Compositions comprising lipase and sulfite

Abstract

The present invention relates to a composition comprising: (a) a sulfite source; and (b) a lipase variant of a parent lipase, wherein one or more amino acids in the parent lipase within 20 angstrom # imgabs0 # of the cysteine bridge are replaced with more negatively charged amino acids.

Description

[0001] The present invention is a divisional application based on a Chinese invention patent application filed on May 4, 2018, with application number “201880028849.6” and invention name “Composition comprising lipase and sulfite”.

[0002] References to sequence listings

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0005] The present invention relates to a composition comprising a sulfite source and a lipase variant. The present invention also relates to a lipase variant and the use of the composition of the present invention for cleaning, for example, a surface. In addition, the present invention also relates to a polynucleotide, a nucleic acid construct comprising the polynucleotide of the present invention, an expression vector comprising the polynucleotide of the present invention, a host cell comprising the polynucleotide of the present invention, a method for producing the lipase variant of the present invention, and a method for obtaining the lipase variant of the present invention. Background Art

[0006] Sulfites are naturally occurring substances in some foods and in the human body. They are used in foods (as preservatives or fortifiers) as well as in non-food products (such as, for example, household products) and are therefore included in various compositions.

[0007] Lipases have been used in compositions for removing lipid stains by hydrolyzing triglycerides to produce fatty acids. Current detergents, cleaning and / or fabric care compositions contain many active ingredients that interfere with the ability of lipase to remove lipid stains. In some compositions, sulfite sources are added as antioxidants for more unstable functional ingredients (such as, but not limited to, pigments, polymers, spices), or as traces / impurities in sulfonated surfactants. However, sulfite can affect the stability of lipase.

[0008] WO 2017 / 005640 relates to a composition comprising: (a) a sulfite source; and (b) a lipase variant of a parent lipase having improved stability towards sulfite.

[0009] There is a need for compositions, including detergent compositions, comprising sulfite and lipase enzymes that are stable and active in the presence of sulfite. Summary of the invention

[0010] The present invention is concerned with the problem of reducing the stability of lipases in compositions comprising a sulfite source.

[0011] Sulfite (SO3 2- ). Sulfite can disrupt disulfide bonds (RSS-R') and reduce the stability of lipases containing one or more cysteine ​​bridges. The inventors surprisingly found that the reduction in stability can be reduced by shielding the cysteine ​​bridge with a negatively charged amino acid close to the cysteine ​​bridge. It is believed that this effect is due to the fact that sulfite is negatively charged and therefore sulfite is less likely to bind to negatively charged lipase regions.

[0012] Therefore, in a first aspect, the present invention relates to a composition comprising:

[0013] (a) sulfite source;

[0014] (b) a parent lipase shown as SEQ ID NO: 2 or a lipase variant of a parent lipase having at least 60% identity to SEQ ID NO: 2;

[0015] The 20 angstroms of the cysteine ​​bridge One or more amino acids in the parent lipase in the expression vector are replaced by more negatively charged amino acids.

[0016] In one embodiment, the parent lipase is a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0017] In a preferred embodiment, the replaced, preferably substituted amino acid is within 15 angstroms, preferably within 10 angstroms, more preferably within 5 angstroms of the cysteine ​​bridge (ie, from the carboxyl group to the sulfur atom).

[0018] In a preferred embodiment, the sulfite source is selected from: sulfites, such as sodium sulfite, potassium sulfite, calcium sulfite; bisulfite, such as potassium bisulfite, sodium bisulfite, calcium bisulfite; metabisulfite, such as potassium metabisulfite, sodium metabisulfite, calcium metabisulfite; or any combination thereof.

[0019] In a preferred embodiment, sulfite (SO3 2-) or pyrosulfite (S2O5 2- ) is from 0.1wt% to 3wt%; from 0.2wt% to 2wt%; from 0.5wt% to 2wt%.

[0020] In particular embodiments, the lipase variant comprises one or more substitutions corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0021] According to the present invention, the parent lipase may comprise one or more cysteine ​​bridges. The cysteine ​​bridges are preferably located on the surface of the parent lipase.

[0022] In a particular embodiment of the invention, the cysteine ​​bridge is or corresponds to one or more of the cysteine ​​bridges at the following positions:

[0023] SEQ ID NO:2

[0024] C22-C268,

[0025] C36-C41, and

[0026] C104-C107.

[0027] In an embodiment of the present invention, the lipase variant of the present invention has improved stability in the presence of a sulfite source compared to the parent lipase.

[0028] In a preferred embodiment, the composition further comprises a surfactant.

[0029] In one aspect, the invention relates to the use of a composition of the invention for cleaning, for example, a surface.

[0030] The present invention also relates to lipase variants of a parent lipase, wherein the variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2 or any fragment thereof having lipase activity, and comprises one or more (e.g., several) substitutions at positions corresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184 and 267 of SEQ ID NO: 2.

[0031] In a particular embodiment, the lipase variant comprises one or more substitutions corresponding to substitutions selected from the group consisting of Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, and T267D, E of SEQ ID NO:2.

[0032] In one aspect, the invention relates to a polynucleotide encoding a variant of the invention.

[0033] In one aspect, the invention relates to a nucleic acid construct comprising a polynucleotide of the invention.

[0034] In one aspect, the invention relates to an expression vector comprising a polynucleotide of the invention.

[0035] In one aspect, the present invention relates to a host cell comprising a polynucleotide of the present invention, in particular an expression vector of the present invention.

[0036] In one aspect, the invention relates to a method for producing a lipase variant, the method comprising: (a) culturing a host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.

[0037] In one aspect, the present invention relates to a method for obtaining a lipase variant of the present invention, the method comprising: adding a 20 angstrom residue at a cysteine ​​bridge to a More negatively charged amino acids are introduced into the parent lipase shown as SEQ ID NO: 2 or a parent lipase having at least 60% sequence identity thereto.

[0038] In one embodiment, the parent lipase is a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0039] In a preferred embodiment, one or more substitutions corresponding to the following substitutions in SEQ ID NO:2 are introduced into the parent lipase: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E.

[0040] definition

[0041] Lipase: The terms "lipase", "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide" and "lipolytic protein" refer to an enzyme in the EC3.1.1 class as defined in the enzyme nomenclature. It may 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 section: The hydrolytic activity may be determined using the PnP assay using substrates of varying chain lengths.

[0042] On the one hand, variant of the present invention has at least 20% of the lipase activity of parent lipase, for example at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100%. On the one hand, parent lipase is the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12, or its fragment with lipase activity.

[0043] Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0044] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0045] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are usually determined by an open reading frame that begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0046] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) to the polynucleotide encoding the variant, or native or exogenous to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter, and transcription and translation termination signals. The control sequence may be provided with a linker for the purpose of introducing specific restriction sites that facilitate the connection of the control sequence to the coding region of the polynucleotide encoding the variant.

[0047] Expression: The term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048] Expression vector: The term "expression vector" means a linear or circular DNA molecule that includes a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.

[0049] 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 terminus of a polypeptide; wherein the fragment has lipase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids present in a parent lipase. In one aspect, the parent lipase is a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the parent lipase is amino acids 1-269 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12.

[0050] High stringency conditions: The term "high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42° C. for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide. The carrier material is finally washed three times at 65° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

[0051] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0052] Improved properties: The term "improved properties" means characteristics associated with a variant that are improved relative to a parent lipase. Such improved properties include, but are not limited to, stability (such as, for example, stability in the presence of a sulfite source, stability in a detergent composition, stability in a detergent composition comprising a sulfite source, stability under storage conditions, stability under storage conditions in the presence of a sulfite source), thermostability, and thermostability in the presence of a sulfite source.

[0053] Isolated: The term "isolated" means a substance in a form or environment that does not exist in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including but not limited to any enzyme, variant, nucleic acid, protein, peptide or cofactor, which is at least partially removed from one or more or all naturally occurring components associated with its properties; (3) any substance that has been artificially modified relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). The isolated substance may be present in a fermentation broth sample.

[0054] Low stringency conditions: The term "low stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide. The carrier material is finally washed three times at 50°C using 2X SSC, 0.2% SDS for 15 minutes each time.

[0055] Mature polypeptide: The term "mature polypeptide" or "mature portion of a polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 269 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. It is known in the art that a host cell can 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.

[0056] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide having lipase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 807 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0057] Moderate stringency conditions: The term "moderate stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42° C. for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide. The carrier material is finally washed three times at 55° C. with 2X SSC, 0.2% SDS for 15 minutes each time.

[0058] Medium-high stringency conditions: The term "medium-high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 ug / mL sheared and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Finally, the carrier material is washed three times at 60° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

[0059] Mutant: The term "mutant" means a polynucleotide encoding a variant.

[0060] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified to contain a segment of nucleic acid in a manner that does not exist in nature, or is synthetic, and that contains one or more control sequences.

[0061] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0062] Parent or parent lipase: The term "parent" or "parent lipase" means a lipase that is altered to produce a lipase variant of the present invention. The parent may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof. Examples of such parent lipases are those having the amino acid sequence given in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0063] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0064] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453), as implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite (The European Molecular Biology Open Software Suite), Rice et al., 2000, TrendsGenet. [Genetics Trend] 16:276-277) (preferred 5.0.0 version or updated version). The parameter used is a gap opening penalty of 10, a gap extension penalty of 0.5, and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output (obtained using non-simplified (-nobrief) option) of the "longest identity" marked by Needle is used as the identity percentage and is calculated as follows:

[0065] (identical residues x 100) / (alignment length - total number of gaps in the alignment)

[0066] For the purpose of the present invention, the sequence identity between two deoxynucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), which is implemented by the Needleman program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferred 5.0.0 version or updated version). The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output (obtained using the non-simplified (-nobrief) option) of the Needleman mark is used as the identity percentage and is calculated as follows:

[0067] (number of identical deoxyribonucleotides x 100) / (alignment length - total number of gaps in the alignment)

[0068] Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides deleted from the 5' end and / or 3' end of a polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity. On the one hand, the subsequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of nucleotides 1 to 807 encoding the parent lipase. On the one hand, the nucleotides encoding the parent lipase comprise SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or a subsequence thereof encoding a fragment having lipase activity, or consisting thereof.

[0069] Sulfite: The term "sulfite" means containing sulfite ions [SO3] 2- The term "bisulfite" (bisulfite or hydrogen sulfite) means a compound containing bisulfite ions [HSO3] - The term "metabisulfite" or "disulfite" means a compound containing the metabisulfite ion [S2O5] 2- For the purposes of the present invention, the term "sulfite source" is used to cover any sulfite source that provides the compounds mentioned in the paragraphs of the present invention. The present invention includes intentionally added sulfite sources.

[0070] Variant: The term "variant" means a polypeptide having lipase activity, comprising an alteration (i.e., substitution, insertion and / or deletion) at one or more (e.g., several) positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding an 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 the parent lipase. In one aspect, the parent lipase comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or a variant thereof having lipase activity, or consists thereof.

[0071] Very high stringency conditions: The term "very high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 ug / mL sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. The carrier material is finally washed three times at 70° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

[0072] Very low stringency conditions: The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 ug / mL sheared and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. The carrier material is finally washed three times at 45° C. using 2X SSC, 0.2% SDS for 15 minutes each time.

[0073] Wild-type lipase: The term "wild-type" lipase means a lipase expressed by a naturally occurring microorganism found in nature, such as a bacterium, yeast, or filamentous fungus.

[0074] Variant naming conventions

[0075] For the purpose of the present invention, the polypeptide of SEQ ID NO:2 is used to determine the corresponding amino acid residue in another lipase. The amino acid sequence of another lipase is compared with the lipase of SEQ ID NO:2, and based on the comparison, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) implemented in the Needle program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, TrendsGenet. [Genetics Trend] 16:276-277) (preferably 5.0.0 version or later) is used to determine the amino acid position number corresponding to any amino acid residue in the polypeptide of SEQ ID NO:2. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0076] The identity of the corresponding amino acid residue in another lipase can be determined by aligning multiple polypeptide sequences using several computer programs using their corresponding default parameters, including but not limited to MUSCLE (Multiple Sequence Comparison by Logarithmic Expectations; 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 23:372-374. Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and EMBOSS EMMA using ClustalW (version 1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:4673-4680).

[0077] When other enzymes deviate from the polypeptide of SEQ ID NO:2 so that traditional sequence-based comparison methods cannot detect their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. [Journal of Molecular Biology] 295: 613-615), other pairwise sequence comparison algorithms can be used. Higher sensitivity in sequence-based searches can be obtained using search programs that use probabilistic representations (profiles) of families of polypeptides to search databases. For example, the PSI-BLAST program generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. [Nucleic Acids Research] 25: 3389-3402). Even higher sensitivity can be achieved if a family or superfamily of polypeptides has one or more representatives in a protein structure database. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) use information from a variety of sources (PSI-BLAST, secondary structure predictions, structural alignment spectra, and solvation potential) as input to a neural network that predicts the structural fold of a query sequence. Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align sequences of unknown structure with superfamily models present in the SCOP database. These alignments, in turn, can be used to generate homology models for polypeptides, and the accuracy of such models can be assessed using a variety of tools developed for this purpose.

[0078] For proteins of known structure, several tools and resources can be used to retrieve and generate structural alignments. For example, the SCOP superfamily of proteins has been structurally aligned, and those alignments are accessible and downloadable. Multiple algorithms such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combinatorial extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747) can be used to align two or more protein structures, and the implementation of these algorithms can be used in addition to query structural databases with structures of interest to find possible structural homologues (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0079] In describing the variants of the present invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter abbreviations for amino acids are used.

[0080] replace .For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, the substitution of threonine at position 226 by alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represents the substitution of glycine (G) and serine (S) at position 205 and position 411 by arginine (R) and phenylalanine (F), respectively.

[0081] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position, * Thus, a deletion of glycine at position 195 is indicated as "Gly195*" or "G195*". Multiple deletions are separated by plus signs ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0082] insert For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Thus, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0083] In such cases, the inserted amino acid residue(s) are numbered by adding lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would therefore be:

[0084] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0085] Multiple changes. Variants containing multiple changes are separated by plus signs ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively. Multiple variants can also be disclosed, for example, as "R170Y G195E" (ie, without "+" between changes).

[0086] Different changes. In the case where different changes can be introduced at one position, the different changes are separated by commas, for example, "Arg170Tyr,Glu" represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0087] "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala". DETAILED DESCRIPTION

[0088] The present invention relates to a composition comprising: (a) a sulfite source, and (b) a lipase variant of a parent lipase; and the use of the composition for cleaning, for example, a surface. The present invention also relates to a lipase variant, a polynucleotide encoding the variant; a nucleic acid construct, a vector, and a host cell comprising the polynucleotide; and a method for producing the variant.

[0089] Composition

[0090] The present invention provides a composition comprising:

[0091] (a) sulfite source;

[0092] (b) a parent lipase shown as SEQ ID NO: 2 or a lipase variant of a parent lipase having at least 60% identity to SEQ ID NO: 2;

[0093] The 20 angstroms of the cysteine ​​bridge One or more amino acids in the parent lipase in the expression vector are replaced by more negatively charged amino acids.

[0094] In one embodiment, the parent lipase is a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0095] In a preferred embodiment, the replaced, preferably substituted amino acid is within 15 angstroms, preferably within 10 angstroms, more preferably within 5 angstroms of the cysteine ​​bridge (ie, from the carboxyl group to the sulfur atom).

[0096] In an embodiment, the sulfite source is selected from sulfite, including sodium sulfite, potassium sulfite, calcium sulfite; bisulfite, such as potassium bisulfite, sodium bisulfite, calcium bisulfite; pyrosulfite / metabisulfite, such as potassium pyrosulfite, sodium pyrosulfite, calcium pyrosulfite; or any combination thereof.

[0097] In the embodiment, sulfite (SO3 2- ) or pyrosulfite (S2O5 2- ) in an amount ranging from 0,005wt% to 5wt%, such as from 0,01wt% to 3wt%, such as from 0.1wt% to 2wt%, such as from 0.2wt% to 1wt%, such as from 0.4wt% to 0.6wt%, such as from 0.45wt% to 0.55wt%, such as 0.005wt%, such as 0,01wt%, such as 0.1wt%, such as 0.25wt%, such as 0.5wt%, or any combination of these ranges or values.

[0098] In an embodiment, a composition of the invention comprises a lipase variant comprising one or more (e.g., several) substitutions at positions corresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO:2.

[0099] In an embodiment, the variant in the composition of the invention comprises a substitution corresponding to one or more of: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0100] In an embodiment, within 20 angstroms of the cysteine ​​bridge The amino acid in the parent lipase is replaced by a negatively charged amino acid selected from D or E.

[0101] In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0102] In an embodiment, the composition of the present invention comprises a lipase variant of a parent lipase shown as SEQ ID NO: 2, or a lipase variant of a lipase 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%, but less than 100% sequence identity with the parent lipase, and comprising one or more (e.g., several) substitutions at positions corresponding to amino acid residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2.

[0103] In embodiments, the parent lipase has 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:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0104] In an embodiment, the variant of a parent lipase is selected from the group consisting of:

[0105] (a) a polypeptide encoded by a polynucleotide that hybridizes under 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, or (ii) the full-length complementary sequence of (i);

[0106] (b) 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% identity to the polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; and

[0107] (c) A fragment of the polypeptide of (a) or (b), in particular a fragment of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12, wherein the fragment has lipase activity.

[0108] In another embodiment, the variants included in the compositions of the present invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more substitutions.

[0109] In one aspect, the present invention relates to a composition comprising a lipase variant, wherein the variant further comprises one or more (e.g., several) substitutions corresponding to any one of the positions selected from: 4, 27, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 231, 233, 249, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO:2. In one aspect, the present invention relates to a composition comprising a lipase variant, wherein the variant further comprises one or more (e.g., several) substitutions corresponding to any one of the positions selected from: 4V, 27R, 38A, 57G, 58A, 60S, 83T, 86V, 91A / N / Q, 94K / R, 97M, 99K, 111A, 150G, 163K, 210K / Q, 216P, 225R, 227G, 231R, 233R, 249R, 254S, 255A, 256K / T / V, 263Q, 264A, 265T, 266D, 267A, and 269N of SEQ ID NO:2.

[0110] In another embodiment, the variants included in the compositions of the invention further comprise a set of substitutions corresponding to the following group (numbered using SEQ ID NO: 2), the group consisting of:

[0111]

[0112]

[0113]

[0114] In one aspect, the variant has increased stability compared to the parent lipase. In one aspect, stability is stability in the presence of a sulfite source, stability in a detergent composition, stability in a detergent composition comprising a sulfite source, stability under storage conditions, stability under storage conditions in the presence of a sulfite source, thermostability, and thermostability in the presence of a sulfite source.

[0115] The non-limiting list of composition components set forth below is suitable for use in the composition, and the method herein may be desirably incorporated into certain aspects of the present invention, such as to assist or enhance cleaning performance, for processing substrates to be cleaned, or to modify the aesthetics of the composition, as with spices, colorants, dyes or the like. The level of any such component incorporated into any composition is in addition to any material previously cited for incorporation. The precise nature of these other components and their incorporation levels will depend on the physical form of the composition and the properties of the cleaning operations in which the composition will be used. Although the components mentioned below are classified by general headings according to specific functionality, this is not to be construed as limiting, because as will be understood by those of ordinary skill, components may include other functionality.

[0116] Unless otherwise indicated, amounts in percentages are by 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, catalytic materials, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, clay removal / anti-redeposition agents, brighteners, foam inhibitors, dyes, hueing dyes, perfumes, perfume delivery systems, structural elastic agents, fabric softeners, carriers, hydrotropes, processing aids, solvents, antioxidants (e.g., sulfite sources), alkalis, preservatives, and / or pigments. In addition to the disclosure below, suitable examples of such other components and usage levels are found in US5576282, US6306812, and US6326348, which are hereby incorporated by reference.

[0117] Thus, in certain aspects, the present invention does not comprise one or more of the following adjunct materials: surfactants, soaps, builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay removal / anti-redeposition agents, brighteners, foam inhibitors, dyes, perfumes, perfume delivery systems, structural elastic agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and / or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

[0118] Surfactants - The composition according to the invention may comprise a surfactant or a surfactant system, wherein the surfactant may be chosen 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 from 0.1 wt% to 60wt%, from 0.2wt% to 40wt%, from 0.5wt% to 30wt%, from 1wt% to 50wt%, from 1wt% to 40wt%, from 1wt% to 30wt%, from 1wt% to 20wt%, from 3wt% to 10wt%, from 3wt% to 5wt%, from 5wt% to 40wt%, from 5wt% to 30wt%, from 5wt% to 15wt%, from 3wt% to 20wt%, from 3wt% to 10wt%, from 8wt% to 12wt%, from 10wt% to 12wt%, from 20wt% to 25wt%, or from 25wt%-60wt%.

[0119] Suitable anionic detersive surfactants include sulfate and sulphonate detersive surfactants.

[0120] Suitable sulfonate detersive surfactants include alkyl benzene sulfonates, in one aspect C 10-13 Alkylbenzene sulfonate. Suitable alkylbenzene sulfonate (LAS) can be obtained by sulfonating commercially available linear alkylbenzene (LAB); suitable LABs include low 2-phenyl LABs, such as or Other suitable LABs include high 2-phenyl LABs, such as Suitable anionic detersive surfactants are alkylbenzene sulfonates obtained by the DETAL catalytic process, but other synthetic routes (such as HF) may also be suitable. In one aspect, the magnesium salt of LAS is used.

[0121] Suitable sulfate detersive surfactants include alkyl sulfates, in one aspect C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfate.

[0122] Other suitable sulfate detersive surfactants are alkyl alkoxylated sulfates, in one aspect alkyl ethoxylated sulfates, in one aspect C 8-18 Alkyl alkoxylated sulfate, in one aspect C 8-18 Alkyl ethoxylated sulfates, typically the alkyl alkoxylated sulfates have an average degree of alkoxylation of from 0.5 to 20 or from 0.5 to 10, typically the alkyl alkoxylated sulfates are C 8-18Alkyl ethoxylated sulfates having an average degree of ethoxylation of from 0.5 to 10, from 0.5 to 7, from 0.5 to 5, or from 0.5 to 3.

[0123] The alkyl sulfates, alkyl alkoxylated sulfates and alkyl benzene sulfonates may be linear or branched, substituted or unsubstituted.

[0124] The detersive surfactant can be a mid-chain branched detersive surfactant, in one aspect a mid-chain branched anionic detersive surfactant, in one aspect a mid-chain branched alkyl sulfate and / or a mid-chain branched alkylbenzene sulfonate, such as a mid-chain branched alkyl sulfate. In one aspect, the mid-chain branch is C 1-4 The alkyl group is typically methyl and / or ethyl.

[0125] Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS) , alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soap, and combinations thereof. Anionic surfactants can be added as the corresponding acid or as a salt, or can be used as derivatives with ethanolamine, for example, monoethanolamine-linear alkylbenzene sulfonate (MEA-LAS).

[0126] Suitable nonionic detersive surfactants are selected from the group consisting of: C8-C 18 Alkyl ethoxylates, e.g. C6-C 12 Alkylphenol alkoxylate, wherein the alkoxylate unit may be an ethyleneoxy unit, a propyleneoxy unit or a mixture thereof; C 12 -C 18 Alcohol and C6-C 12 Condensation products of alkylphenols with ethylene oxide / propylene oxide block polymers, e.g. C 14 -C 22 Medium chain branched alcohol; C 14-C 22 Mid-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation of from 1 to 30; alkyl polysaccharides, in one aspect alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

[0127] Suitable nonionic detersive surfactants include alkyl polyglycosides and / or alkyl alkoxylated alcohols.

[0128] In one aspect, the nonionic detersive surfactant comprises an alkyl alkoxylated alcohol, in one aspect C 8-18 Alkyl alkoxylated alcohols, such as C 8-18 The alkyl ethoxylated alcohol may have an average degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C 8-18 Alkyl ethoxylated alcohols having an average degree of ethoxylation of from 1 to 10, from 1 to 7, more preferably from 1 to 5 or from 3 to 7. The alkyl alkoxylated alcohols may be linear or branched, and substituted or unsubstituted. Suitable nonionic surfactants include

[0129] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)), methyl ester ethoxylate (MEE), as well as products available under the SPAN and TWEEN trade names, and combinations thereof.

[0130] Suitable cationic detersive surfactants include alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds and mixtures thereof.

[0131] Suitable cationic detersive surfactants are quaternary ammonium compounds having the general formula: (R)(R1)(R2)(R3)N + X - , wherein R is a linear or branched, substituted or unsubstituted C 6-18An 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 providing charge neutrality, suitable anions include halides, such as chlorides; sulfates; and sulfonates. Suitable cationic detersive surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. Highly suitable cationic detersive surfactants are mono-C 8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C 10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

[0132] Non-limiting examples of cationic surfactants include alkyl dimethylethanol quaternary ammonium (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearylammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium, and combinations thereof.

[0133] Suitable amphoteric surfactants / zwitterionic surfactants include amine oxides and betaines (such as alkyl dimethyl betaines, sulfobetaines), or combinations thereof. The amine-neutralized anionic surfactants of the present invention-anionic surfactants and auxiliary anionic co-surfactants can be present in acid form, and the acid form can be neutralized to form surfactant salts that are desired for use in the detergent composition of the present invention. Typical agents for neutralization include metal counterion bases, such as hydroxides, such as NaOH or KOH. Other preferred agents for neutralizing the anionic surfactants of the present invention and auxiliary anionic surfactants or co-surfactants in their 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. Amine neutralization may be performed to a complete or partial extent, for example, part of the anionic surfactant mixture may be neutralized with sodium or potassium, and part of the anionic surfactant mixture may be neutralized with an amine or alkanolamine.

[0134] Non-limiting examples of semi-polar surfactants include amine oxides (AO), such as alkyl dimethyl amine oxides.

[0135] Surfactant systems comprising a mixture of one or more anionic surfactants, and additionally one or more nonionic surfactants, and optionally additional surfactants such as cationic surfactants may be preferred. Preferred weight ratios of anionic to nonionic surfactants are at least 2:1, or at least 1:1 to 1:10.

[0136] In one aspect, the surfactant system can comprise a mixture of isoprenoid surfactants represented by Formula A and Formula B:

[0137]

[0138] wherein Y is CH2 or absent, and Z may be selected such that the resulting surfactant is selected from the group consisting of alkyl carboxylates, alkyl polyalkoxys, alkyl anionic polyalkoxy sulfates, alkyl glyceryl sulfonates, alkyl dimethyl amine oxides, alkyl polyhydroxy-based surfactants, alkyl phosphates, alkyl glyceryl sulfonates, alkyl polyglucosides, alkyl polyphosphates, alkyl phosphonates, alkyl polyglycosides, alkyl monoglycosides, alkyl diglycosides, alkyl sulfosuccinates, alkyl disulfates, alkyl disulfonic acids. Ester surfactants, alkyl sulfonated succinate surfactants, alkyl glucose amide surfactants, alkyl taurate surfactants, alkyl sarcosinate surfactants, alkyl glycine ester surfactants, alkyl hydroxyethyl sulfonate surfactants, alkyl dialkanolamide surfactants, alkyl monoalkanolamide surfactants, alkyl monoalkanolamide sulfate surfactants, alkyl dihydroxyacetamide surfactants, alkyl dihydroxyacetamide sulfate surfactants, alkyl glyceride surfactants, alkyl glyceride sulfate surfactants, alkyl glyceryl ether surfactants, alkyl glyceryl ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglyceryl ether surfactants, alkyl polyglyceryl ether sulfate surfactants Surfactants, alkyl sorbitan ester surfactants, alkyl amino alkane sulfonate surfactants, alkyl amide propyl betaine surfactants, alkyl allylated quaternary ammonium salt-based surfactants, alkyl monohydroxyalkyl-di-alkylated quaternary ammonium salt-based surfactants, alkyl di-hydroxyalkyl monoalkyl quaternary ammonium salt-based surfactants, alkylated quaternary ammonium salt surfactants, alkyl trimethyl ammonium quaternary ammonium salt surfactants, alkyl polyhydroxyalkyloxypropyl quaternary ammonium salt-based surfactants, alkyl glyceride quaternary ammonium salt surfactants, alkyl glycol amine quaternary ammonium salt surfactants, alkyl monomethyl dihydroxyethyl quaternary ammonium surfactants, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactants, alkyl trimethyl ammonium surfactants, alkyl imidazoline-based surfactants, olefin-2-yl-succinate surfactants, alkyl α-sulfonated carboxylic acid surfactants, alkyl α-sulfonated carboxylic acid alkyl ester surfactants, α-olefin sulfonate surfactants, alkyl phenol ethoxylate surfactants, alkyl benzene sulfonate surfactants, alkyl sulfobetaine surfactants, alkyl hydroxysulfobetaine surfactants, alkyl ammonium carboxylic acid betaine surfactants, alkyl sucrose ester surfactants, alkyl alkanolamide surfactants, alkyl di(polyoxyethylene)monoalkylammonium surfactants, alkyl mono(polyoxyethylene)dialkylammonium surfactants, alkyl benzyldimethylammonium surfactants, alkylaminopropionate surfactants, alkylamidopropyldimethylamine surfactants, or mixtures thereof;And if Z is a charged moiety, Z is charge balanced by a suitable metal or organic counterion. Suitable counterions include metal counterions, amines, or alkanolamines, such as C1-C6 alkanol ammonium. More specifically, suitable counterions include Na+, Ca+, Li+, K+, Mg+, such as monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-l-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, l-amino-3-propanol, or mixtures thereof. In one aspect, the composition contains from 5% to 97% of one or more non-isoprenoid surfactants; and one or more auxiliary cleaning additives; wherein the weight ratio of the surfactant of formula A to the surfactant of formula B is 50:50 to 95:5. ;

[0139] soap -The compositions herein may contain soap. Without being limited by theory, it may be desirable to include soap because it acts partly as a surfactant and partly as a builder, and can be used to suppress foam, and in addition, may advantageously interact with the various cationic compounds of the composition to enhance the softness of textile fabrics treated with the composition of the present invention. Any soap known in the art for use in laundry detergents may be utilized. On the one hand, the composition contains from 0wt% to 20wt%, from 0.5wt% to 20wt%, from 4wt% to 10wt%, or from 4wt% to 7wt% soap. On the one hand, the composition contains from 0wt% to 5wt%; from 0wt% to 2wt%, or from 0wt% to 1wt%.

[0140] Examples of soaps useful herein include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps having varying chain lengths and degrees of substitution. One such mixture is topped palm kernel fatty acid.

[0141] In one aspect, soaps are selected from free fatty acids. Suitable fatty acids are saturated and / or unsaturated and can be obtained from natural sources such as plant or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tall oil, castor oil, tallow and fish oils, greases, and mixtures thereof), or synthetically prepared (e.g., via oxidation of petroleum or via hydrogenation of carbon monoxide by the Fischer Tropsch process).

[0142] The example of the applicable saturated fatty acid used in the present composition comprises capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. The unsaturated fatty acid kind that is applicable comprises: palmitoleic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. The example of preferred lipid acid is saturated Cn fatty acid, saturated C12-C14 fatty acid and saturated or undersaturated Cn to C18 fatty acid and mixture thereof.

[0143] When present, the weight ratio of fabric softening cationic co-surfactant to fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.

[0144] The levels of soap and non-soap anionic surfactants herein are percentages by weight of the detergent composition specified on an acid basis. However, as is generally understood in the art, in practice anionic surfactants and soaps are neutralized using sodium, potassium or alkanolammonium bases such as sodium hydroxide or monoethanolamine.

[0145] Water-soluble additives - The composition of the invention may comprise one or more hydrotropes. A hydrotrope is a compound that dissolves hydrophobic compounds in aqueous solution (or, conversely, polar substances in a non-polar environment). Typically, a hydrotrope has both hydrophilic and hydrophobic characteristics (so-called amphiphilic properties, as known from surfactants); however, the molecular structure of a hydrotrope is generally not conducive to 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 show a critical concentration above which self-aggregation occurs, as found for surfactants, and lipids form micelles, lamellae or other well-defined mesophases. Instead, many hydrotropes show a continuous type of aggregation process, in which the size of the aggregates grows with increasing concentration. However, many hydrotropes modify the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oils, surfactants, and polymers. Hydrotropes are routinely used in various industries ranging from pharmacy, personal care, food to technical applications. The use of hydrotropes in detergent compositions allows, for example, more concentrated surfactant formulations (such as in the process of compacting liquid detergents by removing water) without causing undesirable phenomena, such as phase separation or high viscosity.

[0146] The detergent may contain from 0 to 10 wt%, such as from 0 to 5 wt%, 0.5 wt% to 5 wt%, or from 3 wt% to 5 wt% of a hydrotrope. Any hydrotrope known in the art for use in a detergent may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfonate, and combinations thereof.

[0147] Detergent -The compositions of the present invention may comprise one or more builders, co-builders, builder systems or mixtures thereof. When a builder is used, the cleaning composition will typically comprise from 0 to 65 wt%, at least 1 wt%, from 2 wt% to 60 wt% or from 5 wt% to 10 wt% of the builder. In dishwashing cleaning compositions, the level of builder is typically 40 wt% to 65 wt% or 50 wt% to 65 wt%. The composition may be substantially free of builder; substantially free means "no intentionally added" zeolite and / or phosphate. Typical zeolite builders include zeolite A, zeolite P and zeolite MAP. A typical phosphate builder is sodium tripolyphosphate.

[0148] Builder and / or co-builder can specifically be the chelating agent that forms the water-soluble complex with Ca and Mg.Any builder and / or co-builder known in the art for use in detergent can be used.The limiting examples of builder include zeolite, diphosphate (pyrophosphate), triphosphate such as sodium triphosphate (STP or STPP), carbonate such as sodium carbonate, soluble silicate such as sodium metasilicate, layered silicate (for example from the SKS-6 of Hoechst), ethanolamine (for example 2-amino 2-ethanol (MEA), iminodiethanol (DEA) and 2,2',2 "-nitrilotriethanol (TEA)) and carboxymethyl inulin (CMI), and combination thereof.

[0149] The cleaning composition can include a co-builder individually or in combination with a builder (e.g., a zeolite builder). Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or co-(acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents (e.g., aminocarboxylates, aminopolycarboxylates, and phosphates), and alkyl succinic acids or alkenyl succinic acids. Further specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SE AS), 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), para-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Additional exemplary builders and / or co-builders are described, for example, in WO 09 / 102854, US 5977053.

[0150] In one aspect, the present invention relates to a composition comprising a lipase variant of a parent lipase, wherein the variant has lipase activity, comprises a substitution at positions corresponding to positions 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of the parent lipase, the composition comprises up to 10 wt% or 15 wt% aluminosilicate (on anhydrous basis) and / or phosphate builder, and the composition has a reserve alkalinity greater than 4 or 7.5.

[0151] In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the substitution is selected from position Q15D, E, G23, D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E or T267D, E of a parent lipase (particularly SEQ ID NO: 2).

[0152] As used herein, the term "reserve alkalinity" is a measure of the buffering capacity of a composition determined by titrating a 1% (w / v) solution of the composition with hydrochloric acid to pH 7.5 in order to calculate the reserve alkalinity (g / NaOH / 100 g composition). The reserve alkalinity can be calculated as disclosed on page 9 in WO 2006 / 090335.

[0153] In one aspect, the present invention relates to a composition comprising a lipase variant, wherein the variant further comprises one or more (e.g., several) substitutions corresponding to any one of the positions selected from: 4, 27, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 230, 231, 233, 249, 250, 254, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the present invention relates to a composition comprising a lipase variant, wherein the variant further comprises one or more (e.g., several) substitutions corresponding to any one of the positions selected from: 4V, 27R, 38A, 57G, 58A, 60S, 83T, 86V, 91A / N / Q, 94K / R, 97M, 99K, 111A, 150G, 163K, 210K / Q, 216P, 225R, 227G, 231R, 233R, 249R, 254S, 255A, 256K / T / V, 263Q, 264A, 265T, 266D, 267A, and 269N of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, or SEQ ID NO:12.

[0154] Chelating agents and crystal growth inhibitors - The compositions herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, ion and / or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediamine disuccinic acid (EDDS), ethylenediaminetetraacetic acid (EDTA), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethylinulin (CMI) and 2-phosphonobutane 1,2,4-tricarboxylic acid. Typically, the composition may contain from 0.005 wt % to 15 wt %, or from 3.0 wt % to 10 wt % of the chelating agent or crystal growth inhibitor.

[0155] Bleaching components- Suitable bleaching components for incorporation into the methods and compositions of the present invention include one or a mixture of more than one bleaching components. Suitable bleaching components include bleaching catalysts, photobleaches, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids and mixtures thereof. Typically, when a bleaching component is used, the composition of the present invention may contain from 0 to 30 wt%, from 0.00001 wt% to 90 wt%, 0.0001 wt% to 50 wt%, from 0.001 wt% to 25 wt% or from 1 wt% to 20 wt%. Examples of suitable bleaching components include:

[0156] (1) Preformed Peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of: preformed peroxyacids or salts thereof, typically peroxycarboxylic acids or salts thereof, or peroxysulfuric acid or salts thereof.

[0157] The preformed peroxyacid or salt thereof is preferably a peroxycarboxylic acid or salt thereof, typically having a chemical structure corresponding to the following formula:

[0158]

[0159] Where: R 14 is selected from an alkyl group, an aralkyl group, a cycloalkyl group, an aryl group or a heterocyclic group; R 14 The group may be linear or branched, substituted or unsubstituted; and Y is any suitable counterion to achieve charge neutrality, preferably, Y is selected from hydrogen, sodium or potassium. Preferably, R 14 is a linear or branched, substituted or unsubstituted C 6-9 Preferably, the peroxy acid or its salt is selected from peroxycaproic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and salts thereof, or any combination thereof. Particularly preferred peroxy acid is phthalimido-peroxy-alkanoic acid, in particular ε-phthalimidoperoxycaproic acid (PAP). Preferably, the peroxy acid or its salt has a melting point in the range of from 30°C to 60°C.

[0160] The preformed peroxyacid or salt thereof may also be peroxysulfuric acid or a salt thereof, typically having a chemical structure corresponding to the following formula:

[0161]

[0162] Where: R 15 is selected from an alkyl group, an aralkyl group, a cycloalkyl group, an aryl group or a heterocyclic group; R 15 The group may be linear or branched, substituted or unsubstituted; and Z is any suitable counterion to achieve charge neutrality, preferably, Z is selected from hydrogen, sodium or potassium. Preferably, R 15 is a linear or branched, substituted or unsubstituted C 6-9Preferably, such bleaching components may be present in the composition of the present invention in an amount of from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

[0163] (2) Hydrogen peroxide sources include, for example, inorganic perhydrate salts, including alkali metal salts, such as perborate (usually monohydrate or tetrahydrate), percarbonate, persulfate, perphosphate, sodium salt of persilicate and mixtures thereof. In one aspect of the present invention, inorganic perhydrate salts are, for example, those selected from the group consisting of perborate, sodium salt of percarbonate and mixtures thereof. When used, inorganic perhydrate salts are typically present in an amount of 0.05wt% to 40wt% or 1wt% to 30wt% of the overall composition and are typically incorporated into such compositions as crystalline solids that can be coated. Suitable coatings include: inorganic salts, such as alkali metal silicates, carbonates or borates or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils or fatty soaps. Preferably, such bleaching components may be present in the composition of the present invention in an amount of 0.01wt% to 50wt%, or 0.1wt% to 20wt%.

[0164] (3) The term bleach activator means herein a compound which reacts with hydrogen peroxide to form a peracid via a perhydrolysis reaction. The peracid formed in this way constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters, amides, imides or anhydrides. Suitable bleach activators are those having R-(C=O)-L, wherein R is an alkyl group (preferably branched) having from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms when the bleach activator is hydrophobic, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleach activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives - especially benzene sulfonates. Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS)), and / or those disclosed in WO 98 / 17767. A family of bleach activators is disclosed in EP 624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or short chain triglycerides (like triacetin) have the advantage that it is environmentally friendly. In addition, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product during storage and are effective bleach activators. Finally, ATC is multifunctional because the citrate released in the perhydrolysis reaction can act as a builder. Alternatively, the bleaching system can contain peroxy acids of the amide, imide or sulfone type, for example. The bleaching system can also contain peracids, such as 6-(phthalimido)perhexanoic acid (PAP). Suitable bleach activators are also disclosed in WO 98 / 17767. Although any suitable bleach activator can be used, in one aspect of the present invention, the subject cleaning composition can contain NOBS, TAED or a mixture thereof. When present, peracids and / or bleach activators are typically present in the composition in an amount of 0.1 wt % to 60 wt %, 0.5 wt % to 40 wt % or 0.6 wt % to 10 wt % based on the fabric and home care composition. One or more hydrophobic peracids or their precursors can be used in combination with one or more hydrophilic peracids or their precursors. Preferably, such bleaching components may be present in the compositions of the present invention in an amount of from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

[0165] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0166] (4) Diacyl Peroxides - Preferred diacyl peroxide bleaching species include those selected from the group consisting of diacyl peroxides having the following general formula: R 1 -C(O)-OO-(O)CR 2 , where R 1 Indicates C6-C 18 An alkyl group, preferably a straight chain having at least 5 carbon atoms and optionally containing one or more substituents (e.g., -N + (CH3)3, -COOH or -CN) and / or one or more interrupting moieties (e.g., -CONH- or -CH=CH-) inserted between adjacent carbon atoms of the alkyl group. 12 alkyl group, and R 2 represents an aliphatic group that is compatible with the peroxide portion so that R 1 and R 2 Together they contain a total of 8 to 30 carbon atoms. In a preferred aspect, R 1 and R 2 It is a straight chain unsubstituted C6-C 12 Most preferably, R 1 and R 2 are the same. 1 and R 2 All are C6-C 12 alkyl groups) are particularly preferred. Preferably, at least one, most preferably only one of the R groups (R1 or R2) does not contain a branched or pendant ring at the α position, or preferably does not contain a branched or pendant ring at either the α position or the β position, or most preferably does not contain a branched or pendant ring at either the α position, the β position, or the γ position. In an additional preferred aspect, the DAP may be asymmetric, such that the R1 acyl group preferably hydrolyzes rapidly to produce a peracid, but the R2 acyl group hydrolyzes slowly.

[0167] The tetraacyl peroxide bleaching species are preferably selected from tetraacyl peroxides of the following general formula: R 3 -C(O)-OO-C(O)-(CH2)nC(O)-OO-C(O)-R 3 , where R 3 represents a C1-C9 alkyl group or a C3-C7 group, and n represents an integer from 2 to 12 or 4 to 10 both inclusive.

[0168] Preferably, the diacyl and / or tetraacyl peroxide bleaching species is present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm of washing liquid by weight. In a preferred aspect, the bleaching species is present in an amount sufficient to provide from 0.5 ppm to 300 ppm, from 30 ppm to 150 ppm of washing liquid by weight.

[0169] Preferably, the bleach component comprises a bleach catalyst (5 and 6).

[0170] (5) Preferred are organic (non-metallic) bleach catalysts, including bleach catalysts capable of accepting an oxygen atom from a peroxyacid and / or its salt and transferring the oxygen atom to an oxidizable substrate. Suitable bleach catalysts include, but are not limited to, iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonyl imides; N-phosphoryl imides; N-acyl imides; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones and mixtures thereof.

[0171] Suitable iminium cations and polyions include, but are not limited to, N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (see, e.g., Compound 4, page 433); N-methyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in US 5360569 (e.g., column 11, Example 1); and n-octyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in US 5360568 (e.g., column 10, Example 3).

[0172] Suitable iminium zwitterions include, but are not limited to, N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared as described in US 5576282 (e.g., column 31, Example II); N-[2-(sulfooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in US 5817614 (e.g., column 32, Example V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in WO 05 / 047264 (e.g., page 18, Example 8), and 2-[3-[(2-butyloctyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, inner salt.

[0173] Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can be prepared according to the procedure described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfer catalysts include, but are not limited to, sodium 1-hydroxy-N-oxo-N-[2-(sulfoxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0174] Suitable N-sulfonylimine oxygen transfer catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, which can be prepared according to the procedures described in Journal of Organic Chemistry (1990), 55(4), 1254-61.

[0175] Suitable N-phosphonyl imine oxygen transfer 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 can be prepared according to the procedures described in Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70.

[0176] Suitable N-acylimine oxygen transfer catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, which can be prepared according to the procedure described in Polish Journal of Chemistry (2003), 77(5), 577-590.

[0177] Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, which can be prepared according to the procedure described in US Pat. No. 5,753,599 (column 9, Example 2).

[0178] Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butyrimidinium fluoride, which can be prepared according to the procedure described in Tetrahedron Letters (1994), 35(34), 6329-30.

[0179] Suitable cyclic sugar ketone oxygen transfer catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose as prepared in US 6649085 (column 12, example 1).

[0180] Preferably, the bleaching catalyst comprises an iminium ion and / or a carbonyl functional group and is typically capable of forming an oxaziridinium and / or a dioxirane functional group upon acceptance of an oxygen atom, especially from a peroxyacid and / or its salt. Preferably, the bleaching catalyst comprises an oxaziridinium functional group and / or is capable of forming an oxaziridinium functional group upon acceptance of an oxygen atom, especially from a peroxyacid and / or its salt. Preferably, the bleaching catalyst comprises a cyclic iminium functional group, preferably wherein the cyclic moiety has a ring size of from five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleaching catalyst comprises an aryl iminium functional group, preferably a dicyclic aryl imine functional group, preferably a 3,4-dihydroisoquinolinium functional group. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary oxaziridinium functional group upon accepting an oxygen atom, especially upon accepting an oxygen atom from a peroxyacid and / or a salt thereof. In one aspect, the detergent composition comprises a logP of no greater than 0, no greater than -0.5, no greater than -1.0, no greater than -1.5, no greater than -2.0, no greater than -2.5, no greater than -3.0, or no greater than -3.5. o / w The following is a more detailed description of the method for determining logP o / w method.

[0181] Typically, the bleaching ingredients are capable of producing an X of from 0.01 to 0.30, from 0.05 to 0.25, or from 0.10 to 0.20. SO The following is a more detailed description of the method for determining X SO For example, a bleaching component having an isoquinolinium structure can generate a bleaching species having an oxaziridinium structure. In this example, X SO It is a type of oxadiazine bleaching SO .

[0182] Preferably, the bleach catalyst has a chemical structure corresponding to the following formula:

[0183]

[0184] Wherein: n and m are independently 0 to 4, preferably n and m are both 0; each R 1are independently selected from substituted or unsubstituted groups selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocycle, fused heterocycle, nitro, halide, cyano, sulfonate, alkoxy, keto, carboxyl and alkoxycarbonyl; and any two vicinal R 1 The substituents may be combined to form a fused aryl, fused carbocyclic or fused heterocyclic ring; each R 2 independently selected from substituted or unsubstituted groups, the groups independently selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylene, heterocycle, alkoxy, arylcarbonyl, carboxyalkyl and amide groups; any R 2 Can be used with any other R 2 Combined together to form part of a common ring; any geminal R 2 can combine to form a carbonyl group; and any two R 2 can be combined to form a substituted or unsubstituted fused unsaturated moiety; R 3 C1 to C 20 Substituted or unsubstituted alkyl; R 4 is hydrogen or Q t -A moiety, wherein: Q is a branched or unbranched olefin, t=0 or 1, and A is an anionic group selected from the group consisting of: OSO3 - 、SO3 - 、CO2 - 、OCO2 - 、OPO3 2- 、OPO3H - and OPO2 - ; R 5 is hydrogen or -CR 11 R 12 -YG b -Y c -[(CR 9 R 10 ) y -O] k -R 8 moiety, wherein: each Y is independently selected from the group consisting of: O, S, NH or NR 8 ; and each R 8 is independently selected from the group consisting of alkyl, aryl and heteroaryl, the moiety being substituted or unsubstituted, and whether substituted or unsubstituted, the moiety has less than 21 carbons; each G is independently selected from the group consisting of CO, SO2, SO, PO and PO2; R 9 and R 10 are independently selected from the group consisting of: H and C1-C4 alkyl; R 11 and R 12independently selected from the group consisting of: H and alkyl, or when put together can combine to form a carbonyl; b = 0 or 1; c can be = 0 or 1, but if 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 is substituted or unsubstituted; and if X is present, it is a suitable charge-balancing counterion, when R 4 When X is hydrogen, it is preferably present. Suitable X include, but are not limited to, chloride, bromide, sulfate, methylsulfate, sulfonate, p-toluenesulfonate, boron tetrafluoride phosphate.

[0185] In one aspect of the invention, the bleach catalyst has a structure corresponding to the following general formula:

[0186]

[0187] Where R 13 is a branched alkyl group containing from three to 24 carbon atoms (including branched carbon atoms) or a straight chain alkyl group containing from one to 24 carbon atoms; preferably, R 13 is a branched alkyl group containing from eight to 18 carbon atoms or a straight chain alkyl group containing from eight to eighteen carbon atoms; preferably, R 13 is selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl; preferably, R 13 Selected from the group consisting of 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

[0188] Preferably, the bleaching component comprises, in addition to a bleaching catalyst, in particular an organic bleaching catalyst, a peracid source. The peracid source may be selected from (a) preformed peracids; (b) percarbonates, perborates or persulfates (hydrogen peroxide sources), preferably in combination with a bleach activator; and (c) perhydrolases and esters for in situ formation of peracids in the presence of water in the textile or hard surface treatment step.

[0189] When present, the peracid and / or bleach activator is typically present in the composition in an amount of from 0.1 wt% to 60 wt%, from 0.5 wt% to 40 wt% or from 0.6 wt% to 10 wt%, based on the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof.

[0190] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

[0191] (6) Metal-containing bleach catalysts - the bleaching component can be provided by a catalytic metal complex. One type of metal-containing bleach catalyst is a catalytic system comprising a transition metal cation with defined bleach catalytic activity (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum or manganese cation), an auxiliary metal cation with little or no bleach catalytic activity (e.g., zinc or aluminum cation), and a spacer with defined stability constants for the catalytic and auxiliary metal cations, in particular ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and its water-soluble salts. Such catalysts are disclosed in US 4430243. Preferred catalysts are described in WO 09 / 839406, US 6218351 and WO 00 / 012667. Particularly preferred are transition metal catalysts or ligands thereof as cross-linked multidentate N-donor ligands.

[0192] If desired, the compositions herein may be catalyzed with the aid of manganese compounds. Such compounds and usage levels are well known in the art and include, for example, the manganese-based catalysts disclosed in US 5576282.

[0193] Cobalt bleach catalysts useful herein are known and described, for example, in US 5597936, US 5595967. Such cobalt catalysts may be readily prepared by known procedures, such as, for example, those taught in US 5597936 and US 5595967.

[0194] The compositions herein may also suitably include transition metal complexes of ligands such as bispidone (US 7501389) and / or polycyclic rigid ligands - abbreviated as "MRLs". As a practical matter and not by way of limitation, the compositions and methods herein may be adjusted to provide about at least one part per hundred million of active MRL species in an aqueous wash medium, and will typically provide from 0.005 ppm to 25 ppm, from 0.05 ppm to 10 ppm, or from 0.1 ppm to 5 ppm of MRL in the wash liquor.

[0195] Suitable transition metals in the transition metal bleach catalyst 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, for example, US 6225464 and WO 00 / 32601.

[0196] (7) Photobleaches - Suitable photobleaches include, for example, sulfonated zinc phthalocyanine, sulfonated aluminium phthalocyanine, xanthene dyes and mixtures thereof. Preferred bleaching components for use in the compositions of the present invention comprise a hydrogen peroxide source, a bleach activator and / or an organic peroxyacid, optionally generated in situ by reaction of a hydrogen peroxide source and a bleach activator in combination with a bleach catalyst. Preferred bleaching components comprise a bleach catalyst, preferably an organic bleach catalyst as described above.

[0197] Particularly preferred bleaching components are bleach catalysts, especially organic bleach catalysts.

[0198] Exemplary bleaching systems are also described in, for example, WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242.

[0199] Fabric Toner -The composition may comprise a fabric hueing agent. Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes belonging to the following color index (CI) classification: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet and basic red or a mixture thereof.

[0200] In one aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: Colorimetric Index (Society of Dyers and Colorists, Bradford, England) numbers Direct Violet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, Acid Violet 50 , Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75, Basic Blue 159 and mixtures thereof. In one aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: Color Index (Royal Society of Colorists, Bradford, England) No. Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, Acid Red 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, Acid Blue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51, and mixtures thereof. In one aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: Color Index (Royal Society of Colorists, Bradford, England) No. Acid Violet 17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113, or mixtures thereof.

[0201] Suitable polymeric dyes include polymeric dyes selected from the group consisting of polymers containing a conjugated chromogen (dye-polymer conjugate) and polymers in which the chromogen is copolymerized into the polymer backbone, and mixtures thereof.

[0202] In one aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of: (Milliken), the colorant being a dye-polymer conjugate formed from at least one reactive dye and a polymer selected from the group consisting of the following polymers, the polymer comprising a moiety selected from the group consisting of: a hydroxyl moiety, a primary amine moiety, a secondary amine moiety, a thiol moiety, and mixtures thereof. In still another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of: Violet CT, carboxymethylcellulose (CMC) conjugated with reactive blue, reactive violet or reactive red dyes such as CMC conjugated with CI Reactive Blue 19 (sold by Megazyme, Wicklow, Ireland, under the product name AZO-CM-CELLULOSE, product code S-ACMC), alkoxylated triphenyl-methane polymeric colorants, alkoxylated thiophene polymeric colorants, and mixtures thereof.

[0203] Preferred hueing dyes include the brighteners found in WO08 / 87497. These brighteners can be characterized by the following structure (I):

[0204]

[0205] Wherein R1 and R2 can be independently selected from:

[0206] a)[(CH2CR'HO) x (CH2CR"HO) y H]

[0207] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

[0208] b) R1 = alkyl, aryl or arylalkyl, and R2 = [(CH2CR'HO) x (CH2CR"HO) y H]

[0209] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

[0210] c) R1=[CH2CH2(OR3)CH2OR4] and R2=[CH2CH2(OR3)CH2OR4]

[0211] Wherein R3 is selected from the group consisting of: H, (CH2CH2O) z H and mixtures thereof; and wherein z = 0 to 10;

[0212] Wherein R4 is selected from the group consisting of: (C1-C 16) alkyl groups, aryl groups, and mixtures thereof; and

[0213] d) wherein R1 and R2 may be independently selected from the amino addition products of styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, tert-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl hexadecyl ether, followed by the addition of from 1 to 10 alkylene oxide units.

[0214] The preferred brighteners of the present invention can be characterized by the following structure (II):

[0215]

[0216] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

[0217] Another preferred brightener of the present invention can be characterized by the following structure (III):

[0218]

[0219] Typically a mixture having a total of 5 EO groups is included. Suitable preferred molecules are those in structure I having the following pendant groups in "part a" above.

[0220] Table 1

[0221] R1 R2 R’ R” X y R’ R” X y A H H 3 1 H H 0 1 B H H 2 1 H H 1 1 c=b H H 1 1 H H 2 1 d=a H H 0 1 H H 3 1

[0222] Additional brighteners for use include those described in US 2008 / 34511 (Unilever). A preferred agent is "Violet 13".

[0223] Suitable dye clay conjugates include dye clay conjugates selected from the group consisting of at least one cationic / basic dye and smectite and mixtures thereof. In one aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: a cationic / basic dye selected from the group consisting of CI Basic Yellow 1 to 108, CI Basic Orange 1 to 69, CI Basic Red 1 to 118, CI Basic Violet 1 to 51, CI Basic Blue 1 to 164, CI Basic Green 1 to 14, CI Basic Brown 1 to 23, CI Basic Black 1 to 11; and a clay selected from the group consisting of montmorillonite clay, hectorite clay, saponite clay and mixtures thereof. In still 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 CI42040 conjugate, montmorillonite basic red R1 C.I.45160 conjugate, montmorillonite CI basic black 2 conjugate, hectorite basic blue B7 C.I.42595 conjugate, hectorite basic blue B9 CI52015 conjugate, hectorite basic violet V3 CI42555 conjugate, hectorite basic green G1 CI42040 conjugate, hectorite basic red R1 CI45160 conjugate. CI45160 conjugate, hectorite CI basic black 2 conjugate, saponite basic blue B7 CI42595 conjugate, saponite basic blue B9 CI52015 conjugate, saponite basic violet V3 CI42555 conjugate, saponite basic green G1 CI42040 conjugate, saponite basic red R1 C.I.45160 conjugate, saponite CI basic black 2 conjugate and mixtures thereof.

[0224] Suitable pigments include pigments selected from the group consisting of flavonol, indanthrone, chlorine-containing indanthrone containing from 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide (wherein the imide group may be unsubstituted or substituted by C1-C3-alkyl or phenyl or heterocyclic groups, and wherein the phenyl and heterocyclic groups may additionally carry substituents which do not impart solubility in water), anthrapyrimidinecarboxylic acid amides, anthrone violet, isoanthrone violet, dioxazine pigments, copper phthalocyanine which may contain up to 2 chlorine atoms per molecule, polychloro-copper phthalocyanine or polybrominated chloro-copper phthalocyanine containing up to 14 bromine atoms per molecule, and mixtures thereof.

[0225] In one aspect, suitable pigments include pigments selected from the group consisting of Ultramarine Blue (CI Pigment Blue 29), Ultramarine Violet (CI Pigment Violet 15), and mixtures thereof.

[0226] The above fabric hueing agents may be used in combination (any mixture of fabric hueing agents may be used). Suitable hueing agents are described in more detail in US 7208459. The preferred level of dye in the composition of the present invention is 0.00001wt% to 0.5wt%, or 0.0001wt% to 0.25wt%. The concentration of dye preferably used in the treatment and / or cleaning step in water is from 1ppb to 5ppm, 10ppb to 5ppm or 20ppb to 5ppm. In a preferred composition, the concentration of surfactant will be from 0.2 to 3g / l.

[0227] Encapsulated - The composition may comprise an encapsulate. In one aspect, the encapsulate comprises a core, a shell having an inner surface and an outer surface, said shell encapsulating said core.

[0228] In one aspect of the encapsulate, the core may comprise a material selected from the group consisting of: fragrance brighteners; dyes; insect repellents; silicones; waxes; flavorings; vitamins; fabric softeners; skin care agents; in one aspect, paraffin; enzymes; antibacterial agents; bleaches; sensates; and mixtures thereof; and the shell may comprise a material selected from the group consisting of: polyethylene; polyamides; polyvinyl alcohol, optionally containing other comonomers; polystyrene; polyisoprene; polycarbonates; polyesters; polyacrylates; aminoplasts, in one aspect, the aminoplasts may comprise polyureas, polyurethanes and / or polyurea polyurethanes, in one aspect, the polyureas may comprise polyoxymethylene ureas and / or melamine formaldehyde; polyolefins; polysaccharides, in one aspect, the polysaccharides may comprise alginate and / or chitosan; gelatin; shellac; epoxy resins; vinyl polymer water-insoluble inorganics; silicones; and mixtures thereof.

[0229] In one aspect of the encapsulate, the core may comprise a fragrance.

[0230] In one aspect of the encapsulate, the shell may comprise melamine formaldehyde and / or cross-linked melamine formaldehyde.

[0231] In one aspect, it is disclosed that suitable encapsulated materials may include a core material and a shell, the shell at least partially surrounding the core material. At least 75%, 85% or 90% of the encapsulated materials may have a breaking strength of from 0.2 MPa to 10 MPa, from 0.4 MPa to 5 MPa, from 0.6 MPa to 3.5 MPa or from 0.7 MPa to 3 MPa; and have a beneficial agent leakage of from 0% to 30%, from 0% to 20% or from 0% to 5%.

[0232] In one aspect, at least 75%, 85%, or 90% of the encapsulates may have a particle size of from 1 micron to 80 microns, from 5 microns to 60 microns, from 10 microns to 50 microns, or from 15 microns to 40 microns.

[0233] In one aspect, at least 75%, 85%, or 90% of the encapsulates may have a particle wall thickness of from 30 to 250 nm, from 80 to 180 nm, or from 100 to 160 nm.

[0234] In one aspect, the core material of the encapsulated object may include a material selected from the group consisting of flavor raw materials, and / or optionally includes a material selected from the group consisting of: vegetable oils, including pure vegetable oils and / or blended vegetable oils, including castor oil, coconut oil, cottonseed oil, grapeseed oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, oil), lemon oil and mixtures thereof; esters of vegetable oils, esters including dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, octyl benzyl adipate, tricresyl phosphate, trioctyl phosphate and mixtures thereof; straight or branched chain hydrocarbons including those having a boiling point above about 80°C; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls (including monoisopropyl biphenyl), alkylated naphthalenes (including dipropyl naphthalene), petroleum spirits (including kerosene), mineral oils and mixtures thereof; aromatic solvents including benzene, toluene and mixtures thereof; silicone oils; and mixtures thereof.

[0235] On the one hand, the wall material of the encapsulated object can include a suitable resin, the resin includes the reaction product of an aldehyde and an amine, and a suitable aldehyde includes formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril and mixtures thereof. Suitable melamines include methylol melamine, methylated methylol melamine, imino melamine and mixtures thereof. Suitable ureas include dimethylol urea, methylated dimethylol urea, urea-resorcinol and mixtures thereof.

[0236] In one aspect, suitable formaldehyde scavengers can be used with and / or added to the composition, for example in a capsule slurry, before, during or after the encapsulate is added to the composition. Suitable capsules can be made by following the teachings of US2008 / 0305982, and / or US2009 / 0247449.

[0237] In a preferred aspect, the composition may further comprise a deposition aid, preferably consisting of the group comprising cationic or nonionic polymers. Suitable polymers include cationic starch, cationic hydroxyethylcellulose, polyvinyl formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene glycol terephthalate, and polymers containing dimethylaminoethyl methacrylate, optionally with one or more monomers selected from the group comprising acrylic acid and acrylamide.

[0238] spices- In one aspect, the composition comprises a perfume, the perfume comprising one or more perfume raw materials selected from the group consisting of: 1,1'-oxybis-2-propanol; diethyl 1,4-cyclohexanedicarboxylate; (ethoxymethoxy)cyclododecane; 1,3-nonanediol monoacetate; (3-methylbutoxy)acetic acid 2-propenyl ester; β-methylcyclododecaneethanol; 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol; oxacyclohexadecane-2-one; α-methyl-benzyl alcohol acetate; trans-3-ethoxy-1,1,5-trimethylcyclohexane; 4-(1,1-dimethylethyl)cyclohexanol acetate; dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; β-methylphenylpropanal; β-methyl-3-(1-methylethyl)phenylpropanal; 4-phenyl-2-butanone; ethyl 2-methylbutyrate; benzaldehyde; 1-methylethyl 2-methylbutyrate; dihydro-5-pentyl-2(3H)furanone; (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal; undecanal; 2-ethyl-α,α-dimethylphenylpropanal; decanal; α,α-dimethylphenylethanol acetate; 2 -(phenylmethylene)octanal; methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one; 2-pentylcyclopentanone; methyl 3-oxo-2-pentylcyclopentaneacetate; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde; 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; phenylethyl alcohol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde; 10-undecenal; benzyl propionate; β-methylphenylpentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene; α,α-dimethylphenylethyl alcohol; (2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; benzyl acetate; 2-propenyl cyclohexylpropionate; 2-propenyl hexanoate; 1,2-dimethoxy-4-(2-propenyl)benzene; 1,5-dimethyl-bicyclo[3.2.1] octane-8-one oxime; 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carbaldehyde; 3-butene-2-ol; 2-[[[2,4(or 3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoic acid methyl ester; 8-cyclohexadecene-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol; 2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-octadien-1-ol ; 2-hydroxy-benzoic acid (3Z)-3-hexenyl ester; 2-tridecenenitrile; 4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; (2-methylbutoxy)acetic acid-2-propenyl ester; 2-hydroxy-3-methylbutyl benzoate; (Z)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; 2- Hexyl-3-oxocyclopentanecarboxylic acid methyl ester; 4-ethyl-α,α-dimethyl-phenylpropanal; 3-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carbaldehyde; 1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-oxodimethanoazulene-5-yl)-[3R-(3α,3aβ,7β,8aα)]-ethanone; 2-methyl-2H-pyran-2-one 6-butyltetrahydro-undecanal; 4-(1,1- dimethylethyl)-α-methyl-phenylpropanal; 5-heptyldihydro-2(3H)-furanone; 2-[(7-hydroxy-3,7-dimethyloctylene)amino]benzoic acid methyl ester; 2-hydroxy-benzoic acid benzyl ester; 2-methoxynaphthalene; 2-hexyl-2-cyclopenten-1-one; 5-hexyldihydro-2(3H)-furanone; 3-methyl-3-phenyl-oxiranecarboxylic acid ethyl ester; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane; phenylpentanol, .γ.-methyl-; 3,7-dimethyl-3-octanol; 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-methylene-1H-inden-6-ol propionate; 3-methyl-2-butene-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-inden-5-ylidene)-butyraldehyde; 3-2,4-dimethyl-cyclohexene-1-carbaldehyde; 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalene)-ethanone; 2-hydroxy-benzoic acid methyl ester; 2-hydroxy-benzoic acid hexyl ester; 2-phenoxy-ethanol; 2 -Hydroxy-pentyl benzoate; 2,3-heptanedione; 2-hexen-1-ol; 2,6-dimethyl-6-octen-2-ol; damascenone (α, β, γ or δ or a mixture thereof), 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-ol acetate; 9-undecenal; 8-undecenal; isocitral; 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2- Naphthalene)-ethanone; 3,5-dimethyl-3-cyclohexene-1-carbaldehyde; 2,4-dimethyl-3-cyclohexene-1-carbaldehyde; 3,7-dimethyl-1,6-octadien-3-ol; 3,7-dimethyl-1,6-octadien-3-ol acetate; pt-Bucinal, and 2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]-cyclopentanone and 1-methyl-4-(1-methylvinyl)cyclohexene and mixtures thereof.

[0239] In one aspect, the composition may comprise encapsulated fragrance particles comprising water-soluble hydroxyl compounds or melamine-formaldehyde or modified polyvinyl alcohol. In one aspect, the encapsulate comprises (a) an at least partially water-soluble solid matrix comprising one or more water-soluble hydroxyl compounds, preferably starch; and (b) a fragrance oil encapsulated by the solid matrix.

[0240] In another aspect, the fragrance may be pre-complexed with a polyamine, preferably polyethyleneimine, to form a Schiff base.

[0241] polymer- The composition may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates (such as polyacrylates), maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0242] The composition may include one or more amphiphilic cleaning polymers, such as compounds having the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + -(CH3)-bis((C2H5O)(C2H4O)n), wherein n=from 20 to 30, and x=from 3 to 8, or sulfated or sulfonated variants thereof.

[0243] The compositions may comprise amphiphilic alkoxylated grease cleaning polymers having balanced hydrophilic and hydrophobic properties such that they remove grease particles from fabrics and surfaces. Specific aspects of the amphiphilic alkoxylated grease cleaning polymers of the invention comprise a core structure and a plurality of alkoxylated groups attached to that core structure. These may comprise alkoxylated polyalkylenimines, preferably having inner polyethylene oxide blocks and outer polypropylene oxide blocks.

[0244] Alkoxylated polycarboxylates (e.g., those prepared from polyacrylates) may be used herein to provide additional grease removal properties. Such materials are described in WO 91 / 08281 and PCT 90 / 01815. Chemically, these materials include polyacrylates having one ethoxy side chain per 7-8 acrylate units. The side chains have the formula -(CH2CH2O) m (CH2) n CH3, wherein m is 2-3 and n is 6-12. The side chains are ester-linked to the polyacrylate "backbone" to provide a "comb-like" polymer type structure. The molecular weight may vary, but is typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylates may comprise from 0.05 wt % to 10 wt % of the composition herein.

[0245] The isoprenoid-derived surfactants of the present invention, and mixtures thereof with other co-surfactants and other adjuvant ingredients, are particularly suitable for use with amphiphilic graft copolymers, preferably comprising (i) a polyethylene glycol backbone; and (ii) and at least one pendant portion selected from polyvinyl acetate, polyvinyl alcohol and mixtures thereof. A preferred amphiphilic graft copolymer is Sokalan HP22 supplied by BASF. Suitable polymers include random graft copolymers, preferably polyvinyl acetate grafted polyethylene oxide copolymers, having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000, and the weight ratio of polyethylene oxide to polyvinyl acetate is 40 to 60, and there are no more than 1 grafting point per 50 ethylene oxide units.

[0246] Carboxylate polymers - The composition of the invention further comprises one or more carboxylate polymers, such as maleate / acrylate random copolymers or polyacrylate homopolymers. In one aspect, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of from 4,000Da to 9,000Da or from 6,000Da to 9,000Da.

[0247] Soil Release Polymers - The compositions of the present invention may also include one or more soil release polymers having a structure as defined by one of the following structures (I), (II) or (III):

[0248] (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d

[0249] (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e

[0250] (III)-[(OCHR 5 -CHR 6 ) c -OR 7 ] f

[0251] in:

[0252] a, b and c are from 1 to 200;

[0253] d, e and f are from 1 to 50;

[0254] Ar is 1,4-substituted phenylene;

[0255] sAr is a 1,3-substituted phenylene group, wherein the phenylene group is substituted at the 5-position with SO3Me;

[0256] Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, monoalkylammonium, dialkylammonium, trialkylammonium or tetraalkylammonium, wherein the alkyl group is C1-C 18 Alkyl or C2-C 10 Hydroxyalkyl, or a mixture thereof;

[0257] R 1 , R 2 , R 3 , R 4 , R 5 and R 6 Independently selected from H or C1-C 18 n-alkyl or isoalkyl; and

[0258] R 7 Is a straight or branched C1-C 18 Alkyl, or straight or branched C2-C 30 alkenyl, or a cycloalkyl group having 5 to 9 carbon atoms, or a C8-C 30 Aryl, or C6-C 30 Arylalkyl.

[0259] Suitable soil release polymers are polyester soil release polymers, such as Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6, supplied by Rhodia. Other suitable soil release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, supplied by Clariant. Other suitable soil release polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0260] Cellulose polymer - The composition of the present invention further comprises one or more cellulosic polymers, including those selected from alkyl celluloses, alkyl alkoxyalkyl celluloses, carboxyalkyl celluloses, alkyl carboxyalkyl celluloses. In one aspect, the cellulosic polymer is selected from the group comprising 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 from 0.5 to 0.9 and a molecular weight of from 100,000 Da to 300,000 Da.

[0261] Enzymes -Compositions can include one or more enzymes that provide cleaning performance and / or fabric care benefits.The example of suitable enzyme includes but is not limited to hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosylase, malanase (malanase), beta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase or its mixture.Typical combination is enzyme mixture, can include for example protease and lipase together with amylase.When present in composition, aforementioned other enzyme can exist by the weight of composition with the level of from 0.00001wt% to 2wt%, from 0.0001wt% to 1wt% or from 0.001wt% to 0.5wt% enzyme protein.

[0262] Generally, the properties of the enzyme(s) selected should be compatible with the selected detergent (ie, pH optimum, compatibility with other enzymes and non-enzyme ingredients, etc.), and the enzyme(s) should be present in an effective amount.

[0263] On the one hand, preferred enzymes will include cellulases. Suitable cellulases include those of bacterial or fungal origin. Include chemically modified mutants or protein engineered mutants. Suitable cellulases include cellulases from the genus Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, such as the fungal cellulases produced by Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4435307, US 5648263, US 5691178, US 5776757 and WO 89 / 09259.

[0264] Particularly suitable cellulases are alkaline or neutral cellulases having color care benefits. Examples of such cellulases are cellulases described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0531315, US 5457046, US 5686593, US 5763254, WO 95 / 24471, WO 98 / 12307 and PCT / DK98 / 00299.

[0265] Commercially available cellulases include Celluzyme TM and Carezyme TMCelluclean, Carezyme Premium, Whitezyme (Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor International Inc.), and KAC-500(B) TM (Kao Corporation).

[0266] On the one hand, preferred enzymes will include proteases. Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, such as plant or microbial origin. Microbial origin is preferred. Chemically modified mutants or protein engineered mutants are included. It can be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease can be, for example, a S1 family (such as trypsin) or a S8 family (such as subtilisin). Metalloproteases can be, for example, thermolysin or other metalloproteases from, for example, an M4 family, such as those from an M5, M7 or M8 family.

[0267] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Engng. [Protein Engineering] 4 (1991) 719-737 and Siezen et al., Protein Science [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 divided into 6 subclasses, namely, the subtilisin family, the thermophilic protease family, the proteinase K family, the lanthionine antibiotic peptidase family, the Kexin family and the Pyrolysin family.

[0268] Examples of subtilases are those derived from Bacillus, such as B. lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, B. pumilus and Bacillus gibsonii described in US 7262042 and WO 09 / 021867; and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, B. licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89 / 06279 and protease PD138 described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024 and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease (described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372), and chymotrypsin from Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0269] Further preferred proteases are the alkaline protease from Bacillus lentus DSM 5483 (as described, for example, in WO 95 / 23221) and variants thereof (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0270] Examples of metalloproteases are neutral metalloproteases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0271] Examples of useful proteases are the 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, WO 11 / 036264, in particular variants having substitutions at one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, using BPN' for numbering. More preferably, the subtilase variant may comprise the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (use BPN' for numbering).

[0272] Suitable commercially available proteases include those sold under the following trade names: Duralase Tm 、Durazym Tm , as well as All of these can be or (Novozymes); those sold under the following trade names: Purafect Prefer Tm , Effect Tm , as well as (Danisco / DuPont), Axapem TM (Gist-Brocases N.V.), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (alkaliphilic Bacillus subtilisin) from Kao Corporation (Kao).

[0273] In one aspect, preferred enzymes will include amylases. Suitable amylases may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, such as the alpha-amylases of a particular strain of Bacillus licheniformis described in more detail in GB1296839.

[0274] Suitable amylases include those having SEQ ID NO: 3 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are described in WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and SEQ ID NO: 4 in WO 99 / 019467, for example variants having substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0275] Various suitable amylases include those having SEQ ID NO: 6 in WO 02 / 010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having deletions in positions 181 and 182 and a substitution in position 193.

[0276] Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of a Bacillus licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid alpha-amylase are those having a substitution, deletion or insertion in one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209 and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 are those having the following substitutions:

[0277] M197T;

[0278] H156Y+A181T+N190F+A209V+Q264S; or

[0279] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0280] Suitable additional amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having substitutions, deletions or insertions in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those having deletions in positions R181 and G182, or positions H183 and G184.

[0281] Additional amylases that may be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873 or variants thereof having 90% sequence identity thereto. 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 in 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. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 7 are those having deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0282] Other amylases that can be used are those having SEQ ID NO: 2 in WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those having substitutions, deletions or insertions in one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211 and 264.

[0283] Additional suitable amylases are those having SEQ ID NO: 2 in WO 09 / 061380, or variants thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having C-terminal truncations and / or substitutions, deletions or insertions in 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 with 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 those with deletions at positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO: 2 are those with the following substitutions:

[0284] N128C+K178L+T182G+Y305R+G475K;

[0285] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0286] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0287] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein the variant is C-terminally truncated and optionally further comprises a substitution at position 243 and / or a deletion at position 180 and / or position 181.

[0288] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01 / 66712 or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions or insertions in one or more of the following positions in SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K and R458K, and variants additionally having substitutions in one or more positions selected from the group consisting of: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferably variants additionally having substitutions in all of these positions.

[0289] Other examples are amylase variants as described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0290] Commercially available amylase is Duramyl TM Termamyl TM TermamylUltra TM 、Fungamyl TM 、Ban TM 、Stainzyme TM 、StainzymePlus TM , Supramyl TM 、Natalase TM 、Liquozyme X、BAN TM , Resillience and Everest (from Novozymes), AT 9000 Biozym Biotech Trading GmbH (Wehlistrasse, Vienna, Austria, 27bA-1200, and Rapidase TM 、Purastar TM / EffectTM ,Powerase,Preferenz S100,PreferenxS110, OPTISIZE HT and PURASTAR (Danisco / DuPont) and (Kao Co., Ltd.).

[0291] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipases from thermophilic fungi, e.g., from Thermomyces lanuginosus (formerly named Humicola lanuginosus) as described in EP 258068 and EP 305216; cutinases from Humicola, e.g., Humicola insolens (WO 96 / 13580); lipases from strains of Pseudomonas (some of these now renamed Burkholderia), e.g., Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (EP 331376), Pseudomonas strain SD705 (WO 95 / 06720 & WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (WO 10 / 065455); a cutinase from Magnaporthe grisea (WO 10 / 107560); a cutinase from Pseudomonas mendocinae (US 5,389,536); a lipase from Thermobifida fusca (WO 11 / 084412, WO 13 / 033318); a Geobacillus stearothermophilus lipase (WO 11 / 084417); a lipase from Bacillus subtilis (WO 11 / 084599); and a lipase from Streptomyces griseus (WO 11 / 150157) and Streptomyces pristinae spiralis (WO 12 / 137147).

[0292] Other examples are lipase variants as 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.

[0293] Preferred commercial lipase products include Lipolase TM 、Lipex TM 、LipexEvity TM 、Lipolex TM , Lipoclean TM and LipexEvity 100L (Novozymes), Lumafast (DuPont), and Lipomax TM (From Gist-Brocades).

[0294] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, such as acyltransferases with homology to Candida antarctica lipase A (WO 10 / 111143), acyltransferases from Mycobacterium smegmatis (WO05 / 56782), perhydrolases from the CE 7 family (WO09 / 67279) and variants of the Mycobacterium smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0295] In one aspect, other preferred enzymes include endoglucanases (EC 3.2.1.4) of microbial origin exhibiting endo-β-1,4-glucanase activity, including bacterial polypeptides endogenous to members of the genus Bacillus (said polypeptide having a sequence at least 90%, 94%, 97% or 99% identical to the amino acid sequence SEQ ID NO: 2 in US 7141403) and mixtures thereof. Suitable endoglucanases are sold under the trade name and (Novozymes) sales.

[0296] Other preferred enzymes include those sold under the trade name Pectin lyases sold under the trade name (Novozymes) and Mannanase sold by (Danisco / DuPont).

[0297] One or more detergent enzymes can be included in the detergent composition by adding a separate additive containing one or more enzymes, or by adding a combination additive comprising all or more of these enzymes. The detergent additive of the present invention, i.e., a separate or combined additive, can be formulated into, for example, granules, liquids, slurries, bars, etc. Preferred detergent additive formulations are granules, especially non-dusting granules; liquids, especially stabilized liquids; or slurries.

[0298] Dust-free particles can be manufactured as disclosed in, for example, US 4106991 and US 4661452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; alcohols therein containing ethoxylated fatty alcohols with 12 to 20 carbon atoms and 15 to 80 ethylene oxide units therein; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for use by fluidized bed technology are given in GB1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method disclosed in EP238216.

[0299] Dye transfer inhibitors - The compositions of the present invention may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in the composition, the dye transfer inhibitors may be present at a level of from 0.0001 wt% to 10 wt%, from 0.01 wt% to 5 wt% or from 0.1 wt% to 3 wt%.

[0300] Brightener - The compositions of the invention may also contain additional ingredients which may impart colour to the items being cleaned, such as optical brighteners.

[0301] The composition may include a CI fluorescent brightener 260 in α-crystal form having the following structure:

[0302]

[0303] In one aspect, the brightener is a cold water soluble brightener, such as a CI fluorescent brightener in α-crystalline form 260. In one aspect, the brightener is primarily in α-crystalline form, meaning typically at least 50 wt%, at least 75 wt%, at least 90 wt%, at least 99 wt%, or even substantially all of the CI fluorescent brightener 260 is in α-crystalline form.

[0304] The brightener is typically in the form of micronized particles having a weighted average primary particle size of from 3 to 30 microns, from 3 to 20 microns, or from 3 to 10 microns.

[0305] The composition may include a CI fluorescent brightener 260 in β-crystal form, and the weight ratio of (i) CI fluorescent brightener 260 in α-crystal form to (ii) CI fluorescent brightener 260 in β-crystal form may be at least 0.1 or at least 0.6. BE 680847 relates to a method for preparing a CI fluorescent brightener 260 in α-crystal form.

[0306] Commercial optical brighteners that can be used in the present invention can be divided into several subgroups, including but not necessarily limited to: diphenylethylene, pyrazoline, coumarin, carboxylic acid, methine cyanine, dibenzothiophene-5,5-dioxide, azoles, derivatives of 5- and 6-membered heterocycles, and other miscellaneous 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 optical brighteners that can be used in the compositions of the present invention are those identified in US 4790856 and US 3646015.

[0307] Another suitable brightener has the following structure:

[0308]

[0309] Suitable fluorescent brightener levels include lower levels of from 0.01 wt%, from 0.05 wt%, from 0.1 wt% or from 0.2 wt% to higher levels of 0.5 wt% or 0.75 wt%.

[0310] In one aspect, the brightener can be loaded on the clay to form particles. Silicate - The composition of the present invention may also contain a silicate, such as sodium silicate or potassium silicate. The composition may contain from 0 wt% to less than 10 wt% silicate, to 9 wt%, or to 8 wt%, or to 7 wt%, or to 6 wt%, or to 5 wt%, or to 4 wt%, or to 3 wt%, or even to 2 wt%, and from above 0 wt%, or from 0.5 wt%, or from 1 wt% silicate. A suitable silicate is sodium silicate.

[0311] Dispersants - The compositions of the invention may also contain a dispersant. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0312] Enzyme Stabilizer -The enzymes used in the composition can be stabilized by various techniques. The enzymes used herein can be stabilized by the presence of a water-soluble source of calcium and / or magnesium ions. Examples of conventional stabilizers are, for example, polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, and lactic acid. The detergent composition may include a protease inhibitor, which is a reversible inhibitor with protease activity (e.g., serine protease activity). Preferably, the protease inhibitor is a (reversible) subtilisin inhibitor. In particular, the protease inhibitor may be a peptide aldehyde, boric acid or boronic acid; or a derivative of any of these.

[0313] The inhibition constant K of protease inhibitors against serine proteases i (mol / L) can be from 1E-12 to 1E-03; more preferably from 1E-11 to 1E-04; even more preferably from 1E-10 to 1E-05; even more preferably from 1E-10 to 1E-06; and most preferably from 1E-09 to 1E-07.

[0314] The protease inhibitor may be boronic acid or a derivative thereof; preferably phenylboronic acid or a derivative thereof.

[0315] In one aspect, the phenylboronic acid derivative has the formula:

[0316]

[0317] Wherein R is selected from the group consisting of hydrogen, hydroxyl, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkenyl and substituted C1-C6 alkenyl. Preferably, R is hydrogen, CH3, CH3CH2 or CH3CH2CH2.

[0318] In a preferred aspect, the protease inhibitor (phenylboronic acid derivative) is 4-formyl-phenyl-boronic acid (4-FPBA).

[0319] In another specific aspect, the protease inhibitor is selected from the group consisting of:

[0320] Thiophene-2 ​​Boric Acid, Thiophene-3 Boric Acid, Acetamidophenyl Boric Acid, Benzofuran-2 Boric Acid, Naphthalene-1 Boric Acid, Naphthalene-2 ​​Boric Acid, 2-FPBA, 3-FBPA, 4-FPBA, 1-Thianthrene Boric Acid, 4-Dibenzofuran Boric Acid, 5-Methylthiophene-2 ​​Boric Acid, Thianaphthene Boric Acid, Furan-2 Boric Acid, Furan-3 Boric Acid, 4,4-Biphenyl-Diboric Acid, 6-Hydroxy-2-Naphthalene, 4-(Methylthio)phenyl Boric Acid, 4(Trimethyl-Silyl)phenyl Boric Acid, 3-Bromothiophene Boric Acid, 4-Methylthiophene Boric Acid, 2-Naphthyl Boric Acid, 5-Bromothiophene Boric Acid, 5-Chlorothiophene Boric Acid, Dimethylthiophene Boric Acid, 2-Bromophenyl Boric Acid, 3-Chlorophenylboronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thianthreneboronic acid, dibenzothiopheneboronic acid, 4-carboxyphenylboronic acid, 9-anthrylboronic acid, 3,5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, p-tolylboronic acid, o-tolylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-bis-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, 4-methoxyphenylboronic acid.

[0321] Other boronic acid derivatives suitable as protease inhibitors in detergent compositions are described in US 4963655, US 5159060, WO 95 / 12655, WO 95 / 29223, WO 92 / 19707, WO 94 / 04653, WO 94 / 04654, U5442100, US 5488157 and US 5472628.

[0322] The protease inhibitor may also be a protein having the formula XB 1 -B 0 -H peptide aldehydes, wherein the groups have the following meanings:

[0323] a) H is hydrogen;

[0324] b)B 0 It is a single amino acid residue, either in L- or D-configuration and has the chemical formula: NH-CHR'-CO;

[0325] c)B 1 is a single amino acid residue; and

[0326] d) X consists of one or more amino acid residues (preferably one or two), optionally including an N-terminal protecting group.

[0327] NH-CHR'-CO(B 0 ) is an L- or D-amino acid residue, wherein R' may be an aliphatic or aromatic side chain, for example an aralkyl group, such as benzyl, wherein R' may be optionally substituted. More particularly, B 0 The residues may be bulky, neutral, polar, hydrophobic and / or aromatic. Examples are Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, norvaline (Nva), Leu, Ile or norleucine (Nle) in D- or L-form.

[0328] XB 1 -B 0 -H, B 1 The residues may be particularly small, aliphatic, hydrophobic and / or neutral. Examples are alanine (Ala), cysteine ​​(Cys), glycine (Gly), proline (Pro), serine (Ser), threonine (Thr), valine (Val), norvaline (Nva) and norleucine (Nle), in particular alanine, glycine or valine.

[0329] Specifically, X may be one or two amino acid residues, with an optional N-terminal protecting group (ie, the compound is a tripeptide aldehyde or a tetrapeptide aldehyde, with or without a protecting group). Thus, X may be B 2 , B 3 -B 2 , ZB 2 or ZB 3 -B 2 , where B 3 and B 2 Each represents an amino acid residue, and Z is an N-terminal protecting group. 2 The residue may be particularly small, aliphatic and / or neutral, such as Ala, Gly, Thr, Arg, Leu, Phe or Val. In particular, B 3 The residue may be bulky, hydrophobic, neutral and / or aromatic, for example Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle or lie.

[0330] The N-terminal protecting group Z (if present) may be selected from formyl, acetyl, benzoyl, trifluoroacetyl, fluoromethoxycarbonyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups, benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or methylaminocarbonyl / methylurea groups. In the case of a tripeptide aldehyde having a protecting group (i.e., X=ZB 2 ), Z is preferably a small aliphatic group, such as formyl, acetyl, fluoromethoxycarbonyl, tert-butoxycarbonyl, methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or methylaminocarbonyl / methylurea groups. In the case of a tripeptide aldehyde having a protecting group (i.e., X = ZB 3 -B 2 ), Z is preferably a bulky aromatic group, such as benzoyl, benzyloxycarbonyl, p-methoxybenzylcarbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP).

[0331] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO98 / 13460, WO 98 / 13461, WO 98 / 13461, WO 98 / 13462, WO07 / 141736, WO 07 / 145963, WO 09 / 118375, WO 10 / 055052 and WO 11 / 036153. More specifically, the peptide aldehyde may be Cbz-RAY-H, Ac-GAY-H, Cbz-GAY-H, Cbz-GAL-H, Cbz-VAL-H, Cbz-GAF-H, Cbz-GAV-H, Cbz-GGY-H, Cbz-GGF- H, Cbz-RVY-H, Cbz-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGAL-H, Ac-FGAF-H, Ac-FGVY-H, Ac-FGAM-H, Ac-WL VY-H, MeO-CO-VAL-H, MeNCO-VAL-H, MeO-CO-FGAL-H, MeO-CO-FGAF-H, MeSO2-FGAL-H, MeSO2-VAL-H, PhCHO(OH)(O)P-VAL-H, EtSO2-FGAL-H, PhCH2SO2-VAL-H, PhCHO(OH)(O)P-LAL-H, PhCHO(OH)(O)P-FAL-H, or MeO(OH)(O)P-LGAL-H. Here, Cbz is benzyloxycarbonyl, Me is methyl, Et is ethyl, Ac is acetyl, H is hydrogen, and the other letters represent amino acid residues designated by standard single letter notation (e.g., F=Phe, Y=Tyr, L=Leu).

[0332] Alternatively, the peptide aldehyde may have the formula as described in WO 11 / 036153:

[0333] PO-(A i -X') n -A n+1 -Q

[0334] wherein Q is hydrogen, CH3, CX"3, CHX"2, or CH2X", wherein X" is a halogen atom;

[0335] One of X' is a "bis-N-capping group" CO, CO-CO, CS, CS-CS or CS-CO, most preferably urido(CO), and the other X' is empty,

[0336] Where n = 1-10, preferably 2-5, most preferably 2,

[0337] Among them A i and A n+1 Each is an amino acid residue having the following structure:

[0338] For the residue to the right of X'=-CO-, it is -NH-CR"-CO-, or

[0339] For the residue to the left of X'=-CO-, it is -CO-CR"-NH-

[0340] wherein R" is H- or an optionally substituted alkyl or alkaryl group which may optionally include heteroatoms and may optionally be attached to an N atom, and

[0341] wherein P is hydrogen or any C-terminal protecting group.

[0342] Examples of such peptide aldehydes include α-MAPI, β-MAPI, F-urea-RVY-H, F-urea-GGY-H, F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H, F-urea-GA-Nle-H, γ-urea-RVY-H, γ-urea-GAY-H, F-CS-RVF-H, F-CS-RVY-H, F-CS-GAY-H, antipain, GE20372A, GE20372B, chymostatin A, chymostatin B, and chymostatin C. Further examples of peptide aldehydes are disclosed in WO 2010 / 055052 and WO 2009 / 118375, WO 94 / 04651, WO 98 / 13459, WO 98 / 13461, WO 98 / 13462, WO07 / 145963, which are hereby incorporated by reference.

[0343] Alternatively, for peptide aldehydes, the protease inhibitor may be a peptide having the formula XB 1 -NH-CHR-CHOH-SO3M bisulfite adduct, where X, B 1 and R are as defined above, and M is H or an alkali metal, preferably Na or K, as described in WO 13 / 004636.

[0344] The peptide aldehyde can be converted into a water-soluble bisulfite adduct by reaction with sodium bisulfite, as described in textbooks, for example March, J., Advanced Organic Chemistry, 4th edition, Wiley-Interscience, USA, 1992, p. 895.

[0345] The aqueous solution of the bisulfite adduct can be prepared by reacting the corresponding peptide aldehyde with an aqueous solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO3) or potassium bisulfite (KHSO3) by known methods, for example, as described in WO 98 / 47523; US 6500802; US 5436229; J.Am.Chem.Soc. [Journal of the American Chemical Society] (1978) 100, 1228; Org.Synth., Coll. [Collection of Organic Synthesis] Vol. 7: 361.

[0346] The molar ratio of the above-mentioned peptide aldehyde (or bisulfite adduct) to the protease can be at least 1:1 or 1.5:1, and it can be less than 1000:1, more preferably less than 500:1, even more preferably from 100:1 to 2:1 or from 20:1 to 2:1, or most preferably, the molar ratio is from 10:1 to 2:1.

[0347] Formate (e.g. sodium formate) and formic acid have also shown good effects as inhibitors of protease activity. Formate can be used in conjunction with the above-mentioned protease inhibitors, as shown in WO 13 / 004635. The formate is present in the detergent composition in an amount of at least 0.1% w / w or 0.5% w / w, such as at least 1.0%, at least 1.2% or at least 1.5%. The amount of the salt is typically less than 5% w / w, less than 4% or less than 3%.

[0348] In one aspect, the protease is a metalloprotease and the inhibitor is a metalloprotease inhibitor, such as a protein hydrolysate based inhibitor (eg, as described in WO 08 / 134343).

[0349] Solvents - Suitable solvents include water and other solvents such as lipophilic fluids. Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerol derivatives (e.g., glycerol ethers), perfluorinated amines, perfluorinated and hydrofluoroether solvents, low volatility non-fluorinated organic solvents, glycol solvents, other environmentally friendly solvents, and mixtures thereof.

[0350] Structuring agent / thickener-Structured liquids can be structured from the inside, whereby the structure is formed by primary ingredients (e.g., surfactant materials), and / or structured from the outside by providing a three-dimensional matrix structure using secondary ingredients (e.g., polymers, clays and / or silicate materials). The composition can include from 0.01wt% to 5wt%, or from 0.1wt% to 2.0wt% of a structurant. The structurant is typically selected from the group consisting of: diglycerides and triglycerides, stearic acid ethylene glycol diesters, microcrystalline cellulose, cellulose-based materials, microfiber cellulose, hydrophobically modified alkali swellable emulsions (e.g., Polygel W30 (3V Sigma)), biopolymers, xanthan gum, gellan gum and mixtures thereof. Suitable structurants include hydrogenated castor oil and non-ethoxylated derivatives thereof. Suitable structurants are disclosed in US 6855680. Such structurants have a thread-like structured system having a certain range of aspect ratios. Other suitable structurants and methods for making them are described in WO 10 / 034736.

[0351] Conditioner -The composition of the present invention may include a high melting point fatty compound. The high melting point fatty compound useful herein has a melting point of 25°C or higher and is selected from the group consisting of: fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives and mixtures thereof. Such compounds with low melting points are not intended to be included in this section. Non-limiting examples of high melting point compounds are found in International Cosmetic Ingredient Dictionary [International Cosmetic Ingredient Dictionary], Fifth Edition, 1993, and CTFA Cosmetic Ingredient Handbook [CTFA Cosmetic Ingredient Handbook], Second Edition, 1992.

[0352] In view of providing improved conditioning benefits such as slippery feel during application to wet hair, softness and moisturized feel to dry hair, high melting point fatty compounds are included in the composition at levels of from 0.1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1.5 wt% to 16 wt%, from 1.5 wt% to 8 wt%.

[0353] The composition of the present invention can contain cationic polymers. The concentration of cationic polymers in the composition typically ranges from 0.05wt% to 3wt%, from 0.075wt% to 2.0wt%, or from 0.1wt% to 1.0wt%. At the pH of the intended use composition, suitable cationic polymers will have a cationic charge density of at least 0.5meq / gm, at least 0.9meq / gm, at least 1.2meq / gm, at least 1.5meq / gm, or less than 7meq / gm, and less than 5meq / gm, and the range of pH will generally be from pH 3 to pH 9 or between pH 4 and pH 8. 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 will generally be between 10,000 and 10,000,000, between 50,000 and 5,000,000, or between 100,000 and 3,000,000.

[0354] Suitable cationic polymers for use in the compositions of the present invention contain cationic nitrogen-containing moieties, such as quaternary ammonium or cationic protonated amino moieties. Any anionic counterions may be used in association with the cationic polymers, as long as the polymer remains dissolved in water, in the composition, or in the condensed phase of the composition, and as long as the counterions are physically and chemically compatible with the major components of the composition or otherwise do not unduly impair composition performance, stability, or aesthetics. Non-limiting examples of such counterions include halides (e.g., chlorides, fluorides, bromides, iodides), sulfates, and methylsulfates.

[0355] Non-limiting examples of such polymers are described in the CTFA Cosmetic Ingredient Dictionary, 3rd Edition, edited by Estrin, Crosley, and Haynes, The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, DC (1982).

[0356] Other suitable cationic polymers for use in the composition include polysaccharide polymers, cationic guar gum derivatives, cellulose ethers containing quaternary nitrogen, synthetic polymers, copolymers of etherified cellulose, guar gum and starch. When used, the cationic polymers herein are soluble in the composition or in the complex coacervate phase soluble in the composition, and the coacervate phase is formed by the cationic polymers described above and anionic, amphoteric and / or zwitterionic surfactant components. The complex coacervate of the cationic polymer can also be formed with other charged materials in the composition. Suitable cationic polymers are described in US 3962418, US 3958581 and US2007 / 0207109.

[0357] The compositions of the present invention may contain 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:

[0358]

[0359] Where R 95 Selected from the group consisting of: H, methyl and mixtures thereof. Regulators, and particularly silicones, may be included in the composition. Regulators for the composition of the present invention typically include water-insoluble, water-dispersible, non-volatile liquids that form emulsified liquid particles. Suitable regulators for use in the composition are those that are generally characterized as follows: silicones (e.g., silicone oils, cationic silicones, silicone gums, high-refractive silicones and silicone resins), organic conditioning oils (e.g., hydrocarbon oils, polyolefins and fatty esters) or combinations thereof, or those regulators that otherwise form liquid dispersed particles in the aqueous surfactant matrix of this article. Such regulators should be physically and chemically compatible with the main components of the composition, and should not otherwise improperly damage composition stability, aesthetics or performance.

[0360] The concentration of the conditioning agent in the composition should be sufficient to provide the desired conditioning benefit. Such concentration may vary depending on the conditioning agent, the desired conditioning properties, the average size of the conditioning agent particles, the type and concentration of other components, and other similar factors.

[0361] The concentration of silicone conditioning agents typically ranges from 0.01 wt% to 10 wt%. Non-limiting examples of suitable silicone conditioning agents and optional suspending agents for silicones are described in U.S. Reissued Patent Nos. 34,584; 5104646; 5106609; 4152416; 2826551; 3964500; 4364837; 6607717; 6482969; 5807956; 5981681; 6207782; 7465439; 7041767; 7217777; 2007 / 0286837A1; 2005 / 0048549A1; 2007 / 0041929A1; GB 849433; DE 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 List 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 Ed., pp. 204-308, John Wiley & Sons, Inc. (1989).

[0362] The composition of the present invention may also contain from 0.05 wt % to 3 wt % of at least one organic conditioning oil as a conditioning agent, alone or in combination with other conditioning agents such as silicones (described herein). Suitable conditioning oils include hydrocarbon oils, polyolefins, and fatty esters. Also suitable for use in the composition herein are conditioning agents described in US 5674478 and US 5750122 or in US 4529586; US 4507280; US 4663158; US 4197865; US 4217914; US 4381919; and US 4422853.

[0363] Hygiene and bad smell - The composition of the present invention may also contain zinc ricinoleate, thymol, quaternary ammonium salts (such as ), polyethyleneimine (e.g. from BASF) ) and zinc complexes thereof, silver and silver compounds (especially those designed to slowly release Ag+ or nanosilver dispersions).

[0364] Prebiotics - The composition may comprise a prebiotic, such as those described in WO 09 / 043709.

[0365] Foam enhancer - If high foaming is desired, a foaming agent (e.g. C 10 -C 16 Alkanolamide or C 10 -C 14 Alkyl sulfate) can typically be incorporated into the composition at a level of 1 wt% to 10 wt%. 10 -C 14 Monoethanol and diethanolamides illustrate typical classes of such suds boosters. Such suds boosters are also advantageously used with high foaming adjunct surfactants (e.g., the amine oxides, betaines, and sultaines mentioned above). If desired, water-soluble magnesium and / or calcium salts (e.g., MgCl2, MgSO4, CaCl2, CaSO4, etc.) may be added typically at levels of 0.1 wt% to 2 wt% to provide additional foam and to enhance grease removal performance.

[0366] Foam Inhibitor - Compounds for reducing or inhibiting foam formation can be incorporated into the compositions of the present invention. Foam inhibition may be particularly important in so-called "high concentration cleaning processes" as described in US 4489455 and US 4489574 and in front-loading-style washing machines. A wide variety of materials can be used as foam inhibitors, and foam inhibitors are well known to those skilled in the art. See, for example, Kirk Othmer Encyclopedia of Chemical Technology, 3rd edition, Vol. 7, pp. 430-447 (John Wiley & Sons, Inc., 1979). Examples of foam inhibitors 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 monovalent alcohols, aliphatic C 18 -C 40Ketones (e.g., stearone), N-alkylated aminotriazines, wax hydrocarbons preferably having a melting point below about 100° C., silicone foam inhibitors, and secondary alcohols. Foam inhibitors are described in US 2954347, US 4265779, US 4265779, US 3455839, US 3933672, US 4652392, US 4978471, US 4983316, US 5288431, US 4639489, US 4749740, US 4798679, US 4075118, EP 89307851.9, EP 150872, and DOS 2,124,526.

[0367] For any detergent composition to be used in an automatic washing machine, suds should not form to the extent that they overflow the washing machine. When used, suds suppressors are preferably present in a "suds suppressing amount." "Suds suppressing amount" means that the formulator of the composition can select an amount of such suds controlling agent that will adequately control suds to result in a low sudsing laundry detergent for use in an automatic washing machine.

[0368] The compositions herein will typically contain from 0 to 10 wt% of a foam suppressor. When used as a foam suppressor, monocarboxylic fatty acids and salts thereof will typically be present in amounts up to 5 wt%. Preferably, from 0.5 wt% to 3 wt% of a fatty monocarboxylic ester foam suppressor is used. Silicone foam suppressors are typically used in amounts up to 2.0 wt%, although higher amounts may be used. Monostearoyl phosphate foam suppressors are typically used in amounts ranging from 0.1 wt% to 2 wt%. Hydrocarbon foam suppressors are typically used in amounts ranging from 0.01 wt% to 5.0 wt%, although higher levels may be used. Alcohol foam suppressors are typically used at 0.2 wt% to 3 wt%.

[0369] The compositions herein can have cleaning activity in a wide range of pH. In certain aspects, the compositions have cleaning activity from pH 4 to pH 11.5. In other aspects, the compositions have activity from pH 6 to pH 11, from pH 7 to pH 11, from pH 8 to pH 11, from pH 9 to pH 11, or from pH 10 to pH 11.5.

[0370] The compositions herein may have cleaning activity over a wide range of temperatures, for example from 10°C or lower to 90°C. Preferably, the temperature will be below 50°C or 40°C or even 30°C. In certain aspects, the optimum temperature range for the composition is from 10°C to 20°C, from 15°C to 25°C, from 15°C to 30°C, from 20°C to 30°C, from 25°C to 35°C, from 30°C to 40°C, from 35°C to 45°C, or from 40°C to 50°C.

[0371] In one aspect, the detergent composition may comprise: a sulfite source, such as, for example, sodium bisulfite; a lipase, such as, for example, a lipase of the present invention; a linear alkylbenzene sulfonate; C12-16 Pareth-9; propylene glycol; alcohol ethoxysulfate, water, polyethyleneimine ethoxylate, glycerol; fatty acid salts; PEG-136 polyvinyl acetate; ethylenediamine disuccinate; monoethanolamine citrate; sodium ethylenediamine pentaacetate; disodium distyryl diphenyl disulfonate; calcium formate; sodium formate, hydrogenated castor oil; dyes; benzisothiazoline; perfume; and optionally one or more enzymes. In one aspect, the detergent composition may comprise: a sulfite source, such as, for example, potassium sulfite; a lipase, such as, for example, a lipase of the present invention; MEA-dodecylbenzenesulfonate; propylene glycol; C12-14 Pareth-7; water; MEA laureth sulfate; MEA-palm kernel oleate; PEI ethoxylate; glycerol; MEA citrate; PARFUM; dodecylbenzenesulfonic acid; magnesium chloride; disodium distyrylbiphenyl disulfonate; PEG / PPG-10 / 2 propylheptyl ether; hydrogenated castor oil; ethanolamine; polystyrene; sodium formate; sorbitol; tripropylene glycol; 2-acrylic acid, polymer with vinylbenzene; butylphenylmethylpropionic acid; sulfuric acid; and optionally more enzymes.

[0372] Form of composition

[0373] The compositions of the invention described herein can be advantageously used, for example, in laundry applications, hard surface cleaning, dishwashing applications, as well as cosmetic applications such as dentures, teeth, hair and skin. The compositions of the invention are particularly solid or liquid cleaning and / or treatment compositions. In one aspect, the present invention relates to a composition, wherein the form of the composition is selected from the group consisting of: a regular, compressed or concentrated liquid; a gel; a paste; a soap bar; a regular or compressed powder; a granular solid; a uniform or multilayer tablet having two or more layers (same or different phases); a bag having one or more chambers; a single or multi-chamber unit dosage form; or any combination thereof.

[0374] The form of the composition can physically separate components in multiple compartments (such as water-soluble bags) or in different layers of a tablet from each other. Thus, undesirable storage interactions between components can be avoided. Different dissolution profiles in each compartment can also cause delayed dissolution of selected components in the wash solution.

[0375] The bag can be configured as a single chamber or multiple chambers. It can have any form, shape and material suitable for holding the composition, such as not allowing the composition to be released from the bag before contact with water. The bag is made of a water-soluble film, which includes an internal volume. The internal volume can be divided into the chambers of the bag. The preferred film is a polymer material, preferably a polymer forming a film or sheet. Preferred polymers, copolymers or derivatives thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, 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 will typically be about 20,000 to about 150,000. The film may also be a blended composition comprising a hydrolytically degradable and water-soluble polymer blend such as polylactic acid and polyvinyl alcohol (known under trade reference M8630, such as sold by MonoSol LLC, Indiana, USA) plus a plasticizer like glycerol, ethylene glycol, propylene glycol, sorbitol and mixtures thereof. The bag may contain a solid laundry cleaning composition or partial components and / or a liquid cleaning composition or partial components separated by a water-soluble film. The chamber for the liquid component may be different in composition from the chamber containing the solid (US2009 / 0011970 A1).

[0376] Method for making the composition

[0377] The compositions of the present invention may be formulated into any suitable form and may be prepared by any method selected by the formulator, non-limiting examples of which are described in the applicant's examples and in US 4990280; US20030087791A1; US20030087790A1; US20050003983A1; US20040048764A1; US ​​4762636; US6291412; US20050227891A1; EP 1070115A2; US 5879584; US 5691297; US 5574005; US5569645; US 5565422; US 5516448; US 5489392; US 5486303, all of which are incorporated herein by reference. Compositions of the invention or compositions prepared according to the invention include cleaning and / or treatment compositions, including but not limited to compositions for treating fabrics, hard surfaces and any other surfaces in the field of fabric and home care, including: air care (including air fresheners and odor delivery systems), car care, dishwashing, fabric conditioning (including softening and / or freshening), laundry descaling, laundry and rinse additives and / or care, hard surface cleaning and / or treatment (including floor and toilet bowl cleaners), all-purpose or "heavy duty" detergents in granular or powder form, especially cleaning detergents; all-purpose detergents in liquid, gel or paste form, especially the so-called heavy duty liquid types; liquid delicate fabric detergents; manual dishwashing detergents or light duty dishwashing detergents, especially those of the high foaming type; machine dishwashing detergents, including different tablet, granular, liquid and rinse aid types for use in homes and institutions: car or carpet shampoos, bathroom cleaners (including toilet bowl cleaners); and cleaning adjuvants, such as bleach additives and "stain sticks" or pre-treatment types, compositions loaded with substrates (e.g., tablets with added desiccant). Preferred are compositions and methods for cleaning and / or treating textiles and / or hard surfaces (most preferably textiles). The composition is preferably a composition used in a pre-treatment step or in the main wash step (most preferably for textile wash steps) of a wash process.

[0378] As used herein, the term "fabric and / or hard surface cleaning and / or treatment composition" is a subset of cleaning and treatment compositions, which, unless otherwise indicated, includes general-purpose or "heavy-duty" detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, gel or paste form, especially the so-called heavy-duty liquid types; liquid fine fabric detergents; manual dishwashing detergents or light-duty dishwashing detergents, especially those of the high-sudsing type; machine dishwashing detergents, including different tablet, granular, liquid and rinse aid types for use in households and institutions; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners (including toilet bowl cleaners); fabric conditioning compositions (including softening and / or refreshing), which may be in liquid, solid and / or dryer tablet form; and cleaning adjuvants, such as bleach additives and "stain sticks" or pre-treatment types, substrate-loaded compositions (e.g., dryer-added tablets). All applicable such compositions may be in standard, concentrated or even highly concentrated form, even to the extent that such compositions may be non-aqueous in some respects.

[0379] Lipase variants of the present invention

[0380] The present invention also relates to lipase variants of a parent lipase, wherein the variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2 or any fragment thereof having lipase activity, and comprises one or more (e.g., several) substitutions at positions corresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of the parent lipase (particularly SEQ ID NO: 2).

[0381] In a preferred embodiment, the lipase variant has one or more substitutions corresponding to the following substitutions: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40, DE, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E or T267D, E of SEQ ID NO:2.

[0382] In embodiments, the variant of a parent lipase comprises 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, 40, 41, or 42 substitutions.

[0383] In one aspect, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the amino acid sequence of the parent lipase.

[0384] In one aspect, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12, or a fragment thereof having lipase activity.

[0385] In one aspect, the number of substitutions in a variant of the invention is 1-40, 1-30, 1-20, 1-10, 1-5, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions.

[0386] In one aspect, the variant comprises a substitution at a position corresponding to position 15 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 15 of SEQ ID NO: 2 is substituted with E or D.

[0387] In one aspect, the variant comprises a substitution at a position corresponding to position 23 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 23 of SEQ ID NO: 2 is substituted with E or D.

[0388] In one aspect, the variant comprises a substitution at a position corresponding to position 35 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 35 of SEQ ID NO: 2 is substituted with E or D.

[0389] In one aspect, the variant comprises a substitution at a position corresponding to position 37 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 37 of SEQ ID NO: 2 is substituted with E or D.

[0390] In one aspect, the variant comprises a substitution at a position corresponding to position 39 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 239 of SEQ ID NO: 2 is substituted with E or D.

[0391] In one aspect, the variant comprises a substitution at a position corresponding to position 40 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 40 of SEQ ID NO: 2 is substituted with E or D.

[0392] In one aspect, the variant comprises a substitution at a position corresponding to position 42 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 42 of SEQ ID NO: 2 is substituted with E or D.

[0393] In one aspect, the variant comprises a substitution at a position corresponding to position 101 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 101 is substituted with E or D.

[0394] In one aspect, the variant comprises a substitution at a position corresponding to position 105 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 105 of SEQ ID NO: 2 is substituted with E or D.

[0395] In one aspect, the variant comprises a substitution at a position corresponding to position 106 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 106 of SEQ ID NO: 2 is substituted with E or D.

[0396] In one aspect, the variant comprises a substitution at a position corresponding to position 184 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 184 is substituted with E or D.

[0397] In one aspect, the variant comprises a substitution at a position corresponding to position 267 of the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2; SEQ ID NO: 4; SEQ ID NO: 6; SEQ ID NO: 8; or SEQ ID NO: 10; or SEQ ID NO: 12. In one aspect, the amino acid at the position corresponding to position 267 of SEQ ID NO: 2 is substituted with E or D.

[0398] In one aspect, the variant of the parent lipase comprises a substitution at two positions corresponding to any one of the following positions: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E or T267D, E of a parent lipase (particularly SEQ ID NO: 2). In one aspect, the parent lipase has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0399] In one aspect, a variant of a parent lipase comprises or consists of two substitutions at positions corresponding to Q15D+G23D, Q15D+G23E, Q15D+T35D, Q15D+T35E, Q15D+T37D, Q15D+T37E, Q15D+N39D, Q15D+N39E, Q15D+A40D, Q15D+A40E, Q15D+P42D, Q15D+P42E, Q15D+N101D, Q15D+N101E, Q15D+S105D, Q15D+S105E, Q15D+G106D, Q15D+G106E, Q15D+F184D, Q15D+F184E, Q15D+ T267D, Q15D+T267E, Q15E+G23D, Q15E+G23E, Q15E+T35D, Q15E+T35E, Q15E+ T37D, Q15E+T37E, Q15E+N39D, Q15E+N39E, Q15E+A40D, Q15E+A40E, Q15E+P4 2D, Q15E+P42E, Q15E+N101D, Q15E+N101E, Q15E+S105D, Q15E+S105E, Q15E+ G106D, Q15E+G106E, Q15E+F184D, Q15E+F184E, Q15E+T267D, Q15E+T267E, G2 3D+T35D, G23D+T35E, G23D+T37D, G23D+T37E, G23D+N39D, G23D+N39E, G23D +A40D、G23D+A40E、G23D+P42D、G23D+P42E、G23D+N101D、G23D+N101E、G23D +S105D, G23D+S105E, G23D+G106D, G23D+G106E, G23D+F184D, G23D+F184E, G23D+T267D, G23D+T267E, G23E+T35D, G23E+T35E, G23E+T37D, G23E+T37E, G 23E+N39D, G23E+N39E, G23E+A40D, G23E+A40E, G23E+P42D, G23E+P42E, G23 E+N101D, G23E+N101E, G23E+S105D, G23E+S105E, G23E+G106D, G23E+G106E , G23E+F184D, G23E+F184E, G23E+T267D, G23E+T267E, T35D+T37D, T35D+T3 7E, T35D+N39D, T35D+N39E, T35D+A40D, T35D+A40E, T35D+P42D, T35D+P42E,<h2 style=";text-align:left;direction:ltr">T35D+N101D、T35D+N101E、T35D+S105D、T35D+S105E、T35D+G106D、T35D+G1 06E、T35D+F184D、T35D+F184E、T35D+T267D、T35D+T267E、T35E+T37D、T35E +T37E、T35E+N39D、T35E+N39E、T35E+A40D、T35E+A40E、T35E+P42D、T35E+P 42E、T35E+N101D、T35E+N101E、T35E+S105D、T35E+S105E、T35E+G106D、T35 E+G106E、T35E+F184D、T35E+F184E、T35E+T267D、T35E+T267E、T37D+N39D、 T37D+N39E、T37D+A40D、T37D+A40E、T37D+P42D、T37D+P42E、T37D+N101D、T 37D+N101E, T37D+S105D, T37D+S105E, T37D+G106D, T37D+G106E, T37D+F184D, T37D+F184E, T37D+T267D, T37D+T267E, T37E+N39D, T37E+N39E, T37E+A 40D, T37E+A40E, T37E+P42D, T37E+P42E, T37E+N101D, T37E+N101E, T37E+S105D, T37E+S105E, T37E+G106D, T37E+G106E, T37E+F184D, T37E+F184E, T3 7E+T267D、T37E+T267E、N39D+A40D、N39D+A40E、N39D+P42D、N39D+P42E、N3 9D+N101D、N39D+N101E、N39D+S105D、N39D+S105E、N39D+G106D、N39D+G106 E, N39D+F184D, N39D+F184E, N39D+T267D, N39D+T267E, N39E+A40D, N39E+A40E, N39E+P42D, N39E+P42E, N39E+N101D, N39E+N101E, N39E+S105D, N39E+ S105E、N39E+G106D、N39E+G106E、N39E+F184D、N39E+F184E、N39E+T267D,N 39E+T267E、A40D+P42D、A40D+P42E、A40D+N101D、A40D+N101E、A40D+S105D、A40D+S105E, A40D+G106D, A40D+G106E, A40D+F184D, A40D+F184E, A40D+T267D, A40D+T267E, A40E+P42D, A40E+P42E, A40E+N101D, A40E+N1 01E, A40E+S105D, A40E+S105E, A40E+G106D, A40E+G106E, A40E+F184D, A40E+F184E, A40E+T267D, A40E+T267E, P42D+N101D, P42D+N101E, P 42D+S105D, P42D+S105E, P42D+G106D, P42D+G106E, P42D+F184D, P42D+F184E, P42D+T267D, P42D+T267E, P42E+N101D, P42E+N101E, P42E+S 105D, P42E+S105E, P42E+G106D, P42E+G106E, P42E+F184D, P42E+F184E, P42E+T267D, P42E+T267E, N101D+S105D, N101D+S105E, N101D+G10 6D, N101D+G106E, N101D+F184D, N101D+F184E, N101D+T267D, N101D+T267E, N101E+S105D, N101E+S105E, N101E+G106D, N101E+G106E, N101 E+F184D, N101E+F184E, N101E+T267D, N101E+T267E, S105D+S105E, S105D+G106D, S105D+G106E, S105D+F184D, S105D+F184E, S105D+T267D 、S105D+T267E、S105E+G106D、S105E+G106E、S105E+F184D、S105E+F184E、S105E+T267D、S105E+T267E、G106D+F184D、G106D+F184E、G106D+T267D、G106D+T267E、G106E+F184D、G106E+F184E、G106E+T267D、G106E+T267E、F184D+T267D、F184D+T267E、F184E+T267D、F184E+T267E。、

[0400] In one aspect, the substitutions of the parent lipase variant further include a set of substitutions at the following positions (using SEQ ID NO: 2 for numbering), the positions corresponding to:

[0401]

[0402]

[0403]

[0404]

[0405] In one aspect, the parent lipase has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0406] The variant may further comprise one or more additional substitutions at one or more (eg, several) other positions.

[0407] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0408] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL 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.

[0409] Alternatively, the amino acid change has such a property that the physicochemical properties of the polypeptide are changed. For example, the amino acid change can improve the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0410] In another embodiment, the variant further comprises one or more (e.g., several) changes, such as substitutions corresponding to any one of the positions selected from: 4, 27, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 231, 233, 249, 254, 255, 256, 263, 264, 265, 266, 267 and 269 of a parent lipase (particularly SEQ ID NO: 2). In one aspect, the variant further comprises one or more (e.g., several) changes (e.g., substitutions) corresponding to any one of the positions selected from: 4V, 27R, 38A, 57G, S58A, 60S, 83T, 86V, 91A / N / Q, 94K / R, 97M, 99K, 111A, 150G, 163K, 210K / Q, 216P, 225R, L227G, 231R, 233R, 249R, 254S, 255A, 256K / T / V, 263Q, 264A, 265T, 266D, 267A, and 269N of the parent lipase. In one aspect, the parent lipase has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12, or a fragment thereof having lipase activity.

[0411] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues that are critical to 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, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, together with mutations to putative contact site amino acids. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identities of essential amino acids can also be inferred from alignments with related polypeptides.

[0412] The variant may consist of or contain 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.

[0413] In one aspect, the variant has improved stability compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12. In one aspect, the variant has improved stability in the presence of a sulfite source compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12. The improved stability in the presence of a sulfite source can be determined by the "Stability Assay" as described in Example 1.

[0414] In one aspect, the variant has improved stability in a detergent composition compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12. In one aspect, the variant has improved stability in a detergent composition comprising a sulfite source compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12.

[0415] In one aspect, the variant has improved stability under storage conditions compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12. In one aspect, the variant has improved stability under storage conditions in the presence of a sulfite source compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0416] In one aspect, the variant has improved thermostability compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12. In one aspect, the variant has improved thermostability in the presence of a sulfite source compared to the parent lipase. In one aspect, the parent lipase is SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0417] Parent lipase

[0418] The parent lipase can be (a) a polypeptide having at least 60% sequence identity with a polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12; (b) a polypeptide encoded by the following polynucleotide, which hybridizes under low stringency conditions with (i) a polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11, (ii) the full-length complementary sequence of (i); or (c) a polypeptide encoded by the following polynucleotide, which hybridizes with a polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0419] In one aspect, the parent has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12, and the polypeptide has lipase activity. In one aspect, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12 by up to 40 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0420] In one aspect, the parent comprises or consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0421] In one aspect, the parent is a fragment of the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12, which contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0422] In one aspect, the parent is an allelic variant of the polypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0423] In one aspect, the parent is encoded by a polynucleotide that 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, (ii) the full complement of (i) (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York).

[0424] The polynucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 or SEQ ID NO: 11 or their subsequences, together with the polypeptides of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 or SEQ ID NO: 12 or their fragments, can be used to design nucleic acid probes to identify and clone the DNA encoding the parent from strains of different genera or species according to methods well known in the art. In particular, such probes can be used to hybridize with the genomic DNA or cDNA of cells of interest, following standard Southern blot procedures, so as to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the entire sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, for example at least 200 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 32 P. 3 H. 35 S, biotin, or avidin) for detecting the corresponding gene. Such probes are encompassed in the present invention.

[0425] Genomic DNA or cDNA libraries prepared by such other strains can be screened for DNA that hybridizes with the probe described above and encodes the parent. Genomic DNA or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. DNA from the library or the separated DNA can be transferred to and fixed on nitrocellulose or other suitable carrier materials. In order to identify clones or DNA that hybridize with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11 or its subsequences, the carrier material is used in Southern blots.

[0426] For purposes of the present invention, hybridization indicates that a polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; (ii) a polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; (iii) a full-length complementary sequence thereof; or (iv) a subsequence thereof under very low to very high stringency conditions. Molecules 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.

[0427] In one aspect, the nucleic acid probe is a polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, or SEQ ID NO: 11. In one 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, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11. In one aspect, the nucleic acid probe is a polynucleotide encoding a polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12; a polypeptide thereof; or a fragment thereof. In one aspect, the nucleic acid probe is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11.

[0428] In one aspect, the parent is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

[0429] 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 another polypeptide.

[0430] The parent may be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include connecting the coding sequences encoding the polypeptides so that they are in frame and the expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides may also be constructed using intein technology, wherein the fusion polypeptide is produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779).

[0431] The fusion polypeptide may further comprise a cleavage site between the two polypeptides. When the fusion protein is secreted, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. [Industrial Microbiology Journal] 3:568-576; Svetina et al., 2000, J. Biotechnol. [Journal of Biotechnology] 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. [Applied and Environmental Microbiology] 63:3488-3493; Ward et al., 199 5, 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.

[0432] The parent can be obtained from any genus of microorganism. For the purposes of the present invention, as used herein in conjunction with a given source, the term "obtained from" should mean that the parent encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

[0433] The parent can be a bacterial lipase. For example, the parent can be a gram-positive bacterial polypeptide, such as Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus, Streptomyces or Thermospora lipase; or a gram-negative bacterial polypeptide, such as Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silvae, Neisseria, Pseudomonas, Salmonella or Ureaplasma lipase.

[0434] In one aspect, the parent is a 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 lipase.

[0435] In one aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus lipase.

[0436] In one aspect, the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans lipase.

[0437] In one aspect, the parent is a Thermobifida alba or Thermobifida fusca (formerly Thermobifida fusca) lipase.

[0438] The parent can be a fungal lipase. For example, the parent can be a yeast lipase, such as Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia lipase; or a filamentous fungal lipase, such as Acremonium, Agaricus, Alternaria, Aspergillus, Short-stalked Atractylodes, Botryospaeria, Pseudomonas, Trichosporon, Chrysosporium, Claviceps, Coprinus, Coprinus, Coptis, Corydoraspermum, Claviceps, Cryptococcus, Cryptococcus, Diplosporus, Black Ear, Filosporin, etc. , Fusarium, Gibberellum, Holomagnem, Humicola, Rake Tooth, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Flat Leather, Ruminochytrium, Poitrasia, Pseudomonas, Pseudo-Trichoderma, Rhizomucor, Schizophyllum, Columnar Acremonium, Talaromyces, Thermoascus, Thielavia, Toxoplasma, Trichoderma, Trichoderma, Verticillium, Agrocybe, or Xylaria lipase.

[0439] In one aspect, the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis lipase.

[0440] In one aspect, the parent is Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, oryzae), Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicalum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum), Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusariumtorulosum, Fusariumtrichothecioides), Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum purpurogenum), Phanerochae techrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielaviafimeti, Thielavia microspora, Thielavia iovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii koningii), Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

[0441] In one aspect, the parent is a Thermomyces lanuginosus lipase, such as the lipase of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10, or SEQ ID NO:12.

[0442] It should be understood that for the aforementioned species, the present invention encompasses perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0443] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), the Netherlands Type Culture Collection (Centraalbureau Voor Schimmelcultures, CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0444] The above probes can be used to identify the parent and obtain the parent from other sources including the microorganism separated from nature (for example, soil, compost, water, etc.), or directly obtain the DNA sample from natural materials (for example, soil, compost, water, etc.). The technology for directly separating microorganisms and DNA from natural habitats is well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, the polynucleotide can be separated or cloned by utilizing technology known to those of ordinary skill in the art (see, for example, Sambrook et al., 1989, the same).

[0445] Preparation of variants

[0446] The present invention also relates to a method for obtaining a lipase variant of a parent lipase, wherein the variant has lipase activity and comprises one or more (e.g., several) substitutions at positions corresponding to positions 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of the parent lipase, and the parent lipase may have the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or a fragment thereof having lipase activity.

[0447] In an embodiment, the substitution is selected from the positions corresponding to: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0448] Variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semisynthetic gene construction, random mutagenesis, shuffling, and the like.

[0449] Site-directed mutagenesis is a technique for introducing one or more (eg, several) mutations at one or more defined sites in the polynucleotide encoding the parent.

[0450] Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cutting at a site in a plasmid comprising a polynucleotide encoding a parent by a restriction enzyme and then connecting an oligonucleotide containing the mutation to the polynucleotide. Typically, the restriction enzymes that digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to connect to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0451] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, for example, US 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).

[0452] Any site-directed mutagenesis procedure may be used in the present invention.There are many commercially available kits that can be used to prepare variants.

[0453] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode the polypeptide of interest. Gene synthesis can be performed using a variety of techniques, such as multiplexed microchip-based techniques described by Tian et al. (2004, Nature 432: 1050-1054), and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0454] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods followed by a relevant screening procedure, such as those described by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0455] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the importance of individual amino acid residues in a polypeptide to be rapidly determined.

[0456] The many aspects of semi-synthetic gene construction are realized by combining synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or reorganization. Semi-synthetic construction typically utilizes the process of synthetic polynucleotide fragments in conjunction with PCR technology. Therefore, the restricted area of ​​gene can be synthesized from scratch, and other regions can use site-specific mutagenic primers to increase, and other regions can carry out fallibility PCR or non-fallibility PCR amplification. Then the polynucleotide subsequence can be reorganized.

[0457] Polynucleotide

[0458] The present invention also relates to polynucleotides encoding the variants of the present invention.

[0459] Nucleic acid construct

[0460] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0461] Polynucleotides can be manipulated in a variety of ways to provide expression of variants. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0462] The control sequence can be a promoter, i.e. a polynucleotide recognized by a host cell for expressing the polynucleotide. The promoter comprises a transcriptional control sequence that mediates the expression of the variant. The promoter can be any polynucleotide that shows transcriptional activity in a host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0463] Examples of suitable promoters for directing transcription of the nucleic acid construct of the invention in a bacterial host cell are promoters obtained from the following genes: Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agar hydrolase gene (dagA) and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in Gilbert et al., 1980, "Useful proteins from recombinant bacteria," Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0464] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the invention in a filamentous fungal host cell are promoters obtained from the genes for 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). 00 / 56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma β-xylosidase, and the NA2-tpi promoter (a modified promoter from an Aspergillus neutral α-amylase gene in which the untranslated leader sequence is replaced by the untranslated leader sequence of an Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter from an Aspergillus niger neutral α-amylase gene in which the untranslated leader sequence has been replaced with the untranslated leader sequence from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.

[0465] In yeast hosts, useful promoters are obtained from the genes for the following enzymes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0466] The control sequence may also be a transcription terminator that is 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.

[0467] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0468] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0469] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0470] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0471] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0472] The control sequence may also be a leader sequence, i.e., a non-translated region of an mRNA that 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 sequence that is functional in the host cell may be used.

[0473] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0474] Suitable leaders for yeast host cells are obtained from the genes for 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).

[0475] The control sequence may also be a polyadenylation sequence, a sequence that is operably linked to the 3'-terminus of the variant coding sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenylic acid residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0476] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger-alpha glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0477] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0478] The control sequence can also be a signal peptide coding region, which encodes a signal peptide connected to the N-terminus of the variant, and guides the variant to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence, which is naturally connected to the segment of the coding sequence of the coding variant in the translation reading frame. Alternatively, the 5'-end of the coding sequence may include a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally include a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the variant. However, any signal peptide coding sequence that guides the expressed variant to enter the secretory pathway of the host cell can be used.

[0479] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for maltogenic amylase from Bacillus NCIB 11837, subtilisin from Bacillus licheniformis, beta-lactamase from Bacillus stearothermophilus, alpha-amylase from Bacillus stearothermophilus, neutral protease from Bacillus stearothermophilus (nprT, nprS, nprM), and prsA from Bacillus subtilis. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0480] Effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0481] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0482] The control sequence can also be a propeptide coding sequence encoding a propeptide at the N-terminus of a variant. The resulting polypeptide is referred to as a proenzyme or propolypeptide (or in some cases as a zymogen). Propolypeptides are generally inactive and can be converted into active polypeptides by catalytic cleavage or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), Rhizomucor manhei aspartic protease, and Saccharomyces cerevisiae alpha factor.

[0483] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0484] It is also desirable to add regulatory sequences that regulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to chemical or physical stimulation, including the presence of regulatory compounds. Regulatory systems in prokaryotic systems include lac, tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operably connected to the regulatory sequence.

[0485] Expression vector

[0486] The present invention further relates to an expression vector (preferably a recombinant expression vector) comprising a polynucleotide, a promoter, and a transcription and translation termination signal encoding a variant of the present invention. Various nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more suitable restriction sites to allow the polynucleotides encoding variants to be inserted or replaced at such sites. Alternatively, the polynucleotides may be expressed by inserting a polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. When producing the expression vector, the encoding sequence is located in the vector so that the encoding sequence is operably connected to the suitable control sequence for expression.

[0487] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be easily subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0488] The vector can be an autonomously replicating vector, i.e. a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome when it is introduced into the host cell and replicates with one or more chromosomes into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids can be used, which together comprise the total DNA to be introduced into the host cell genome, or a transposon can be used.

[0489] The vector preferably contains one or more selectable markers that permit easy selection of transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0490] Examples of bacterial selective markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as their equivalents. Preferred for use in Aspergillus cells are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0491] The vector preferably contains one or more elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0492] In order to be integrated into the host cell genome, the vector can rely on the polynucleotide sequence of the encoding variant or be used for being integrated into any other vector element in the genome by homologous or non-homologous recombination.Alternately, the vector may include other polynucleotides at the precise position for guiding the chromosome to be integrated into the host cell genome by homologous recombination.In order to increase the possibility of integration at the precise position, the integration element should include 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, and the nucleic acid has a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination.The integration element can be any sequence homologous to the target sequence in the host cell genome.In addition, the integration element can be a non-coding or coded polynucleotide.On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0493] For autonomous replication, the vector may also additionally comprise an origin of replication, which enables autonomous replication of the vector in the host cell in question. The origin of replication may be any plasmid replicon mediating autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0494] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0495] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0496] 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 a plasmid or vector containing the gene can be accomplished according to the method disclosed in WO 00 / 24883.

[0497] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the production of variants. The increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing amplified copies of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0498] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

[0499] Host cells

[0500] The present invention also relates to recombinant host cells comprising polynucleotides encoding variants of the present invention, operably linked to one or more control sequences, which instruct the production of variants of the present invention. The construct or vector comprising the polynucleotides is introduced into the host cell so that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier. The term "host cell" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of host cells will depend to a large extent on the gene encoding the variant and its source.

[0501] The host cell may be any cell useful in the recombinant production of the variant, eg, a prokaryotic or eukaryotic cell.

[0502] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. 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, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silvae, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0503] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus fluorescens, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0504] The bacterial host cell may also be any Streptococcus cell, including but not limited to Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

[0505] 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.

[0506] Introduction of DNA into Bacillus cells can be achieved by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), transformation of competent cells (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 can be achieved 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 can be achieved 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 can be achieved 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 can be achieved by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. [infection and immunity] 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios [microbiology] 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. [application and environmental microbiology] 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. [microbiology review] 45: 409-436). However, any method known in the art for introducing DNA into a host cell may be used.

[0507] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0508] The host cell may be a fungal cell. "Fungi" as used herein includes Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitotic spore fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0509] The fungal host cell can be a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of the present invention, yeast should be defined as described in Biology and Activities of Yeast [Biology and Activity of Yeast] (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9 [Society for Applied Bacteriology Special Paper Collection Series 9], 1980).

[0510] The yeast host cell can be a Candida cell, a Hansenula cell, a Kluyveromyces cell, a Pichia cell, a Saccharomyces cell, a Schizosaccharomyces cell, or a Yarrowia cell, such as a Kluyveromyces lactis cell, a Saccharomyces carlsbergensis cell, a Saccharomyces cerevisiae cell, a Saccharomyces diastaticus cell, a Saccharomyces douglasii cell, a Saccharomyces kluyveri cell, a Saccharomyces norbensis cell, a Saccharomyces ovalbumin cell, a Saccharomyces cerevisiae cell, a Saccharomyces diastaticus cell, a Saccharomyces douglasii cell, a Saccharomyces kluyveri cell, a Saccharomyces norbensis cell, a Saccharomyces oviformis cells or Yarrowia lipolytica cells.

[0511] The fungal host cell may be a filamentous fungal cell. "Filamentous fungi" include all filamentous forms of the subphyla of 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, chitin, mannan and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeasts (such as Saccharomyces cerevisiae) is by budding of a unicellular thallus, and carbon catabolism may be fermentative.

[0512] The filamentous fungal host cell can be an Acremonium, Aspergillus, Bjerkandera, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neotrichia, Neurospora, Paecilomyces, Penicillium, Pseudocerviphora, Phlebia, Chytridium, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxicodendron, Trametes, or Trichoderma cell.

[0513] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis sa neirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis rubrum ... sissubrufa), Ceriporiopsissubvermispora, Chrysosporiuminops, Chrysosporiumhoratis, Chrysosporiumlucnowense, Chrysosporiummerdarium, Chrysosporiumqueenslandicum, Chrysosporiumtropicalis, Chrysosporiumzonatum, Coprinus cinereus), Coriolushirsutus, rod-shaped fusarium, graminum fusarium, Kuwei fusarium, broad-knife fusarium, graminum fusarium, red fusarium (Fusarium graminum), heterosporous fusarium, albizia fusarium, sharp spore fusarium, multi-branched fusarium, pink fusarium, elder fusarium, color fusarium, pseudo-branched fusarium, sulfur-colored fusarium, round fusarium, pseudo-spore fusarium, embellished fusarium, Humicola insolens, sparse cotton-like fusarium, rice black mucor, thermophilic myceliophthora, rough neurospora, purple Penicillium, Phanerochaetechrysosporium, radiata fusarium, Pleurotuseryngii, terrestrial fusarium, long domain hair Trametes villosa, Trametes versicolor (Trametes versicolor), Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0514] Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474 and Christensen et al., 1988, Bio / Technology [Biology / Technology] 6: 1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene [Gene] 78: 147-156, and WO 96 / 00787. Yeast can be transformed using procedures described in Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1920.

[0515] Generation method

[0516] The present invention also relates to a method for producing a variant of a parent lipase, the method comprising: (a) culturing a host cell of the present invention under conditions suitable for expressing the variant; and (b) recovering the variant.

[0517] Host cells can be cultured in a nutrient medium suitable for producing variants using methods known in the art. For example, cells can be cultured by shaking bottles, or in a suitable medium and under conditions allowing variant expression and / or separation, small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, batch-fed fermentation or solid-state fermentation) can be carried out in a laboratory or industrial fermentor tank to culture cells. Using procedures as known in the art, cultivation occurs in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable substratum can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of American Type Culture Collection). If variant is secreted into the nutrient medium, variant can be directly recovered from the substratum. If variant is not secreted, it can be recovered from cell lysate.

[0518] The variants can be detected using methods known in the art that are specific for the variants. 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 can be used to determine the activity of the variant (such as those described in the Examples).

[0519] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium by a variety of conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation or precipitation.

[0520] Variants may be purified by a variety of procedures known in the art to obtain substantially pure variants, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, eds., VCH Publishers, New York, 1989).

[0521] In alternative aspects, the variant is not recovered, but rather a host cell of the invention expressing the variant is used as a source of the variant.

[0522] Lipase Granules

[0523] The lipase variant of the present invention may be included in a water-soluble film as a lipase granule. The lipase granule may contain one or more additional enzymes, as described below.

[0524] Lipase granules are any form of lipase variants in the form of solid microparticles. Lipase granules can be lipase crystals, lipase precipitations, sprayed or freeze-dried lipase or any form of granular lipase, which is a suspension in a powder or liquid. Typically, the particle size of the lipase granules measured as equivalent spherical diameter (average particle size based on volume) is less than 2 mm, preferably less than 1 mm, less than 0.5 mm, less than 0.25 mm or less than 0.1 mm; and higher than 0.05 μm, preferably higher than 0.1 μm, higher than 0.5 μm, higher than 1 μm, higher than 5 μm or higher than 10 μm. In a preferred aspect, the particle size of the lipase granules is from 0.5 μm to 100 μm.

[0525] The lipase granules comprise at least 1% w / w lipase protein, preferably at least 5% w / w lipase protein, at least 10% w / w lipase protein, at least 20% w / w lipase protein, at least 30% w / w lipase protein, at least 40% w / w lipase protein, at least 50% w / w lipase protein, at least 60% w / w lipase protein, at least 70% w / w lipase protein, at least 80% w / w lipase protein, or at least 90% w / w lipase protein.

[0526] In a preferred embodiment, the lipase particles are lipase crystals, or the lipase protein is in crystalline form. Enzyme crystallization can be performed in a variety of ways as known in the art (eg, as described in WO 91 / 09943 or WO 94 / 22903).

[0527] The lipase can be formulated in lipase granules as known in the art for solid enzyme formulations, e.g., formulations for reducing dust, improving stability and or altering the release rate of the enzyme. The lipase granules can also be formulated in a matrix or coated with an agent that inhibits the dissolution of the enzyme granules in the PVOH / film solution used to prepare the water-soluble film.

[0528] The lipase molecules on the surface of the lipase particles may also be cross-linked, like CLEC (cross-linked enzyme crystals) or CLEA (cross-linked enzyme aggregates).

[0529] Water soluble film

[0530] Water-soluble films, optional ingredients therein, and methods for preparing them are well known in the art. In one class of embodiments, the water-soluble film comprises PVOH. PVOH is a synthetic resin typically prepared by alcoholysis (commonly referred to as hydrolysis or saponification) of polyvinyl acetate. Completely hydrolyzed PVOH (wherein almost all acetoxy groups have been converted into alcohol groups) is a highly crystalline polymer of strong hydrogen bonding that dissolves only in hot water (above about 140°F (60°C)). If a sufficient number of acetoxy groups are allowed to remain after the hydrolysis of polyvinyl acetate, the PVOH polymer is referred to as partially hydrolyzed, and its hydrogen bonding is weaker and the crystallinity is lower and is soluble in cold water (below about 50°F (10°C)). Intermediate cold / hot water soluble films may include, for example, intermediate partially hydrolyzed PVOH (e.g., having a hydrolysis degree of about 94% to about 98%), and are easily soluble only in warm water (e.g., rapidly dissolving at a temperature of about 40°C and above). Both fully and partially hydrolyzed PVOH types are often referred to as PVOH homopolymers, although the partially hydrolyzed types are technically vinyl alcohol-vinyl acetate copolymers.

[0531] The degree of hydrolysis of the PVOH included in the water-soluble film can be about 75% to about 99%. When the degree of hydrolysis decreases, the film made of the resin will have a reduced mechanical strength but will dissolve faster at temperatures below about 20°C. When the degree of hydrolysis increases, the film made of the resin will tend to have higher mechanical strength and thermoformability will tend to decrease. The degree of hydrolysis of the PVOH can be selected so that the water solubility of the resin is temperature-dependent, and therefore the solubility of the film made of the resin, the compatibilizing agent, and the other ingredients is also affected. In one class of embodiments, the film is cold water soluble. Cold water soluble films (soluble in water at temperatures below 10°C) can include PVOH with a degree of hydrolysis in the range of about 75% to about 90%, or in the range of about 80% to about 90%, or in the range of about 85% to about 90%. In another class of embodiments, the film is hot water soluble. Hot water soluble films (soluble in water at a temperature of at least about 60°C) can include PVOH with a degree of hydrolysis of at least about 98%.

[0532] In addition to PVOH or in the alternative of PVOH, other film-forming resins used may include but are not limited to modified polyvinyl alcohol, polyacrylate, water-soluble acrylic copolymer, polyacrylate, polyacrylamide, polyvinyl pyrrolidone, pullulan, water-soluble natural polymers include but are not limited to guar gum, xanthan gum, carrageenan, and starch, water-soluble polymer derivatives include but are not limited to ethoxylated starch and hydroxypropylated starch, poly (acrylamide-2-methylpropane sodium sulfonate), polymethyl maleate, its copolymers and any combination of the foregoing items. In one class of embodiments, the film-forming resin is a terpolymer composed of vinyl alcohol, vinyl acetate and sodium acrylamide-2-methylpropane sulfonate. Surprisingly, water-soluble films based on terpolymers of vinyl alcohol, vinyl acetate and sodium acrylamide-2-methylpropane sulfonate have demonstrated high percentages of enzyme recovery.

[0533] The water-soluble resin may be included in the water-soluble film in any suitable amount, such as in an amount in the range of about 35 wt % to about 90 wt %. The preferred weight ratio of the amount of the water-soluble resin compared to the combined amount of all enzymes, enzyme stabilizers and auxiliary additives may be any suitable ratio, such as in a ratio in the range of about 0.5 to about 5, or about 1 to about 3, or about 1 to about 2.

[0534] The water-soluble resins used in the films described herein, including but not limited to PVOH resins, can be characterized by any suitable viscosity for the desired film properties, optionally a viscosity in the range of about 5.0 cP to about 30.0 cP, or about 10.0 cP to about 25 cP. The viscosity of the PVOH resin is determined by measuring a freshly prepared solution using a Brookfield LV viscometer with a UL adapter, as described in the British Standard EN ISO 15023-2:2006 Annex E Brookfield Test Method. It is international practice to state the viscosity of a 4% aqueous solution of polyvinyl alcohol at 20°C. All PVOH viscosities specified herein in cP should be understood to refer to the viscosity of a 4% aqueous solution of polyvinyl alcohol at 20°C, unless otherwise stated.

[0535] It is well known in the art that the viscosity of PVOH resin is related to the weight average molecular weight of the same PVOH resin. related, and the viscosity is often used as Therefore, the weight average molecular weight of the water-soluble resin can optionally be in the range of about 35,000 to about 190,000, or about 80,000 to about 160,000. The molecular weight of the resin only needs to be sufficient to enable it to be molded into a plastic film by a suitable technique.

[0536] The water-soluble film may include other optional additive ingredients, including, but not limited to, plasticizers, surfactants, defoamers, film formers, antiblocking agents, internal mold release agents, anti-yellowing agents, and other functional ingredients, for example, in amounts suitable for its intended purpose.

[0537] Water is considered to be a very effective plasticizer for PVOH and other polymers; however, the volatility of water limits its effectiveness, as polymer films need to have at least some tolerance (robustness) to a variety of environmental conditions including low and high relative humidity. Glycerol is much less volatile than water and has been well established as an effective plasticizer for PVOH and other polymers. If the level used in the film formulation is too high, glycerol or other such liquid plasticizers themselves can cause surface "sweating" and greasy. This can cause the following problems in the film, such as giving the consumer's hands an unacceptable touch, and if sweating is not alleviated in a certain way (e.g., surface dusting), the film may even be blocked on a roller or in a pile of sheets. This can be characterized as overplasticization. However, if too little plasticizer is added to the film, the film may lack sufficient ductility and flexibility for many end uses, such as being converted into an end-use type, such as a bag.

[0538] The plasticizer used in the water-soluble film of the present disclosure includes but is not limited to sorbitol, glycerol, diglycerol, propylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol (up to MW 400), 2 methyl 1,3 propanediol, lactic acid, monoacetin, triacetin, triethyl citrate, 1,3-butylene glycol, trimethylolpropane (TMP), polyether triol and combination thereof. As described above, polyols can be used as plasticizers generally. The less plasticizer is used, the more brittle the film may become, and the more plasticizer is used, the more the film may lose tensile strength. Plasticizer can be included in the water-soluble film by, for example, about 25phr to about 50phr or from about 30phr to about 45phr or from about 32phr to about 42phr in an amount within the range.

[0539] The surfactant used in the water-soluble film is well known in the art. Optionally, surfactant is included to help the dispersion of resin solution when casting. Suitable surfactants for the water-soluble film of the present disclosure include but are not limited to the lactic acid esters of dialkyl sulfosuccinate, glycerol and propylene glycol, fatty acid lactyl esters, sodium alkyl sulfates, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkyl polyoxyethylene ether, lecithin, acetylated fatty acid esters of glycerol and propylene glycol, sodium lauryl sulfate, fatty acid acetylated esters, myristyl dimethyl amine oxide, trimethyl tallow alkyl ammonium chloride, quaternary ammonium compounds, its salt and the combination of any of the above items. Therefore, surfactant can be, for example, less than about 2phr, for example, less than about 1phr or less than about 0.5phr amount and be included in the water-soluble film.

[0540] One type of secondary component contemplated for use is a defoamer. Defoamers can help coalesce foam bubbles. Suitable defoamers for use in water-soluble films according to the present disclosure include, but are not limited to, hydrophobic silica, such as fine particle size silica or fumed silica, including Foam Defoamers (available from Emerald Performance Materials), including as well as They are proprietary non-mineral oil defoamers. In embodiments, defoamers may be used in amounts of 0.5 phr or less, such as 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr. Preferably, significant amounts of silica will be avoided to avoid stress whitening.

[0542] Methods for preparing water-soluble articles (including films) include casting, blow molding, extrusion or blow extrusion, as known in the art. One class of contemplated embodiments is characterized by the water-soluble film described herein being formed by casting, such as by mixing the ingredients described herein with water to produce an aqueous mixture (e.g., a solution with optionally dispersed solids), applying the mixture to a surface, and drying to remove water to produce a film. Similarly, other compositions can be formed by drying the mixture while confining it to a desired shape.

[0543] In one class of contemplated embodiments, the water-soluble film is formed by casting a water-soluble mixture, wherein the water-soluble mixture is prepared according to the following steps:

[0544] (a) providing a mixture of a water-soluble resin, water and any optional additives excluding plasticizers;

[0545] (b) boiling the mixture for 30 minutes;

[0546] (c) degassing the mixture in an oven at a temperature of at least 40°C; optionally in the range of 40°C to 70°C, for example about 65°C;

[0547] (d) adding one or more enzymes, a plasticizer and additional water to the mixture at a temperature of 65°C or less; and

[0548] (e) stirring the mixture without vortexing until the mixture appears substantially uniform in color and consistency; optionally for a period of time in the range of 30 minutes to 90 minutes, optionally for at least 1 hour; and

[0549] (f) Casting the mixture quickly after the stirring period (e.g., within 4 hours, or 2 hours, or 1 hour).

[0550] If the enzyme is added to the mixture too early (e.g., with an auxiliary additive or resin), the enzyme activity may decrease. Without intending to be bound by any particular theory, it is believed that boiling the mixture with the enzyme causes the enzyme to denature and storage in solution for an extended period of time also causes the enzyme activity to decrease.

[0551] In one class of embodiments, the water-soluble film according to the present disclosure is kept high enzyme activity by rapidly drying the film under moderate to mild conditions. As used herein, rapid drying refers to less than 24 hours, optionally less than 12 hours, optionally less than 8 hours, optionally less than 2 hours, optionally less than 1 hour, optionally less than 45 minutes, optionally less than 30 minutes, optionally less than 20 minutes, optionally less than 10 minutes, such as a drying time in the range of about 6 minutes to about 10 minutes or 8 minutes. As used herein, moderate to mild conditions refer to less than 170 ° F (77 ° C), optionally in the range of about 150 ° F to about 170 ° F (about 66 ° C to about 77 ° C), for example, 165 ° F (74 ° C) drying temperature. As the drying temperature increases, the enzyme tends to denature faster, and as the drying temperature decreases, the drying time increases, thereby exposing the enzyme to the solution in an extended period of time.

[0552] The film can be used to produce a bag to hold a composition, for example, laundry or dishwashing composition, thus forming a bag. Film as herein described can also be used to prepare a bag with two or more chambers, and the bag is made of the same film or in conjunction with the film of other polymeric materials. Other films can be, for example, obtained by casting, blow molding, extruding or blowing extrusion of the same or different polymeric materials, as known in the art. In one type of embodiment, the polymer, copolymer or its derivatives suitable for use as other films are selected from polyvinyl alcohol, polyvinyl pyrrolidone, polyalkylene oxide, polyacrylic acid, cellulose, cellulose ether, cellulose ester, cellulose amide, polyvinyl acetate, polycarboxylic acid and salt, polyamino acid or peptide, polyamide, polyacrylamide, maleic acid / acrylic acid copolymer, polysaccharide (including starch and gelatin), natural gum (such as xanthan gum and carrageenan). For example, the polymer can be selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, sodium carboxymethylcellulose, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates and combinations thereof, or from polyvinyl alcohol, polyvinyl alcohol copolymers and hydroxypropylmethylcellulose (HPMC) and combinations thereof.

[0553] Bag and / or bag can comprise at least one sealed chamber.Therefore, bag can comprise single chamber or multiple chambers.Described bag can have zone containing enzyme and not containing enzyme.In the embodiment comprising multiple chambers, each chamber can contain identical and / or different compositions.And then, described composition can take any suitable form, including but not limited to liquid, solid and combination thereof (for example, solid suspended in liquid).In certain embodiments, described bag comprises first, second and third chamber, and wherein each chamber holds different first, second and third compositions respectively.In certain embodiments, as described in EP 2258820, composition can be visually different.

[0554] The chambers of a multi-chamber bag and / or bag can have one or more identical or different sizes and / or volumes. The chambers of a multi-chamber bag of the present invention can be separate or combined in any suitable manner. In certain embodiments, the second and / or third and / or subsequent chambers are superimposed on the first chamber. On the one hand, the third chamber can be superimposed on the second chamber, which in turn is superimposed on the first chamber in a sandwich configuration. Alternatively, the second and third chambers can be superimposed on the first chamber. However, it can also be similarly envisioned that the first, second and optionally third and subsequent chambers can be attached to each other in a side-by-side relationship. The chambers can be packaged into a string, each chamber being separable individually by a perforated line. Therefore, each chamber can be torn off individually from the remainder of the string by the end user.

[0555] In some embodiments, the multi-chamber bag and / or bag includes three chambers, which are composed of a large first chamber and two smaller chambers. The smaller second and third chambers are superimposed on the large first chamber. The size and geometry of the chambers are selected so that this arrangement is achievable. The geometry of the chambers can be the same or different. In some embodiments, the second and optionally the third chambers each have different geometries and shapes compared to the first chamber. In these embodiments, the second and optionally the third chambers are arranged on the first chamber in a certain design. The design can be decorative, instructive, or illustrative, for example to illustrate a concept or guidance, and / or for indicating the source of the product. In some embodiments, the first chamber is the largest chamber, which has two large faces sealed around the periphery, while the second chamber is smaller, and it covers less than about 75%, or less than about 50% of the surface area of ​​one face of the first chamber. In the embodiment where there is a third chamber, the above structure can be the same, but the second and third chambers cover less than about 60%, or less than about 50%, or less than about 45% of the surface area of ​​one face of the first chamber.

[0556] The bag and / or bag can comprise one or more different films. For example, in the embodiment of a single chamber, the bag can be made of a wall folded onto itself and sealed at the edge, or alternatively, made of two walls sealed together at the edge. In the embodiment of a plurality of chambers, the bag can be made of one or more films so that any given bag chamber can comprise a wall made of a single film or a plurality of films with different compositions. In one aspect, the multi-chamber bag comprises at least three walls: an outer upper wall, an outer lower wall and a partition. The outer upper wall and the outer lower wall are usually relative and form the outside of the bag. The partition is fixed to the outer wall usually relative in the inside of the bag and along the sealing line. The partition is separated into at least one first chamber and a second chamber by the partition. In a class of embodiments, the partition can be the only film containing enzyme to minimize the exposure of consumers to enzymes.

[0557] Bags and bags can be made with any suitable equipment and method. For example, a single chamber bag can be made with vertical filling, horizontal filling or drum filling technology known in the art. Such methods can be continuous or intermittent. The film can be moistened and / or heated to improve its ductility. The method can also involve using vacuum to draw the film into a suitable mold. Once the film is on the horizontal portion of the surface, the vacuum drawn into the mold can be implemented for about 0.2 to about 5 seconds or about 0.3 to about 3 or about 0.5 to about 1.5 seconds. The vacuum can make it provide a downward pressure, for example, in the range of 10 mbar to 1000 mbar or in the range of 100 mbar to 600 mbar.

[0558] The mold (in which the bag can be made) can have any shape, length, width and depth, depending on the desired dimensions of the bag. If desired, the molds can also be different in size and shape from each other. For example, the volume of the final bag can be about 5 mL to about 300 mL, or about 10 mL to 150 mL, or about 20 mL to about 100 mL, and the mold size can be adjusted accordingly.

[0559] In one aspect, the bag comprises a first sealed chamber and a second sealed chamber. Typically, the second chamber is in a superimposed relationship with the first sealed chamber, so that the second sealed chamber and the first sealed chamber share a dividing wall inside the bag.

[0560] In one aspect, the package comprising the first chamber and the second chamber further comprises a third sealed chamber. Typically, the third sealed chamber is in a superimposed relationship with the first sealed chamber, such that the third sealed chamber and the first sealed chamber share a dividing wall inside the bag.

[0561] In various aspects, the first composition and the second composition are selected from one of the following combinations: liquid, liquid; liquid, powder; powder, powder; and powder, liquid.

[0562] In various aspects, the first, second, and third compositions are selected from one of the following groups: solid, liquid, liquid, and liquid, liquid, liquid.

[0563] In one aspect, a single chamber or multiple sealed chambers contain a composition. The multiple chambers can each contain the same or different compositions. The composition is selected from a liquid, a solid or a combination thereof.

[0564] In the method, heat can be applied to the film, commonly referred to as thermoforming. Heat can be applied by any suitable means. For example, before the film is supplied to the surface or once on the surface, it can be directly heated by placing it under a heating element or by hot air. Alternatively, for example, it can be heated indirectly by heating the surface or applying a hot article to the film. Infrared light can be used to heat the film. The film can be heated to a temperature of at least 50°C, for example, about 50°C to about 150°C, about 50°C to about 120°C, about 60°C to about 130°C, about 70°C to about 120°C, or about 60°C to about 90°C.

[0565] Alternatively, the film may be wetted by any suitable means, for example, directly by spraying a wetting agent (including water, a solution of the film composition, a plasticizer for the film composition, or any combination of the foregoing) onto the film before it is supplied to the surface or once on the surface, or indirectly by wetting the surface or by applying a wet article to the film.

[0566] Once the film has been heated and / or moistened, it can be drawn into a suitable mold, preferably using vacuum. For example, the film can be thermoformed by a stretch ratio of at least about 1.5, and for example, optionally up to a stretch ratio of 2. The filling of the molded film can be completed by using any suitable means. In certain embodiments, the most preferred method will depend on the product form and the required filling speed. In certain embodiments, the molded film is filled by an online filling technique. Then by any suitable method, the filled open bag is closed using the second film to form a bag. This can be completed when in a horizontal position and in continuous uniform motion. Closure can be completed in the following manner: the second film (preferably a water-soluble film) is continuously supplied to and above the open bag, and then preferably the first film and the second film are sealed together, typically in the area between the molds and therefore between the bags.

[0567] Any suitable method for sealing the bag and / or its independent chamber can be utilized. Non-limiting examples of such means include heat sealing, solvent welding, solvent sealing or liquid sealing and combinations thereof. The water-soluble bag and / or its independent chamber can be heat-sealed at a temperature of at least 200°F (93°C), for example, in the range of about 220°F (about 105°C) to about 290°F (about 145°C), or about 230°F (about 110°C) to about 280°F (about 140°C). Typically, only heat or solvent treatment will form the sealed area. Typically, heat or solvent can be applied to the enclosed material by any method, and typically, only applied to the area where the seal will be formed. If solvent sealing or liquid sealing or welding is used, it can be preferably applied to heat as well. Preferred liquid sealing or solvent sealing / welding methods include selectively applying solvent to the area between the molds or the enclosed material, by, for example, spraying or printing it on these areas, and then applying pressure to these areas to form a seal. For example, sealing rolls and belts as described above may be used (optionally also providing heat).

[0568] The formed bag may then be cut by a cutting device. The cutting may be done using any known method. It may be preferred that the cutting may also be done in a continuous manner, and preferably at a constant speed and preferably when in a horizontal position. The cutting device may be, for example, a sharp object, or a hot object, or a laser, whereby, in the latter case, the hot object or laser "burns" through the film / sealing area.

[0569] The different chambers of the multi-chamber bag can be made together in a side-by-side fashion, wherein the resulting one-piece bag may or may not be separated by cutting. Alternatively, the chambers may be made separately.

[0570] In some embodiments, the bag may be made according to a method comprising the steps of:

[0571] a) forming a first chamber (as described above);

[0572] b) forming recesses in some or all of the enclosed chamber formed in step (a) to produce a second molding chamber superimposed on the first chamber;

[0573] c) filling and closing the second chamber by means of a third membrane;

[0574] d) sealing said first, second and third films; and

[0575] e) Cutting the film to produce multi-chamber bags.

[0576] The recess formed in step (b) may be achieved by applying a vacuum to the chamber prepared in step (a).

[0577] In some embodiments, the second and / or third chamber may be made in separate steps and then combined with the first chamber as described in EP 2088187 or WO 2009 / 152031.

[0578] In other embodiments, the bag may be made according to a method comprising the steps of:

[0579] a) forming a first chamber using a first film on a first forming machine, optionally using heat and / or vacuum;

[0580] b) filling the first chamber with a first composition;

[0581] c) deforming the second film on a second molding machine, optionally using heat and vacuum, to produce a second and optionally a third molding chamber;

[0582] d) filling the second and optionally third chamber;

[0583] e) sealing the second and optionally third chamber using a third membrane;

[0584] f) placing the sealed second and optionally third chamber onto the first chamber;

[0585] g) sealing said first, second and optionally third chambers; and

[0586] h) Cutting the film to produce multi-chamber bags.

[0587] The first and second forming machines can be selected based on their suitability for performing the above method. In some embodiments, the first forming machine is preferably a horizontal forming machine, and the second forming machine is preferably a rotary drum forming machine, preferably located above the first forming machine.

[0588] It will be appreciated that by using appropriate feed stations, it is possible to manufacture multi-chamber bags incorporating a variety of different or unique compositions and / or different or unique liquid, gel or paste compositions.

[0589] How to use

[0590] The present invention is included in the method for cleaning any surface (including processing textiles or hard surfaces or other surfaces) in the field of fabric and / or household care. It is considered that cleaning as described can be both small-scale (in such as family housework) and large-scale (such as in such as industrial and professional environments). In one aspect of the present invention, the method is included in the step of contacting the surface to be treated in the pre-treatment step or main washing step of the washing process (most preferably for use in the textile washing step or alternatively for use in dishwashing (including manual and automatic / mechanical dishwashing)). In one aspect of the present invention, the lipase variant and other components are sequentially added to the method for cleaning and / or processing the surface. Alternatively, the lipase variant and other components are added simultaneously.

[0591] As used herein, washing includes, but is not limited to, scrubbing and mechanical agitation. Washing can be performed with a foam composition (as described in WO 08 / 101958), and / or by applying alternating pressure (pressure / vacuum) as an additional method or alternative to scrubbing and mechanical agitation. Drying such surfaces or fabrics can be accomplished by any of the common means employed in a household or industrial environment. The cleaning compositions of the present invention are ideally suited for use in laundry and dishwashing applications. Therefore, the present invention includes methods for cleaning objects (including, but not limited to, fabrics, tableware, knives, and kitchenware). The method includes the step of contacting the object to be cleaned with the cleaning composition, which includes at least one of the cleaning compositions, cleaning additives, or mixtures thereof of the applicant. Fabrics can include most any fabrics that can be washed under conventional consumer or institutional use conditions. The solution can have a pH from 8 to 10.5. Compositions can be used in a solution at a concentration from 500ppm to 15.000ppm. The water temperature range is typically from 5°C to 90°C. The ratio of water to fabric is typically from 1:1 to 30:1.

[0592] In one aspect, the present invention relates to a method for producing a composition using a lipase variant of a parent lipase having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12, wherein the variant has lipase activity and comprises a substitution at a position corresponding to position 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2.

[0593] In one aspect, the substitution is selected from position Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO: 2 of a parent lipase. In one aspect, the invention relates to use of a composition for cleaning an object.

[0594] In one aspect, the present invention relates to a method for cleaning a surface, the method comprising contacting a lipid stain present on the surface to be cleaned with the cleaning composition. In one aspect, the present invention relates to a method for hydrolyzing a stain present on a surface and / or lipids in the stain, the method comprising contacting the stain and / or stain with a cleaning composition. In one aspect, the present invention relates to the use of the composition in the hydrolysis of carboxylic acid esters. In one aspect, the present invention relates to the use of the composition in the hydrolysis, synthesis or exchange of esters. In one aspect, the present invention relates to the use of the composition for the manufacture of a stable formulation.

[0595] The invention is described in the following paragraphs, Examples:

[0596] 1. A composition comprising:

[0597] (a) sulfite source;

[0598] (b) a parent lipase shown as SEQ ID NO: 2 or a lipase variant of a parent lipase having at least 60% identity to SEQ ID NO: 2;

[0599] The 20 angstroms of the cysteine ​​bridge One or more amino acids in the parent lipase in the expression vector are replaced by more negatively charged amino acids.

[0600] 2. A composition as described in paragraph 1, wherein the replaced, preferably substituted amino acid is within 15 angstroms, preferably within 10 angstroms, more preferably within 5 angstroms of the cysteine ​​bridge (ie, from the carboxyl group to the sulfur atom).

[0601] 3. The composition of paragraph 1 or 2, wherein the sulfite source is selected from: sulfite, such as sodium sulfite, potassium sulfite, calcium sulfite; bisulfite, such as potassium bisulfite, sodium bisulfite, calcium bisulfite; pyrosulfite / metabisulfite, such as potassium pyrosulfite, sodium pyrosulfite, calcium pyrosulfite; or any combination thereof.

[0602] 4. A composition as described in any of paragraphs 1-3, wherein sulfite (SO3 2- ) or pyrosulfite (S2O5 2- ) is from 0.1 wt% to 3 wt%, from 0.2 wt% to 2 wt%, from 0.5 wt% to 2 wt%.

[0603] 5. The composition of any of paragraphs 1-4, wherein the lipase variant is a variant of the parent lipase shown as SEQ ID NO: 2, or a variant of a lipase 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%, but less than 100% sequence identity with the parent lipase, particularly any of the parent lipases shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12; or a fragment thereof having lipase activity.

[0604] 6. A composition as described in any of paragraphs 1-4, wherein the lipase variant further comprises one or more (e.g., several) substitutions at positions corresponding to amino acid residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO: 2.

[0605] 7. A composition as described in any of paragraphs 1-6, wherein at 20 angstroms of the cysteine ​​bridge The amino acid in the parent lipase is replaced by a negatively charged amino acid selected from D or E.

[0606] 8. A composition as described in any of paragraphs 1-7, wherein the lipase variant comprises a substitution corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO: 2.

[0607] 9. The composition of any of paragraphs 1-8, wherein the parent lipase has 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: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 12.

[0608] 10. The composition of any of paragraphs 1-9, wherein the cysteine ​​bridge is on the surface of the parent lipase.

[0609] 11. The composition of any of paragraphs 1-10, wherein the cysteine ​​bridge is or corresponds to one or more of the cysteine ​​bridges at the following positions:

[0610] SEQ ID NO:2

[0611] C22-C268,

[0612] C36-C41, and

[0613] C104-C107.

[0614] 12. The composition of any of paragraphs 1-11, wherein the lipase variant has improved stability in the presence of a sulfite source compared to the parent lipase, particularly SEQ ID NO: 2.

[0615] 13. The composition of any of paragraphs 1-12, wherein the variant of a parent lipase is selected from the group consisting of:

[0616] (a) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary sequence of (i);

[0617] (b) 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%, but less than 100% sequence identity to the polypeptide coding sequence of SEQ ID NO: 1; and

[0618] (c) A fragment of the polypeptide of (a) or (b) having lipase activity.

[0619] 14. The composition of any of paragraphs 1-13, wherein the variant of a parent lipase comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 substitutions.

[0620] 15. The composition of any of paragraphs 1-14, wherein the composition further comprises a surfactant.

[0621] 16. Use of a composition as described in any of paragraphs 1 to 15 for cleaning a surface including a fabric, a textile or a hard surface.

[0622] 17. A lipase variant of a parent lipase, wherein the variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO:2 or any fragment thereof having lipase activity, and comprises one or more (e.g., several) substitutions at positions corresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO:2.

[0623] 18. The variant of paragraph 17, wherein the variant has one or more substitutions corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0624] 19. A polynucleotide encoding the variant of any one of paragraphs 17 or 18.

[0625] 20. A nucleic acid construct comprising the polynucleotide of paragraph 19.

[0626] 21. An expression vector comprising the polynucleotide of paragraph 19.

[0627] 22. A host cell comprising the polynucleotide of paragraph 19.

[0628] 23. A method of producing a lipase variant, the method comprising: (a) culturing the host cell of paragraph 22 under conditions suitable for expression of the variant; and (b) recovering the variant.

[0629] 24. A method for obtaining a lipase variant as described in any one of paragraphs 17 or 18, the method comprising: adding a 20 angstrom More negatively charged amino acids are introduced into the parent lipase shown as SEQ ID NO: 2 or a parent lipase having at least 60% sequence identity thereto.

[0630] 25. The method of paragraph 24, wherein the introduced, preferably substituted, amino acid is within 15 angstroms, preferably within 10 angstroms, more preferably within 5 angstroms of the cysteine ​​bridge (ie, from the carboxyl group to the sulfur atom).

[0631] 26. The method of paragraph 24 or 25, wherein the substitution of a more negatively charged amino acid is made at positions corresponding to positions 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO:2.

[0632] 27. The method of any of paragraphs 24-26, wherein the negatively charged amino acid is D or E.

[0633] 28. The method of any of paragraphs 24-27, wherein one or more substitutions corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO: 2 are introduced into the parent lipase.

[0634] The present invention also relates to the following embodiments:

[0635] 1. A composition comprising:

[0636] (a) sulfite source;

[0637] (b) a parent lipase shown as SEQ ID NO: 2 or a lipase variant of a parent lipase having at least 60% identity to SEQ ID NO: 2;

[0638] The 20 angstroms of the cysteine ​​bridge One or more amino acids in the parent lipase in the expression vector are replaced by more negatively charged amino acids.

[0639] 2. A composition as described in embodiment 1, wherein the lipase variant is a variant of the parent lipase shown as SEQ ID NO: 2, or a variant of a lipase 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%, but less than 100% sequence identity with the parent lipase.

[0640] 3. A composition as described in embodiment 1 or 2, wherein the lipase variant comprises one or more (e.g., several) substitutions at positions corresponding to amino acid residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO:2.

[0641] 4. A composition as described in any of embodiments 1-3, wherein at 20 angstroms of the cysteine ​​bridge The amino acid in the parent lipase in the expression vector is replaced by a negatively charged amino acid selected from D or E, in particular, wherein the lipase variant comprises a substitution corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO: 2.

[0642] 5. A composition as described in any of embodiments 1-4, wherein the parent lipase has 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:2.

[0643] 6. The composition of any one of embodiments 1-5, wherein the cysteine ​​bridge is or corresponds to one or more of the cysteine ​​bridges at the following positions:

[0644] SEQ ID NO:2

[0645] C22-C268,

[0646] C36-C41, and

[0647] C104-C107.

[0648] 7. The composition of any one of embodiments 1-6, wherein the lipase variant has improved stability in the presence of a sulfite source compared to the parent lipase, in particular SEQ ID NO: 2.

[0649] 8. The composition of any one of embodiments 1-7, wherein the variant of the parent lipase is selected from the group consisting of:

[0650] (a) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1, or (ii) the full-length complementary sequence of (i);

[0651] (b) 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%, but less than 100% sequence identity to the polypeptide coding sequence of SEQ ID NO: 1; and

[0652] (c) A fragment of the polypeptide of (a) or (b) having lipase activity.

[0653] 9. The composition of any one of embodiments 1-8, wherein the composition comprises a surfactant.

[0654] 10. Use of the composition of any one of embodiments 1 to 9 for cleaning a surface.

[0655] 11. A lipase variant of a parent lipase, wherein the variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO:2 or any fragment thereof having lipase activity, and comprises one or more (e.g., several) substitutions at positions corresponding to residues 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, and 267 of SEQ ID NO:2.

[0656] 12. The variant of embodiment 11, wherein the variant has one or more substitutions corresponding to Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0657] 13. A polynucleotide encoding a variant as described in any one of embodiments 11 or 12.

[0658] 14. A nucleic acid construct comprising the polynucleotide according to embodiment 13.

[0659] 15. An expression vector comprising the polynucleotide according to embodiment 13.

[0660] 16. A host cell comprising the polynucleotide of embodiment 13.

[0661] 17. A method for producing a lipase variant, the method comprising: (a) culturing the host cell of embodiment 16 under conditions suitable for expressing the variant; and (b) recovering the variant.

[0662] 18. A method for obtaining a lipase variant as described in any one of embodiments 11 or 12, the method comprising: adding a 20 angstrom More negatively charged amino acids are introduced into the parent lipase shown as SEQ ID NO: 2 or a parent lipase having at least 60% sequence identity thereto.

[0663] 19. A method as described in embodiment 18, wherein the introduction of more negatively charged amino acids is completed at positions corresponding to one or more of the following positions: 15, 23, 35, 37, 39, 40, 42, 101, 105, 106, 184, 267 of SEQ ID NO:2, in particular, wherein the more negatively charged amino acid is D or E.

[0664] 20. A method as described in embodiment 18 or 19, wherein one or more of the substituted / introduced amino acids correspond to the following substitutions: Q15D, E, G23D, E, T35D, E, T37D, E, N39D, E, A40D, E, P42D, E, N101D, E, S105D, E, G106D, E, F184D, E, or T267D, E of SEQ ID NO:2.

[0665] Materials and Methods

[0666] p-Nitrophenyl (pNP) determination :

[0667] The hydrolytic activity of the lipase can be determined by a kinetic assay using p-nitrophenyl acyl ester as substrate.

[0668] 100 mM stock solutions of the following substrates in DMSO can be diluted to a final concentration of 1 mM in assay buffer (50 mM Tris; pH 7.7; 0.4% Triton X-100): p-nitrophenyl butyrate (C4), p-nitrophenyl hexanoate (C6), p-nitrophenyl decanoate (C10), p-nitrophenyl laurate (C12) and p-nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark A / S, Kirkebjerg Allé 84, 2605 Brondby Catalog numbers: C4: N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752).

[0669] The lipase of the present invention, the parent lipase and appropriate controls (e.g., buffer (negative), Lipolase TM &Lipex TM (positive)) is added to the substrate solution in a 96-well NUNC plate (Catalog No. 260836, Kamstrupvej 90, DK-4000, Roskilde) at a final concentration of 0.01 mg / mL; 5x10-3 mg / mL; 2.5x10-4 mg / mL; and 1.25x10-4 mg / ml. The p-nitrophenol released by the hydrolysis of the p-nitrophenyl acyl group can be monitored at 405 nm for 5 minutes at 10 second intervals on a Spectra max 190 (Molecular Devices GmbH, Bismarckring 39, Biberach an der Riss 88400, Germany). The hydrolysis activity of the variant on one or more substrates can be compared with the hydrolysis activity of the parent lipase on one or more substrates.

[0670] Examples

[0671] Example 1

[0672] Stability of lipase variants in detergent compositions containing and without sulfites

[0673] A lipase variant of SEQ ID NO: 2 was constructed and expressed in Aspergillus oryzae as described in WO 2016 / 050661 (incorporated by reference).

[0674] Growth of variants:

[0675] Transformants expressing the lipase variants were inoculated into 96-well MTPs containing 200 μl YP + 2% maltose. They were grown at 34°C for 120 hours (without shaking).

[0676] Filter variants by:

[0677] 1 g of sodium sulfite was added to 100 g of a detergent composition (TIDE purchased from P&G, USA in March 2017). TMOriginal Scent HE Turbo Clean Liquid Laundry Detergent) and agitate for at least 10 minutes. TM

[0678] An aliquot of 90 μL of detergent containing sulfite was added to a 96-well plate (with 500 μL space).

[0679] A 90 μL aliquot of sulfite-free detergent was added to a 96-well plate (with 500 μL space).

[0680] Add magnets to the board.

[0681] Add 10 µL of medium from the growth plate to each detergent plate.

[0682] The plate was sealed with an aluminum seal and shaken on a magnetic plate stirrer for at least 10 min.

[0683] The plates were incubated in a heating box at 49°C in a plastic box for 19 hours.

[0684] Stability determination:

[0685] Dilution buffer: 0.1M Tris-HCl, 9mM CaCl2, 0.0225% Brij-30, pH 8.0

[0686] Assay buffer: AOS substrate buffer: 100 mM Tris, 6.5 mM deoxycholate, 1.4 g / L AOS buffer, pH 8.0

[0687] Substrate: 100 mL AOS buffer, 0,5 mM pNP-palmitate, 1 mM CaCl2

[0688] Add 230 μL of dilution buffer to the incubated plate. Agitate the detergent plate for 10 min.

[0689] 5 μL from the detergent plate was added to a new plate containing 295 μL of dilution buffer and shaken for 10 min.

[0690] Transfer 10 μL from the first two plates (with and without sulfite) to a 384-well plate.

[0691] 40 μL of substrate solution was added to the plate and run in the Elisa reader for 30 min.

[0692] The following variants of SEQ ID NO: 2 have improved stability (ie, increased residual activity) in detergent compositions with sulfite: T35D, N39E, P42D, N101D, S105E, F184D.

[0693] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure including definitions shall prevail.

Claims

1. A composition comprising a surfactant, a sulfite source and a lipase variant of SEQ ID NO: 2, (1)-(a) In which the cysteine ​​bridge The asparagine (N) at position 39 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; (1)-(b) where the cysteine ​​bridge The proline (P) at position 42 in SEQ ID NO: 2 is replaced by the more negatively charged amino acid aspartic acid (D); (1)-(c) where the cysteine ​​bridge The asparagine (N) at position 101 in SEQ ID NO: 2 is replaced by aspartic acid (D), an amino acid with a more negative charge; (1)-(d) where the cysteine ​​bridge The serine (S) at position 105 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; or (1)-(e) where the cysteine ​​bridge The phenylalanine (F) at position 184 in SEQ ID NO: 2 is replaced by aspartic acid (D), an amino acid with a more negative charge; and (2) wherein the lipase variant has improved stability in the presence of a sulfite source compared to the parent lipase in SEQ ID NO:

2.

2. The composition of claim 1, wherein the cysteine ​​bridge is or corresponds to one or more of the cysteine ​​bridges at the following positions: C22-C268 of SEQ ID NO: 2, C36-C41, and C104-C107.

3. The composition of claim 1 or 2, wherein the variant of the parent lipase is selected from a polypeptide encoded by a polynucleotide having at least 95% but less than 100% sequence identity with the polypeptide encoding sequence of SEQ ID NO:

1.

4. Use of a composition as claimed in any one of claims 1 to 3 for cleaning a surface.

5. A lipase variant of a parent lipase, wherein the variant has lipase activity, (1)-(a) In which the cysteine ​​bridge The asparagine (N) at position 39 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; (1)-(b) where the cysteine ​​bridge The proline (P) at position 42 in SEQ ID NO: 2 is replaced by the more negatively charged amino acid aspartic acid (D); (1)-(c) where the cysteine ​​bridge The asparagine (N) at position 101 in SEQ ID NO: 2 is replaced by aspartic acid (D), an amino acid with a more negative charge; (1)-(d) where the cysteine ​​bridge The serine (S) at position 105 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; or (1)-(e) where the cysteine ​​bridge The phenylalanine (F) at position 184 in SEQ ID NO: 2 is replaced by aspartic acid (D), an amino acid with a more negative charge; and (2) wherein the lipase variant has improved stability in the presence of a sulfite source compared to the parent lipase in SEQ ID NO:

2.

6. A polynucleotide encoding the variant according to claim 5.

7. A nucleic acid construct comprising the polynucleotide according to claim 6. An expression vector comprising the polynucleotide according to claim 6.

9. A host cell comprising the polynucleotide of claim 6, wherein the host cell is not a plant variety.

10. A method for producing a lipase variant, the method comprising: (a) culturing the host cell of claim 9 under conditions suitable for expressing the variant; and (b) recovering the variant.

11. A method for obtaining the lipase variant according to claim 5, the method comprising: (a) Among them, the The asparagine (N) at position 39 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; (b) where the cysteine ​​bridge The proline (P) at position 42 in SEQ ID NO: 2 is replaced by the more negatively charged amino acid aspartic acid (D); (c) where the cysteine ​​bridge The asparagine (N) at position 101 in SEQ ID NO: 2 is replaced by aspartic acid (D), an amino acid with a more negative charge; (d) Among them, The serine (S) at position 105 in SEQ ID NO: 2 is replaced by glutamic acid (E), an amino acid with a more negative charge; or (e) Among them, The phenylalanine (F) at position 184 in SEQ ID NO: 2 is replaced by the more negatively charged amino acid aspartic acid (D).

12. The composition of claim 1, wherein the substituted amino acid is within 15 angstroms from the carboxyl group to the sulfur atom of the cysteine ​​bridge.

13. The composition of claim 12, wherein the substituted amino acid is within 10 angstroms of a cysteine ​​bridge.

14. The composition of claim 12, wherein the substituted amino acid is within 5 angstroms of a cysteine ​​bridge.

15. The composition of claim 12, wherein the replaced amino acid is a substituted amino acid.

16. The composition of claim 1 or 12, wherein the sulfite source is selected from: sulfites; bisulfites; metabisulfites; or any combination thereof.

17. The composition of claim 16, wherein the sulfite is sodium sulfite, potassium sulfite or calcium sulfite.

18. The composition of claim 16, wherein the bisulfite is potassium bisulfite, sodium bisulfite or calcium bisulfite.

19. The composition of claim 16, wherein the metabisulfite is potassium metabisulfite, sodium metabisulfite or calcium metabisulfite.

20. The composition of claim 16, wherein sulfite (SO3 2- ) or metabisulfite (S2O5 2- ) is in an amount of from 0.1 wt % to 3 wt %.

21. The composition of claim 20, wherein sulfite (SO3 2- ) or pyrosulfite (S2O5 2- ) is in an amount of from 0.2 wt % to 2 wt %.

22. The composition of claim 20, wherein sulfite (SO3 2- ) or pyrosulfite (S2O5 2- ) is in an amount of from 0.5 wt % to 2 wt %.

23. The composition of claim 1, wherein the cysteine ​​bridge is on the surface of the parent lipase.

24. The composition of claim 1, wherein the variant of the parent lipase is a polypeptide encoded by a polynucleotide having at least 90% but less than 100% sequence identity with the polypeptide coding sequence of SEQ ID NO:

1.

25. Use of a composition as claimed in any one of claims 1 or 12 to 24 for cleaning surfaces including textiles or hard surfaces.

26. The use according to claim 25, wherein the textile is a fabric.

27. The method of claim 11, wherein the substituted amino acid is within 15 angstroms from the carboxyl group to the sulfur atom of the cysteine ​​bridge.

28. The method of claim 27, wherein the substituted amino acid is within 10 angstroms of a cysteine ​​bridge.

29. The method of claim 27, wherein the substituted amino acid is within 5 angstroms of a cysteine ​​bridge.

30. The method of claim 27, wherein the replaced amino acid is a substituted amino acid.

Images