containing interior in the capsule. The collapse of the membrane can also result in a decrease in permeability. This can be further exploited by adding stabilizers / polymers, especially those that cannot permeate through the membrane. The collapse and resulting viscosity increase reduce / hinder the diffusion of inappropriate ingredients (e.g., surfactants or scavengers) into the capsules, thereby enhancing the storage stability of the enzyme in the liquid detergent. Ingredients in the liquid detergent that are sensitive to enzymes (e.g., ingredients that act as substrates for the enzyme) are also protected from degradation by the enzyme. During washing, the liquid detergent is diluted with water, thereby increasing the water activity. Water then diffuses into the capsules (osmosis). The capsules will expand and the membrane will become permeable to the enzyme, allowing the capsules to either survive or simply rupture, thus releasing the enzyme. The concept is very efficient in stabilizing enzymes against undesirable ingredients in liquid detergents, and vice versa, protecting enzyme-sensitive ingredients in liquid detergents from the enzyme.

[0394] Examples of detergent ingredients that are sensitive to and can be degraded by enzymes (relevant enzymes in parentheses) include xanthan gum (xanthanase), polymers with ester bonds (lipase), hydrogenated castor oil (lipase), fragrances (lipase), methyl ester sulfonate surfactants (lipase), cellulose and cellulose derivatives (e.g., CMC) (cellulase), and dextrins and cyclodextrins (amylase).

[0395] Sensitive detergent ingredients can also be encapsulated and thus stabilized in the microcapsules of the present invention. Sensitive detergent ingredients tend to decompose during storage. Such detergent ingredients include bleaching compounds, bleach activators, perfumes, polymers, builders, surfactants, etc.

[0396] In general, the microcapsules of the present invention can be used to separate incompatible ingredients / compounds in detergents.

[0397] The addition of microcapsules to detergents can be used to affect the appearance of the detergent product, for example to create an opacifying effect (small microcapsules) or to create a clearly visible particle effect (large microcapsules). The microcapsules may also be colored.

[0398] Microcapsules can be used to reduce enzyme dust levels during handling and processing of enzyme products.

[0399] Unless otherwise indicated, all percentages are given throughout this application as percent by weight (% w / w).

[0400] microcapsules Microcapsules are typically produced by forming water droplets into a water-immiscible continuum, typically by preparing a water-in-oil emulsion, followed by the formation of a membrane by interfacial polymerization via the addition of a crosslinking agent. After final hardening, the capsules can be collected and further rinsed and formulated by methods known in the art. The capsule formulation is then added to a detergent.

[0401] The payload to be encapsulated, the main membrane components, and finally additional components are found in the aqueous phase. In the continuum, components that stabilize the water droplets toward coalescence (emulsifiers, emulsion stabilizers, surfactants, etc.) are found, and cross-linkers are also added through the continuum.

[0402] Emulsions can be prepared by any method known in the art, such as mechanical stirring, dripping processes, membrane emulsification, microfluidics, sonication, etc. In some cases, simple mixing of the phases will automatically result in an emulsion (often referred to as self-emulsification). It is advantageous to use a method that results in a narrow size distribution.

[0403] The crosslinker is then typically added to the emulsion either directly, or more typically by preparing a solution of the crosslinker in a solvent that is soluble in the continuous phase. The emulsion and crosslinker or a solution thereof can be mixed by conventional methods used in the art, such as by simple mixing or by carefully controlling the flow of the emulsion and crosslinker solution through an in-line mixer.

[0404] In some cases, hardening of the capsules is required to complete film formation. Hardening often consists of simply stirring the capsules for a period of time to allow the interfacial polymerization reaction to complete. In other cases, film formation can be stopped by the addition of a reaction quencher.

[0405] The capsules may be post-modified, for example by reacting components onto the membrane, to prevent or reduce flocculation of the particles in detergents, as described in WO 99 / 01534.

[0406] The produced capsules may be isolated or concentrated by methods known in the art, for example, by filtration, centrifugation, distillation or decantation of the capsule dispersion.

[0407] The resulting capsules can be further formulated, for example, by the addition of surfactants to give the product desired properties for storage, shipping, and subsequent handling and addition to detergents. Other microcapsule formulations include rheology modifiers, biocides (e.g., Proxel), acids / bases for adjusting the pH (which will also adjust the interior of the microcapsules), and water for adjusting the water activity.

[0408] The capsule formation process involves the following steps: - preparation of the initial water and oil phase, -water-in-oil emulsions, -Film formation by interfacial polymerization, - optional post-modification, - optional isolation and / or formulation, -Addition to detergents may include:

[0409] The process can be either a batch process or a continuous or semi-continuous process.

[0410] Microcapsules according to the present invention are small globules of water with a uniform membrane surrounding them. The material inside the microcapsules is sometimes referred to as the core, internal phase, or fill, while the membrane is sometimes referred to as the shell, coating, or wall. Microcapsules of the present invention have diameters between 0.5 μm and 2 mm. Preferably, the average diameter of the microcapsules is in the range of 1 μm to 1000 μm, more preferably in the range of 5 μm to 500 μm, even more preferably in the range of 10 μm to 500 μm, even more preferably in the range of 50 μm to 500 μm, and most preferably in the range of 50 μm to 200 μm. Alternatively, the diameter of the microcapsules is in the range of 0.5 μm to 30 μm; or in the range of 1 μm to 25 μm. The diameter of the microcapsules is measured in the oil phase after polymerization is complete. The diameter of the capsules may vary depending on the water activity of the surrounding chemical environment.

[0411] Microencapsulation of enzymes, as used in the present invention, may be carried out by interfacial polymerization, in which the two reactants in the polymerization reaction meet at an interface and react rapidly. This method is based on the reaction of a polyamine with an acid derivative, usually an acid halide, which acts as a crosslinker. The polyamine is preferably substantially water-soluble (when in its free base form). Under the correct conditions, a thin, mobile film rapidly forms at the interface. One method of polymerization is to use an aqueous solution of the enzyme and polyamine emulsified in a non-aqueous solvent (and emulsifier), to which a solution containing the acid derivative is added. An alkalizing agent may be present in the enzyme solution to neutralize the acid formed during the reaction. A polymer (polyamide) film quickly forms at the interface of the emulsion droplets. The polymer film of the microcapsules is typically cationic and therefore can bind / complex with anionic compounds.

[0412] The diameter of the microcapsules is determined by the size of the emulsion droplets, which is controlled, for example, by the stirring speed.

[0413] emulsion An emulsion is a temporary or permanent dispersion of one liquid phase in a second liquid phase. The second liquid is generally referred to as the continuous phase. Surfactants are generally used to contribute to the formation and stabilization of emulsions. Not all surfactants are equally capable of stabilizing emulsions. The type and amount of surfactant must be selected to optimize emulsion utility, particularly with regard to emulsion preparation and physical stability, as well as stability during dilution and further processing. Physical stability refers to maintaining the emulsion in a dispersed liquid form. Processes such as coalescence, flocculation, adsorption to container walls, sedimentation, and creaming are forms of physical instability and must be avoided. Examples of suitable surfactants are described in WO 97 / 24177, pp. 19-21; and WO 99 / 01534.

[0414] Emulsions can be further classified as either simple emulsions, where the dispersed liquid phase is a simple homogeneous liquid, or more complex emulsions, such as double or multiple emulsions, where the dispersed liquid phase is a heterogeneous combination of liquid or solid phases. For example, water-in-oil double or multiple emulsions can be formed where the aqueous phase itself further contains an emulsified oil phase; this type of emulsion can be identified as an oil-in-water-in-oil (o / w / o) emulsion. Alternatively, water-in-oil emulsions can be formed where the aqueous phase contains a dispersed solid phase, often referred to as a suspension-emulsion. Other more complex emulsions can be described. Due to the inherent difficulty of describing such systems, the term emulsion is used to describe both simple and more complex emulsions, without necessarily limiting the emulsion's form or the type and number of phases present.

[0415] Polyamines The rigidity / mobility and permeability of the membrane are mainly influenced by the choice of polyamine. The polyamine is in accordance with the hyperbranched polyamine of the present invention. Each branch, preferably the terminus having a primary amino group, serves as an anchoring point in the membrane network, thereby achieving the preferred properties of the present invention. The hyperbranched polyamine is in accordance with the polyamine of the present invention having more than two branch points and more than two reactive amino groups (capable of reacting with crosslinkers, i.e., primary and secondary amino groups). The hyperbranched polyamine is used as a starting material when the emulsion is prepared (it is not formed in situ from other starting materials). To achieve the attractive properties of the present invention, the hyperbranched structure of the polyamine must be present as a starting material.

[0416] A primary amine will always be located at the end of a branch: since a linear amine can have only two primary amines, there is a close relationship between the number of branch points and the number of primary amines. Hypothetically, each branch point introduced into such a linear diamine allows for the introduction of one or more primary amines at the end of the introduced branch. In this context, we understand a primary amino group as part of a branch, i.e., as the end point of the branch. For example, we consider both tris(2-aminoethyl)amine and 1,2,3-propanetriamine to be molecules with one branch point. In the present invention, a polyamine has at least four primary amines. Branch points can be introduced from an aliphatic hydrocarbon chain, such as in the examples above, or from an unsaturated carbon bond, such as in 3,3'-diaminobenzidine, or from a tertiary amino group, such as in N,N,N',N'-tetrakis-(2-aminoethyl)ethylenediamine.

[0417] In addition to the number of branching points, we have found that the compactness of the reactive amino groups is very important. For example, a substance such as N,N,N',N'-tetrakis-(12-aminododecyl)ethylenediamine would be unsuitable. Neither peptides nor proteins, such as enzymes, would be suitable for membrane formation. Therefore, hyperbranched polyamines are not peptides or proteins.

[0418] In one embodiment, the reactive amino groups account for at least 15% of the molecular weight of the hyperbranched polyamine, e.g., more than 20% or more than 25%. Preferably, the molecular weight of the hyperbranched polyamine is at least 1 kDa; more preferably, the molecular weight of the hyperbranched polyamine is at least 1.3 kDa.

[0419] In a preferred embodiment, the hyperbranched polyamine is polyethyleneimine (PEI) and its modifications, which have more than two branch points and more than two reactive amino groups, and the reactive amino groups account for at least 15%, e.g., more than 20%, or more than 25% of the molecular weight of the PEI. Preferably, the molecular weight of the PEI is at least 1 kDa.

[0420] Combinations of different hyperbranched polyamines may be used to prepare the microcapsules according to the present invention.

[0421] The advantageous properties of the microcapsules of the present invention (e.g., enzyme storage stability, reduced enzyme leakage, reduced detergent ingredient flux) may be improved by adding one or more small amines having a molecular weight of less than 1 kDa. The small amine is preferably substantially water-soluble (when in free base form) and can be a material such as ethylenediamine, hexamethylenediamine, hexanediamine, diethylenetetramine, ethylenetetramine, diaminobenzene, piperazine, tetramethylenepentamine, or, preferably, diethylenetriamine (DETA). The small amine may be added in an amount of up to 50%, preferably up to 40%, up to 30%, up to 20%, up to 10%, or up to 5% by weight of the total content of the small amine and hyperbranched polyamine when preparing the microcapsules of the present invention.

[0422] Crosslinking agent A crosslinker, as used in the present invention, is a molecule that has at least two groups / sites capable of reacting with an amine to form a covalent bond.

[0423] The crosslinking agent is preferably oil-soluble and can be in the form of an acid anhydride or an acid halide, preferably an acid chloride, such as adipoyl chloride, sebacoyl chloride, dodecane diacid chloride, phthaloyl chloride, terephthaloyl chloride, isophthaloyl chloride, or trimesoyl chloride, but preferably the crosslinking agent is terephthaloyl chloride or trimesoyl chloride.

[0424] enzyme In one embodiment, the composition or microcapsule composition of the present invention may further comprise an enzyme selected from the group consisting of proteases, amylases, lipases, cellulases, mannanases, pectinases, deoxyribonucleases, laccases, peroxidases, haloperoxidases, perhydrolases, and combinations thereof.

[0425] The enzymes in the compositions or microcapsules of the present invention may, for example, comprise one or more enzymes suitable for use in laundry or dishwashing detergents (detergent enzymes), such as proteases (e.g., subtilisins or metalloproteases), lipases, cutinases, amylases, carbohydrases, cellulases, pectinases, mannanases, arabinases, galactanases, xanthanases, xylanases, deoxyribonucleases, perhydrolases, oxidoreductases (e.g., laccases, peroxidases, peroxygenases and / or haloperoxidases). Preferred detergent enzymes are proteases (e.g., subtilisins or metalloproteases), lipases, amylases, lyases, cellulases, pectinases, mannanases, deoxyribonucleases, perhydrolases, and oxidoreductases (e.g., laccases, peroxidases, peroxygenases, and / or haloperoxidases); or combinations thereof. More preferred detergent enzymes are proteases (e.g., subtilisins or metalloproteases), lipases, amylases, cellulases, pectinases, and mannanases; or combinations thereof.

[0426] The composition or microcapsule composition of the present invention may comprise more than 0.1% (w / w) active enzyme protein, in particular a lipase variant of the present invention; preferably more than 0.25%, more preferably more than 0.5%, more preferably more than 1%, more preferably more than 2.5%, more preferably more than 5%, more preferably more than 7.5%, more preferably more than 10%, more preferably more than 12.5%, more preferably more than 15%, even more preferably more than 20%, and most preferably more than 25% (w / w) active enzyme protein.

[0427] Protease: The protease used in the present invention is a serine protease, such as a subtilisin, a metalloprotease, and / or a trypsin-like protease. Preferably, the protease is a subtilisin or a metalloprotease; more preferably, the protease is a subtilisin.

[0428] Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds, in which an essential serine residue is present in the active site (White, Handler and Smith, 1973, "Principles of Biochemistry," Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272). Subtilisins include, and preferably consist of, the I-S1 and I-S2 subgroups as defined by Siezen et al., Protein Eng. 4 (1991) 719-737; and Siezen et al., Protein Science 6 (1997) 501-523. Due to the highly conserved structure of the active site of serine proteases, the subtilisins described in the present invention may be functionally equivalent to the subtilases designated as the subgroups proposed by Siezen et al. (supra).

[0429] The subtilisin may be of animal, plant or microbial origin, preferably an alkaliphilic microbial subtilisin, including chemically or genetically recombinant mutants (protein-modified variants). Exemplary subtilisins include those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89 / 06279), and protease PD138 (WO 93 / 18140). Examples are described in WO 98 / 020115, WO 01 / 44452, WO 01 / 58275, WO 01 / 58276, WO 03 / 006602 and WO 04 / 099401. Examples of trypsin-like proteases include trypsin (e.g., of porcine or bovine origin) and the Fusarium proteases described in WO 89 / 06270 and WO 94 / 25583. Other examples are variants described in WO 92 / 19729, WO 88 / 08028, WO 98 / 20115, WO 98 / 20116, WO 98 / 34946, WO 2000 / 037599, WO 2011 / 036263, in particular variants having substitutions at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.

[0430] The metalloproteases may be of animal, plant or microbial origin, preferably alkaliphilic microbial metalloproteases, including chemically or genetically recombinant mutants (protein engineered variants). Examples are described in WO 2007 / 044993, WO 2012 / 110562 and WO 2008 / 134343.

[0431] Examples of commercially available subtilisins include Kannase™, Everlase™, Relase™, Esperase™, Alcalase™, Durazym™, Savinase™, Ovozyme™, Liquanase™, Coronase™, Polarzyme™, Pyrase™, pancreatic trypsin NOVO (PTN), Bio-Feed™ Pro and Clear-Lens™ Pro; Blaze (all available from Novozymes A / S, Bagsvaerd, Denmark). Other commercially available proteases include Neutrase™, Ronozyme™ Pro, Maxatase™, Maxacal™, Maxapem™, Opticlean™, Properase™, Purafast™, Purafect™, PurafectOx™, Purafact Prime™, Excellase™, FN2™, FN3™, and FN4™ (available from Novozymes, Genencor International Inc., Gist-Brocades, BASF, or DSM). Other examples are Primase™ and Duralase™. Examples include Blap R, Blap S, and Blap X, available from Henkel.

[0432] Lyase: The lyase may be a pectate lyase derived from Bacillus, in particular B. licheniformis or B. agaradhaerens, or a variant derived from any of these, as described, for example, in U.S. Pat. No. 6,124,127, WO 99 / 027083, WO 99 / 027084, WO 02 / 006442, WO 02 / 092741, WO 03 / 095638; commercially available pectate lyases are XPect; Pectawash and Pectaway (Novozymes A / S).

[0433] Mannanase: The mannanase may be an alkaline mannanase of family 5 or 26. It may be a wild-type mannanase from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99 / 064619. A commercially available mannanase is Mannaway (Novozymes A / S).

[0434] Cellulases: Suitable cellulases include those of bacterial or fungal origin, including chemically or protein-modified mutants. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, and Acremonium, such as fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum, as disclosed in U.S. Pat. Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757, and WO 89 / 09259.

[0435] Particularly suitable cellulases are alkaline or neutral cellulases with color care properties. 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, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK98 / 00299.

[0436] Commercially available cellulases include Celluzyme™ and Carezyme™ (Novozymes A / S), Clazinase™ and PuradaxHA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation).

[0437] In addition to the lipase variants of the present invention, the composition or microcapsule composition may include other lipases.

[0438] Other lipases and cutinases: Suitable lipases and cutinases include those of bacterial or fungal origin, including chemically or protein-modified mutants. Examples include those derived from Thermomyces, such as T. lanuginosus (formerly Humicola lanuginosus), as described in EP 258068 and EP 305216. Lipases from Humicola, for example H. insolens as described in WO 96 / 13580, such as P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas species, Examples of lipases that can be used include Pseudomonas lipases derived from Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), and Bacillus lipases derived from, for example, B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta, 1131:253-360), B. stearothermophilus (JP 64 / 744992 A) or B. pumilus (WO 91 / 16422 A).

[0439] Other examples are lipase variants such as those described in WO 92 / 05249, WO 94 / 01541, EP 407225, EP 260105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 060063, WO 2007 / 087508 and WO 2009 / 109500.

[0440] Other commercially available lipase enzymes include Lipolase™, Lipolase Ultra™, and Lipex™; Lipex Evity 100L, Lecitase™, Lipolex™; Lipoclean™, Lipoprime™ (Novozymes A / S). Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist-Brocades / Genencor Int Inc) and Bacillus sp. lipase from Solvay.

[0441] Amylases: Suitable amylases include those of bacterial or fungal origin, including chemically or protein-modified mutants. Examples of amylases include alpha-amylases obtained from Bacillus, such as specialized strains of Bacillus licheniformis (described in more detail in GB Patent No. 1,296,839).

[0442] Examples of suitable amylases include amylases having SEQ ID NO: 2 of WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, such as variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408, and 444.

[0443] Different suitable amylases include the amylase having SEQ ID NO: 6 of WO 02 / 010355, or a variant thereof having 90% sequence identity to SEQ ID NO: 6. A preferred variant of SEQ ID NO: 6 has deletions at positions 181 and 182 and a substitution at position 193. Another amylase that is suitable is a hybrid alpha-amylase comprising residues 1 to 33 of the alpha-amylase from B. amyloliquefaciens as set forth in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36 to 483 of the B. licheniformis alpha-amylase as set forth in SEQ ID NO: 4 of WO 2006 / 066594, or a variant thereof having 90% sequence identity. A preferred variant of this hybrid alpha-amylase has substitutions, deletions or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. A most preferred variant of the hybrid alpha-amylase comprises residues 1-33 of the alpha-amylase from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4. M197T; H156Y+A181T+N190F+A209V+Q264S; or G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S has a substitution in

[0444] A further suitable amylase is the amylase having SEQ ID NO: 6 of WO 99 / 019467 or a variant thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those which have 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 which have deletions at positions R181 and G182, or H183 and G184.

[0445] Further amylases that can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those that have 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 that have deletions at positions 181 and 182 or 183 and 184. 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 substitutions at one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0446] Other amylases that may be used are those having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 of WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 of WO 01 / 66712. Preferred variants of SEQ ID NO: 10 of WO 01 / 66712 are those that have substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

[0447] A further preferred amylase is the amylase having SEQ ID NO: 2 of WO 09 / 061380 or a variant thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 have C-terminal truncations and / or substitutions, deletions or insertions at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475. More preferred variants of SEQ ID NO: 2 are those which have 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 deletions at positions R180 and / or S181 or T182 and / or G183. Most preferred amylase variants of SEQ ID NO: 2 have substitutions: N128C+K178L+T182G+Y305R+G475K; N128C+K178L+T182G+F202Y+Y305R+D319T+G475K; S125A+N128C+K178L+T182G+Y305R+G475K; or S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0448] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 of WO 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants have substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 of 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 a deletion of D183 and G184 and substitutions of R118K, N195F, R320K and R458K, as well as variants further having substitutions at one or more positions selected from the group of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferably at all of these positions.

[0449] Other examples are amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0450] Commercially available amylases are Stainzyme; Stainzyme Plus; Duramyl™, Termamyl™, Termamyl Ultra; Natalase, Fungamyl™ and BAN™ (Novozymes A / S), Rapidase™ and Purastar™ / Effectenz™, Powerase and Preferenz S100 (Genencor International Inc. / DuPont).

[0451] Deoxyribonuclease (DNase): A suitable deoxyribonuclease (DNase) is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester bonds in the DNA backbone, thereby degrading DNA. According to the present invention, DNases obtainable from bacteria are preferred; in particular, DNases obtainable from Bacillus are preferred; in particular, DNases obtainable from Bacillus subtilis or Bacillus licheniformis are preferred. Examples of such DNases are described in patent application WO 2011 / 098579 or PCT / EP2013 / 075922.

[0452] Perhydrolase: Suitable perhydrolases are capable of catalyzing the perhydrolysis reaction, which results in the production of peracids from carboxylic acid ester (acyl) substrates in the presence of a peroxygen source (e.g., hydrogen peroxide). While many enzymes perform this reaction at low levels, perhydrolases exhibit high perhydrolysis (often with a hydrolysis ratio greater than 1). Suitable perhydrolases may be of plant, bacterial, or fungal origin. Chemically or protein-engineered mutants are included.

[0453] Examples of useful perhydrolases include naturally occurring Mycobacterium perhydrolase enzymes or variants thereof. An exemplary enzyme is derived from Mycobacterium smegmatis. Such enzymes, their enzymatic properties, structures, and variants are described in WO 2005 / 056782, WO 2008 / 063400, U.S. Patent No. 2008 / 145353, and U.S. Patent No. 2007167344.

[0454] Oxidases / Peroxidases: Suitable oxidases and peroxidases (or oxidoreductases) include various sugar oxidizing enzymes, laccases, peroxidases and haloperoxidases.

[0455] Suitable peroxidases include those encompassed by the enzyme classification EC 1.11.1.7 as set forth by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom, which exhibits peroxidase activity.

[0456] Suitable peroxidases include those of plant, bacterial, or fungal origin, and include chemically or protein-modified mutants. Examples of useful peroxidases include peroxidases from Coprinopsis, such as peroxidase from C. cinerea (EP 179,486), and variants thereof (such as those described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257).

[0457] The peroxidases used in the present invention also include haloperoxidase enzymes, such as chloroperoxidase, bromoperoxidase, and compounds that exhibit chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (EC 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

[0458] In one embodiment, the haloperoxidase is a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase. In a preferred method of the invention, the vanadate-containing haloperoxidase is combined with a source of chloride ions.

[0459] Haloperoxidases have been isolated from a variety of fungi, particularly from the dark filamentous fungi imperfecti, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium, and Botrytis fungal groups.

[0460] Haloperoxidases have also been isolated from bacteria such as Pseudomonas, eg, P. pyrrocinia, and Streptomyces, eg, S. aureofaciens.

[0461] In a preferred embodiment, the haloperoxidase is from a Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, for example C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or Drechslera hartlebii as described in WO 01 / 79459, Dendryphiella salina as described in WO 01 / 79458, or from a Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, for example C. inaequalis CBS 102.42 as described in WO 95 / 27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97 / 04102; or Drechslera hartlebii as described in WO 01 / 79459, Dendryphiella salina as described in WO 01 / 79458, or from a Curvularia sp., in particular Curvularia verruculosa CBS 102.42 as described in WO 95 / 27046; or C. inaequalis ... salina, Phaeotrichoconis crotalarie as described in WO 01 / 79461, or Geniculosporium sp. as described in WO 01 / 79460.

[0462] Oxidases according to the invention include in particular any laccase enzyme encompassed by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting similar activity, 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).

[0463] Preferred laccase enzymes are of microbial origin: the enzyme may be derived from plants, bacteria or fungi (including filamentous fungi and yeasts).

[0464] Suitable examples of fungal origin include Aspergillus, Neurospora, for example, N. crassa, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Trametes, for example, T. villosa and T. versicolor, Rhizoctonia, for example, R. solani, Coprinopsis, for example, C. cinerea, C. comatus, C. friesii, and C. plicatilis, Examples of laccases that can be derived from strains of Psathyrella, such as P. condelleana, Panaeolus, such as P. papilionaceus, Myceliophthora, such as M. thermophila, Schytalidium, such as S. thermophilum, Polyporus, such as P. pinsitus, Phlebia, such as P. radiata (WO 92 / 01046), or Coriolus, such as C. hirsutus (JP Patent No. 2238885).

[0465] A suitable example of bacterial origin is laccase inducible from a strain of Bacillus.

[0466] Laccases derived from Coprinopsis or Myceliophthora; in particular from Coprinopsis cinerea as disclosed in WO 97 / 08325; or from Myceliophthora thermophila as disclosed in WO 95 / 33836 are preferred.

[0467] Other examples of oxidases include, but are not limited to, amino acid oxidase, glucose oxidase, lactate oxidase, galactose oxidase, polyol oxidase (e.g., WO 2008 / 051491), and aldose oxidase. An oxidase and its corresponding substrate may be used as a hydrogen peroxide-generating enzyme system and thus a source of hydrogen peroxide. Some enzymes, such as peroxidases, haloperoxidases, and perhydrolases, require a source of hydrogen peroxide. Other examples of such combinations of oxidases and substrates are readily apparent to those skilled in the art by reviewing EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._ or similar classes (under the International Union of Biochemistry).

[0468] Enzyme stabilizers and / or rheology modifiers The composition or microcapsules may also contain enzyme stabilizers as are known in the art, such as polyols, polymers, reversible enzyme inhibitors, divalent cations, enzyme substrates, antioxidants, etc. Water-soluble stabilizers are preferred.

[0469] The addition of slowly dissolving stabilizers can be used to create a local environment inside the capsule that is more "friendly" to the encapsulated enzyme / compound and therefore improves stability during storage.

[0470] Examples of reversible protease inhibitors include boric acid, peptide aldehydes and their derivatives, and polymeric protein-type inhibitors (such as BASI / RASI inhibitors, see WO 2009 / 095425). Examples of metalloprotease inhibitors are described in WO 2008 / 134343. Protease inhibitors are described in more detail under the heading "Protease Inhibitors."

[0471] Stabilizing polymers can be based, for example, on polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and their copolymers. Stabilizing polyols can be smaller molecules such as glycerol, sorbitol, propylene glycol, etc., but also larger molecules such as polyethylene glycol, polysaccharides, etc.

[0472] Among the stabilized divalent cations, Ca 2+ , Mg 2+ and Zn 2+ is well known in the art. Thus, in one embodiment, the composition of the present invention comprises Ca 2+ , Mg 2+ or Zn 2+ A source of ions, preferably Ca 2+ , Mg 2+ or Zn 2+ The source of the ions is Ca 2+ , Mg 2+ or Zn 2+ Weakly soluble means that the solubility in pure water at 20°C is less than 5 g / L, 2 g / L, 1 g / L, 0.5 g / L, 0.2 g / L, 0.1 g / L, or 0.05 g / L. 2+ , Mg 2+ or Zn 2+Preferred salts of are calcium carbonate, magnesium carbonate, zinc carbonate, calcium sulfate, calcium sulfite, magnesium sulfite, zinc sulfite, calcium phosphate, dicalcium phosphate, magnesium phosphate, zinc phosphate, calcium citrate, magnesium citrate, zinc citrate, calcium oxalate, magnesium oxalate, zinc oxalate, calcium tartrate, magnesium tartrate, or zinc tartrate.

[0473] Also, slowly dissolving acids or bases can be used to create a local pH inside the microcapsules that is more "friendly" to the encapsulated enzyme / compound.

[0474] Enzymes are often stabilized by the addition of their substrates (e.g., proteins for proteases, starches for amylases, etc.). For example, antioxidants or reducing agents such as thiosulfate or ascorbate can be applied to reduce enzyme oxidation. The required net dosage of these stabilizers per gram of detergent is much lower than when stabilizers are added to the continuous detergent phase, since they are concentrated in the internal capsule phase, and in many cases they either do not diffuse during storage or only diffuse gradually depending on the structure and molecular weight of the stabilizer. Stabilizers with particularly high molecular weights (e.g., greater than 1 kDa, or greater than 2 kDa, more preferably greater than 5 kDa) will result in improved net efficiency. Therefore, high molecular weight inhibitors, polymers, polyols, cations, enzyme substrates, and antioxidants are preferred.

[0475] Enzymes may be protected by the addition of "scavenger" proteins. Thus, enzymes whose components have been destabilized by reacting with amino acid groups (e.g., amines) on the protein may react with the added scavenger or sacrificial protein. Scavenger proteins with a sufficiently high molecular weight so that they remain inside the capsule are preferred.

[0476] A slightly different approach to improving enzyme stability is to add a rheology-modifying component that increases the viscosity of the internal capsule phase. Increasing the internal viscosity retards the diffusion of enzyme destabilizers into the capsule (and / or retards the diffusion of enzyme stabilizers out of the capsule), thereby extending the enzyme's shelf life. Examples of such viscosity modifiers include polymers such as polyethylene glycol (PEG), polyethylene oxide (PEO), hydrophilic polyurethanes, polyvinylpyrrolidone (PVP) and PVP-vinyl acetate copolymers; starch; hyaluronic acid; water-soluble cellulose derivatives such as carboxymethylcellulose; water-soluble gums such as gum arabic, locust bean gum, guar gum, or xanthan gum; and combinations or copolymers thereof. Nonionic high-molecular-weight polymers with molecular weights greater than 1 kDa, or greater than 2 kDa, and more preferably greater than 5 kDa, are most preferred. Nonionic polymers are preferred because they are often more compatible with reactive membrane polymers than ionic polymers.

[0477] High viscosity can be achieved by forming capsules using a high-viscosity aqueous phase, or even more so, by forming capsules where an initial viscosity increase occurs after emulsion / capsule formation. This "induced" viscosity increase is preferred when emulsions are prepared with a high-viscosity aqueous phase, which can be difficult. Induced viscosity increase can occur in situ upon addition to a detergent due to the internal capsule phase having a higher water activity than the detergent to which it is added, causing water (excluding rheology modifiers) to diffuse out of the capsules, increasing the viscosity of the internal phase after addition to the detergent. This can also be achieved by using the diffusion of salts or other low-molecular-weight components, for example, by having a component that increases viscosity when the salt concentration decreases upon addition to the detergent (e.g., a polymer that precipitates at an initial high salt content but becomes soluble when the salt concentration decreases due to salt diffusion upon addition to the detergent). Another method for inducing viscosity increase is to use a component whose viscosity is pH-dependent. In some interfacial polymerization processes (e.g., amine-acid halide reactions), the pH of the internal phase changes during encapsulation; in the case of amine-acid halide reactions, the pH will decrease during interfacial polymerization. This can be used to induce an increase in viscosity. Many rheology modifiers, such as polyacrylates, exhibit a maximum viscosity at a specific pH or pH range. Carbopol 934 from Lubrizol and Texipol 63-258 from Scott-Bader are examples of rheology modifiers whose viscosity increases significantly when the pH is decreased from 11 to 8 or increased from 4 to 8. Another polymer type with different viscosities at low and high pH is partially hydrolyzed polyacrylamide. Yet another possibility is to use a rheology modifier that is temperature-dependent, allowing the emulsion / encapsulation to occur at one temperature and then increasing the viscosity with a change in temperature. Light- or ultrasound-induced viscosity is also available. Yet another method is to use a shear-thinning rheology modifier, so that when the emulsion is formed, the viscosity decreases with high shear and increases with decreasing shear.

[0478] Another stabilization technique is to ensure that the enzyme precipitates within the capsule during storage, for example, by the addition of a precipitating agent such as salt or polyethylene glycol (PEG). The same "induced stabilization" as above can be used, for example, by adding PEG, which, after addition to the detergent, becomes concentrated by diffusion of water to such an extent that the enzyme precipitates. In this way, the enzyme may be in solution during processing of the capsule, but may be precipitated when added to the detergent.

[0479] When preparing microcapsules, the enzyme can also be used in precipitated or crystalline form.

[0480] In a specifically contemplated embodiment, the microcapsule composition of the invention is that described in WO 2014 / 177709 (herein incorporated by reference) which comprises a lipase variant of the invention.

[0481] In another embodiment, the present invention relates to a microcapsule composition comprising a lipase variant of the present invention encapsulated in a compartment formed by a membrane, wherein the membrane is produced by crosslinking (a) a hyperbranched polyamine having a molecular weight of more than 800 Da and (b) an aliphatic or aromatic amine having a molecular weight of less than 300 Da; wherein the weight ratio of (a) / (b) is in the range of 0.1 to 1000.

[0482] As noted above, the microcapsules may further comprise an enzyme selected from the group consisting of proteases, metalloproteases, subtilisins, amylases, lipases, cutinases, cellulases, mannanases, pectinases, xanthanases, deoxyribonucleases, laccases, peroxidases, haloperoxidases, perhydrolases, and combinations thereof. Other enzymes mentioned above are also contemplated.

[0483] In one embodiment, the reactive amino groups of the hyperbranched polyamine account for at least 15% of the molecular weight. In one embodiment, the diameter of the compartment is at least 50 μm. In a preferred embodiment, the compartment contains at least 1% by weight of the active enzyme, particularly the lipase variant of the present invention, of the total compartment. Furthermore, as mentioned above, the microcapsules may further comprise an alcohol, such as a polyol.

[0484] In a preferred embodiment, (a) is polyethyleneimine.

[0485] In one embodiment, (b) is an ethyleneamine or an alkanolamine. In a preferred embodiment, (b) is selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetraamine, bis(3-aminopropyl)amine, monoethanolamine, diethanolamine, triethanolamine, hexamethylenediamine, diaminobenzene, piperazine, and tetraethylenepentamine. In a more preferred embodiment, (b) is selected from the group consisting of diethylenetriamine, triethylenetetraamine, bis(3-aminopropyl)amine, monoethanolamine, and diethanolamine.

[0486] According to the present invention, the compartments of the microcapsules contain Mg 2+ , Ca 2+ , or Zn 2+ A source of ions, e.g., Mg 2+ , Ca 2+ , or Zn 2+ Contains weakly soluble salts of

[0487] In a preferred embodiment, the membrane is produced by using an acid chloride, such as isophthaloyl chloride, terephthaloyl chloride, or trimesoyl chloride, as the cross-linking agent.

[0488] In a preferred embodiment, the membrane is produced by interfacial polymerization.

[0489] In a specifically contemplated embodiment, the microcapsule composition of the invention is that described in WO 2015 / 1144784 (herein incorporated by reference) which comprises a lipase variant of the invention.

[0490] liquid products In a final aspect, the present invention relates to a liquid product comprising the microcapsule composition of the present invention. In a preferred embodiment, the liquid product comprises water or at least a significant amount of water.

[0491] The following paragraphs describe embodiments of the present invention: 1. A variant of a parent lipase having lipase activity and having at least 60% but less than 100% sequence identity with SEQ ID NO:2 and comprising one or more (e.g., several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R, N233R, T244E, and P256T.

[0492] 2. The variant of paragraph 1, comprising a substitution at a position corresponding to T231R+N233R and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

[0493] 3. A variant of paragraph 1 or 2, comprising a substitution corresponding to any of the following sets of substitutions:

[0494] [Table 4]

[0495] 4. The variant of any of paragraphs 1 to 3, comprising a substitution corresponding to E56R+T231R+N233R and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T.

[0496] 5. The variant of any of paragraphs 1 to 4, comprising a substitution at a position corresponding to R118F+T231R+N233R and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

[0497] 6. The variant of any of paragraphs 1 to 5, comprising substitutions at positions corresponding to E56R+R118F+T231R+N233R and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T.

[0498] 7. The variant of any of paragraphs 1 to 6, comprising substitutions at positions corresponding to E56R+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0499] 8. The variant of any of paragraphs 1 to 7, comprising substitutions at positions corresponding to F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0500] 9. The variant of any of paragraphs 1 to 8, comprising substitutions at positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0501] 10. The variant of any of paragraphs 1 to 9, comprising substitutions at positions corresponding to D27N+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0502] 11. The variant of any of paragraphs 1 to 10, comprising substitutions at positions corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0503] 12. The variant of any of paragraphs 1 to 11, comprising substitutions at positions corresponding to F51I,L+E56R+D57N+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0504] 13. The variant of any of paragraphs 1 to 12, comprising substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E.

[0505] 14. The variant of any of paragraphs 1 to 13, comprising substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, Y220F, and T244E.

[0506] 15. The variant of any of paragraphs 1 to 14, comprising substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+T244E+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, and Y220F.

[0507] 16. The variant of any of paragraphs 1 to 15, comprising substitutions at positions corresponding to F51I, L+E56R+D57N+V60E, K+K98I+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, N101D, G163S, Y220F, and T244E.

[0508] 17. The variant of any of paragraphs 1-16, comprising substitutions at positions corresponding to F51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E.

[0509] 18. The variant of any of paragraphs 1 to 17, comprising substitutions at positions corresponding to F51I, L+E56R+D57N+V60E, K+K98I+N101D+R118F+T231R+N233R+P256T, and one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, G163S, Y220F, and T244E.

[0510] 19. Replace the following set of substitutions: E56R+R118F+T231R+N233R; R118F+T231R+N233R+P256T; A40I+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R; E56R+D57N+R118F+T231R+N233R; E56R+V60K+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T; <h2 style=";text-align:left;direction:ltr">G23S+F51I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D27N+F51I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+F51L+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+D57N+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> A40I+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+D57N+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> F51L+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+K98I+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> D57N+E56R+R118F+T231R+N233R+P256T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> K98I+G163S+E56R+R118F+T231R+N233R;<h2 style=";text-align:left;direction:ltr"> K98I+E56R+R118F+T231R+N233R+P256T; G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R; A40I+F51L+G163S+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+E56R+R118F+T231R+N233R; A40I+D57N+G163S+E56R+R118F+T231R+N233R; A40I+D57N+E56R+R118F+T231R+N233R+P256T; A40I+K98I+G163S+E56R+R118F+T231R+N233R; A40I+K98I+E56R+R118F+T231R+N233R+P256T; A40I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+E56R+R118F+T231R+N233R; F51L+D57N+G163S+E56R+R118F+T231R+N233R; F51L+D57N+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R; F51L+K98I+E56R+R118F+T231R+N233R+P256T; F51L+G163S+E56R+R118F+T231R+N233R+P256T; D57N+K98I+G163S+E56R+R118F+T231R+N233R; D57N+K98I+E56R+R118F+T231R+N233R+P256T; D57N+G163S+E56R+R118F+T231R+N233R+P256T; K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R+P256T; D27N+A40I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; D27N+E56R+R118F+T231R+N233R+P256T; A40I+F51I+E56R+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R+T244E; A40I+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+P256T; E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; G23S+E56R+R118F+T231R+N233R+T244E+P256T; G23S+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+E56R+V60E+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R; D27N+A40I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; D27N+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E; A40I+F51I+E56R+R118F+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E: G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; F51I+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R; K98I+E56R+R118F+T231R+N233R; Y220F+E56R+R118F+T231R+N233R; E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; G23S+K98I+E56R+R118F+T231R+N233R; G23S+Y220F+E56R+R118F+T231R+N233R; G23S+E56R+R118F+T231R+N233R+T244E; G23S+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R; D27N+K98I+E56R+R118F+T231R+N233R; D27N+Y220F+E56R+R118F+T231R+N233R; D27N+E56R+R118F+T231R+N233R+T244E; D27N+E56R+R118F+T231R+N233R+P256T; F51I+K98I+E56R+R118F+T231R+N233R; F51I+Y220F+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+P256T; K98I+Y220F+E56R+R118F+T231R+N233R; K98I+E56R+R118F+T231R+N233R+T244E; K98I+E56R+R118F+T231R+N233R+P256T; Y220F+E56R+R118F+T231R+N233R+T244E; Y220F+E56R+R118F+T231R+N233R+P256T; E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+K98I+E56R+R118F+T231R+N233R; G23S+D27N+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+E56R+R118F+T231R+N233R+T244E; G23S+D27N+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+E56R+R118F+T231R+N233R; G23S+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+K98I+E56R+R118F+T231R+N233R+T244E; G23S+K98I+E56R+R118F+T231R+N233R+P256T; G23S+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R; D27N+F51I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R; D27N+K98I+E56R+R118F+T231R+N233R+T244E; D27N+K98I+E56R+R118F+T231R+N233R+P256T; D27N+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R; F51I+K98I+E56R+R118F+T231R+N233R+T244E; F51I+K98I+E56R+R118F+T231R+N233R+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; K98I+Y220F+E56R+R118F+T231R+N233R+T244E; K98I+Y220F+E56R+R118F+T231R+N233R+P256T; K98I+E56R+R118F+T231R+N233R+T244E+P256T; Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+K98I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T. 19. A variant of any of paragraphs 1 to 18, comprising at a position corresponding to one of:

[0511] 20. a) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO:2; b) a polypeptide encoded by a polynucleotide that hybridizes to (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i) under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions; c) a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:1; and d) a fragment of the polypeptide of SEQ ID NO: 2 20. The variant of any of paragraphs 1 to 19, which is a variant of a parent lipase selected from the group consisting of:

[0512] 21. The variant of any of paragraphs 1-20, having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to SEQ ID NO:2.

[0513] 22. The variant of any of paragraphs 1 to 21, wherein the number of substitutions is 1 to 40, such as 1 to 30, for example 1 to 20, such as 1 to 12, for example 1 to 11, such as 1 to 10, for example 1 to 9, for example 1 to 8, such as 1 to 7, for example 1 to 6, such as 1 to 5, for example 1 to 4, such as 1 to 3, or 1, 2, 3 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions.

[0514] 23. The variant of any of paragraphs 1 to 22, having one or more of the following properti...

Claims

1. A variant of a parent lipase having lipase activity and having at least 90% but less than 100% sequence identity with SEQ ID NO:2 and comprising the substitutions T231R+N233R+A40I.

2. 2. The variant of claim 1, comprising the substitutions T231R+N233R+A40I and one or more of G23S, D27N, F51I, F51L, E56R, D57N, V60E, V60K, K98I, N101D, G163S, Y220F, T244E, and P256T.

3. The mutant has the following set of substitutions: A40I+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R; A40I+D57N+E56R+R118F+T231R+N233R; A40I+K98I+E56R+R118F+T231R+N233R; A40I+G163S+E56R+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R; A40I+F51L+G163S+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+E56R+R118F+T231R+N233R; A40I+D57N+G163S+E56R+R118F+T231R+N233R; A40I+D57N+E56R+R118F+T231R+N233R+P256T; A40I+K98I+G163S+E56R+R118F+T231R+N233R; A40I+K98I+E56R+R118F+T231R+N233R+P256T; A40I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R; D27N+A40I+E56R+R118F+T231R+N233R; A40I+F51I+E56R+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R+T244E; A40I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R; G23S+A40I+F51I+E56R+R118F+T231R+N233R; G23S+A40I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+E56R+R118F+T231R+N233R+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R; D27N+A40I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+E56R+R118F+T231R+N233R+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E; A40I+F51I+E56R+R118F+T231R+N233R+P256T; A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E: G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E; G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; 3. The mutant of claim 1, comprising any one of:

4. The parent lipase is: a) a polypeptide having at least 95% identity to SEQ ID NO:2; and b) a fragment of the polypeptide of SEQ ID NO: 2; The mutant according to any one of claims 1 to 3, selected from the group consisting of:

5. 5. The variant of any one of claims 1 to 4, having at least 95% but less than 100% sequence identity to SEQ ID NO:

2.

6. A composition comprising the mutant of any one of claims 1 to 5.

7. Use of a variant according to any one of claims 1 to 5 for hydrolysing a lipase substrate.

8. A method for cleaning a surface to be cleaned, comprising contacting the surface to be cleaned with a mutant according to any one of claims 1 to 5.

9. A method for hydrolyzing a lipase substrate, comprising treating the lipase substrate with a lipase variant according to any one of claims 1 to 5.

10. A polynucleotide encoding the variant according to any one of claims 1 to 5.

11. 11. A nucleic acid construct comprising the polynucleotide of claim 10, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the lipase variant in a recombinant host cell.

12. 12. An expression vector comprising the polynucleotide of claim 10 or the nucleic acid construct of claim 11.

13. A host cell comprising the nucleic acid construct of claim 11 or the expression vector of claim 12.

14. a) culturing the host cell of claim 13 under conditions suitable for expression of said variant; b) recovering said mutant; A method for producing a lipase variant, comprising: