Lipase variant and microcapsule composition containing such lipase variant

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] Reference to Sequence Listing This application includes a sequence listing in computer-readable form, which is incorporated herein by reference.

Background Art

[0002] The present invention relates to a lipase variant, a polynucleotide encoding the variant, and a method for producing the variant. The present invention also relates to a composition and a microcapsule composition containing the lipase variant of the present invention, and a liquid product containing the microcapsule composition of the present invention.

[0003] Lipase is an important biocatalyst that has been shown to be useful for various applications. The wild-type Thermomyces lanuginosus lipase (synonymous with Humicola lanuginosa) and its variants sold under the trade name LIPOLASE™ are commercially available as active ingredients in detergent compositions that remove lipid stains by hydrolyzing triglycerides to produce fatty acids.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Detergent, cleaning and / or fabric care compositions contain active ingredients that interfere with the ability of lipase to remove lipid stains. A number of known Thermomyces lanuginosus lipase variants with good washing performance form short-chain fatty acids that produce odors during washing and / or have short storage stability.

[0005] Therefore, there is still a need for and a demand for lipases with improved washing performance, reduced odor generation and / or improved storage stability / longer shelf life / enhanced thermal stability.

Means for Solving the Problems

[0006] The present invention relates to a variant of a parent lipase having lipase activity, 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.

[0007] The present invention further relates to a composition comprising the lipase variant of the present invention, and its use for hydrolyzing a lipid substrate. Further, the present invention relates to a polynucleotide encoding the variant of the present invention; a nucleic acid construct, vector, and host cell comprising the polynucleotide.

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

[0009] In a further aspect, the present invention relates to a microcapsule composition comprising the lipase variant of the present invention encapsulated within a compartment formed by a membrane, wherein the membrane is produced by crosslinking (a) a multi-branched 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.

[0010] Finally, the present invention relates to a liquid product comprising the microcapsule composition 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 EC3.1.1 as defined by enzyme nomenclature. This can have lipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13), and / or wax-ester hydrolase activity (EC3.1.1.50). For the purposes of the present invention, lipase activity is determined according to the procedure described in the examples. In one aspect, the variant of the present 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" means any of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic mutations occur naturally through mutation and may result in polymorphisms within a population. Gene mutations can be silent (no change in the encoded polypeptide) or may encode a polypeptide with an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of the gene.

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

[0014] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by the open reading frame, which starts 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 sequence: The term "control sequence" means all nucleic acid sequences necessary for the expression of a polynucleotide, encoding the variant of the present invention. Each control sequence may be endogenous (i.e., from the same gene), exogenous (i.e., from a different gene), or a combination of endogenous and exogenous with respect to the polynucleotide encoding the variant. Such control sequences include, but are not limited to, leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, as well as transcription and translation stop signals. The control sequences may be provided with a linker to introduce specific restriction sites that facilitate the ligation reaction 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, and specifically includes, but is not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

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

[0018] Fragment: The term "fragment" means a polypeptide in which one or more (e.g., several) amino acids are absent from the amino and / or carboxyl termini of the polypeptide; where the fragment has lipase activity. In one embodiment, 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 to 269 of SEQ ID NO: 2.

[0019] High stringency conditions: The term "high stringency conditions" means, for a probe of at least 100 nucleotides in length, 42°C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and sheared salmon sperm DNA, and prehybridization and hybridization in 50% formamide, followed by standard Southern blotting for 12 - 24 hours. The carrier material is finally washed 3 times at 65°C for 15 minutes each using 2×SSC, 0.2% SDS.

[0020] Host cell: The term "host cell" means any cell type that is sensitive to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "host cell" includes any progeny of a parent cell that differs from the parent cell due to a mutation that occurs during replication.

[0021] Improved property: The term "improved property" means a characteristic associated with an improved variant as 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" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include: (1) any non-natural substance; (2) any enzyme, variant, nucleic acid, protein, peptide, or cofactor that is at least partially removed from one or more or all of the naturally occurring components that are naturally associated, including but not limited to these; (3) any substance that has been artificially modified with respect to a naturally occurring substance; or (4) any substance that has been modified by increasing the amount of the substance (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance) compared to other components that are naturally associated. An isolated substance can be present in a fermentation broth sample.

[0023] Low stringency conditions: The term "low stringency conditions" means, for a probe of at least 100 nucleotides in length, 42 °C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, and prehybridization and hybridization in 25% formamide, followed by standard Southern blotting for 12 - 24 hours. The carrier material is finally washed 3 times at 50 °C for 15 minutes each using 2×SSC, 0.2% SDS.

[0024] Mature polypeptide: The term "mature polypeptide" means the polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal cleavage, 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" means 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" means, for a probe of at least 100 nucleotides in length, 42 °C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and sheared salmon sperm DNA, and prehybridization and hybridization in 35% formamide, followed by standard Southern blotting for 12 - 24 hours. The carrier material is finally washed 3 times at 55 °C for 15 minutes each using 2×SSC, 0.2% SDS.

[0027] Medium - high stringency conditions: The term "medium - high stringency conditions" means, for a probe of at least 100 nucleotides in length, 42 °C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and sheared salmon sperm DNA, prehybridization and hybridization in 35% formamide, followed by standard Southern blotting for 12 - 24 hours. The carrier material is finally washed 3 times at 60 °C for 15 minutes each using 2×SSC, 0.2% SDS.

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

[0029] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule that is single - stranded or double - stranded and that has been modified to contain segments of nucleic acid isolated from natural genes, segments of nucleic acid that would not otherwise occur naturally, or segments of synthetic nucleic acid, and includes one or more control sequences.

[0030] Operably linked: The term "operably linked" means a configuration in which a control sequence is positioned at an appropriate position relative to a coding sequence of a polynucleotide such that the control sequence brings about the expression of the coding sequence.

[0031] Parent or parent lipase: The term "parent" or "parent lipase" means a lipase that has been modified to give rise to an enzyme variant of the present invention. The parent lipase can be a naturally occurring (wild-type) polypeptide or a variant thereof.

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

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

[0034] For the purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably the Needle program 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 of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows: (Number of identical deoxyribonucleotides × 100) / (Length of alignment - Total number of gaps in the alignment)

[0035] Stability: The stability of the lipase variants of the present invention may be represented as the residual activity or residual performance of the lipase during or after exposure to various test conditions (stress conditions), such as storage in a detergent composition in the presence of different components such as various temperatures, various pHs, proteases, chemicals, and / or oxidizing substances, or during use within a washing process. The stability of the lipase variant can be measured relative to the known activity or performance of the parent lipase, for example, the lipase represented by SEQ ID NO: 2, or alternatively, relative to the known activity or performance of the lipase variant when first added to a detergent composition stored at low temperature or frozen, or relative to a lipase variant stored at low temperature or frozen (non-stress conditions).

[0036] Subarray: The term "subarray" means a polypeptide in which one or more (e.g., several) nucleotides are absent from the 5' and / or 3' ends of positions 1 to 807 of Array No. 1; here, the subarray encodes a fragment having lipase activity. In one aspect, the subarray of the present 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 coding sequence.

[0037] Variant: The term "variant" means a polypeptide having lipase activity that contains modifications, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means that the amino acid occupying a position has been removed; insertion means adding one amino acid adjacent to and immediately following the amino acid occupying a position. The variant of the present invention has 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" means, for a probe having a length of at least 100 nucleotides, 42°C in 5×SSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, and prehybridization and hybridization in 50% formamide, followed by standard Southern blotting for 12 to 24 hours. The carrier material is finally washed 3 times at 70°C for 15 minutes each using 2×SSC, 0.2% SDS.

[0039] Ultra-low stringency conditions: The term "ultra-low stringency conditions" means, for a probe of at least 100 nucleotides in length, 42 °C in 5× SSPE, 0.3% SDS, 200 micrograms / ml fragmented and modified salmon sperm DNA, and prehybridization and hybridization in 25% formamide, followed by standard Southern blotting for 12 - 24 hours. The carrier material is finally washed 3 times at 45 °C for 15 minutes each using 2× SSC, 0.2% SDS.

[0040] Wild-type lipase: The term "wild-type" lipase means a lipase expressed by a naturally occurring microorganism such as bacteria, yeast, filamentous fungi, etc. found in nature.

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

[0042] Identification of the corresponding amino acid residues in the lipase of the other party can be determined by alignment of multiple polypeptide sequences using several computer programs including, but 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] When other enzymes have diverged from the polypeptide of SEQ ID NO:2 and their relatedness cannot be detected by comparison based on traditional sequences (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used. By searching a database using a search program that utilizes a probabilistic representation (profile) of the polypeptide family, higher sensitivity in sequence-based searches can be achieved. For example, the PSI-BLAST program has the ability to generate a profile through an iterative database search process and detect remote homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even higher sensitivity can be achieved if a family or superfamily in the polypeptide has one or more representatives in the protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment profile, and solvation potential) as input to a neural network that predicts the structural fold for the query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align the sequence of unknown structure with superfamily models present in the SCOP database. These alignments can then be used to create a homology model for the polypeptide, and such models can be evaluated for accuracy using various 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 superfamilies of proteins are structurally aligned and those alignments are accessible and downloadable. Two or more protein structures can be aligned using various algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), and in addition, it is possible to utilize the execution of these algorithms to search a structural database having the structure of interest in order to find promising structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0045] In the description of the variants of the present invention, the following nomenclature is applied to facilitate reference. The recognized one-letter or three-letter amino acid abbreviations of the IUPAC are used.

[0046] Substitution. In amino acid substitutions, the following nomenclature is used for the original amino acid, position, and substituted amino acid. Thus, the substitution of threonine with alanine at position 226 is named "Thr226Ala" or "T226A". Multiple mutations are separated by attaching the symbol ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represents the substitution of glycine (G) with arginine (R) and the substitution of serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0047] Deletion. In amino acid deletions, the following nomenclature is used for the original amino acid, position, * For example, the deletion of glycine at position 195 is named "Gly195 * " or "G195 * ". Multiple deletions are separated by attaching the symbol ("+"), for example, "Gly195* +Ser411 * 」 or 「G195 * +S411 * 」 may be mentioned.

[0048] Insertion. In amino acid insertion, the following nomenclature is used for the original amino acid, position, original amino acid, and inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is named 「Gly195GlyLys」 or 「G195GK」. The insertion of multiple amino acids is named as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2, etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is shown as 「Gly195GlyLysAla」 or 「G195GKA」.

[0049] In such cases, the inserted amino acid residues are numbered by attaching a lowercase letter to the position number of the amino acid residue preceding the inserted amino acid residue. Therefore, in the above example, the sequence is as follows.

[0050]

Table 1

[0051] Multiple modifications. Variants containing multiple modifications are separated by attaching a symbol (「+」). For example, 「Arg170Tyr+Gly195Glu」 or 「R170Y+G195E」 represents the substitution 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 the substitution of arginine with tyrosine or glutamic acid at position 170. Therefore, 「Tyr167Gly,Ala+Arg170Gly,Ala」 represents the following variants: 「Tyr167Gly+Arg170Gly」, 「Tyr167Gly+Arg170Ala」, 「Tyr167Ala+Arg170Gly」, and 「Tyr167Ala+Arg170Ala」 refer to.

Mode for Carrying Out the Invention

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

[0054] Variant The present invention provides a variant that is a variant of a parent lipase having lipase activity, having at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and containing 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 variant has at least 60% but less than 100% sequence identity with SEQ ID NO: 2 and contains 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 variant further contains one or both substitutions at positions corresponding to T231R and / or N233R.

[0056] Preferred variants include a substitution at the position corresponding to G23S. Preferred variants include a substitution at the position corresponding to D27N. Preferred variants include a substitution at the position corresponding to A40l. Preferred variants include a substitution at the position corresponding to F51I,L. Preferred variants include a substitution at the position corresponding to E56R. Preferred variants include a substitution at the position corresponding to D57N. Preferred variants include a substitution at the position corresponding to V60E,K. Preferred variants include a substitution at the position corresponding to K98I. Preferred variants include a substitution at the position corresponding to N101D. Preferred variants include a substitution at the position corresponding to R118F. Preferred variants include a substitution at the position corresponding to G163S. Preferred variants include a substitution at the position corresponding to Y220F. Preferred variants include a substitution at the position corresponding to T244E. Preferred variants include a substitution at the position corresponding to P256T.

[0057] Preferred variants include one or more substitutions at the position corresponding to F51I,L, E56R and / or R118F.

[0058] In a preferred embodiment, the variant of the present invention includes any one of the following substitution sets.

[0059] [Table 2]

[0060] In one embodiment, the variant includes a substitution at the position corresponding to T231R+N233R, and one or more (e.g., several) substitutions at the 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, 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.

[0062] In a preferred embodiment, the variant comprises substitutions at positions 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.

[0063] In a more preferred embodiment, the variant comprises 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.

[0064] In an even more preferred embodiment, the variant comprises 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.

[0065] In an even more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+R118F+T231R+N233R, and 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 has 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.

[0067] In another more preferred embodiment, the variant has substitutions at positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R, and 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 has 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.

[0069] In an even more preferred embodiment, the variant has substitutions at positions corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R, and 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 has 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.

[0071] In an even more preferred embodiment, the variant contains 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.

[0072] In an even more preferred embodiment, the variant contains 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.

[0073] In a more preferred embodiment, the variant contains 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. In an even more preferred embodiment, the variant contains 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, V60EK, N101D, Y220F, and T244E.

[0074] In an even more preferred embodiment, the variant contains 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.

[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, and 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 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. In a further preferred embodiment, the variant comprises 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.

[0077] Particularly preferred embodiments are the following sets 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; A40I + G163S + E56R + R118F + T231R + N233R; A40I + E56R + R118F + T231R + N233R + P256T; F51L + D57N + E56R + R118F + T231R + N233R; F51L + K98I + E56R + R118F + T231R + N233R; F51L + G163S + E56R + R118F + T231R + N233R; F51L + E56R + R118F + T231R + N233R + P256T; D57N + K98I + E56R + R118F + T231R + N233R; D57N + G163S + E56R + R118F + T231R + N233R; D57N + E56R + R118F + T231R + N233R + P256T; K98I + G163S + E56R + R118F + T231R + N233R; 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 It includes variants containing substitutions at positions corresponding to one of the following.

[0078] The lipase variant of the present invention has a sequence identity of 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%, or at least 99% with respect to the parent lipase, but less than 100%.

[0079] In a preferred embodiment, the variant of the present invention has a sequence identity of 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%, or at least 99% with respect to SEQ ID NO: 2, but less than 100%.

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

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

[0082] The lipase variant of the present invention may further include one or more additional substitutions at one or more (e.g., several) other positions.

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

[0084] Examples of conservative substitutions are included among groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that 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 change is a property such that the physicochemical properties of the polypeptide are modified. For example, the amino acid change may improve the thermal stability of the polypeptide, modify substrate specificity, change the optimal pH, and the like.

[0086] The 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, a single alanine mutation is introduced into 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 activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure as measured by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling in relation to mutations of putative contact site amino acids. See, for example, 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 an alignment with related polypeptides.

[0087] The 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% of the 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%, 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 with (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 ultra-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 may be selected from the group consisting of.

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

[0090] In one aspect, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 2 by at most 40 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40.

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

[0092] In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 2 that comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of 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 hybridizes, under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions, to (i) the polypeptide coding sequence of SEQ ID NO: 1, (ii) a polynucleotide encoded by a polynucleotide that hybridizes to the complete complement of (i) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

[0095] To design nucleic acid probes to identify and clone DNA encoding parents derived from strains of different genera or species according to methods well known in the art, 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. In particular, such probes can be used to hybridize with genomic DNA or cDNA of target cells according to standard Southern blotting procedures to identify and isolate the corresponding genes therein. Such probes may be significantly shorter than the full sequence, but should be at least 15 nucleotides in length, for example, at least 25 nucleotides, at least 35 nucleotides, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probe is typically labeled (e.g., with 32P, 3H, 35S, biotin, or avidin) to detect the corresponding gene. Such probes are encompassed by the present invention.

[0096] Genomic DNA or cDNA libraries prepared from such other strains may be hybridized with the above probes and screened for DNA encoding the parents. 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 the separated DNA may be transferred to nitrocellulose or other suitable carrier materials and immobilized. Carrier materials are used in Southern blots to identify clones or DNA that hybridize with SEQ ID NO: 1 or a subsequence thereof.

[0097] As an object of the present invention, hybridization indicates that a polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1; (ii) the polypeptide coding sequence of SEQ ID NO: 1; (iii) its full-length complement; or (iv) a subsequence thereof under ultra-low to ultra-high stringency conditions. The target molecule to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.

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

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

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

[0101] The parent lipase may be a fusion polypeptide or a cleavable fusion polypeptide in which another polypeptide is fused to the N-terminus or C-terminus of the polypeptide of the present invention. The fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating coding sequences encoding polypeptides such that they are in-frame and the expression of the fusion polypeptide is under the control of the same promoter and terminator. The fusion polypeptide may also be constructed using intein technology such that the fusion polypeptide is created 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 include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved and the two polypeptides are released. Examples of cleavage sites include, but are not limited to, those disclosed 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., 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 microorganisms of any genus. For the purposes of the present invention, the term "obtained from" when used herein in connection with a given source means that the parent encoded by the polynucleotide is made by the source or by the strain in the case where a polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

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

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

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

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

[0108] In another aspect, 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, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,It may be a lipase of Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria.

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

[0111] In another aspect, 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, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusariumreticulatum), Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex 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, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, ThielaviaIt is a lipase of Thermomyces lanuginosus, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

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

[0113] In the above species, it will be understood that the present invention includes, in addition to both the perfect and imperfect states, other taxonomic equivalents, such as anamorphs, regardless of the known species names. Those skilled in the art will readily understand the equivalence of the appropriate equivalents.

[0114] Strains of these species are readily publicly available 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., contaminants, compost, water, etc.), or the DNA sample may be obtained directly from natural materials (e.g., contaminants, compost, water, etc.) using the above probes. Techniques for directly isolating DNA from microorganisms and natural habitats are well known in the art. Next, the polynucleotide encoding the parent may also be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected using a probe, the polynucleotide can be isolated or cloned by utilizing 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 mutant 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 a mutant having lipase activity and having one of the desired properties described above as compared to the parent lipase; and (c) recovering the mutant.

[0117] Mutants 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, etc.

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

[0119] Site-directed mutagenesis can be achieved in vitro by PCR, which involves the use of oligonucleotide primers having the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves cleavage by a restriction enzyme at a site within a plasmid containing a polynucleotide encoding the parental lipase, followed by ligation of an oligonucleotide having a mutation in the polynucleotide. Usually, since the restriction enzyme that digests the plasmid and the oligonucleotide is the same, it is possible to ligate the sticky ends of the plasmid and the insert to each other. See, for example, 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, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0121] In the present invention, any site-directed mutagenesis method can be used. Many commercially available kits are available and can be used to prepare mutants.

[0122] Synthetic gene construction requires the in vitro synthesis of polynucleotide molecules designed to encode the polypeptide of interest. Gene synthesis can be performed using several techniques, such as the multiple microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques where oligonucleotides are synthesized and constructed 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 related screening methods, e.g., 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-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

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

[0125] Semi-synthetic gene construction is achieved by combining synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. The semi-synthetic composition is typically represented by a process that utilizes synthesized polynucleotide fragments in combination with PCR technology. Thus, while defined regions of a gene may be newly synthesized, other regions may be amplified using site-directed mutagenesis primers, while other regions may be subjected to error-prone PCR or non-error-prone PCR amplification. The polynucleotide partial sequences may then be shuffled.

[0126] Polynucleotide The present invention also relates to an isolated polynucleotide encoding a lipase variant of the present invention. In certain embodiments, the present invention relates to a nucleic acid construct comprising the polynucleotide of the present invention. In certain embodiments, the present invention relates to an expression vector comprising the polynucleotide of the present invention. In certain embodiments, the present invention relates to a host cell comprising the polynucleotide of the present invention. In certain embodiments, the present invention relates to a method for producing a lipase variant, comprising: (a) culturing the host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.

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

[0128] The polynucleotide may be manipulated in various ways to effect the expression of the variant. Manipulation of the 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 array may be a promoter that is a polynucleotide recognized by a host cell for the expression of a polynucleotide. The promoter has a transcriptional control array that mediates the expression of the variant. The promoter may be any polynucleotide that exhibits transcriptional activity within the host cell, including mutant, truncated, and hybrid promoters, and may also be obtained from a gene encoding an extracellular or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0130] Examples of promoters suitable for inducing transcription of the nucleic acid constructs of the invention in bacterial host cells are 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 the prokaryotic beta - lactamase gene (Villa - Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727 - 3731), and promoters derived from the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21 - 25). Further promoters are described in Gilbert et al., 1980, Scientific American 242:74 - 94; and Sambrook et al., 1989 (supra) in "Useful Proteins from Recombinant Bacteria". Examples of tandem promoters are disclosed in WO 99 / 43835 pamphlet.

[0131] Examples of promoters suitable for inducing transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells are 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 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 cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reeseiPromoters obtained from genes in 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 an Aspergillus neutral alpha-amylase gene in which the untranslated leader is replaced by the untranslated leader from the Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader is replaced by the untranslated leader from the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and truncated mutants, and their hybrid promoters.

[0132] In yeast hosts, useful promoters are obtained from genes in 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 in 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 the 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 in bacterial host cells are obtained from genes in Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

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

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

[0137] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene which enhances the 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 an untranslated leader region of mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' end 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 in Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0141] Leaders suitable 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 that is an operably linked sequence at the 3' end of the variant coding sequence, and when transcribed, is recognized by the host cell as a signal for adding 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 in Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0144] A polyadenylation sequence useful for yeast host cells is 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 cell's secretory pathway. The 5' end of the coding sequence of the polynucleotide may essentially have a signal peptide coding sequence naturally linked to a segment of the coding sequence encoding the variant within the translation reading frame. Alternatively, the 5' end of the coding sequence may have a signal peptide coding sequence that is foreign to the coding sequence. If the coding sequence does not naturally have a signal peptide coding sequence, a foreign signal peptide coding sequence may be required. Alternatively, to enhance the secretion of the variant, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of the host cell may be used.

[0146] Signal peptide coding sequences effective for bacterial host cells are signal peptide coding sequences obtained from the genes in Bacillus NCIB11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additionally, signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0147] Signal peptide coding sequences effective for filamentous fungal host cells are signal peptide coding sequences obtained from genes in 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] Signal peptides useful in yeast host cells are obtained from genes in Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992 (supra).

[0149] The control array may also be a propeptide coding array encoding a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or in some cases a zymogen). The propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding array may be obtained from genes in 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 also be desirable to add control sequences that regulate the expression of the variant with respect to the growth of the host cell. Examples of control systems are those that result in the turning on or off of gene expression in response to chemical or physical stimuli, including the presence of a regulatory compound. Control systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or the 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 control sequences are those that enable gene amplification. In eukaryotic systems, these control sequences include the dihydrofolate reductase gene amplified in the presence of methotrexate and the metallothionein gene amplified using heavy metals. In these cases, the polynucleotide encoding the variant will be operably linked to the control sequence.

[0152] Expression vector The present invention also relates to a recombinant expression vector comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcription and translation termination signals. To produce a recombinant expression vector that may include one or more beneficial restriction sites to allow for the insertion or substitution of the polynucleotide encoding the variant at such sites, various nucleotide and control sequences may be ligated together. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. When constructing an expression vector, the coding sequence is positioned within the vector such that the coding sequence is operably linked to an appropriate control sequence for expression.

[0153] A recombinant expression vector can advantageously be one that can follow recombinant DNA methods and can be any vector (e.g., plasmid or virus) that can bring about the expression of a polynucleotide. The choice of vector will typically depend on the compatibility between the vector and the host cell into which the vector is to be introduced. The vector can be a linear or circular plasmid.

[0154] The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, and its replication does not depend on chromosomal replication, such as a plasmid, episome, minichromosome, or artificial chromosome. The vector can contain any means for ensuring self - replication. Alternatively, the vector can be one that is integrated into the genome when introduced into the host cell and is replicated together with the chromosome into which it is integrated. Further, a single vector or plasmid, or two or more vectors or plasmids having all the DNA to be introduced into the host cell's genome, or a transposon together, can be used.

[0155] The vector preferably has one or more selectable markers that enable easy selection of transformed cells, transfected cells, transduced cells, or similar cells. A selectable marker is a gene whose product confers biocide or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, etc.

[0156] Examples of bacterial selectable markers include the Bacillus licheniformis or Bacillus subtilis dal gene, or markers conferring antibiotic resistance, such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance. Markers suitable for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers used 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 use of the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae, and the bar gene of Streptomyces hygroscopicus is preferred.

[0157] The vector preferably has elements that allow for integration of the vector into the genome of the host cell or autonomous replication independent of the genome of the vector within the cell.

[0158] In the case of integration into the host cell genome, the vector may depend 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 induce integration into the host cell genome by homologous recombination at an exact position in the chromosome. To increase the likelihood of integration at an exact position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, having 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 homologous to the target sequence in the host cell genome. Furthermore, the integration element may be a non-coding or coding polynucleotide. On the other hand, the vector may be integrated into the host cell genome by non-homologous recombination.

[0159] In autonomous replication, the vector may further contain an origin of replication that enables the vector to replicate autonomously within the host cell in question. The origin of replication may be any plasmid replicon that mediates autonomous replication functioning within the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

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

[0161] Examples of origins of replication used within 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 useful origins of replication 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 plasmids or vectors containing the gene can be made according to the methods disclosed in WO 00 / 24883.

[0163] To increase the generation of mutants, two or more copies of the polynucleotide of the present invention may be inserted into the host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including a selectable marker gene amplifiable together with the polynucleotide, when cells having amplified copies of the selectable marker gene and thereby additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selectable agent.

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

[0165] Host cell The present invention also relates to a recombinant host cell comprising a polynucleotide encoding a mutant of the present invention operably linked to one or more regulatory sequences that induce the generation of the mutant of the present invention. When a construct or vector containing the polynucleotide is introduced into a host cell, the construct or vector is maintained as an integrated chromosomal entity or as a self-replicating extrachromosomal vector as described above. The term "host cell" includes 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 often depend on the gene encoding the mutant and its source.

[0166] The host cell may be any cell useful in the recombinant production of variants, such as a prokaryotic or eukaryotic cell.

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

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

[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] The 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 accomplished 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 accomplished 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 accomplished 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 accomplished 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 accomplished 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 a host cell 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 the Oomycota and all mitosporic fungi (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 yeasts belonging to the ascomycetous yeasts (Endomycetales), basidiomycetous yeasts, and the Fungi Imperfecti (Blastomycetes). Since the classification of yeasts may change in the future, for the purposes of the present invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0175] The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, for example, a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0176] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include 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 unicellular thalli and carbon catabolism may be fermentative.

[0177] The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[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 gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bacteridioides (Fusariumbactridioides), Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, 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] The fungal cells may be transformed by processes including protoplast formation, protoplast transformation, and cell wall regeneration, in essentially known ways. Methods suitable for the transformation of Aspergillus and Trichoderma host cells are described in European Patent No. 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 International Publication No. 96 / 00787 pamphlet. 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, pp182-187, 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] Production method The present invention also relates to a method for producing a lipase mutant of the present invention, comprising: (a) culturing the host cell of the present invention under conditions suitable for the expression of the mutant; and (b) recovering the mutant.

[0181] The host cell is cultured in a nutrient medium suitable for the production of the mutant using methods known in the art. For example, the cells may be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in a laboratory or industrial fermenter carried out in a suitable medium or under conditions that allow for the expression and / or isolation of the mutant. The culture is carried out in a suitable nutrient medium containing a carbon and nitrogen source 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 mutant is secreted into the nutrient medium, the mutant can be recovered directly from the medium. If the mutant is not secreted, it can be recovered from the cell lysate.

[0182] The mutant may be detected using methods known in the art that are specific to the mutant. 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, an enzyme assay may be used to measure the activity of the mutant as described in the examples.

[0183] The mutant may be recovered using methods known in the art. For example, the mutant 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 variant may be purified by various methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, isoelectric focusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain a substantially pure variant.

[0185] In an alternative embodiment, not only is the variant not recovered, but the host cell of the invention that expresses the variant is used as a source of the variant.

[0186] Composition The present invention also includes a composition comprising a lipase variant of the present invention.

[0187] In certain embodiments, the present invention relates to a composition comprising a variant of a parent lipase having lipase activity, 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 comprises substitutions at positions corresponding to R118F+T231R+N233R, and 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 variant has improved washing performance, reduced odor generation, and / or improved storage stability / longer shelf life / enhanced thermal stability.

[0190] The following non-limiting list of composition components described hereinafter is suitable for use in the present composition, and the methods herein may desirably be incorporated into particular embodiments of the present invention, for example, to assist or enhance cleaning performance for the treatment of a substrate to be cleaned, or to modify the aesthetics of the composition, as in the case of fragrances, colorants, dyes, etc. The level of any such optional component incorporated into any composition is in addition to any of the materials previously listed for incorporation. The exact nature of these additional components and the level of their incorporation will depend on the physical form of the composition and the nature of the cleaning operation in which it is used. The following components are classified 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 since a component may include additional functions.

[0191] Unless otherwise indicated, percent amounts are based on the weight of the composition (wt%). Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes, and enzyme stabilizers, catalyst materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay soil removal / redeposition inhibitors, optical brighteners, antifoaming agents, dyes, tone dyes, fragrances, fragrance delivery systems, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments. In addition to the following disclosure, suitable examples and levels of use of other such components can be found in U.S. Patent No. 5,576,282, U.S. Patent No. 6,306,812, and U.S. Patent No. 6,326,348, which are incorporated herein by reference.

[0192] Therefore, in certain embodiments, the present invention does not contain one or more of the following auxiliary materials: surfactants, soaps, builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalyst materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymeric dispersants, clay soil removal / redeposition inhibitors, optical brighteners, antifoaming agents, dyes, fragrances, fragrance delivery systems, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments. However, when one or more components are present, one or more components as detailed below may be present.

[0193] Surfactants - The compositions according to the invention can comprise a surfactant or a surfactant system, which surfactant can 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 wt%, 0.2 to 40 wt%, 0.5 to 30 wt%, 1 to 50 wt%, 1 to 40 wt%, 1 to 30 wt%, 1 to 20 wt%, 3 to 10 wt%, 3 to 5 wt%, 5 to 40 wt%, 5 to 30 wt%, 5 to 15 wt%, 3 to 20 wt%, 3 to 10 wt%, 8 to 12 wt%, 10 to 12 wt%, 20 to 25 wt%, or 25 to 60 wt%.

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

[0195] Suitable sulfonate detergent surfactants include alkylbenzene sulfonates, in one embodiment, C 10~13It contains alkylbenzene sulfonate. 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®, and other suitable LABs include higher 2-phenyl LABs such as Hyblene®. Suitable anionic detergent surfactants are alkylbenzene sulfonates obtained by the DETAL catalyst process, although other synthetic routes such as HF may also be suitable. In one aspect, the magnesium salt of LAS is used.

[0196] Suitable sulfate detergent surfactants include alkyl sulfates. In one aspect, C 8~18 alkyl sulfates or mainly C 12 alkyl sulfates are included.

[0197] Other suitable sulfate detergent surfactants are alkyl alkoxylated sulfates, in one aspect alkyl ethoxylated sulfates, in one aspect C 8~18 alkyl alkoxylated sulfates, and in another aspect C 8~18 alkyl ethoxylated sulfates. Typically, the average degree of alkoxylation of alkyl alkoxylated sulfates is 0.5 - 20 or 0.5 - 10. Typically, alkyl alkoxylated sulfates are C 8~18 alkyl ethoxylated sulfates with an average degree of ethoxylation of 0.5 - 10, 0.5 - 7, 0.5 - 5, or 0.5 - 3.

[0198] Alkyl sulfates, alkyl alkoxylated sulfates, and alkylbenzene sulfonates may be linear or branched and may or may not be substituted.

[0199] The cleaning surfactant is a middle-chain branched cleaning surfactant, in one aspect, a middle-chain branched anionic cleaning surfactant, in one aspect, a middle-chain branched alkyl sulfate, and / or a middle-chain branched alkylbenzene sulfonate, for example, it may be a middle-chain branched alkyl sulfate. In one aspect, the middle-chain branch is C 1~4 alkyl group, typically a methyl group and / or an ethyl group.

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

[0201] Suitable nonionic cleaning surfactants are selected from the group consisting of: C8-C 18 alkyl ethoxylates such as NEODOL®; C6-C where the alkoxylate units can 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 condensates; C 14 ~C 22 mid-chain branched alcohols; typically C having an average degree of alkoxylation of 1 to 30 14 ~C 22 mid-chain branched alkyl alkoxylates; alkyl polysaccharides, in one aspect, alkyl polyglycosides; polyhydroxy fatty acid amides; ether-capped poly(oxyalkylated) alcohol surfactants; and mixtures thereof.

[0202] Suitable nonionic cleaning surfactants include alkyl polyglycosides and / or alkyl alkoxylated alcohols.

[0203] In one aspect, the nonionic cleaning surfactant includes an alkyl alkoxylated alcohol, in one aspect, C 8~18 alkyl alkoxylated alcohol, for example C 8~18 alkyl ethoxylated alcohol is included, and the alkyl alkoxylated alcohol may 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 alcohol has an average ethoxylation degree of 1 to 10, 1 to 7, further 1 to 5 or 3 to 7 C 8~18 alkyl ethoxylated alcohol may be. The alkyl alkoxylated alcohol may be linear or branched, 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 (PFA), 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), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

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

[0206] Suitable cationic cleansing surfactants are quaternary ammonium compounds having the following general formula: (R)(R1)(R2)(R3)N + X - , wherein R is a straight-chain or branched substituted or unsubstituted C 6~18 alkyl or alkenyl moiety, 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 imparts electrical neutrality. Suitable anions include halides, such as chloride; sulfates; and sulfonates. A suitable cationic cleansing surfactant is mono-C 6~18 alkylmonohydroxyethyldimethyl quaternary ammonium chloride. A highly suitable cationic cleansing surfactant is mono-C 8~10 alkylmonohydroxyethyldimethyl quaternary ammonium chloride, mono-C 10~12Alkylmonohydroxyethyldimethylquaternary ammonium chloride, and mono C 10 is alkylmonohydroxyethyldimethylquaternary ammonium chloride.

[0207] Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, esterquats, and combinations thereof.

[0208] Suitable zwitterionic / amphoteric surfactants include amine oxides and betaines such as alkyldimethylbetaine, sulfobetaine, or combinations thereof. Amine-neutralized anionic surfactants - The anionic surfactants and auxiliary anionic co-surfactants of the present invention may exist in acid form, and the acid form can be neutralized to form surfactant salts desirable for use in the present detergent composition. Typical neutralizing agents include hydroxides, such as metal counterion bases like 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. For example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Neutralization with amines can be carried out in whole or in part. For example, a part of the anionic surfactant mixture can be neutralized with sodium or potassium, and a part of the anionic surfactant mixture can also be neutralized with amines or alkanolamines.

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

[0210] In some cases, a surfactant system comprising one or more anionic surfactants and, in addition thereto, a mixture of one or more nonionic surfactants optionally accompanied by 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 aspect, the surfactant system may comprise a mixture of isoprenoid surfactants represented by the following formulas A and B: [Chemical formula] In the formula, Y is CH2 or absent, and Z can be selected such that the resulting surfactant is selected from the following surfactants: alkyl carboxylate surfactant, alkyl polyalkoxy surfactant, alkyl anionic polyalkoxy sulfate surfactant, alkyl glycerol ester sulfonate surfactant, alkyl dimethylamine oxide surfactant, alkyl polyhydroxy-based surfactant, alkyl phosphate ester surfactant, alkyl glycerol sulfonate surfactant, alkyl polygluconate surfactant, alkyl polyphosphate ester surfactant, alkyl phosphonate surfactant, alkyl polyglycoside surfactant, alkyl monoglycoside surfactant, alkyl diglycoside surfactant, alkyl sulfosuccinate surfactant, alkyl disulfate surfactant, alkyl disulfonate surfactant, alkyl sulfosuccinamate surfactant, alkyl glucamide surfactant, alkyl taurinate surfactant, alkyl sarcosinate surfactant, alkyl glycinate surfactant, alkyl isethionate surfactant, alkyl dialkanolamide surfactant, alkyl monoalkanolamide surfactant, alkyl monoalkanolamide sulfate surfactant, alkyl diglycolamide surfactant, alkyl diglycolamide sulfate surfactant, alkyl glycerol ester surfactant, alkyl glycerol ester sulfate surfactant, alkyl glycerol ether surfactant, alkyl glycerol ether sulfate surfactant, alkyl methyl ester sulfonate surfactant, alkyl polyglycerol ether surfactant, alkyl polyglycerol ether sulfate surfactant, alkyl sorbitan ester surfactant, alkyl ammonioalkane sulfonate surfactant, alkyl amidopropyl betaine surfactant, alkyl allylated quat-based surfactant, alkyl monohydroxyalkyl-di-alkylated quat-based surfactant, alkyl dihydroxyalkyl monoalkyl quat-based surfactant, alkylated quat surfactant, alkyl trimethylammonium quat surfactant, alkyl polyhydroxyalkyloxypropyl quat-based surfactant,Alkyl glycerol ester quaternary surfactant, alkyl glycol amine quaternary surfactant, alkyl monomethyl dihydroxyethyl quaternary ammonium surfactant, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactant, alkyl trimethyl ammonium surfactant, alkyl imidazoline-based surfactant, alkene-2-yl-succinate surfactant, alkyl a-sulfonated carboxylic acid surfactant, alkyl a-sulfonated carboxylic acid alkyl ester surfactant, alpha olefin sulfonate surfactant, alkyl phenol ethoxylate surfactant, alkyl benzene sulfonate surfactant, alkyl sulfobetaine surfactant, alkyl hydroxysulfobetaine surfactant, alkyl ammoniocarboxylate betaine surfactant, alkyl sucrose ester surfactant, alkyl alkanolamide surfactant, alkyl di(polyoxyethylene) monoalkyl ammonium surfactant, alkyl mono(polyoxyethylene) dialkyl ammonium surfactant, alkyl benzyl dimethyl ammonium surfactant, alkyl aminopropionate surfactant, alkyl amidopropyldimethylamine surfactant, or a mixture thereof; also when Z is the charged part, 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+, for example, monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, 1-amino-3-propanol, or a mixture 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 of the present specification can contain soap. Without being bound by theory, as a partial surfactant and as a partial builder, it can be useful for suppressing foam and can further advantageously interact with various cationic compounds of the composition to enhance the softness of the fabric rubbed with the composition of the present invention. It may be desirable to include soap. Any soap known in the art for use in laundry detergents can be utilized. In one embodiment, the composition contains 0 wt% to 20 wt%, 0.5 wt% to 20 wt%, 4 wt% to 10 wt%, or 4 wt% to 7 wt% 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 mixtures of fatty acid soaps having various chain lengths and degrees of substitution. One such mixture is top - palm kernel fatty acid.

[0214] In one embodiment, the soap is selected from free fatty acids. Suitable fatty acids are saturated and / or unsaturated and can be obtained from natural sources such as vegetable or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil, tallow, and fish oil, 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 process).

[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. Examples of 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 C12 - C14 fatty acids, and saturated or unsaturated Cn - C18 fatty acids, and mixtures thereof.

[0216] When present, the weight ratio of the cationic co-surfactant to the fatty acid for softening the fabric 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 levels of the soaps and non-soap anionic surfactants herein are given as weight percentages of the detergent composition specified on an acid form basis. However, as is generally understood in the art, the anionic surfactants and soaps are actually neutralized using sodium, potassium, or alkanolammonium bases such as sodium hydroxide or monoethanolamine.

[0218] Hydrotropes - The compositions of the present invention can include one or more hydrotropes. A hydrotrope is a compound that solubilizes a hydrophobic compound into an aqueous solution (or conversely, a polar substance into a nonpolar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characteristics (so-called amphiphilic like 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 for self-aggregation as seen in surfactants and lipids that form micellar phases, lamellar phases, or other well-defined intermediate phases. Instead, many hydrotropes exhibit a continuous type of aggregation process where the size of the aggregates increases with increasing concentration. However, many hydrotropes change the phase behavior, stability, and colloidal properties of systems containing polar and nonpolar substances, including mixtures of water, oil, surfactants, and polymers. Hydrotropes have been conventionally used in industries ranging from pharmaceuticals, personal care, and food to technical applications. By using hydrotropes in detergent compositions, it is possible to have more concentrated formulations of surfactants without causing undesirable phenomena such as phase separation or high viscosities (such as in the process of compacting liquid detergents by removing water).

[0219] The detergent can contain 0 to 10% by weight, for example, 0 to 5% by weight, 0.5 to 5% by weight, or 3 to 5% by weight of a hydrotrope. Any hydrotrope known in the art used 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 oxide, alcohol and polyglycol ether, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexyl sulfate, and combinations thereof.

[0220] Builder - The compositions of the present invention can include one or more builders, cobuilders, builder systems, or mixtures thereof. When a builder is used, the cleaning composition will typically contain 0 to 65% by weight, at least 1% by weight, 2 to 60% by weight, or 5 to 10% by weight of the builder. In dishwashing compositions, the level of the builder is typically 40 to 65% by weight or 50 to 65% by weight. The composition may substantially not contain a builder; substantially not containing means 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 cobuilder may in particular be a chelating agent that forms a water-soluble complex with Ca and Mg. Any builder and / or cobuilder known in the art for use in detergents can be utilized. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as trisodium phosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 manufactured by Hoechst), ethanolamines such as 2-aminoethane-1-ol (MEA), iminodiethanol (DEA), and 2,2’,2”-nitrilotriethanol (TEA), as well as carboxymethyl inulin (CMI), and combinations thereof.

[0222] The cleaning composition can contain the cobuilder alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of cobuilders include homopolymers or copolymers of polyacrylates, such as poly(acrylic acid) (PAA) or copoly(acrylic acid / maleic acid) (PAA / PMA). Further non-limiting examples include citrate, chelating agents such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenyl succinic acids.As further specific examples, there may be mentioned 2,2’,2”-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodiacetic succinic 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), iminodiacetic 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 sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanol glycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and / or cobuilders are described, for example, in WO 09 / 102854 pamphlet, US Patent No. 5977053 specification.

[0223] Chelating Agents and Crystal Growth Inhibitors - The compositions of the present specification can contain a chelating agent and / or a crystal growth inhibitor. Suitable molecules include chelating agents for copper, iron, and / or manganese, and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), disodium salt hydrate of 1,2-dihydroxybenzene-3,5-disulfonic acid, 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 a crystal growth inhibitor.

[0224] Bleaching Components - Suitable bleaching components 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 bleaching catalysts, photo-bleaching agents, bleach activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, and mixtures thereof. Generally, when a bleaching agent component is used, the compositions of the present invention can contain 0 to 30% by weight, 0.00001 to 90% by weight, 0.0001 to 50% by weight, 0.001 to 25% by weight, or 1 to 20% by weight. Suitable bleaching components include the following: (1) Pre-formed Peracids: Suitable pre-formed peracids include, but are not limited to, compounds selected from the group consisting of pre-formed peracids or salts thereof, typically either a peroxycarboxylic acid or a salt thereof, or a peroxymonosulfonic acid or a salt thereof.

[0225] The pre-formed peracid or its salt is preferably a peroxycarboxylic acid or its salt, which typically has a chemical structure corresponding to the following chemical formula: [Chemical formula] and has wherein R 14 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; the R 14 group may be linear or branched, substituted or unsubstituted; Y is any suitable counterion to achieve charge neutrality, preferably, Y is selected from hydrogen, sodium or potassium. Preferably, R 14 is linear or branched, substituted or unsubstituted C 6~9 alkyl. Preferably, the peracid or its salt is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydodecanoic acid, any of their salts, or any combination thereof. A particularly preferred peracid is phthalimide-peroxy-alkanoic acid, especially ε-phthalimide-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 pre-formed peracid or its salt may also be a peroxymonosulfonic acid or its salt, which typically has a chemical structure corresponding to the following chemical formula: [Chemical formula] and has wherein R 15 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; the R 15 group may be linear or branched, substituted or unsubstituted; Z is any suitable counterion to achieve charge neutrality, preferably, Z is selected from hydrogen, sodium or potassium. Preferably, R 15 is linear or branched, substituted or unsubstituted C 6~9It is alkyl. Preferably, such a bleaching component may be present in the composition of the present invention in an amount of 0.01 to 50% by weight, or 0.1 to 20% by weight.

[0227] (2) Examples of the hydrogen peroxide source include inorganic peroxide salts containing alkali metal salts such as sodium salts of perboric acid (usually monohydrate or tetrahydrate), percarbonic acid, persulfuric acid, perphosphoric acid, and metasilicic acid, and mixtures thereof. In one embodiment of the present invention, it is selected from the group consisting of inorganic peroxide salts such as perboric acid, percarbonic acid, and mixtures thereof. When used, the inorganic peroxide salts are present in an amount of 0.05 to 40% by weight, or 1 to 30% by weight of the whole composition, and are typically incorporated into a composition such as a crystalline solid, but this solid 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 a bleaching component can be present in the composition 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 "bleaching activator" as used herein means a compound that reacts with hydrogen peroxide to form a peracid via perhydrolysis. The peracid thus formed constitutes an activated bleaching agent. Suitable bleaching activators used herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable bleaching activators are those having R-(C=O)-L, wherein R is an optionally branched alkyl group having 6 to 14 carbon atoms or 8 to 12 carbon atoms when the bleaching activator is hydrophobic, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleaching activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives, especially benzenesulfonate. Suitable bleaching 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 pamphlet. A family of bleaching activators is disclosed in EP 624154, and among them, particularly preferred is acetyltriethyl citrate (ATC). Short-chain triglycerides such as ATC or triacetin have the advantage of being environmentally friendly. Furthermore, acetyltriethyl citrate and triacetin have good hydrolysis stability in the product during storage and are also efficient bleaching 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. Any suitable bleach activator can be used, but in one aspect of the invention, the cleaning composition of interest can comprise NOBS, TAED, or mixtures thereof. When present, the peracid and / or bleach activator is generally present in the composition in an amount of from 0.1 to 60 wt%, from 0.5 to 40 wt%, or from 0.6 to 10 wt% based on the fabric and home care compositions. 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 can be present in the composition of the invention in an amount of from 0.01 to 50 wt%, or from 0.1 to 20 wt%.

[0229] The amounts of the hydrogen peroxide source and the peracid or bleach activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to the peracid is 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 of the general formula: R 1 -C(O)-OO-(O)C-R 2 (wherein R 1 contains a straight chain of at least 5 carbon atoms and optionally one or more substituents (e.g., -N + (CH3)3, -COOH or -CN) and / or one or more interrupting moieties (e.g., -CONH- or -CH=CH-) inserted between adjacent carbon atoms of the alkyl group, C6-C 18 alkyl, preferably C6-C 12 alkyl group; R 2 represents an aliphatic group compatible with the peroxide moiety, such that R 1 and R 2 together contain a total of 8 to 30 carbon atoms) are selected from diacyl peroxides. In a preferred aspect, R 1 and R 2 are straight-chain unsubstituted C6-C 12 alkyl chains. Most preferably, R 1 and R2 is the same. R 1 and R 2 Both of the diacyl peroxides in which are C6-C12 alkyl groups are particularly preferred. Preferably, at least one of the R groups (R1 or R2), most preferably only one of them, is at the alpha position, or preferably at both the alpha and beta positions, or most preferably at the alpha, beta, and gamma positions, without any branches or pendant rings. In a further preferred embodiment, preferably, the hydrolysis of the R1 acyl group is rapid to produce a peracid, while the hydrolysis of the R2 acyl group is slow, so that the DAP can be made asymmetric.

[0231] The tetraacyl peroxide bleaching species is preferably of the general formula: R 3 -C(O)-OO-C(O)-(CH2)n-C(O)-OO-C(O)-R 3 (wherein R 3 represents a C1-C9 alkyl group or a C3-C7 alkyl group; n represents an integer from 2 to 12 or from 4 to 10 (including both end values)) and is selected from tetraacyl peroxides.

[0232] Preferably, the diacyl and / or tetraacyl peroxide bleaching species are present in an amount sufficient to supply at least 0.5 ppm, at least 10 ppm, or at least 50 ppm based on the weight of the cleaning liquid. In a preferred embodiment, the bleaching species are present in an amount sufficient to supply 0.5-300 ppm, 30-150 ppm based on the weight of the cleaning liquid.

[0233] Preferably, the bleaching component contains a bleaching catalyst (5 and 6).

[0234] (5) Preferred organic (non-metal) bleaching catalysts include those that can accept an oxygen atom from a peracid and / or its salt and transport the oxygen atom to a substrate that can be oxidized. Preferred bleaching catalysts include, but are not limited to, the following: 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] Preferred iminium cations and polyions include, but are not limited to, the following: 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. Patent No. 5,360,569 (e.g., Column 11, Example 1); and N-octyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in U.S. Patent 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. Patent 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. Patent No. 5,817,614 (e.g., Column 32, Ex. V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfoxy)propyl]-3,4-dihydroisoquinolinium, an inner salt prepared as described in International Publication No. 05 / 047264 (e.g., page 18, Ex. 8); and 2-[3-[(2-butyloctyl)oxy]-2-(sulfoxy)propyl]-3,4-dihydroisoquinolinium, an inner salt.

[0237] Suitable modified amino acid oxygen transport catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can be produced according to the method described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amino acid 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", which is 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", which is 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", which is prepared according to the method described in U.S. Patent 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 produced 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", which is prepared according to the method described in U.S. Patent No. 6,649,085 (Column 12, Ex. 1).

[0244] Preferably, the bleaching catalyst contains an iminium and / or a carbonyl functional group, and typically can form an oxaziridinium and / or a dioxolane functional group upon receiving an oxygen atom, especially upon receiving an oxygen atom from a peracid and / or its salt. Preferably, the bleaching catalyst contains an oxaziridinium functional group and / or can form an oxaziridinium functional group upon receiving an oxygen atom, especially upon receiving an oxygen atom from a peracid and / or its salt. Preferably, the bleaching component contains a cyclic iminium functional group, and preferably the cyclic moiety has a ring size of 5 to 8 atoms (including the nitrogen atom), preferably 6 atoms. Preferably, the bleaching catalyst contains an allyliminium functional group, preferably a bicyclic allyliminium functional group, preferably a 3,4-dihydroisoquinolinium functional group. Typically, the imine functional group is a quaternary imine functional group, and typically can form a quaternary oxaziridinium functional group upon receiving an oxygen atom, especially upon receiving an oxygen atom from a peracid and / or its salt. In another aspect, the detergent composition has a bleaching component with a logP of 0 or less o / w , a logP of -0.5 or less o / w , a logP of -1.0 or less o / w , a logP of -1.5 or less o / w , a logP of -2.0 or less o / w , a logP of -2.5 or less o / w , a logP of -3.0 or less o / w , or a logP of -3.5 or less o / w . The method for determining logP o / w will be described in detail below.

[0245] Typically, the bleaching component can generate a bleaching species having an X of 0.01 to 0.30, 0.05 to 0.25, or 0.10 to 0.20 SO . The method for determining X SO will be described in detail below. For example, a bleaching component having an isoquinolinium structure can generate a bleaching species having an oxaziridinium structure. In this example, X SO is that of the oxaziridinium bleaching species.

[0246] Preferably, the bleaching catalyst has the following chemical formula: [Chem.] and has a chemical structure corresponding to wherein n and m are independently 0 to 4, preferably both n and m are 0; each R 1 is independently selected from a substituted or unsubstituted radical 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 substituents may be bonded to form a fused aryl, fused carbocyclic, or fused heterocyclic ring; each R 2 is independently selected from a substituted or unsubstituted radical selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylene, heterocycle, alkoxy, arylcarbonyl group, carboxyalkyl group, and amide group; any R 2 may be bonded to any other R 2 to form part of a common ring; any geminal R 2 may be bonded to form a carbonyl; also, any two R 2 may be bonded to form a substituted or unsubstituted fused unsaturated moiety; R 3 is C1 - C 20 substituted or unsubstituted alkyl; R 4 is hydrogen or moiety Q t -A, where Q is branched or unbranched alkylene, t = 0 or 1, and A is an anion group selected from the group consisting of OSO3 - SO3 - CO2 - OCO2 - OPO3 2- OPO3H - and OPO2 - ; R 5 is hydrogen or moiety -CR 11 R 12 -Y-G b -Yc -[(CR 9 R 10 ) y -O] k -R 8 wherein each Y is independently selected from the group consisting of O, S, N-H, or N-R 8 ; each R 6 is independently selected from the group consisting of alkyl, aryl and heteroaryl, said moiety being substituted or unsubstituted, and regardless of whether substituted or unsubstituted, said moiety having less than 21 carbons; each G is independently selected from the group consisting of CO, SO2, SO, PO and PO2; R 9 and R 10 are 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 when considered together, may combine to form a carbonyl; b = 0 or 1; c = 0 or 1, provided that when b = 0, c must be 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 being substituted or unsubstituted; X, if present, is a suitable charge-balancing counterion, and when R 4 is hydrogen, it is preferred that X be present. Suitable X includes, but is not limited to, chloride, bromide, sulfate, methosulfate, sulfonate, p-toluenesulfonate, boron tetrafluoride and phosphate.

[0247] In one embodiment of the present invention, the bleaching catalyst has the following general formula:

Chemical formula

[0248] Preferably, the bleaching component contains, in addition to the bleaching catalyst, a peracid source, particularly an organic bleaching catalyst. The peracid source may be: (a) a pre-formed peracid, (b) preferably a percarbonate, perborate or persulfate (perhydrogensulfate source) combined with a bleach activator; and (c) a peroxidase enzyme and an ester that form a peracid in situ in the presence of water in the fabric or hard surface treatment step.

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

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

[0251] (6) The metal-containing bleaching catalyst - the catalyst component may be provided by a catalyst metal complex. One type of metal-containing bleaching catalyst is, for example, a transition metal cation with a defined bleaching catalyst activity such as a copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation, an auxiliary metal cation with little or no bleaching catalyst activity such as a zinc or aluminum cation, and a metal ion sequestrant having a predetermined stability constant for the catalyst and the auxiliary metal cation, in particular a catalyst system containing ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts. Such catalysts are disclosed in U.S. Patent No. 4,430,243. Preferred catalysts are described in International Publication No. 09 / 839406 pamphlet, U.S. Patent No. 6,218,351 specification, and International Publication No. 00 / 012667 pamphlet. Transition metal catalysts, or ligands that are cross-linked polydentate N-donor ligands are particularly preferred.

[0252] If necessary, the compositions herein can be catalyzed using a manganese compound. Such compounds and usage levels are known to those skilled in the art, for example, there is a manganese-based catalyst disclosed in U.S. Patent No. 5,576,282 specification.

[0253] Cobalt bleaching catalysts useful in the present invention are known, for example, described in U.S. Patent No. 5,597,936 specification; U.S. Patent No. 5,595,967 specification. Such cobalt catalysts can be easily prepared by known methods as taught, for example, in U.S. Patent No. 5,597,936 specification and U.S. Patent No. 5,595,967 specification.

[0254] The compositions of the present invention preferably contain a transition metal complex of a ligand such as bispidone (U.S. Patent No. 7,501,389 specification) and / or a macrocyclic rigid ligand (abbreviation "MRL"). In fact, and without limitation, the compositions and methods of the present invention can be adjusted to provide at least about 1 part per hundred million parts of active MRL species in an aqueous cleaning medium, typically 0.005 - 25 ppm, 0.05 - 10 ppm, or 0.1 - 5 ppm of MRL in the cleaning liquid.

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

[0256] (7) Optical bleaching agent - Suitable optical bleaching agents include, for example, sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, 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 the reaction of the hydrogen peroxide source and the bleach activator), in combination with a bleaching catalyst. Preferred bleaching components include, as described above, a bleaching catalyst, preferably an organic bleaching catalyst.

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

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

[0259] Fabric tone - adjusting agent - The composition can include a fabric tone - adjusting agent. Suitable fabric tone - adjusting agents include dyes, dye - clay conjugates, and pigments. Suitable dyes include small - molecule dyes and polymer dyes. Suitable small - molecule dyes include small - molecule dyes selected from the group consisting of dyes corresponding to the Color Index (C.I.) 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 aspect, suitable small molecule dyes include those selected from the group consisting of Direct Violet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, Acid Violet 50, Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, Acid Blue 90, and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75, Basic Blue 159, their Color Index (Society of Dyers and Colorists, Bradford, UK) numbers, and mixtures thereof. In another aspect, suitable small molecule dyes include those 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, their Color Index (Society of Dyers and Colorists, Bradford, UK) numbers, and mixtures thereof.In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of Acid Violet 17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113 Color Index (Society of Dyers and Colorists, Bradford, UK) numbers, 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 having chromogens copolymerized into the polymer backbone, and mixtures thereof.

[0262] In another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of fabric substantive colorants sold under the name Liquitint® (Milliken), polymeric dyes formed from polymers containing at least one reactive dye and a moiety selected from the group consisting of hydroxyl moieties, primary amine moieties, secondary amine moieties, thiol moieties, and mixtures thereof. In yet another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of carboxymethyl cellulose (CMC) conjugated with Liquitint® Violet CT, Reactive Blue, Reactive Violet, or Reactive Red dyes, such as CMC conjugated with C.I. 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 color tone dyes include the optical brighteners found in WO 08 / 87497 Pamphlet. These optical brighteners can be characterized by the following structure (I): [Chemical formula] In the formula, R1 and R2 can independently be selected from the following: a) [(CH2CR’HO) x (CH2CR”HO) y H] wherein R’ is selected from the group consisting of H, CH3, CH2O(CH2CH2O) 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 to 5; b) R1 = alkyl, aryl, or arylalkyl and R2 = [(CH2CR’HO) x (CH2CR”HO) y H] wherein R’ is selected from the group consisting of H, CH3, CH2O(CH2CH2O) 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 to 5; c) R1 = [CH2CH2(OR3)CH2OR4] and R2 = [CH2CH2(OR3)CH2OR4] wherein R3 is selected from the group consisting of H, (CH2CH2O) z H, and mixtures thereof; and z = 0 to 10; R4 is selected from the group consisting of (C1 - C 16 ) alkyl group, aryl group, and mixtures thereof; and d) R1 and R2 can be independently selected from those obtained by adding 1 to 10 alkylene oxide units to the amino adducts of styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, t-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl hexadecyl ether.

[0264] The preferred brighteners of the present invention can be characterized by the following structure (II):

Chemical formula

[0265] A further preferred brightener of the present invention can be characterized by the following structure (III):

Chemical formula

[0266]

Table 3

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

[0268] Suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of at least one cationic / basic dye and smectite clay, and 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 C.I. Basic Yellow 1-108, C.I. Basic Orange 1-69, C.I. Basic Red 1-118, C.I. Basic Violet 1-51, C.I. Basic Blue 1-164, C.I. Basic Green 1-14, C.I. Basic Brown 1-23, CI Basic Black 1-11, and a clay selected from the group consisting of montmorillonite clay, hectorite clay, and saponite clay, and mixtures thereof.In yet another aspect, suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of montmorillonite basic blue B7 C.I. 42595 conjugate, montmorillonite basic blue B9 C.I. 52015 conjugate, montmorillonite basic violet V3 C.I. 42555 conjugate, montmorillonite basic green G1 C.I. 42040 conjugate, montmorillonite basic red R1 C.I. 45160 conjugate, montmorillonite C.I. basic black 2 conjugate, hectorite basic blue B7 C.I. 42595 conjugate, hectorite basic blue B9 C.I. 52015 conjugate, hectorite basic violet V3 C.I. 42555 conjugate, hectorite basic green G1 C.I. 42040 conjugate, hectorite basic red R1 C.I. 45160 conjugate, hectorite C.I. basic black 2 conjugate, saponite basic blue B7 C.I. 42595 conjugate, saponite basic blue B9 C.I. 52015 conjugate, saponite basic violet V3 C.I. 42555 conjugate, saponite basic green G1 C.I. 42040 conjugate, saponite basic red R1 C.I. 45160 conjugate, saponite C.I. basic black 2 conjugate, and mixtures thereof.

[0269] Suitable pigments include flavanthrone, indanthrone, chlorinated indanthrone having 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide (the imide group may or may not be substituted with a C1-C3-alkyl or phenyl or heterocyclic group, and the phenyl and heterocyclic groups may further carry substituents that do not confer water solubility), anthrapyrimidinecarboxylic acid amide, violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyanine (which may contain up to 2 chlorine atoms per molecule), polychloro-copper phthalocyanine or polybromochloro-copper phthalocyanine (which contains up to 14 bromine atoms per molecule), and pigments selected from the group consisting of mixtures thereof.

[0270] In another embodiment, suitable pigments include pigments selected from the group consisting of ultramarine blue (C.I. Pigment Blue 29), ultramarine violet (C.I. Pigment Violet 15), and mixtures thereof.

[0271] The aforementioned fabric color tone agents can be used in combination (any mixture of fabric color tone agents can be used). Suitable color tone agents are described in detail in U.S. Patent No. 7,208,459. The preferred level of the dye in the composition 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 stage is 1 ppb to 5 ppm, 10 ppb to 5 ppm, or 20 ppb to 5 ppm. In a preferred composition, the concentration of the surfactant is 0.2 to 3 g / l.

[0272] The capsule-composition can contain capsules. In one embodiment, the capsule includes a core and a shell having an inner surface and an outer surface, and the shell encloses the core.

[0273] In one aspect of the capsule, the core can include a material selected from the group consisting of perfume; brightening agent; dye; insect repellent; silicone; wax; flavor; vitamins; fabric softener; skin care agent, in one aspect paraffin; enzyme; antibacterial agent; bleaching agent; sensation inducer; and mixtures thereof; the shell can include polyethylene; polyamide; polyvinyl alcohol, optionally containing other comonomers; polystyrene; polyisoprene; polycarbonate; polyester; polyacrylate; aminoplast, in one aspect the aminoplast can include polyurea, polyurethane, and / or polyureaurethane, in one aspect the polyurea can include polyoxymethylene urea and / or melamine formaldehyde; polyolefin; polysaccharide, in one aspect the polysaccharide can include alginate and / or chitosan; gelatin; shellac; epoxy resin; vinyl polymer; water-insoluble inorganic substance; silicone; and mixtures thereof.

[0274] In one aspect of the capsule, the core can include a perfume.

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

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

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

[0278] In one aspect, 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 aspect, the capsule core material can include a material selected from the group consisting of flavor raw materials and / or optionally, a pure and / or blended vegetable oil including, for example, castor oil, coconut oil, cottonseed oil, grape seed 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 including esters of vegetable oils, dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl phosphate, and mixtures thereof; linear or branched hydrocarbons including linear or branched hydrocarbons having a boiling point above about 80°C; alkyl biphenyls including partially hydrogenated terphenyl, dialkyl phthalate, monoisopropyl biphenyl, alkylated naphthalenes including dipropyl naphthalene, petroleum spirits including kerosene, mineral oil, and mixtures thereof; aromatic solvents including benzene, toluene, and mixtures thereof; silicone oil; and materials selected from the group consisting of mixtures thereof.

[0280] In one aspect, the capsule wall material can include a suitable resin including a reaction product of an aldehyde and an amine, and suitable aldehydes include formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril, and mixtures thereof. Suitable melamines include methylol melamine, methylated methylol melamine, iminomelamine, and mixtures thereof. Suitable ureas include dimethylol urea, methylated dimethylol urea, urea - resorcinol, and mixtures thereof.

[0281] In one aspect, a suitable formaldehyde scavenger can be used with 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 aspect, the composition can also include an adhesion aid, preferably selected from the group consisting of a cationic polymer or a nonionic polymer. Suitable polymers include cationic starch, cationic hydroxyethyl cellulose, polyvinyl formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene terephthalate, and polymers containing dimethylaminoethyl methacrylate with one or more monomers selected from the group consisting of acrylic acid and acrylamide, optionally.

[0283] Fragrance - In one aspect, the composition contains a fragrance comprising one or more fragrance raw materials selected from the group consisting of: 1,1'-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol, 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; oxacyclohexadecan-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-methylbenzene propanal; beta-methyl-3-(1-methylethyl)benzene propanal; 4-phenyl-2-butanone; 2-methylbutanoic acid, ethyl ester; benzaldehyde; 2-methylbutanoic acid, 1-methylethyl ester; dihydro-5-pentyl-2(3H)furanone; (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal; undecanal; 2-ethyl-alpha,alpha-dimethylbenzene propanal; 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; 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; Phenylmethyl propanoate; Beta-Methylbenzene pentanol; 1,1-Diethoxy-3,7-dimethyl-2,6-octadiene; Alpha,alpha-Dimethylbenzene ethanol; (2E)-1-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; Phenylmethyl acetate; 2-Propenyl cyclohexanepropanoate; 2-Propenyl hexanoate; 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; Methyl 2-[[[2,4(or 3,5)-dimethyl-3-cyclohexene-1-yl]methylene]amino]benzoate; 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-octadien-1-ol; (3Z)-3-Hexenyl 2-hydroxybenzoate; 2-Tridecenenitrile; 4-(2,2-Dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; Tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; 2-Propenyl (2-methylbutoxy)acetate; 3-Methylbutyl 2-hydroxybenzoate; (Z)-1-(2,6,6-Trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; Methyl 2-hexyl-3-oxocyclopentanecarboxylate; 4-Ethyl-alpha,alpha-dimethylbenzene propanal; 3-Cyclohexene-1-carboxaldehyde (3-(4-hydroxy-4-methylpentyl)-; 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.)]-ethanone; 2-Methyl-2H-pyran-2-one, 6-butyltetrahydro-undecanal;Benzene propanal, 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-; Oxirane carboxylic acid, 3-methyl-3-phenyl-, ethyl ester; 2-Oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; Benzene pentanol,.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-indene-6-ol propanoate; 3-Methyl-2-buten-1-ol acetate; (Z)-3-Hexen-1-ol acetate; 2-Ethyl-4-(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol; 4-(Octahydro-4,7-methano-5H-indene-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-Hydroxy-benzoic acid, methyl ester; 2-Hydroxy-benzoic acid, hexyl ester; 2-Phenoxy-ethanol; 2-Hydroxy-benzoic acid, pentyl ester; 2,3-Heptanedione; 2-Hexen-1-ol; 6-Octen-2-ol, 2,6-dimethyl-; Damascone (alpha, beta, gamma, or delta, or mixtures thereof), 4,7-methano-1H-indene-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; linalool (p-t-businal), and cyclopentanone, 2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]- and 1-methyl-4-(1-methylethenyl)cyclohexene, and mixtures thereof.;

[0284] In one aspect, the composition can include encapsulated fragrance particles that include either a water-soluble hydroxyl compound or melamine-formaldehyde or a modified polyvinyl alcohol. In one aspect, the capsule includes (a) at least a partially water-soluble solid matrix that includes one or more water-soluble hydroxyl compounds, preferably starch; and (b) an essential oil encapsulated by the solid matrix.

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

[0286] The polymer-composition can include one or more polymers. Examples include carboxymethyl cellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinyl pyridine-N-oxide), poly(vinyl imidazole), polycarboxylates such as polyacrylates, maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0287] The composition can include one or more amphiphilic cleansing polymers such as a compound 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 composition can include an amphiphilic alkoxylated oil cleaning polymer having a balance of hydrophilicity and hydrophobicity so as to remove oil particles from the fabric and the surface. Certain embodiments of the amphiphilic alkoxylated oil cleaning polymer 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 polyacrylate, are useful herein to provide additional oil removal performance. Such materials are described in WO 91 / 08281 pamphlet and PCT 90 / 01815 specification. Chemically, these materials include polyacrylates having one ethoxy side chain per 7 to 8 acrylate units. This side chain is of the formula -(CH2CH2O) m (CH2) n CH3 (wherein m is 2 to 3 and n is 6 to 12). This side chain is ester-bonded to the polyacrylate "skeleton" to give a "comb" polymer type structure. The molecular weight can vary but is typically in the range of 2,000 to 50,000. Such alkoxylated polycarboxylates can be included in the composition herein at 0.05 wt% to 10 wt%.

[0290] The isoprenoid-derived surfactants of the present invention, as well as their mixtures with other co-surfactants and other auxiliary components, are particularly suitable for use with amphiphilic graft copolymers. Preferably, the amphiphilic graft copolymer comprises (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 a plurality of 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 graft 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 maleate / acrylate random copolymers or polyacrylate homopolymers. In one aspect, 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] Pollutant release polymers - The compositions of the present invention can also include one or more pollutant 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 wherein a, b, and c are from 1 to 200; d, e, and f are from 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, a mono-, di-, tri-, or tetraalkylammonium where the alkyl group is C1-C 18 alkyl or C2-C 10 hydroxyalkyl, or a mixture thereof; R 1 、R 2 、R 3 、R 4 、R 5 、and R 6 are independently selected from H or C1-C 18 n- or iso-alkyl; and R 7 is a straight-chain or branched C1-C 18 alkyl or a straight-chain or branched C2-C 30 alkenyl, or a cycloalkyl having 5 to 9 carbon atoms, or a C8-C 30 aryl group, or a C6-C 30 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 selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, alkyl carboxyalkyl celluloses. In one aspect, the cellulose polymer is selected from the group including carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof. In one aspect, the carboxymethyl cellulose has a degree of carboxymethyl substitution of 0.5 to 0.9 and a molecular weight of 100,000 to 300,000 Da.

[0295] Enzymes - The compositions can include one or more enzymes that provide benefits in washing performance and / or fabric care. 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, phenol oxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mannanase, β - glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase, or mixtures thereof. A typical combination can be an enzyme cocktail that includes, for example, protease and lipase combined with amylase. When present in the composition, the aforementioned additional enzymes can be present at a level of 0.00001 to 2 wt%, 0.0001 to 1 wt%, or 0.001 to 0.5 wt% of enzyme protein, based on the weight of the composition.

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

[0297] In one aspect, if it is a preferred enzyme, it will include cellulase. Suitable cellulases include those of bacterial or fungal origin. Mutants by chemical modification or protein engineering are included. Suitable cellulases include cellulases derived from the genus Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum disclosed in U.S. Patent No. 4,435,307, U.S. Patent No. 5,648,263, U.S. Patent No. 5,691,178, U.S. Patent No. 5,776,757, and WO 89 / 09259 pamphlet.

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

[0299] Examples of 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 aspect, 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. Also included are mutants by chemical modification or protein engineering. The protease may be an alkaline protease such as a serine protease or a metalloprotease. The serine protease may be, for example, of the S1 family such as trypsin, or of the S8 family such as subtilisin. The metalloprotease may be, for example, thermolysin derived from family M4, or other metalloproteases such as those derived from families M5, M7, or M8.

[0301] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Engng. 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 groups, namely, the subtilisin family, the thermitase family, the proteinase K family, the lantibiotic peptidase family, the kexin family, and the pyrrolysin family.

[0302] Examples of subtilases include those derived from Bacillus species such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus, and Bacillus gibsonii, as described in U.S. Patent No. 7,262,042 and WO 09 / 021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN’, subtilisin 309, subtilisin 147, and subtilisin 168, as described in WO 89 / 06279, and protease PD138, as described in (WO 93 / 18140). Other useful proteases may 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., derived from porcine or bovine), and Fusarium proteases as described in WO 89 / 06270, WO 94 / 25583, and WO 05 / 040372, and chymotrypsin proteases derived from Cellumonas, as described in WO 05 / 052161 and WO 05 / 052146.

[0303] Further preferred proteases are, for example, the alkaline protease from Bacillus lentus DSM5483, 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 metalloprotease described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0305] Examples of useful proteases include variants 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 WO 11 / 036264, in particular, when using the numbering of BPN’, 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. More preferably, the subtilase variant may include the following mutations: S3T, V4I, S9R, A15T, K27R, * 36D, V68A, N76D, N87S, R, *97E, A98S, S99G, D, A, S99AD, S101G, M, R S103A, V104I, Y, N, S106A, G118V, R, H120D, N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, K235L, Q236H, Q245R, N252K, T274A (using the numbering of BPN’).

[0306] Suitable commercially available protease enzymes include those sold under the 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 of which are available as Ultra® or Evity® and are sold under (Novozymes A / S), those sold under the names Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean®, and Optimase® (Danisco / DuPont), those sold under the name Axapem™ (Gist-Brocases N.V.), BLAP (the sequence shown in Figure 29 of U.S. Patent No. 5,352,604) and variants thereof (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao.

[0307] In one aspect, a preferred enzyme may be amylase. Suitable amylases may be alpha - amylase or glucoamylase, and may be of bacterial origin or fungal origin. Mutants by chemical modification or protein engineering are included. As amylases, for example, the alpha - amylase obtained from a specific strain of the genus Bacillus, such as Bacillus licheniformis, described in detail in British Patent No. 1296839, may be mentioned.

[0308] Suitable amylases include the amylase having SEQ ID NO: 3 in WO 95 / 10603 pamphlet, or mutants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred mutants are described in SEQ ID NO: 4 of WO 94 / 02597 pamphlet, WO 94 / 18314 pamphlet, WO 97 / 43424 pamphlet, and WO 99 / 019467 pamphlet, and are, for example, mutants 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] As different suitable amylases, the amylase having SEQ ID NO: 6 in WO 02 / 010355 pamphlet, or mutants thereof having 90% sequence identity to SEQ ID NO: 6, may be mentioned. Preferred mutants of SEQ ID NO: 6 are those in which positions 181 and 182 are deleted and position 193 is substituted.

[0310] Other suitable amylases are a hybrid alpha-amylase comprising residues 1 to 33 of an alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 pamphlet and residues 36 to 483 of B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594 pamphlet, or a variant thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase have 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 to 33 of an alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 pamphlet and residues 36 to 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] Further suitable amylases are an amylase having SEQ ID NO: 6 in WO 99 / 019467 pamphlet or a variant thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 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 have deletions at position R181 and G182, or at position H183 and G184.

[0312] Additional 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 pamphlet, 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 having substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants are those having deletions at positions 181 and 182, or positions 183 and 184. The most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those having deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

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

[0314] More preferred amylases are the amylase having SEQ ID NO: 2 in WO 09 / 061380 pamphlet, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having a C-terminal truncation 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 having substitutions at one or more of the following positions: Q87E, R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E, R, N272E, R, S243Q, A, E, D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or deletions at position R180 and / or S181, or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those having the following substitutions: 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 these variants have a truncated C-terminus and optionally further contain a substitution at position 243 and / or deletions at position 180 and / or position 181.

[0315] Other suitable amylases are the alpha - amylase having SEQ ID NO:12 in WO 01 / 66712 pamphlet, 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 pamphlet: 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 are variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K, and R458K, and 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, and variants further having substitutions at all of these positions are most preferred.

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

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

[0318] Suitable lipases and cutinases include those of bacterial or fungal origin. They include mutant enzymes by chemical modification or protein engineering. Examples include lipases from the genus Thermomyces, such as the lipase from T. lanuginosus (former name, Humicola lanuginosa) described in European Patent No. 258068 and European Patent No. 305216, cutinases from the genus Humicola, such as the cutinase from H. insolens (WO 96 / 13580), lipases from strains of the genus Pseudomonas (some of which are now renamed to the genus Burkholderia), such as P. alcaligenes or P. pseudoalcaligenes (European Patent No. 218272), P. cepacia (European Patent No. 331376), Pseudomonas sp. strain SD705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (Lipases derived from Pseudomonas wisconsinensis (WO 96 / 12012), GDSL-type Streptomyces lipases (WO 10 / 065455), cutinases derived from Magnaporthe grisea (WO 10 / 107560), cutinases derived from Pseudomonas mendocina (U.S. Pat. No. 5,389,536), lipases derived from Thermobifida fusca (WO 11 / 084412, WO 13 / 033318), Geobacillus stearothermophilus lipase (WO 11 / 084417), lipases derived from Bacillus subtilis (WO 11 / 084599), and lipases derived from Streptomyces griseus (WO 11 / 150157) and S. pristinaespiralis (WO 12 / 137147) are available.

[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 made by Genencor) and Lipomax™ (formerly made by Gist-Brocades).

[0321] Still other examples are lipases that may be called acyltransferases or perhydrolases, such as acyltransferases having homology to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO 05 / 56782), perhydrolases from the CE7 family (WO 09 / 67279), and variants of M. smegmatis perhydrolase, in particular the S54V variant used in the product Gentle Power Bleach made by 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) comprising a bacterial polypeptide endogenous to a member of the genus Bacillus having a sequence with at least 90%, 94%, 97%, or 99% identity to amino acid SEQ ID NO: 2 in US 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®, Xpect®, and mannanases sold under the trade names Mannaway® (Novozymes) and Purabrite® (Danisco / DuPont).

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

[0325] The non-dusting granules can be manufactured, for example, as disclosed in U.S. Patent No. 4,106,991 and U.S. Patent No. 4,661,452 and, optionally, can be coated by methods known in the art. Examples of wax coating materials include poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molar weight of 1000 - 20000; ethoxylated nonylphenol having 16 - 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol moiety contains 12 - 20 carbon atoms and there are 15 - 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by the fluidized bed technique are shown 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. Protected enzymes can be prepared by the method disclosed in European Patent No. 238,216.

[0326] Dye migration inhibitor - The composition of the present invention can also contain one or more dye migration inhibitors. Suitable polymeric dye migration inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyl oxazolidone, and polyvinylimidazole, and mixtures thereof. When present in the composition, the dye migration inhibitor can be present at a level of 0.0001 wt% to 10 wt%, 0.01 wt% to 5 wt%, or 0.1 wt% to 3 wt%.

[0327] Whitening agent - The composition of the present invention can also contain additional components such as fluorescent whitening agents that can adjust the color tone of the product to be washed.

[0328] The composition can include C.I. Fluorescent Whitening Agent 260 in the alpha crystalline form having the following structure:

Chemical formula

[0329] In one aspect, the whitening agent is a cold water-soluble whitening agent such as C.I. Fluorescent Whitening Agent 260 in the alpha crystalline form. In one aspect, the whitening agent is predominantly in the alpha crystalline form, which typically means that at least 50 wt%, at least 75 wt%, at least 90 wt%, at least 99 wt%, or substantially all of the C.I. Fluorescent Whitening Agent 260 is in the alpha crystalline form.

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

[0331] The composition can contain C.I. Fluorescent Brightener 260 in the beta crystalline form, and the weight ratio of (i) C.I. Fluorescent Brightener 260 in the alpha crystalline form to (ii) the C.I. Fluorescent Brightener in the beta crystalline form can be at least 0.1, or at least 0.6. Japanese Patent No. BE680847 relates to a method for producing C.I. Fluorescent Brightener 260 in the alpha crystalline form.

[0332] The commercial fluorescent brighteners that can be useful in the present invention can be classified into subgroups, including, but not limited to, stilbene, pyrazoline, coumarin, carboxylic acid, methine cyanine, dibenzothiophene-5,5-dioxide, azole, derivatives of 5-membered and 6-membered heterocyclic rings, and various other agents. Examples of such brighteners are disclosed in “The Production and Application of Fluorescent Brightening Agents”, M. Zahradnik, published by John Wiley & Sons, New York (1982). Specific non-limiting examples of the fluorescent brighteners useful in the present composition are those identified in U.S. Patent No. 4790856 and U.S. Patent No. 3646015.

[0333] More preferred brighteners have the following structure:

Chemical formula

[0334] Suitable fluorescent brightener levels include a lower limit level of 0.01 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt% to an upper limit level of 0.5 wt% or 0.75 wt%.

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

[0336] Dispersant - The composition of the present invention can also contain a dispersant. Suitable water - soluble organic materials include acids or their salts of homopolymers or copolymers, where the polycarboxylic acid contains at least two carboxyl groups separated from each other by 2 or fewer carbon atoms.

[0337] Enzyme stabilizer - The enzyme used in the composition can be stabilized by various methods. The enzyme used herein can be stabilized by the presence of a water - soluble source of calcium ions and / or magnesium ions. Examples of common stabilizers include, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, peptide aldehydes, lactic acid, boric acid, or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4 - formylphenylboronic acid. The composition can be formulated as described, for example, in WO 92 / 19709 pamphlet and WO 92 / 19708 pamphlet. In the case of an aqueous composition containing protease, a reversible protease inhibitor such as a boron compound containing borate, 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 the stability. The peptide aldehyde has the formula B2 - B1 - B0 - R (where R is hydrogen, CH3, CX 3、is CHX2 or CH2X, where X is a halogen atom; B0 is a phenylalanine residue having an OH substituent at the p- and / or m-positions; B1 is a single amino acid residue; B2 can be 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 (wherein Z is benzyloxycarbonyl and Ac is acetyl).

[0338] Solvent - 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] Structuring agent / thickener - structured liquids can be internally structured such that the structure is formed by a primary component (e.g., surfactant substances), and / or externally structured by providing a three-dimensional matrix structure using a secondary component (e.g., polymers, clays, and / or silicate substances). The composition can contain from 0.01 to 5 wt%, or from 0.1 to 2.0 wt% of a structuring agent. Structuring agents are typically selected from the group consisting of diglycerides and triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose-based substances, microfibrillated cellulose, hydrophobically modified alkali-swellable emulsions such as Polygel W30 (3VSigma), biopolymers, xanthan gum, gellan gum, and mixtures thereof. Suitable structuring agents include hydrogenated castor oil and its non-ethoxylated derivatives. Suitable structuring agents are disclosed in U.S. Patent No. 6,855,680. Such structuring agents have filamentous structure systems with a range of aspect ratios. Other suitable structuring agents and methods for making them are described in International Publication No. WO 10 / 034736 pamphlet.

[0340] Conditioning agent - The compositions of the present invention can contain high melting point aliphatic compounds. High melting point aliphatic compounds useful herein have a melting point of 25°C or higher and are selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof. It is not intended to include such compounds with low melting points 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] Considering the advantages of providing improved conditioning, such as a smooth feel when applied to wet hair, flexibility and a moisturized feel in dry hair, the high melting point aliphatic compound is included in the composition at levels of 0.1 to 40% by weight, 1 to 30% by weight, 1.5 to 16% by weight, or 1.5 to 8% by weight.

[0342] The composition of the present invention can contain a cationic polymer. The concentration of the cationic polymer in the composition is typically in the range of 0.05 to 3% by weight, 0.075 to 2.0% by weight, or 0.1 to 1.0% by weight. 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 pH of use of the composition (which generally ranges between pH 3 to pH 9, or between 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 generally ranges between 10,000 and 10 million, between 50,000 and 5 million, or between 100,000 and 3 million.

[0343] Suitable cationic polymers used in the composition 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, D.C. (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 the complex coacervate phase in the composition formed by the cationic polymers and anionic, zwitterionic, and / or amphoteric surfactant components described above herein. The complex coacervates of the 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 nonionic polymers as conditioning agents. Polyalkylene glycols having a molecular weight greater than 1000 are useful herein. Those having the following general formula are useful:

Chemical formula

[0347] The concentration of the conditioning agent in the composition should be sufficient to provide the desired conditioning benefits. Such concentrations can vary depending on the conditioning agent, the desired conditioning performance, the average size of the conditioning agent particles, the type and concentration of other components, 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 No. 34,584; U.S. Patent No. 5,104,646; U.S. Patent No. 5,106,609; U.S. Patent No. 4,152,416; U.S. Patent No. 2,826,551; U.S. Patent No. 3,964,500; U.S. Patent No. 4,364,837; U.S. Patent No. 6,607,717; U.S. Patent No. 6,482,969; U.S. Patent No. 5,807,956; U.S. Patent No. 5,981,681; U.S. Patent No. 6,207,782; U.S. Patent No. 7,465,439; U.S. Patent No. 7,041,767; U.S. Patent 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. 10036533 (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 composition of the present invention can also contain, as a conditioning agent, alone or in combination with other conditioning agents such as silicone (described herein), at least one organic conditioning oil in an amount of 0.05 to 3% by weight. Suitable conditioning oils include hydrocarbon oils, polyolefins, and fatty acid esters. Further, those suitable for use in the composition herein are described in U.S. Patent No. 5,674,478 and U.S. Patent No. 4,529,586, or in U.S. Patent No. 5,750,122; U.S. Patent No. 4,507,280; U.S. Patent No. 4,663,158; U.S. Patent No. 4,197,865; U.S. Patent No. 4,217,914; U.S. Patent No. 4,381,919; and U.S. Patent No. 4,422,853.

[0350] Hygiene and Odor - The composition of the present invention can also contain zinc ricinoleate, thymol, quaternary ammonium salts such as Bardac®, polyethyleneimine (Lupasol® manufactured by BASF and its zinc complexes), silver and silver compounds, especially Ag + designed to release or one or more of silver nano-dispersions.

[0351] Probiotics - The composition can contain probiotics such as those described in WO 09 / 043709 pamphlet.

[0352] Foaming Agent - When high foaming properties are desired, C 10 ~C 16 alkanolamides or C 10 ~C 14 alkyl sulfates and other foaming agents can typically be incorporated into the composition at a level of 1 to 10% by weight. C 10 ~C 14Monoethanol and diethanol amides are examples of typical types of such foam boosters. It is also beneficial to use such foam boosters together with high-foaming auxiliary surfactants such as the amine oxides, betaines, and sultaines described above. If desired, water-soluble magnesium and / or calcium salts such as MgCl2, MgSO4, CaCl2, CaSO4, etc. can be added, typically at levels of 0.1 to 2% by weight, to provide further foaming and enhance the oil and grease removal performance.

[0353] Antifoaming agents - Compounds that reduce or suppress the formation of foam can be incorporated into the compositions of the present invention. Foam suppression can be particularly important in so-called "high-concentration washing processes" described in U.S. Patent No. 4,489,455 and U.S. Patent No. 4,489,574, as well as in front-loading washing machines. A wide variety of substances can be used as antifoaming agents, and antifoaming agents are well known to those skilled in the art. See, for example, Kirk Othmer Encyclopedia of Chemical Technology, 3rd Edition, Volume 7, pp. 430-447 (John Wiley & Sons, Inc., 1979). Examples of antifoaming agents 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, aliphatic C 18 ~C 40Examples include ketones (e.g., stearone), N-alkylated aminotriazines, preferably waxy hydrocarbons having a melting point of less than about 100 °C, silicone antifoaming agents, and secondary alcohols. Antifoaming agents are described in U.S. Patent No. 2,954,347; U.S. Patent No. 4,265,779; U.S. Patent No. 4,265,779; U.S. Patent No. 3,455,839; U.S. Patent No. 3,933,672; U.S. Patent No. 4,652,392; U.S. Patent No. 4,978,471; U.S. Patent No. 4,983,316; U.S. Patent No. 5,288,431; U.S. Patent No. 4,639,489; U.S. Patent No. 4,749,740; U.S. Patent No. 4,798,679; U.S. Patent No. 4,075,118; European Patent No. 89307851.9; European Patent No. 150,872; and DOS 2,124,526.

[0354] In the case of any detergent composition used in an automatic washing machine, foam should not be formed to the extent that it overflows from the washing machine. When used, the antifoaming agent is preferably present in an "antifoaming amount". By "antifoaming amount" is meant that the formulator of the composition can select the amount of this foam control agent that will adequately control the foam to result in a low-foaming laundry detergent for use in an automatic washing machine.

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

[0356] The compositions herein can have cleaning activity over a wide range of pH. In certain embodiments, the composition has cleaning activity at pH 4 to pH 11.5. In other embodiments, the composition is 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 can have cleaning activity over a wide range of temperatures, for example, at 10 °C or lower to 90 °C. Preferably, the temperature is also less than 50 °C or less than 40 °C, or less than 30 °C. In certain embodiments, the optimal temperature range of 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 the 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 in particular solid or liquid cleaning compositions and / or treatment compositions. In one aspect, the present invention relates to compositions wherein the form of the composition is selected from regular, compressed, or concentrated liquids; gels; pastes; bar soaps; regular or compressed powders; granular solids; homogeneous tablets or multilayer tablets having two or more layers (same or different phases); pouches having one or more compartments; single or multiple compartment unit dosage forms; or any combination thereof.

[0359] Depending on the form of the composition, components can be physically separated from each other in different layers of compartments or tablets, such as water-soluble pouches. Thereby, negative storage interactions between the components can be avoided. The different elution profiles of each compartment can also delay the elution 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 holding the composition, for example, without releasing the composition from the pouch before contact with water. The pouch is made from a water-soluble film that encloses an internal volume. The internal volume can be divided into compartments of the pouch. Preferred films are polymers, preferably polymers formed into films or sheets. Preferred polymers, copolymers, or their derivatives are selected polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylate, and most preferably polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC). Preferably, the level of polymer in the film, such as PVA, is at least about 60%. The preferred average molecular weight is typically from about 20,000 to about 150,000. The film can also be a blended composition containing a hydrolyzable and water-soluble polymer blend, such as a blend of polylactic acid and polyvinyl alcohol (known under the trade name M8630 sold by MonoSol LLC, Indiana, USA) with plasticizers such as glycerol, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. The pouch can contain a solid laundry washing composition or sub-components and / or a liquid laundry washing composition or sub-components separated by a water-soluble film. The composition of the compartment for the liquid component can be different from that of the compartment containing solids (U.S. Patent Application Publication No. 2009 / 0011970A1).

[0361] Water-soluble film - The compositions of the present invention can also be encapsulated within a water-soluble film. Preferred film materials are preferably polymeric materials. As is 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, polyvinyl pyrrolidone, polyalkylene oxide, acrylamide, acrylic acid, cellulose, cellulose ether, cellulose ester, cellulose amide, polyvinyl acetate, polycarboxylic acid and salts, polyamino acids or peptides, polyamides, polyacrylamides, copolymers of maleic acid / acrylic acid, polysaccharides including starch and gelatin, and natural rubbers 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, polymethacrylate, and most preferably selected from polyvinyl alcohol, polyvinyl alcohol copolymers, and hydroxypropylmethylcellulose (HPMC), and combinations thereof. Preferably, the level of polymer in the pouch material, such as a PVA polymer, is at least 60% by weight. The polymer can have any weight average molecular weight, but is preferably about 1,000 to 1,000,000, about 10,000 to 300,000, 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. The advantage of selecting different films is that the resulting compartments can exhibit different solubility or release characteristics.

[0363] Preferred film materials are PVA films known as MonoSol product numbers M8630, M8900, H8779, as well as those described in U.S. Patent No. 6,166,117 and U.S. Patent No. 6,787,512, and PVA films having corresponding solubility and deformability characteristics.

[0364] The film materials herein can also include 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 and the like.

[0365] Method for making a composition The compositions of the present invention can be formulated into any suitable form and can be prepared by any method selected by the formulator. Non-limiting examples of such methods are the applicant's examples and U.S. Patent No. 4,990,280; U.S. Patent Application Publication No. 20030087791A1; U.S. Patent Application Publication No. 20030087790A1; U.S. Patent Application Publication No. 20050003983A1; U.S. Patent Application Publication No. 20040048764A1; U.S. Patent No. 4,762,636; U.S. Patent No. 6,291,412; U.S. Patent Application Publication No. 20050227891A1; European Patent Application Publication No. 1070115A2; U.S. Patent No. 5,879,584; U.S. Patent No. 5,691,297; U.S. Patent No. 5,574,005; U.S. Patent No. 5,569,645; U.S. Patent No. 5,565,422; U.S. Patent No. 5,516,448; U.S. Patent No. 5,489,392; U.S. Patent No. 5,486,303, all of which are hereby incorporated by reference herein. The compositions of the present invention, or compositions prepared in accordance with the present invention, include, but are not limited to, compositions, granules or powders for treating fabrics, hard surfaces, and other arbitrary surfaces in the fabric and home care areas (e.g., air care including air fresheners and fragrance delivery systems, automotive care, dishwashing, fabric conditioning (including softening and / or freshening), laundry washing, addition and / or care of washing and rinsing, hard surface cleaning and / or treatment including floor and toilet cleaners), multi-purpose detergents or "heavy-duty" detergents in the form of compositions, granules or powders, particularly washing detergents; multi-purpose detergents in the form of liquids, gels, or pastes, particularly so-called heavy-duty liquid types; liquid detergents for fine fabrics; detergents for hand washing dishes or light-duty dishwashing detergents, particularly high-foaming types; dishwashing detergents for dishwashers including various tablet types, granule types, liquid types, and rinse aid types for household and business use; automotive or carpet shampoos, bathroom cleaners including toilet cleaners; and cleaning aids such as bleaching additives, and "soil-attaching" or pretreatment-type substrate-loading compositions such as dryer sheets, including cleaning and / or treatment compositions. Compositions and methods for washing and / or treating textile products and / or hard surfaces, most preferably textile products, are preferred.The composition is preferably a composition used in a pretreatment step or a main washing step of a washing process, most preferably a composition for use in a washing step of textile products.

[0366] As used herein, the term "washing and / or treatment composition for fabrics and / or hard surfaces" refers, unless otherwise indicated, to all-purpose detergents or "heavy-duty" detergents in granular or powder form, particularly washing detergents; all-purpose detergents in liquid, gel, or paste form, particularly so-called heavy-duty liquid types; liquid detergents for fine fabrics; dishwashing detergents for hand washing dishes or light-duty dishwashing detergents, particularly high-foaming types; dishwashing detergents for dishwashers including various tablet, granule, liquid, and rinse aid types for household and business use; liquid cleaning agents and germicides, bathroom cleaners including shampoos for automobiles or carpets and toilet cleaners; fabric conditioning compositions which may be in the form of liquids, solids, and / or dryer sheets and which include softening and / or freshening; and washing aids such as bleach additives and "soil-attaching" or pretreatment-type substrate loading compositions such as dryer sheets, which are a subset of washing and / or treatment compositions. All such compositions applicable may be in standard form, concentrated form, or in a highly concentrated form which may be non-aqueous in certain embodiments.

[0367] Method of use The present invention includes a method for cleaning any surface, including treating a textile or a hard surface, or other surfaces in the fields of fabrics and / or home care. It is contemplated that such cleaning may be in any case, whether on a small scale, for example in a family household, or on a large scale, for example in industrial and professional situations. In one aspect of the present invention, the method preferably comprises contacting a surface to be treated in a pre-treatment step or a main cleaning step of the cleaning process for use in the cleaning process of textiles, or for use in dishwashing including both hand dishwashing and automatic / mechanical dishwashing. In one embodiment of the present invention, among the methods for cleaning and / or treating a surface, a lipase variant and other components are added sequentially. Alternatively, the lipase variant and other components are added simultaneously.

[0368] As used herein, cleaning includes, but is not limited to, scrubbing and mechanical agitation. Cleaning can be performed using a foam composition by applying pressure alternately (pressure / vacuum), as described in WO 08 / 101958 pamphlet, and / or 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 either in the home or in an industrial environment. The cleaning composition of the present invention is ideally suitable 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 kitchenware. The method includes contacting the cleaning composition, which includes at least one embodiment of the applicant's cleaning composition, cleaning additive, or a mixture thereof, with the object to be cleaned. The fabric can include almost any fabric that can be laundered under normal consumer or facility use conditions. The pH of the solution can be from 8 to 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 ratio of water to fabric is typically from 1:1 to 30:1.

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

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

[0371] plant The present invention also relates to a plant, such as a transgenic plant, a plant part, or a plant cell, comprising the polynucleotide of the present invention for expressing and producing a recoverable amount of a variant. The variant may be recovered from the plant or the plant part. Alternatively, the plant or plant part containing the variant may be used so as to be intended to improve one or more food qualities, for example, to improve nutritional value, palatability, and rheological properties, or to destroy non-nutritional factors.

[0372] The transgenic plant can be a dicotyledon (dicot) or a monocotyledon (monocot). Examples of monocotyledonous plants are grasses, such as pasture grass (green grass, Poa), forage grass, such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, such as wheat, rye, barley, oats, rice, sorghum, and corn (maize).

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

[0374] Examples of plant parts are stems, calli, leaves, roots, fruits, seeds, and tubers, and each tissue containing these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, and meristem. Certain plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm, are also considered to be plant parts. Furthermore, any plant cell, regardless of its tissue origin, is considered to be a plant part. Similarly, plant parts such as specific tissues and cells isolated to facilitate the utilization of the present invention are also considered to be plant parts, such as embryos, endosperm, aleurone and seed coats.

[0375] Furthermore, 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 variant may be constructed according to methods known in the art. Briefly, the plant or plant cell is constructed by integrating one or more expression constructs encoding the variant into the plant host genome or chloroplast genome and growing the resulting modified plant or plant cell into a transgenic plant or plant cell.

[0377] The expression construct is, for convenience, a nucleic acid construct comprising a polynucleotide encoding the variant operably linked to appropriate regulatory sequences required for the expression of the polynucleotide in the selected plant or plant part. Furthermore, the expression construct may include a selectable marker useful for identifying the plant cell into which the expression construct has been integrated and a DNA sequence necessary for the introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

[0378] The selection of control sequences, such as promoter and terminator sequences, and optionally signal or transit sequences, is determined, for example, based on the time, place and manner in which expression of the variant is desired. For example, expression of a gene encoding a variant may be constitutive or inducible, or developmental, stage or tissue-specific, and the gene product may be targeted to specific tissues or plant parts, such as seeds or leaves. Control sequences are described, for example, by Tague et al., 1988, Plant Physiology 86:506.

[0379] In constitutive expression, the 35S-CaMV, maize ubiquitin 1, or rice actin 1 promoter 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 derived from storage sink tissues such as seeds, potato tubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or promoters derived from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24:863-878), seed-specific promoters such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant Cell Physiol. 39:885-889), the Vicia faba promoter from the legumin B4 and an unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152:708-711), the promoter from a seed oil body protein (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 pamphlet, may also be used.Furthermore, the promoter may be a leaf-specific promoter, such as the rbcs promoter derived 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 derived 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, the promoter may be induced by abiotic treatments such as temperature, drying, or salt alteration, or by exogenously applied substances that activate the promoter, such as ethanol, estrogen, 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 parts of the expression construct may be selected from those available in the art.

[0382] The nucleic acid constructs are incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-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 (see, for review, Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:15-38) and is also a method for transforming monocotyledonous plants, although other transformation methods may be used for these plants. A method for generating transgenic monocotyledonous plants is particle bombardment of embryogenic calli or developing embryos (fine gold or tungsten particles coated with the transforming DNA) (Christou, 1992, Plant J. 2:275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio / Technology 10:667-674). Alternative methods for transforming monocotyledonous plants are based on protoplast transformation as described by Omirulleh et al., 1993, Plant Mol. Biol. 21:415-428. Further transformation methods include those described in U.S. Patent No. 6,395,966 and U.S. Patent No. 7,151,204 (both of which are incorporated herein by reference in their entirety).

[0384] After transformation, transformants incorporating the expression construct are selected and regenerated into whole plants according to methods well known in the art. The transformation method is often designed with the intention of selectively removing the selection gene, either during regeneration or, for example, during subsequent production using co-transformation of two separate T-DNA constructs or site-specific excision by a specific recombinase of the selection gene.

[0385] In addition to direct transformation of a particular plant genotype using the constructs of the invention, transgenic plants may also be produced by crossing a plant having the construct with a second plant lacking the construct. For example, constructs encoding mutants can be introduced into a particular plant variety by crossing, without the need to directly transform plants of a given variety heretofore. Thus, the invention encompasses not only plants regenerated directly from cells transformed according to the invention, but also the progeny of such plants. As used herein, progeny may refer to progeny of any generation of a parent plant prepared according to the invention. Such progeny may contain a DNA construct prepared according to the invention. Crossing results in the introduction of the transgene into the plant line by outcrossing 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 produced through the process of backcross conversion. For example, plants include those referred to as backcross-converted genotypes, lines, inbreds, or hybrids.

[0387] A gene marker may be used to assist the transfer 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, gene markers may provide data regarding the relative degree of elite germplasm in each progeny of a particular cross. For example, when a plant having a desired trait, which normally has a non-agronomically desirable genetic background, is crossed with an elite parent, a gene marker may be used to select progeny that have not only the trait of interest but also 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 variant of the present invention, comprising: (a) culturing a transgenic plant or plant cell comprising a polynucleotide encoding the variant under conditions that contribute to the production of the variant; and (b) recovering the variant.

[0389] A microcapsule composition comprising a lipase variant of the present invention In this aspect, the present invention relates to a microcapsule composition in which the membrane of the microcapsule is produced by crosslinking a multi-branched polyamine having a molecular weight of more than 1 kDa, wherein the microcapsule contains the lipase variant of the present invention.

[0390] The membrane formed by crosslinking the multi-branched polyamine has the ability to separate the enzyme from (anionic) surfactants in detergents, which are known to be harmful to the stability of the enzyme.

[0391] A critically important parameter when using an enzyme encapsulated in a capsule in a detergent is the ability to release the enzyme immediately after dilution of the detergent with water, for example in the case of a laundry or dishwashing application. The microcapsules of the present invention have excellent properties in this regard and are capable of releasing all of the encapsulated enzyme within 1 minute.

[0392] The microcapsules do not require the presence of a core polymer that should have the ability to release the enzyme upon dilution with water. Further, the present invention does not require an enzyme that should be in a precipitated form within the core of the microcapsule to control mid-course release, as described in WO 97 / 24177 pamphlet.

[0393] The lipase variants of the present invention, optionally other enzymes, are encapsulated in microcapsules having a semipermeable membrane and, when they have a higher water activity inside these capsules than in the liquid detergent (before addition to the liquid detergent), the capsules undergo (partial) disintegration (with water oozing out) when added to the detergent, and thus a more concentrated and more viscous enzyme containing the interior in the capsule remains. The disintegration of the membrane may also result in a decrease in permeability. This can further be exploited by the addition of stabilizers / polymers, especially those that cannot permeate through the membrane. The disintegration and the resulting increase in viscosity reduce / impede the diffusion of undesired components (e.g., surfactants or scavengers) into the capsules and thus enhance the storage stability of the enzyme in the liquid detergent. Components in the liquid detergent that are sensitive to the enzyme (e.g., components that act as substrates for the enzyme) are also protected against degradation by the enzyme. During washing, the liquid detergent is diluted by water, so the water activity is enhanced. Water will diffuse into the capsules here (osmosis). The capsules will swell and the membrane will become permeable to the enzyme, either allowing the capsules to remain or simply rupture and thus release the enzyme. The concept is very efficient in stabilizing the enzyme against undesired components in the liquid detergent and vice versa, protecting enzyme-sensitive components in the liquid detergent from the enzyme.

[0394] Examples of detergent components that are sensitive to enzymes and can be decomposed by enzymes (the related enzymes are in parentheses) include xanthan gum (xantanase), polymers having an ester bond (lipase), hydrogenated castor oil (lipase), perfume (lipase), methyl ester sulfonate surfactant (lipase), cellulose and cellulose derivatives (e.g., CMC) (cellulase), and dextrin and cyclodextrin (amylase).

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

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

[0397] By using the addition of microcapsules to detergents, it is possible to affect the appearance of detergent products, for example, the effect of making them opaque (small microcapsules) or the effect of clearly visible particles (large microcapsules). The microcapsules may also be colored.

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

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

[0400] Microcapsules Microcapsules are typically formed by forming water droplets in a continuous phase that is immiscible with water, i.e., typically by preparing a water-in-oil emulsion, and then by forming a film through interfacial polymerization via the addition of a crosslinking agent. After final curing, by methods known in the art, the capsules can be collected, further rinsed, and formulated. Subsequently, the capsule formulation is added to the detergent.

[0401] The payload to be encapsulated, the main film components, and the final additional components are found in the aqueous phase. In the continuous phase, components that stabilize the water droplets towards coalescence (emulsifiers, emulsion stabilizers, surfactants, etc.) are found, and the crosslinking agent is also added through the continuous phase.

[0402] The emulsion can be prepared by any method known in the art, such as mechanical stirring, the dripping process, membrane emulsification, microfluidics, sonication, etc. In some cases, the 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] Subsequently, the crosslinking agent is typically added to the emulsion directly or, more typically, by preparing a solution of the crosslinking agent in a solvent that is soluble in the continuous phase. The emulsion and the crosslinking agent or the solution thereof can be mixed by the usual methods used in the art, such as by simple mixing or by carefully controlling the flow of the emulsion and the crosslinking agent solution through an in-line mixer.

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

[0405] The capsule may be post-modified, for example, by reacting components on the membrane, in order to prevent or reduce the flocculent precipitation of particles in the detergent, as described in WO 99 / 01534 pamphlet.

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

[0407] The obtained capsules can be further formulated, for example, by adding surfactants to endow the product with desired properties in storage, transportation, and subsequent handling and addition to the detergent. Other microcapsule formulations include rheology modifiers, biocides (e.g., Proxel), acids / bases for pH adjustment (which will also adjust the inside of the microcapsules), and water for water activity adjustment.

[0408] The capsule formation process may include the following steps: - Preparation of the initial water and oil phases, - Water-in-oil emulsion, - Membrane formation by interfacial polymerization, - Optional post-modification, - Optional isolation and / or formulation, - Addition to the detergent and may be included.

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

[0410] The microcapsules described in the present invention are small water spheres having a uniform film around them. The material inside the microcapsules is referred to as the core, internal phase, or filling, while the film may be referred to as the shell, coating agent, or wall. The microcapsules of the present invention have a diameter between 0.5 μm and 2 mm. Preferably, the average diameter of the microcapsules is within the range of 1 μm to 1000 μm, more preferably within the range of 5 μm to 500 μm, even more preferably within the range of 10 μm to 500 μm, even more preferably within the range of 50 μm to 500 μm, and most preferably within the range of 50 μm to 200 μm. Alternatively, the diameter of the microcapsules is within the range of 0.5 μm to 30 μm; or within the range of 1 μm to 25 μm. The diameter of the microcapsules is measured in the oil phase after the polymerization is completed. The diameter of the capsules may vary depending on the water activity of the surrounding chemical environment.

[0411] When used in the present invention, the microencapsulation of enzymes may be carried out by interfacial polymerization, where the two reactants in the polymerization reaction meet at an interface and react rapidly. The basis of this method is the reaction of a polyamine with an acid derivative, usually an acid halide, which acts as a cross-linking agent. The polyamine is preferably substantially water-soluble (when in the free base form). Under the right conditions, a thin mobile film forms rapidly at the interface. One way to carry out the polymerization is to use an aqueous solution of the enzyme and the polyamine emulsified with a non-aqueous solvent (and an emulsifier), and 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 forms immediately at the interface of the emulsion droplets. The polymer film of the microcapsules is typically cationic and thus binds / complexes 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 can equally stabilize an emulsion. The type and amount of surfactant are necessary for the selection regarding the optimal usefulness of the emulsion, particularly with respect to the preparation and physical stability of the emulsion, and stability during dilution and further processing. Physical stability refers to maintaining the emulsion in a dispersed form. Processes such as coalescence, aggregation, adsorption to container walls, sedimentation, and creaming are forms of physical instability and need to be avoided. Examples of suitable surfactants are described in WO 97 / 24177, pages 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 emulsions or multiple emulsions where the dispersed liquid phase is a heterogeneous combination of liquid or solid phases. For example, a water-in-oil double emulsion or multiple emulsion where the aqueous phase itself further contains an emulsified oil phase may be formed; this type of emulsion may be specified as a water-in-oil-in-water (o / w / o) emulsion. Alternatively, a water-in-oil emulsion where the aqueous phase contains a dispersed solid phase, often referred to as a suspension-emulsion, may be formed. Other more complex emulsions can be described. Since it is essentially difficult to describe such systems, the term emulsion is used without necessarily limiting the form of the emulsion or the type and number of phases present to describe both simple emulsions and more complex emulsions.

[0415] Polyamine The rigidity / mobility and permeability of the membrane are mainly affected by the choice of polyamine. The polyamine follows the multi-branched polyamine of the present invention. Each branch, preferably the terminal having a primary amino group, serves as an anchoring point in the membrane network, thereby obtaining the preferred properties of the present invention. The multi-branched polyamine follows a polyamine having more than two branch points and more than two reactive amino groups (able to react with cross-linking agents, i.e., primary and secondary amino groups) of the present invention. The multi-branched polyamine is used as a starting material when the emulsion is prepared (it is not formed in situ from other starting materials). In order to obtain the attractive properties of the present invention, the multi-branched structure of the polyamine needs to be present as a starting material.

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

[0417] In addition to the number of branch points, we have found that the density of reactive amino groups is very important. Substances such as N,N,N',N'-tetrakis-(12-aminododecyl)ethylenediamine, for example, would not be suitable. Neither peptides nor proteins, such as enzymes, would be suitable for membrane formation. Therefore, the multi-branched polyamine is not a peptide or a protein.

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

[0419] In a preferred embodiment, the multi-branched polyamine is polyethyleneimine (PEI) and its modified forms having more than 2 branch points and more than 2 reactive amino groups; wherein the reactive amino groups account for at least 15% of the molecular weight of the PEI, such as more than 20% or more than 25%. Preferably, the molecular weight of the PEI is at least 1 kDa.

[0420] To prepare the microcapsules described in the present invention, combinations of different multi-branched polyamines may be used.

[0421] The advantageous properties of the microcapsules of the present invention (e.g., enzyme storage stability, reduction of enzyme leakage, reduction of detergent component influx) 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 the 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 the multi-branched polyamine when preparing the microcapsules of the present invention.

[0422] Crosslinking agent When used in the present invention, the crosslinking agent is a molecule having at least two groups / sites capable of reacting with amines to form covalent bonds.

[0423] The crosslinking agent is preferably oil-soluble and can be in the form of an acid anhydride or acid halide, preferably an acid chloride. For example, it can be adipoyl chloride, sebacoyl chloride, dodecanedioyl chloride, phthaloyl chloride, terephthaloyl chloride, isophthaloyl chloride, or trimelloyl chloride, but preferably the crosslinking agent is terephthaloyl chloride or trimelloyl 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 protease, amylase, lipase, cellulase, mannanase, pectinase, deoxyribonuclease, laccase, peroxidase, haloperoxidase, perhydrolase, and combinations thereof.

[0425] The enzyme in the composition of the present invention or encapsulated in the microcapsule may be, for example, one or more enzymes suitable for use in laundry or dishwashing detergents (detergent enzymes), such as protease (e.g., subtilisin or metalloprotease), lipase, cutinase, amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xantanase, xylanase, deoxyribonuclease, perhydrolase, oxidoreductase (e.g., laccase, peroxidase, peroxigenase and / or haloperoxidase). Preferred detergent enzymes are protease (e.g., subtilisin or metalloprotease), lipase, amylase, lyase, cellulase, pectinase, mannanase, deoxyribonuclease, perhydrolase, and oxidoreductase (e.g., laccase, peroxidase, peroxigenase and / or haloperoxidase); or combinations thereof. More preferred detergent enzymes are protease (e.g., subtilisin or metalloprotease), lipase, amylase, cellulase, pectinase, and mannanase; or combinations thereof.

[0426] The composition or microcapsule composition of the present invention may contain more than 0.1% (w / w) of active enzyme protein, particularly the 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) of active enzyme protein.

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

[0428] Serine protease is an enzyme that catalyzes the hydrolysis of peptide bonds, in which an essential serine residue is present at the active site (White, Handler and Smith, 1973 “Principles of Biochemistry” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272). Subtilisin, as defined by Siezen et al., Protein Engng. 4 (1991) 719-737; and Siezen et al., Protein Science 6 (1997) 501-523, preferably consists of, includes the I-S1 and I-S2 subgroups. Due to the highly conserved structure of the active site of serine protease, the subtilisin described in the present invention may be functionally equivalent to subtilase named as the proposed subgroup by Siezen et al. (above).

[0429] Subtilisin may be of animal, plant or microbial origin, including chemical or genetic recombinant mutants (protein-modified mutants), preferably an alkalophilic microbial subtilisin. Examples of subtilisin 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 pamphlet), and protease PD138 (WO 93 / 18140 pamphlet). Examples are described in WO 98 / 020115 pamphlet, WO 01 / 44452 pamphlet, WO 01 / 58275 pamphlet, WO 01 / 58276 pamphlet, WO 03 / 006602 pamphlet and WO 04 / 099401 pamphlet. Examples of trypsin-like proteases include trypsin (e.g., of porcine or bovine origin) and Fusarium protease described in WO 89 / 06270 pamphlet and WO 94 / 25583 pamphlet. Other examples are mutants described in WO 92 / 19729 pamphlet, WO 88 / 08028 pamphlet, WO 98 / 20115 pamphlet, WO 98 / 20116 pamphlet, WO 98 / 34946 pamphlet, WO 2000 / 037599 pamphlet, WO 2011 / 036263 pamphlet, especially mutants 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 metalloprotease may be of animal, plant or microbial origin, including chemical or genetic recombinant mutants (protein-modified mutants), preferably an alkalophilic microbial metalloprotease. Examples are described in WO 2007 / 044993 pamphlet, WO 2012 / 110562 pamphlet and WO 2008 / 134343 pamphlet.

[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™, Purafect Ox™, Purafact Prime™, Excellase™, FN2™, FN3™ and FN4™ (available from Novozymes, Genencor International Inc., Gist-Brocades, BASF, or DSM). Other examples are Primase™ and Duralase™. Also examples are Blap R, Blap S and Blap X available from Henkel.

[0432] Lyase: The lyase may be, for example, a pectate lyase derived from Bacillus, particularly B. licheniformis or B. agaradhaerens, or a variant derived from any of these, as described in, for example, U.S. Patent 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 from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanases are described in WO 99 / 064619. Commercially available mannanase is Mannaway (Novozymes A / S).

[0434] Cellulase: Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein-modified mutants are included. Suitable cellulases include cellulases derived from the genus Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example, fungal cellulases produced from Humicola insolens, Myceliophthora thermophila, and Fusarium oxysporum as disclosed in U.S. Patent No. 4,435,307, U.S. Patent No. 5,648,263, U.S. Patent No. 5,691,178, U.S. Patent No. 5,776,757, and WO 89 / 09259.

[0435] Particularly suitable cellulases are alkaline or neutral cellulases having a color care effect. Examples of such cellulases are those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase mutants as described in WO 94 / 07998, EP 0531315, U.S. Patent No. 5,457,046, U.S. Patent No. 5,686,593, U.S. Patent No. 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK98 / 00299.

[0436] Examples of commercially available cellulases include Celluzyme™, and Carezyme™ (Novozymes A / S), Clazinase™, and Puradax HA™ (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 contain other lipases.

[0438] Other lipases and cutinases: Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein-engineered mutants are included. Examples include lipases derived from Thermomyces, such as T. lanuginosus (formerly known as Humicola lanuginosa) as described in European Patent No. 258068 and European Patent No. 305216, cutinases derived from Humicola, such as H. insolens as described in WO 96 / 13580, Pseudomonas lipases derived from, for example, P. alcaligenes or P. pseudoalcaligenes (European Patent No. 218272), P. cepacia (European Patent No. 331376), P. stutzeri (British Patent No. 1,372,034), P. fluorescens, 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 (Japanese Patent Application Laid-Open No. 64 / 744992), or B. pumilus (WO 91 / 16422).

[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 a Bacillus sp. lipase from Solvay.

[0441] Amylase: Suitable amylases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of amylases include alpha-amylases obtained from Bacillus, such as a special strain of Bacillus licheniformis (described in more detail in GB 1,296,839).

[0442] Examples of suitable amylases include an amylase having SEQ ID NO: 2 of WO 95 / 10603 or a variant having 90% sequence identity to SEQ ID NO: 3 therein. Preferred variants are described in SEQ ID NO: 4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, for example, at 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, which have substitutions at one or more of these positions.

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

[0444] A further preferred amylase is an amylase having SEQ ID NO: 6 of WO 99 / 019467 pamphlet 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 at positions H183 and G184.

[0445] A further amylase that can be used is one having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873 pamphlet or a variant 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 having substitutions, deletions or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those having deletions at positions 181 and 182 or at 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 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 amylases having SEQ ID NO: 2 of WO 08 / 153815 pamphlet, SEQ ID NO: 10 of WO 01 / 66712 pamphlet, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08 / 153815 pamphlet or 90% sequence identity to SEQ ID NO: 10 of WO 01 / 66712 pamphlet. Preferred variants of SEQ ID NO: 10 of WO 01 / 66712 pamphlet are those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

[0447] More suitable amylases are amylases having SEQ ID NO: 2 of WO 09 / 061380 pamphlet or variants having 90% sequence identity to SEQ ID NO: 2 thereof. Preferred variants of SEQ ID NO: 2 are those having C-terminal cleavage 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 having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and / or deletions at position R180 and / or position S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are 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 which has, wherein the variant has a cleaved C-terminus and optionally further comprises a substitution at position 243 and / or a deletion at position 180 and / or 181.

[0448] Other suitable amylases are alpha - amylases having SEQ ID NO: 12 of WO 01 / 66712 pamphlet 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 pamphlet: 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. Specific preferred amylases include variants having deletions of D183 and G184 and substitutions of R118K, N195F, R320K and R458K, and 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 variants further having substitutions at all of these positions.

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

[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 (manufactured by Genencor International Inc. / DuPont).

[0451] Deoxyribonuclease (DNase): A suitable deoxyribonuclease (DNase) is any enzyme that catalyzes the hydrolysis of phosphodiester bonds in the DNA backbone, thereby degrading DNA. According to the present invention, a DNase obtainable from bacteria is preferred; in particular, a DNase obtainable from Bacillus is preferred; in particular, a DNase obtainable from Bacillus subtilis or Bacillus licheniformis is preferred. Examples of such DNases are described in International Publication Pamphlet No. WO 2011 / 098579 of the patent application or in the specification of PCT / EP2013 / 075922.

[0452] Perhydrolase: A suitable perhydrolase has the ability to catalyze a perhydrolysis reaction that results in the formation of a peracid from a carboxylic acid ester (acyl) substrate in the presence of a source of peroxide (e.g., hydrogen peroxide). While a number of enzymes perform this reaction at a low level, perhydrolases exhibit high perhydrolysis (the hydrolysis ratio often exceeds 1). Suitable perhydrolases may be of plant, bacterial or fungal origin. Chemically modified or protein-engineered mutants are included.

[0453] Examples of useful perhydrolases include naturally occurring Mycobacterium perhydrolase enzymes, or variants thereof. Exemplary enzymes are derived from Mycobacterium smegmatis. Such enzymes, their enzymatic properties, their structures, and their variants are described in WO 2005 / 056782, WO 2008 / 063400, US 2008 / 145353, and US 2007 / 167344.

[0454] Oxidase / peroxidase: Suitable oxidases and peroxidases (or oxidoreductases) include various carbohydrate oxidases, laccases, peroxidases, and haloperoxidases.

[0455] Suitable peroxidases include those included by, or any fragment derived therefrom, of enzyme classification EC 1.11.1.7 presented by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) that exhibit peroxidase activity.

[0456] Suitable peroxidases include those of plant, bacterial, or fungal origin. Chemically modified or protein-engineered mutants are included. Examples of useful peroxidases include peroxidases derived from Coprinopsis, such as peroxidase derived from C. cinerea (EP 179,486), and variants thereof (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 exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidase (E.C. 1.11.1.10) catalyzes the formation of hypochlorite from chloride ions.

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

[0459] Haloperoxidases have been isolated from a variety of fungi, particularly from dematiaceous hyphomycetes, 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, e.g., P. pyrrocinia, and Streptomyces, e.g., S. aureofaciens.

[0461] In a preferred embodiment, the haloperoxidase is from the genus Curvularia sp., particularly Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS102.42 as described in WO 95 / 27046 pamphlet; or C. verruculosa CBS147.63 or C. verruculosa CBS444.70 as described in WO 97 / 04102 pamphlet; or it can be derived from Drechslera hartlebii as described in WO 01 / 79459 pamphlet, Dendryphiella salina as described in WO 01 / 79458 pamphlet, Phaeotrichoconis crotalarie as described in WO 01 / 79461 pamphlet, or Geniculosporium sp. as described in WO 01 / 79460 pamphlet.

[0462] The oxidase according to the present invention particularly includes any laccase enzyme included by enzyme classification EC1.10.3.2, or any fragment derived therefrom showing laccase activity, or a compound showing similar activity, such as catechol oxidase (EC1.10.3.1), o-aminophenol oxidase (EC1.10.3.4), or bilirubin oxidase (EC1.3.3.5).

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

[0464] Suitable examples derived from fungi include laccases derivable from strains of 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, Psathyrella, for example, P. condelleana, Panaeolus, for example, P. papilionaceus, Myceliophthora, for example, M. thermophila, Schytalidium, for example, S. thermophilum, Polyporus, for example, P. pinsitus, Phlebia, for example, P. radiata (WO 92 / 01046 pamphlet), or Coriolus, for example, C. hirsutus (Japanese Patent No. 2238885 specification).

[0465] Suitable examples derived from bacteria include laccases derivable from strains of Bacillus.

[0466] Derived from Coprinopsis or Myceliophthora; in particular, derived from Coprinopsis cinerea as disclosed in WO 97 / 08325 pamphlet; or derived from Myceliophthora thermophila as disclosed in WO 95 / 33836 pamphlet, laccase is preferred.

[0467] Examples of other oxidases include, but are not limited to, amino acid oxidase, glucose oxidase, lactate oxidase, galactose oxidase, polyol oxidase (e.g., WO 2008 / 051491 pamphlet), and aldose oxidase. The oxidase and its corresponding substrate may be used as a hydrogen peroxide generating enzyme system, and thus as a source of hydrogen peroxide. Some enzymes, such as peroxidase, haloperoxidase, and perhydrolase, require a source of hydrogen peroxide. By considering 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), other examples of combinations such as oxidase and substrate will be readily understood by those skilled in the art.

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

[0469] To create a local environment inside the capsule that is more "friendly" to the encapsulated enzyme / compound and thus improve stability during storage, the addition of a slowly dissolving stabilizer can be used.

[0470] Examples of reversible protease inhibitors include boric acid, peptide aldehydes and their derivatives, and polymeric proteinaceous inhibitors (similar to BASI / RASI inhibitors, see WO 2009 / 095425 pamphlet). Examples of metalloprotease inhibitors are described in WO 2008 / 134343 pamphlet. Protease inhibitors are described in more detail below the heading "Protease inhibitors".

[0471] Stabilizing polymers can be based on, for example, polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and their copolymers. Stabilizing polyols can be not only smaller molecules such as glycerol, sorbitol, propylene glycol, etc., but also larger molecules such as polyethylene glycol, polysaccharides, etc.

[0472] Among stabilizing divalent cations, Ca 2+ , Mg 2+ and Zn 2+ are well known in the art. Thus, in one embodiment, the composition of the present invention comprises a source of Ca 2+ , Mg 2+ or Zn 2+ ions. Preferably, the source of Ca 2+ , Mg 2+ or Zn 2+ ions is a sparingly soluble (slowly dissolving) salt of Ca 2+ , Mg 2+ or Zn 2+ . Sparingly soluble means having a solubility in pure water at 20 °C of 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. Ca 2+ , Mg 2+ or Zn 2+Preferred salts 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, acids or bases that dissolve slowly can be used to create a local pH within the microcapsules that is more "friendly" to the encapsulated enzyme / compound.

[0474] Enzymes are, in most cases, stabilized by the addition of their substrates (e.g., proteins for proteases, starch for amylases, etc.). For example, antioxidants or reducing agents such as thiosulfate, ascorbate, etc. can be applied to reduce the oxidation of the enzyme. The required net dosage of these stabilizers per gram of detergent is much lower than when adding the stabilizer to the continuous detergent phase since they are concentrated in the internal capsule phase and will, in many cases, either not diffuse during storage or will diffuse only slowly depending on the structure and molecular weight of the stabilizer. In particular, high molecular weight stabilizers (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. Therefore, enzymes whose components are destabilized by reacting on amino acid groups (e.g., amines) on the protein may react with the added scavenger or sacrificial protein. Scavenger proteins having a high enough molecular weight to remain inside the capsule are preferred.

[0476] Slightly different methods for improving enzyme stability involve adding a rheology-modifying component that increases the viscosity of the internal capsule phase. The increase in internal viscosity delays the diffusion of enzyme destabilizers into the capsule (and / or delays the diffusion of enzyme stabilizers out of the capsule), and thus extends the lifespan of the enzyme. Examples of such viscosity-changing agents 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 having a molecular weight greater than 1 kDa, or greater than 2 kDa, more preferably greater than 5 kDa are most preferred. Nonionic polymers are preferred because they are, in most cases, more compatible with the reactive membrane polymer than ionic polymers.

[0477] High viscosity can be achieved by generating capsules using a high-viscosity aqueous phase or, more advanced, by generating capsules when the viscosity increase occurs first after emulsion / capsule generation. This "induced" viscosity increase is preferred when preparing the emulsion with a high-viscosity aqueous phase, which can be difficult. The induced viscosity increase can occur in situ when adding the internal capsule phase, which has a higher water activity than the detergent to which it is added, to the detergent, so that water diffuses out of the capsule (excluding rheology modifiers) and the viscosity of the internal phase increases after addition to the detergent. This can also be utilized by using the diffusion of salts or other low-molecular components, for example, components that increase the viscosity when the salt concentration decreases, such as by addition to the detergent (e.g., a polymer that precipitates at an initial high salt content but is soluble when the salt concentration decreases due to salt diffusion during addition to the detergent). Another way to cause a viscosity increase is to use components where the viscosity is pH-dependent. In some interfacial polymerization processes (e.g., the amine-acid halide reaction), the pH of the internal phase changes during encapsulation, and in the case of the amine-acid halide, the pH decreases during interfacial polymerization. This can be used to cause an increase in viscosity. Many rheology modifiers, such as polyacrylates, exhibit 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 where the viscosity increases significantly when the pH decreases from 11 to 8 or when the pH increases 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, where emulsion / capsule encapsulation is carried out at one temperature and then the temperature is changed to increase the viscosity. Also, light- or ultrasound-induced viscosity is available. Yet another method is to use a shear-thinning rheology modifier such that the viscosity is low at high shear when the emulsion is formed and high when the shear decreases.

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

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

[0480] In a specifically considered embodiment, the microcapsule composition of the present invention is as described in International Publication No. WO 2014 / 177709 pamphlet (incorporated herein by reference) containing the lipase variant of the present invention.

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

[0482] As described above, the microcapsules may further contain an enzyme selected from the group consisting of proteases, metalloproteases, subtilisins, amylases, lipases, cutinases, cellulases, mannases, pectinases, xantanases, deoxyribonucleases, laccases, peroxidases, haloperoxidases, perhydrolases, and combinations thereof. Other enzymes as described above are also considered.

[0483] In one embodiment, the reactive amino groups of the multi-branched 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, in particular the lipase variant of the present invention, of the total compartment. Further as described above, the microcapsules may further contain an alcohol, such as a polyol.

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

[0485] In one embodiment, (b) is an ethylenediamine or an alkanolamine. In a preferred embodiment, (b) is selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, 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, triethylenetetramine, bis(3-aminopropyl)amine, monoethanolamine, and diethanolamine.

[0486] According to the present invention, the compartments of the microcapsules are a source of Mg 2+ , Ca 2+ , or Zn 2+ ions, for example, a sparingly soluble salt of Mg 2+ , Ca 2+ , or Zn 2+ .

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

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

[0489] In a specifically contemplated embodiment, the microcapsule composition of the present invention is as described in International Publication No. WO 2015 / 1144784 (incorporated herein by reference) and includes the lipase variant of the present invention.

[0490] Liquid product 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, 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. A variant of paragraph 1, having a substitution at a position corresponding to T231R+N233R 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, T244E, and P256T.

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

[0494]

Table 4

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

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

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

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

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

[0500] 9. A variant of any of paragraphs 1-8, wherein the substitution is at a position corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R+P256T, and includes one or more (e.g., some) substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0501] 10. A variant of any of paragraphs 1-9, wherein the substitution is at a position corresponding to D27N+F51I,L+E56R+R118F+T231R+N233R+P256T, and includes one or more (e.g., some) substitutions at positions corresponding to G23S, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0502] 11. A variant of any of paragraphs 1-10, wherein the substitution is at a position corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R+P256T, and includes one or more (e.g., some) substitutions at positions corresponding to G23S, D27N, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0503] 12. A variant of any of paragraphs 1-11, wherein the substitution is at a position corresponding to F51I,L+E56R+D57N+R118F+T231R+N233R+P256T, and includes one or more (e.g., some) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, K98I, N101D, G163S, Y220F, and T244E.

[0504] 13. A variant of any of paragraphs 1-12, wherein the substitution is at a position corresponding to F51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T, and includes one or more (e.g., some) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F, and T244E.

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

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

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

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

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

[0510] 19. The substitutions are 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; 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; A40I+G163S+E56R+R118F+T231R+N233R; A40I+E56R+R118F+T231R+N233R+P256T; F51L+D57N+E56R+R118F+T231R+N233R; F51L+K98I+E56R+R118F+T231R+N233R; F51L+G163S+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R+P256T; D57N+K98I+E56R+R118F+T231R+N233R; D57N+G163S+E56R+R118F+T231R+N233R; D57N+E56R+R118F+T231R+N233R+P256T; K98I+G163S+E56R+R118F+T231R+N233R; 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+Y2...

Claims

**Claim 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 containing the substitutions T231R + N233R + A40I. **Claim 2** The variant according to claim 1, containing the substitutions T231R + N233R + A40I and one or more of the substitutions G23S, D27N, F51I, F51L, E56R, D57N, V60E, V60K, K98I, N101D, G163S, Y220F, T244E, and P256T. **Claim 3** The variant is 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; A variant according to any one of claims 1 or 2, comprising any of the above.

4. The parent lipase is as follows: 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; A variant according to any one of claims 1 to 3, selected from the group consisting of. **Claim 5** A variant according to any one of claims 1 to 4, having a sequence identity of at least 95% but less than 100% with SEQ ID NO:

2. **Claim 6** A composition comprising a variant according to any one of claims 1 to 5. **Claim 7** Use of a variant according to any one of claims 1 to 5 for hydrolyzing a lipase substrate. **Claim 8** A method for cleaning a surface to be cleaned, comprising contacting the surface to be cleaned with a variant according to any one of claims 1 to 5. **Claim 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. **Claim 10** A polynucleotide encoding a variant according to any one of claims 1 to 5. **Claim 11** A nucleic acid construct comprising the polynucleotide according to claim 10, wherein the polynucleotide is operably linked to one or more control sequences that induce the production of the lipase variant in a recombinant host cell. **Claim 12** An expression vector comprising the polynucleotide according to claim 10 or the nucleic acid construct according to claim 11. **Claim 13** A host cell comprising the nucleic acid construct according to claim 11 or the expression vector according to claim 12. **Claim 14** a) culturing the host cell according to claim 13 under conditions suitable for the expression of the variant; b) recovering the variant, A method for producing a lipase variant, comprising:

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