Lipase variants and polynucleotides encoding them

Abstract

The present invention relates to variants of a parent lipase, wherein said variants have at least 60% but less than 100% sequence identity to SEQ ID NO: 2, have lipase activity, the variants comprise a substitution at a position corresponding to position 92 and / or 96 of SEQ ID NO: 2; and are unchanged at positions corresponding to positions 231, 233 and 254 of SEQ ID NO: 2. The present invention also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, and methods of using the variants.

Description

[0001] References to sequence lists

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

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

[0004] Related fields description

[0005] Lipases are important biocatalysts that have been shown to be useful in a wide range of applications, and a large number of different lipases have been identified and many have been commercialized. However, new lipases suitable for use in different compositions adapted to the conditions of current use are desirable.

[0006] Lipases are included in detergent compositions to improve washing performance and, in particular, to improve the removal of lipid stains. Current detergent, cleaning, and / or fabric care compositions contain numerous active ingredients that interfere with the ability of lipases to remove lipid stains. Among other things, builders are included in detergent compositions to reduce calcium concentration, as calcium deposition can cause a “graying” appearance on treated surfaces. Low levels of calcium can result in reduced lipase activity.

[0007] In many lipases, the catalytic site is masked by a cap domain (cap region or cap), and studies have indicated that this cap is important for lipase activity and plays an activating role. Enhanced catalytic activity in the presence of the water / lipid interface is termed "interfacial activation," and describes the opening of the amphiphilic surface ring, i.e., the cap, upon contact with the interface. Shu et al. generated four Aspergillus niger lipase (ANL) mutants in Enzyme and Microbial Technology 48(2011)129-133, one without a cap and three with caps in an open conformation, aiming to identify lipase mutants independent of interfacial activation.

[0008] Therefore, there is a need for lipases with improved lipase activity, particularly for use in detergent compositions. Invention Overview

[0010] The present invention relates to variants of parental lipases, wherein the variants have at least 60% but less than 100% sequence identity with SEQ ID NO:2 and have lipase activity, the variants comprising substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2; and remaining unchanged at positions corresponding to positions 231, 233 and 254 of SEQ ID NO:2.

[0011] The present invention also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors and host cells containing these polynucleotides, and methods of using these variants.

[0012] definition

[0013] Lipase: The terms "lipase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to an enzyme in class EC3.1.1 as defined by enzyme nomenclature. It can possess lipase activity (triacylglycerol lipase, EC3.1.1.3), keratinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13), and / or wax ester hydrolase activity (EC3.1.1.50). For the purposes of this invention, lipase activity is determined according to the procedure described in the Examples section: substrates with different chain lengths can be used, and hydrolytic activity is determined using PnP assay. In one aspect, the variants of the invention have at least 20% of the lipase activity of the 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%. In another aspect, the parent lipase is a polypeptide of SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0014] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (without change in the encoded polypeptide) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0015] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from mature, spliced ​​mRNA molecules derived from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0016] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly defines the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (such as ATG, GTG, or TTG) and ends with a stop codon (such as TAA, TAG, or TGA). Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0017] Control Sequences: The term "control sequence" refers to the nucleic acid sequence required for the expression of the polynucleotide encoding the variant of the present invention. Each control sequence may be native (i.e., from the same gene) or exogenous (i.e., from a different gene) for the polynucleotide encoding that variant, or native or exogenous relative to each other. Such control sequences include, but are not limited to, precursors, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. These control sequences may be provided with multiple linkers for the purpose of introducing specific restriction enzyme sites that facilitate the linking of these control sequences to the coding regions of the polynucleotide encoding the variant.

[0018] Expression: The term “expression” includes any step involved in the generation of variants, including (but not limited to) transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0019] Expression vector: The term “expression vector” refers to a straight or circular DNA molecule that includes a polynucleotide encoding a variant and is operatively linked to a control sequence that provides for its expression.

[0020] Fragment: The term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at its amino and / or carboxyl terminus; wherein the fragment has lipase activity. In one aspect, the fragment comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids present in the parental lipase, but less than 100%. In one aspect, the parental lipase is a polypeptide of SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the parental lipase is amino acids 1-269 of SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0021] Highly stringent conditions: The term "highly stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 65°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0022] Host cell: The term "host cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors including the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0023] Improved properties: The term "improved properties" refers to characteristics associated with a variant that is improved relative to the parental lipase. Such improved properties include, but are not limited to, lipase activity and Ca-independence. The lipase activity may be increased lipase activity; increased lipase activity under reduced / low levels of Ca; or increased lipase activity in the presence of EDTA. The Ca-independence may be increased Ca-independence. The term "reduced or low levels of Ca" means that the concentration of Ca in the solution has been reduced or lowered compared to a control solution. Such reduced or low levels of Ca can be obtained by adding a reagent that consumes some or all of the Ca from the solution. Such a reagent may be a washing aid as described in the "Compositions" section of this application.

[0024] Isolated: The term “isolated” means a substance that does not exist in nature in a form or environment. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring; (2) any substance removed at least partially from one or more naturally occurring components associated with it in nature, including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that has been artificially modified relative to the kind of substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; use of a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0025] Low stringency conditions: The term "low stringency conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0026] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 1 to 269 of SEQ ID NO:2, SEQ ID NO:4; or SEQ ID NO:6. It is known in the art that host cells can produce a mixture of two or more different mature polypeptides (i.e., with different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide.

[0027] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide with lipase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 807 of SEQ ID NO:1; SEQ ID NO:3; or SEQ ID NO:5.

[0028] Medium-tough conditions: The term "medium-tough conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0029] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0030] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0031] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a way that does not normally exist in nature to contain segments of nucleic acid, or is synthesized and includes one or more control sequences.

[0032] Operable ligation: The term “operable ligation” means a configuration in which a control sequence is placed in the appropriate position relative to the coding sequence of a polynucleotide so that the control sequence guides the expression of the coding sequence.

[0033] Parental or parental lipase: The term "parental" or "parental lipase" refers to a lipase to which a modification is made to produce the enzyme variant of the present invention. The parent can be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof. Examples of such parental lipases are those having the amino acid sequences given in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0034] Sequence consistency: The correlation between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence consistency".

[0035] For the purposes of this invention, the sequence identity between two amino acid sequences was determined using the Niedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol. 48:443-453), as performed in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The Needle output marked "Longest Consistency" (obtained using the -nobrief option) was used as the percentage consistency and was calculated as follows:

[0036] (Consistent residues x 100) / (Alignment length - Total number of vacancies in the alignment)

[0037] For the purposes of this invention, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, see above), implemented in the Nieder program as in the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 5.0.0 or later), is used to determine sequence consistency between two deoxyribonucleotide sequences. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled “Longest Consistency” (obtained using the -nobrief option) is used as the percentage consistency and is calculated as follows:

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

[0039] Subsequence: The term "subsequence" refers to a polynucleotide in which one or more (e.g., several) nucleotides are omitted from the 5' and / or 3' end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment having lipase activity. In one aspect, the subsequence comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of nucleotides 1 to 807 encoding the parental lipase. In one aspect, the parental lipase comprises or consists of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0040] Variants: The term "variant" refers to a polypeptide having lipase activity that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position. These variants of the invention have at least 20%, for example, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% lipase activity of the parent lipase polypeptide. In one aspect, the parent lipase comprises or consists of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0041] Very High Tough Conditions: The term "very high tough conditions" means that for probes at least 100 nucleotides in length, standard DNA blotting procedures are followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 70°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0042] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 60°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0043] Medium-tough conditions: The term "medium-tough conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 55°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0044] Low stringency conditions: The term "low stringency conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following a standard DNA blotting procedure. Finally, the vector material is washed three times at 50°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0045] Very low stringency conditions: The term "very low stringency conditions" means that for probes of at least 100 nucleotides in length, a standard DNA blotting procedure is followed, including pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Finally, the vector material is washed three times at 45°C with 2X SSC and 0.2% SDS for 15 minutes each time.

[0046] Wild-type lipase: The term "wild-type" lipase refers to a lipase expressed by a naturally occurring microorganism (such as a bacterium, yeast, or filamentous fungus) found in nature.

[0047] Variant Naming Rules

[0048] For the purposes of this invention, the polypeptide disclosed in SEQ ID NO:2 is used to determine the corresponding amino acid residues in another lipase. The amino acid sequence of the other lipase is aligned with the polypeptide disclosed in SEQ ID NO:2, and based on this alignment, the Niederman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Open Software Suite for Molecular Biology, Rice et al., 2000, Trends Genet. 16:276-277) (preferably version 5.0.0 or later) is used to determine the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:2. The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0049] The identification of corresponding amino acid residues in another lipase can be determined by using several computer programs that compare multiple peptide sequences with their corresponding default parameters. These computer programs include, but are not limited to, MUSCLE (multiple sequence comparisons by logarithmic prediction; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Kato et al., 2005, Nucleic Acids Research 33:511-518; Kato, 2007, Bioinformatics 23:372-374; Kato et al., 2009, Methods in Molecular Biology). Biology) 537:39-64; Kato Kazuto, 2010, Bioinformatics 26:1899-1900) and EMBOSS EMMA using ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acid Research 22:4673-4680).

[0050] When other enzymes are dissimilar to the polypeptide of SEQ ID NO:2, making traditional sequence-based comparison methods unable to detect their relationship (Lindahl and Elofsson, 2000, J.Mol.Biol. 295:613-615), other pairwise sequence comparison algorithms can be applied. Greater sensitivity in sequence-based searches can be achieved using search programs that utilize probabilistic representations (profiles) of polypeptide families to search a database. For example, the PSIBLAST program generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acids Res. 25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Procedures such as GenTHREADER (Jones, 1999, J.Mol.Biol. 287:797-815; McGuffin and Jones, 2003, Bioinformatics 19:874-881) utilize information from various sources (PSI-BLAST, secondary structure prediction, structural alignment spectra, and solvation potential) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, J.Mol.Biol. 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0051] For proteins with known structures, several tools and resources are available for retrieving and generating structure alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms, such as distance alignment matrices (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747), can be used to align two or more protein structures. Furthermore, implementations of these algorithms can be used to query structure databases containing structures of interest to discover potential structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16:566-567).

[0052] In the description of variations of the invention, the following nomenclature is used for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are adopted.

[0053] replace For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine for alanine at position 226 would be named "Thr226Ala" or "T226A". Multiple mutations are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F", representing the substitution of glycine (G) for arginine (R) at positions 205 and 411, respectively, and the substitution of serine (S) for phenylalanine (F).

[0054] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Therefore, a deletion of glycine at position 195 is named “Gly195*” or “G195*”. Multiple deletions are separated by a plus sign (“+”), for example, “Gly195*+Ser411*” or “G195*+S411*”.

[0055] insertFor amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [initial amino acid, position, initial 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".

[0056] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0057] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0058] Multiple changes Variants with multiple alterations are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0059] Different changes When different changes can be introduced at a single position, these changes are separated by commas. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variant:

[0060] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”. Invention Details

[0062] This invention relates to lipase variants that, compared to the parent enzyme, have increased lipase activity and / or increased Ca-independence.

[0063] variants

[0064] The present invention provides variants of a parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase and possesses lipase activity, the variant comprising substitutions at positions corresponding to positions 92 and / or 96 of the parental lipase; and remaining unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0065] On the one hand, the variant has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence similarity to the amino acid sequence of the parent lipase.

[0066] In one respect, the variant has at least 60%, for example, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity with the polypeptide of SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0067] In one aspect, the number of substitutions in the variants of the present invention is 1-40, 1-30, 1-20, 1-10, or 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.

[0068] In one aspect, the variant includes a substitution at the position corresponding to position 92 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 92 is substituted with Asp or Lys. In one aspect, the variant includes the substitution G92D or G92K of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 92, wherein the amino acid is D or K.

[0069] In one aspect, the variant includes a substitution at the position corresponding to position 96 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 96 is substituted with Ile, Leu, or Thr. In one aspect, the variant includes the substitution E96I, E96L, or E96T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 96, wherein the amino acid is I, L, or T.

[0070] In one aspect, the variant is unmodified at the position corresponding to position 231 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 231 is Arg, His, or Lys. In one aspect, the variant includes the amino acid R231R, R231H, or R231K of SEQ ID No:2. In one aspect, the variant includes the amino acid at the position corresponding to position 231, wherein the amino acid is R, H, or K.

[0071] In one aspect, the variant is unmodified at the position corresponding to position 233 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 233 is Arg, His, or Lys. In one aspect, the variant includes the amino acid R233R, R233H, or R233K of SEQ ID No:2. In one aspect, the variant includes the amino acid at the position corresponding to position 233, wherein the amino acid is R, H, or K.

[0072] In one aspect, the variant is unmodified at the position corresponding to position 254 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 254 is Ser. In one aspect, the variant includes amino acid S254S of SEQ ID No:2. In one aspect, the variant includes an amino acid at the position corresponding to position 254, wherein the amino acid is S.

[0073] In one respect, the variant includes substitutions at positions corresponding to positions 92 and / or 96 and remains unchanged at positions corresponding to positions 231, 233, and 254, as described above.

[0074] On one hand, the variant includes one or more (e.g., several) substitutions selected from the group consisting of the following items: G92D, G92K, E96I, E96L, and E96T.

[0075] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant includes a substitution for G92D; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0076] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant includes a substitution of G92K; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0077] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant includes the substitution of E96I; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0078] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant includes the substitution of E96L; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0079] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant includes the substitution of E96T; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0080] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution of G92D+E96I; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0081] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution G92D+E96L; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0082] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution G92D+E96T; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0083] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution of G92K+E96I; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0084] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution G92K+E96L; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0085] In one aspect, the variant is a variant of the parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with the parental lipase, possesses lipase activity, and the variant comprises the substitution G92K+E96T; and remains unchanged at positions corresponding to positions 231, 233, and 254 of the parental lipase. In another aspect, the variant has improved Ca-independence compared to the parental enzyme. In another aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6.

[0086] These variants may further include one or more additional substitutions in one or more (e.g., several) other locations.

[0087] In one aspect, the variant includes a substitution at the position corresponding to position 81 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 81 is substituted with Gln. In one aspect, the variant includes the substitution R81Q of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 81, wherein the amino acid is Q.

[0088] In one aspect, the variant includes a substitution at the position corresponding to position 83 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 83 is substituted with Thr. In one aspect, the variant includes the substitution S83T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 83, wherein the amino acid is T.

[0089] In one aspect, the variant includes a substitution at the position corresponding to position 84 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 84 is substituted with His. In one aspect, the variant includes the substitution R84H of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 84, wherein the amino acid is H.

[0090] In one aspect, the variant includes a substitution at the position corresponding to position 85 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 85 is substituted with Thr. In one aspect, the variant includes the substitution S85T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 85, wherein the amino acid is T.

[0091] In one aspect, the variant includes a substitution at the position corresponding to position 86 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 86 is substituted with Leu, Pro, or Trp. In one aspect, the variant includes the substitution I86L, I86P, or I86W of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 86, wherein the amino acid is L, P, or W.

[0092] In one aspect, the variant includes a substitution at the position corresponding to position 87 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 87 is substituted with Ala, Lys, or Thr. In one aspect, the variant includes the substitution E87A, E87K, or E87T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 87, wherein the amino acid is A, K, or T.

[0093] In one aspect, the variant includes a substitution at the position corresponding to position 88 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 88 is substituted with Gln. In one aspect, the variant includes the substitution N88Q of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 88, wherein the amino acid is Q.

[0094] In one aspect, the variant includes a substitution at the position corresponding to position 90 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 90 is substituted with Leu or Met. In one aspect, the variant includes the substitution I90L or I90M of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 90, wherein the amino acid is L or M.

[0095] In one aspect, the variant includes a substitution at the position corresponding to position 91 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 91 is substituted with Ala or Leu. In one aspect, the variant includes the substitution G91A or G91L of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 91, wherein the amino acid is A or L.

[0096] In one aspect, the variant includes a substitution at the position corresponding to position 93 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 93 is substituted with Phe. In one aspect, the variant includes the substitution L93F of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 93, wherein the amino acid is F.

[0097] In one aspect, the variant includes a substitution at the position corresponding to position 94 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 94 is substituted with Asp or Lys. In one aspect, the variant includes the substitution N94D or N94K of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 94, wherein the amino acid is D or K.

[0098] In one aspect, the variant includes a substitution at the position corresponding to position 95 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 95 is substituted with Ala, Leu, or Tyr. In one aspect, the variant includes the substitution F95A, F95L, or F95Y of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 95, wherein the amino acid is A, L, or Y.

[0099] In one aspect, the variant includes a substitution at the position corresponding to position 97 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 97 is substituted with Phe or Pro. In one aspect, the variant includes the substitution L97F or L97P of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 97, wherein the amino acid is F or P.

[0100] In one aspect, the variant includes a substitution at the position corresponding to position 98 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 98 is substituted with Asp or Gln. In one aspect, the variant includes the substitution K98D or K98Q of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 98, wherein the amino acid is D or Q.

[0101] In one aspect, the variant includes a substitution at the position corresponding to position 51 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 51 is substituted with Val. In one aspect, the variant includes the substitution F51V of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 51, wherein the amino acid is V.

[0102] In one aspect, the variant includes a substitution at the position corresponding to position 136 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 136 is substituted with His. In one aspect, the variant includes the substitution P136H of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 136, wherein the amino acid is H.

[0103] In one aspect, the variant includes a substitution at the position corresponding to position 211 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 211 is substituted with Leu. In one aspect, the variant includes the substitution F211L of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 211, wherein the amino acid is L.

[0104] In one aspect, the variant includes a substitution at the position corresponding to position 252 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 252 is substituted with Thr. In one aspect, the variant includes the substitution I252T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 252, wherein the amino acid is T.

[0105] In one aspect, the variant includes a substitution at the position corresponding to position 255 of the parental lipase. In one aspect, the parental lipase is SEQ ID NO:2; SEQ ID NO:4; or SEQ ID NO:6. In one aspect, the amino acid at the position corresponding to position 255 is substituted with Thr. In one aspect, the variant includes the substitution I255T of SEQ ID NO:2. In one aspect, the variant includes an amino acid at the position corresponding to position 255, wherein the amino acid is T.

[0106] In one aspect, the variant further includes substitutions at one or more (e.g., several) locations in the cap region corresponding to locations 81-99 of SEQ ID NO:2, preferably R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q. In another aspect, the variant further includes substitutions at one location corresponding to any one of the following locations: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In one aspect, the variant further includes substitutions at two positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at three positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In one aspect, the variant further includes substitutions at four positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at five positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2.In one aspect, the variant further includes substitutions at six positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at seven positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In one aspect, the variant further includes substitutions at eight positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at nine positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In one aspect, the variant further includes substitutions at ten positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at eleven positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In one aspect, the variant further includes substitutions at twelve positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2.In one aspect, the variant further includes substitutions at thirteen positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2. In another aspect, the variant further includes substitutions at fourteen positions corresponding to any one of the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q of SEQ ID NO:2.

[0107] In one aspect, the variant includes substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2 and remains unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2, and further includes substitutions or constitutes thereof at positions corresponding to one or more (e.g., several) of the following positions: 87, 86, and 91, as described above. In another aspect, the variant includes substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2 and remains unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2, and further includes substitutions or constitutes thereof at positions corresponding to two positions selected from: 87+86; 87+91; or 86+91, as described above. In one aspect, the variant includes substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2 and is unmodified at positions corresponding to positions 231, 233 and 254 of SEQ ID NO:2, and further includes substitutions or constitutes thereof at positions corresponding to the following three positions: 87+86+91, as described above.

[0108] In one aspect, the variant further includes substitutions at one or more (e.g., several) positions corresponding to positions 51, 136, 211, 252, and 255 of SEQ ID NO:2, preferably F51V, P136H, F211L, I252T, and I255T. In another aspect, the variant further includes substitutions at one position corresponding to any one of positions F51V, P136H, F211L, I252T, and I255T of SEQ ID NO:2. In another aspect, the variant further includes substitutions at two positions corresponding to any one of positions F51V, P136H, F211L, I252T, and I255T of SEQ ID NO:2. In another aspect, the variant further includes substitutions at three positions corresponding to any one of positions F51V, P136H, F211L, I252T, and I255T of SEQ ID NO:2. In one aspect, the variant further includes substitutions at four positions corresponding to any one of positions F51V, P136H, F211L, I252T, and I255T in SEQ ID NO:2. In another aspect, the variant further includes substitutions at five positions corresponding to any one of positions F51V, P136H, F211L, I252T, and I255T in SEQ ID NO:2.

[0109] These amino acid changes can be minute in nature, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small amino or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine tracts, antigenic epitopes, or binding domains.

[0110] Examples of conserved substitutions are found in the following group: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific reactivity are known in the art and are described, for example, by H. Neurath and R.R. Hill, 1979, in *The Proteins*, Academic Press, New York. Common substitutes are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0111] Alternatively, these amino acid alterations have the property of changing the physicochemical properties of the peptide. For example, amino acid alterations can improve the peptide's thermal stability, change its substrate specificity, change its optimal pH, and so on.

[0112] For example, these variants may include substitutions at positions corresponding to any of the following: 4, 27, 33, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 249, 255, 256, 263, 264, 265, 266, 267, and 269 of SEQ ID NO:2. In one respect, these variants further include substitutions at one or more (e.g., several) positions corresponding to any of the following: SEQ ID NO:2 at positions 4, 27, 33, 38, 57, 58, 60, 83, 86, 91, 94, 97, 99, 111, 150, 163, 210, 216, 225, 227, 249, 255, 256, 263, 264, 265, 266, 267, and 269. In one aspect, these variants further include one or more (e.g., several) substitutions corresponding to any one of the following: Q4V, D27R, N33Q, G38A, D57G, S58A, V60S, S83T, I86V, G91A / N / Q, N94K / R, D96E / G / L / W, L97M, E99K, D111A, A150G, G163K, E210K / Q, S216P, G225R, L227G, Q249R, I255A, P256K / T / V, G263Q, L264A, I265T, G266D, T267A, and L269N of SEQ ID NO:2.

[0113] For the purposes of this invention, amino acids listed in a single letter code can be divided into the following groups: Group 1 (negatively charged) = ED; Group 2 (positively charged) = KRH; Group 3 (hydrophilic) = TQSN; Group 4 (hydrophobic) = PFYWLI; Group 5 = VAG; and Group 6 = CM. The substitution of amino acids in variations of this invention follows general rules, wherein: in one aspect, amino acids of Group 1 are substituted with amino acids from Group 2, 3, 4, or 5; in one aspect, amino acids of Group 2 are substituted with amino acids from Group 3; in one aspect, amino acids of Group 3 are substituted with amino acids from Group 1 or 3; in one aspect, amino acids of Group 4 are substituted with amino acids from Group 4; and in one aspect, amino acids of Group 5 are substituted with amino acids from Group 5 or 4.

[0114] A cap defined as an amino acid corresponding to residues 81 to 99 of SEQ ID NO:2 can be divided into two parts: part 1 is defined as an amino acid corresponding to residues 81 to 90 of SEQ ID NO:2; and part 2 is defined as an amino acid corresponding to residues 91 to 99 of SEQ ID NO:2. In one aspect, the invention relates to a cap in which amino acid substitution results in a greater overall negative charge. In one aspect, the invention relates to a cap in which amino acid substitution results in a greater overall negative charge in part 1. In one aspect, the invention relates to a cap in which amino acid substitution results in a greater overall negative charge in part 2. In one aspect, the invention relates to a cap in which amino acid substitution results in a more hydrophobic cap. In one aspect, the invention relates to a cap in which amino acid substitution in part 1 results in a more hydrophobic cap. In one aspect, the invention relates to a cap in which amino acid substitution in part 2 results in a more hydrophobic cap. In one aspect, the invention relates to a cap in which a hydrophilic amino acid (group 3) is substituted with a negatively charged amino acid (group 1). In one aspect, the invention relates to a cap in which a hydrophilic amino acid (group 3) in part 2 is substituted with a negatively charged amino acid (group 1).

[0115] Essential amino acids in peptides can be identified using methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, a single alanine mutation is introduced at each residue in the molecule, and the lipase activity of the resulting mutant molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, Journal of Biochemistry 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, such as by techniques including nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutation of the amino acid at the putative contract site. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, Journal of Molecular Biology 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from comparisons with related peptides.

[0116] On one hand, this variant exhibits increased lipase activity compared to the parent enzyme. This increased lipase activity can be determined by the "p-nitrophenyl (pNP) assay" or by the "standard assay," as described in the examples.

[0117] On the one hand, compared to the parent enzyme, this variant exhibits increased lipase activity at reduced / low levels of Ca. The increased lipase activity at reduced Ca levels can be determined by the “standard assay” described in the examples.

[0118] On the one hand, this variant exhibits increased lipase activity in the presence of EDTA compared to the parental enzyme. This increased lipase activity at reduced levels of Ca can be determined by the “standard assay” described in the examples.

[0119] On the one hand, this variant exhibits increased Ca-independence compared to the parent enzyme. This increased Ca-independence can be determined by the "standard assay" described in the examples.

[0120] Parent

[0121] The parental lipase may be (a) a polypeptide having at least 60% sequence identity with the polypeptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the polypeptide encoding sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide that has at least 60% sequence identity with the polypeptide encoding sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5.

[0122] On one hand, the parent has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, and the polypeptide has lipase activity. On the one hand, the amino acid sequence of the parent differs from the polypeptides of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 by up to 40 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 6, 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 different.

[0123] On one hand, the parent includes or consists of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0124] In one respect, the parent is a fragment of a polypeptide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, the fragment comprising 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, or SEQ ID NO:6.

[0125] On the one hand, the parent is an allelic variant of the polypeptide of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0126] On the one hand, the parent is encoded by a polynucleotide that hybridizes with (i) the polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, or (ii) the full-length complement of (i) under very low stringency, low stringency, medium stringency, medium-high stringency, high stringency or very high stringency conditions (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor, New York).

[0127] The polynucleotides or subsequences thereof of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, together with the polypeptides or fragments thereof of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, can be used to design nucleic acid probes to identify and clone parental DNA encoding strains from different genera or species, according to methods well known in the art. Specifically, such probes can be used to hybridize with the genomic DNA or cDNA of cells of interest according to standard DNA blotting procedures to identify and isolate the corresponding genes therein. Such probes can be significantly shorter than the complete sequence, but the length should be at least 15, for example at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe has a length of at least 100 nucleotides, for example at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides. Both DNA and RNA probes can be used. Typically, the probes are labeled (e.g., with...). 32 P, 3 H, 35 This invention covers probes containing biotin (or avidin) to detect corresponding genes.

[0128] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened for DNA that hybridizes to and encodes the parental DNA with the probes described above. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred to nitrocellulose or other suitable carrier material and immobilized thereon. This carrier material is used for DNA blotting to identify clones or DNA that hybridize to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 or their subsequences.

[0129] For the purposes of this invention, hybridization demonstrates that a polynucleotide hybridizes with a labeled nucleic acid probe under very low to very high stringency conditions, the probe corresponding to (i) SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; (ii) the polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5; (iii) its full-length complement; or (iv) its subsequence. Molecules hybridizing with nucleic acid probes under these conditions can be detected using, for example, X-ray film or any other detection method known in the art.

[0130] In one aspect, the nucleic acid probe is a polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In another aspect, the nucleic acid probe is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of nucleotides in SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In another aspect, the nucleic acid probe is a polypeptide encoding SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6; its mature polypeptide; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0131] On one hand, the parent is encoded by a polynucleotide that has at least 60%, for example at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

[0132] The polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0133] The parent peptide can be a fusion peptide or a cleavable fusion peptide, wherein another peptide is fused at the N-terminus or C-terminus of the peptide of the present invention. Fusion peptides are generated by fusing a polynucleotide encoding another peptide with the polynucleotide of the present invention. Techniques for generating fusion peptides are known in the art and include linking the coding sequences of the peptides such that they are within a frame, and that the expression of the fusion peptide is under the control of the same promoter and terminator. Fusion peptides can also be constructed using integrin technology, wherein the fusion peptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0134] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following: Martin et al., 2003, Journal of Industrial Microbiology and Biotechnology (J. Ind. Microbiol. Biotechnol.) 3:568-576; Svetina et al., 2000, Journal of Biotechnology (J. Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Wa... (rd) et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0135] The parent can be obtained from any genus of microorganisms. For the purposes of this invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, the parent is extracellularly secreted.

[0136] 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, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces. Lipases of the genera *Campylobacter*, *Escherichia coli*, *Flavobacterium*, *Fusobacterium*, *Helicobacter*, *Ilyobacter*, *Neisseria*, *Pseudomonas*, *Salmonella*, or *Ureaplasma*.

[0137] On one hand, the parent is alkalophilic Bacillus, amyloliquefaciens, brevis, circular Bacillus, Clausii, coagulant Bacillus, sclerosus, brilliant Bacillus, slow-release Bacillus, licheniformis, megaterium, pumilus, thermophilic lipobacillus, Bacillus subtilis, or Bacillus thuringiensis lipase.

[0138] On one hand, the parent is a lipase of Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus lactis, or Streptococcus equi subsp. Zooepidemicus.

[0139] On the one hand, the parent is a non-color-producing Streptomyces, worm-killing Streptomyces, sky-blue Streptomyces, gray Streptomyces, or light blue-purple Streptomyces lipase.

[0140] The parent can be a fungal lipase. For example, the parent can be a yeast lipase, such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia lipase.Or a filamentous fungal lipase, such as *Acremonium*, *Agaricus*, *Alternaria*, *Aspergillus*, *Aureobasidium*, *Botryospaeria*, *Ceriporiopsis*, *Chaetomidium*, *Chrysosporium*, *Claviceps*, *Cochliobolus*, *Coprinopsis*, *Cop* The genera *totermes*, *Corynascus*, *Cryphonectria*, *Cryptococcus*, *Diplodia*, *Exidia*, *Filibasidium*, *Fusarium*, *Gibberella*, *Holomastigotoides*, *Humicola*, *Irpex*, *Lentinula*, and *Leptospaeria* are all mentioned. The genera *Magnaporthe*, *Melanocarpus*, *Meripilus*, *Mucor*, *Myceliophthora*, *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Piromyces*, *Poitrasia*, *Pseudoplectania*, and *Pseudotr* are all mentioned. Lipases from the genera *Ichonympha*, *Rhizomucor*, *Schizophyllum*, *Scytalidium*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trichoderma*, *Trichophaea*, *Verticillium*, *Volvariella*, or *Xylaria*.

[0141] On one hand, the parent is a lipase from Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis.

[0142] On one hand, the parent species are *Acremonium cellulolyticus*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, and *Chrysosporium queenslandense*. The fungi listed are: queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, and Fusarium sulfide. Fusarium sulphureum, Fusarium torulosum, Fusarium pseudofilariaetrichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thievora achromatica, Thievora albomyces, Thievora albopilosa, Thievora australisinsis, Thievora foetida Thievora fimeti, Thievora microspora, Thievora ovispora, Thievora peruviana, Thievora setosa, Thievora spededonium, Thievora subthermophila, Thievora terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride lipase.

[0143] On one hand, the parent is a lipase of *Pseudomonas spp.*, such as SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0144] It should be understood that, for the aforementioned species, this invention covers perfect and imperfect states, and other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily identify the appropriate equivalents.

[0145] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Netherlands Culture Collection (CentraalbureauVoor Schimmelcultures, CBS), and the Northern Research Center (NRRL) of the Patent Culture Collection of the U.S. Agricultural Research Service.

[0146] The parent can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes mentioned above. Techniques for directly isolating microorganisms and DNA from their natural habitat are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected with one or more probes, it can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, above).

[0147] Preparation of variants

[0148] The present invention also relates to a method for obtaining a variant having lipase activity, the method comprising: (a) introducing a parental lipase by substitution at one or more (e.g., several) positions corresponding to positions 92 and / or 96 of SEQ ID NO:2, wherein the variant has lipase activity; and (b) recovering the variant. In one aspect, the parental lipase comprises or consists of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0149] These variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0150] Site-directed mutagenesis is a technique that introduces one or more (e.g., several) mutations at one or more designated sites in a polynucleotide encoding the parent.

[0151] Site-directed mutagenesis can be achieved in vitro using PCR involving primers containing oligonucleotides with the desired mutation. Site-directed mutagenesis can also be performed in vitro via cassette mutagenesis, which involves cleavage by a restriction enzyme at a site in a plasmid containing a polynucleotide encoding the parent and subsequent ligation of the oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzyme used to digest the plasmid is the same as that used to digest the oligonucleotide to allow the sticky ends of the plasmid and the insert to ligate to each other. See, for example, Scherer and Davis, 1979, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0152] Site-directed mutagenesis can also be achieved in vivo using methods known in the art. See, for example, U.S. Patent Application Publication No. 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0153] Any site-directed mutagenesis procedure can be used in this invention. Many commercially available kits are available for preparing variants.

[0154] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode polypeptides of interest. Gene synthesis can be performed using a variety of techniques, such as the multi-channel microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054), and similar techniques involving the synthesis and assembly of oligonucleotides on optically programmable microfluidic chips.

[0155] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or truncation methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0156] Mutagenesis / reorganization methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenic peptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within the peptide.

[0157] Semi-synthetic gene construction is achieved through a combination of various methods, including synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction typically utilizes the process of synthesizing polynucleotide fragments in conjunction with PCR technology. Therefore, specific regions of the gene can be synthesized de novo, while other regions can be amplified using site-specific mutagenic primers, and still others can undergo error-prone or non-error-prone PCR amplification. The polynucleotide subsequence can then be shuffled.

[0158] Polynucleotides

[0159] The present invention also relates to polynucleotides encoding variants of the invention.

[0160] Nucleic acid constructs

[0161] The present invention also relates to nucleic acid constructs comprising polynucleotides operably linked to one or more control sequences encoding variants of the invention, the one or more control sequences guiding the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0162] The polynucleotide can be manipulated in a variety of ways to provide expression of the variant. Depending on the expression vector, manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0163] The control sequence can be a promoter, which is a polynucleotide recognized by the host cell for the expression of that polynucleotide. The promoter contains a transcriptional control sequence that mediates the expression of that variant. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that of the host cell.

[0164] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of this invention in bacterial host cells are promoters obtained from the following genes: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus maltose amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). (crobiology) 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agar hydrolase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731) and tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in Gilbert et al., 1980, Scientific American 242:74-94, “Useful proteins from recombinant bacteria”; and in Sambrook et al., 1989, see above. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0165] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of the present invention in filamentous fungal host cells are promoters obtained from the genes of: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucosylamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium venenatum amyloglucosidase (WO 00 / 56900), Fusarium venenatum Daria (WO 00 / 56900), Fusarium venenatum Quinn (WO 96 / 00787). 00 / 56900), Rhizomucormiehei lipase, Rhizomucormiehei aspartic protease, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiose hydrolase I, Trichoderma reesei cellobiose hydrolase II, Trichoderma reesei 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 reesei xylanase Mycoplasma β-xylosidase, and the NA2tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-restrictive examples include modified promoters derived from the Aspergillus niger neutral α-amylase gene, wherein the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and its mutant promoters, truncated promoters and heterozygous promoters.

[0166] In yeast hosts, useful promoters are derived from the genes of the following: *Saccharomyces cerevisiae* enolase (ENO-1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8:423-488, describe other useful promoters in yeast host cells.

[0167] The control sequence can also be a transcription terminator recognized by the host cell to terminate transcription. This terminator sequence is operatively linked to the 3' end of the polynucleotide encoding that variant. Any terminator that is functional in the host cell can be used.

[0168] The preferred terminator for bacterial host cells is obtained from genes targeting the following: Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0169] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0170] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described above in Romanos et al., 1992.

[0171] Control sequences can also be mRNA stabilizing regions downstream of the promoter and upstream of the gene's coding sequence, which increase the expression of the gene.

[0172] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0173] This control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation in the host cell. The leader sequence is operatively linked to the 5' end of the polynucleotide encoding that variant. Any leader that functions in the host cell can be used.

[0174] Preferred precursors for use in filamentous fungal host cells were obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0175] Precursors suitable for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0176] The control sequence can also be a polyadenylation sequence, i.e., a sequence operatively linked to the 3' end of the variant-coding sequence and recognized by the host cell during transcription as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0177] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0178] The polyadenylated sequences useful for yeast host cells are described in Guo and Sherman, 1995, Molecular Cellular Biology, 15:5983-5990.

[0179] The control sequence can also be a signal peptide coding region, encoding a signal peptide linked to the N-terminus of the variant and guiding the variant into the cell's secretory pathway. The 5' end of the polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked within the translation reading frame to a segment encoding the variant's coding sequence. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the native signal peptide coding sequence to enhance the variant's secretion. However, any signal peptide coding sequence that guides the expressed variant into the host cell's secretory pathway can be used.

[0180] Effective signal peptide coding sequences for bacterial host cells are obtained from the genes of *Bacillus NCIB 11837* maltose amylase, *Bacillus licheniformis* subtilis protease, *Bacillus licheniformis* calc-lactamase, *Bacillus thermophilus* glutamyl-amylase, *Bacillus thermophilus* neutral proteases (nprT, nprS, nprM), and *Bacillus subtilis* prsA. Other signal sequences are described by Simonen and Palva, 1993, *Microbiological Reviews* 57:109-137.

[0181] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the genes of Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus sparseis lipase, and Rhizopus oryzae aspartic protease.

[0182] Useful signal peptides for yeast host cells were obtained from the genes of Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences were described by Romanos et al. (1992, above).

[0183] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the variant. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is usually inactive and can be converted into an active polypeptide by catalytic cleavage or autocatalytic cleavage of the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Mucor mannii aspartic protease, and Saccharomyces cerevisiae α-factor.

[0184] In the presence of both the signal peptide sequence and the propeptide sequence, the propeptide sequence is positioned immediately adjacent to the N-terminus of the variant, and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0185] It is also desirable to add regulatory sequences that modulate the expression of the variant relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include the lac, tac, and trp operon systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the variant will be operatively linked to the regulatory sequence.

[0186] expression carrier

[0187] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the invention, a promoter, and transcription and translation termination signals. Different nucleotides and control sequences can be linked together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the variant at these sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into a suitable vector for expression. In producing the expression vector, the coding sequence is located within the vector, such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0188] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0189] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, microchromosome, or artificial chromosome. The vector can contain any elements necessary to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0190] The vector preferably contains one or more selective markers that allow for convenient selection of cells such as transformed cells, transfected cells, and transduced cells. A selective marker is a gene whose product provides resistance to biocides or viruses, heavy metal resistance, or auxotrophic prototrophs, etc.

[0191] Examples of bacterial selective markers include the dal gene in *Bacillus licheniformis* or *Bacillus subtilis*, or markers that confer antibiotic resistance (e.g., resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate-amylase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in *Aspergillus* cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces hygroscopicus*.

[0192] The vector preferably contains one or more elements that allow the vector to integrate into the host cell’s genome or to replicate autonomously in the cell independently of the genome.

[0193] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the variant or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can integrate into the host cell genome via non-homologous recombination.

[0194] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicon that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replicon" refer to the polynucleotide that enables a plasmid or vector to replicate in vivo.

[0195] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0196] Examples of replication origins used in yeast host cells include the 2-micron replication origin, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

[0197] 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). The AMA1 gene can be isolated and plasmids or vectors containing the gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0198] More than one copy of the polynucleotide of the present invention can be inserted into host cells to increase the generation of variants. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene along with the polynucleotide, wherein cells containing an amplified copy of the selectable marker gene, and thus additional copies of the polynucleotide, can be selected by culturing cells in the presence of a suitable selectivity reagent.

[0199] The procedures for connecting the above-described elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0200] host cells

[0201] This invention also relates to recombinant host cells comprising a polynucleotide operably linked to one or more control sequences encoding a variant of the invention, the one or more control sequences directing the generation of the variant. A construct or vector comprising the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell will depend largely on the gene encoding the variant and its origin.

[0202] The host cell can be any cell that is useful in the recombinant-generated variants, such as prokaryotic or eukaryotic cells.

[0203] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to: Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to: Campylobacter, Escherichia coli, Flavobacterium, Clostridium, Helicobacter, Coliform, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0204] The bacterial host cell can be any Bacillus genus cell, including but not limited to: Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus croceus, Bacillus coagulans, Bacillus sclerosus, Bacillus splenium, Bacillus stenosis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus thermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0205] The bacterial host cell can also be any streptococcal cell, including but not limited to: Streptococcus equina, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equine subsp. veterinaryis.

[0206] The bacterial host cell can also be any Streptomyces cell, including but not limited to: non-chromogenic Streptomyces, insecticidal Streptomyces, sky blue Streptomyces, gray Streptomyces, and light blue Streptomyces cells.

[0207] DNA can be introduced into Bacillus cells via the following methods: protoplast transformation (see, for example, Chang and Cohen, 1979, Molecular Genetics and Genomics, 168:111-115), and competent cell transformation (see, for example, Young and Spizizen, 1961, Journal of Bacteriology, 81:823-829; or Dubnau and David Dubnau). Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169:5271-5278). DNA can be introduced into *E. coli* cells via protoplast transformation (see, e.g., Hanahan, 1983, Journal of Molecular Biology 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences of the United States of America (Proc. Natl. Acad. Sci. USA) 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, Journal of Microbiological Methods, 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol., 71:51-57).Introducing DNA into Streptococcus cells can be achieved through: native competent cells (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207), electroporation (see, e.g., Buckley et al., 1999, Applied and Environmental Microbiology 65:3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45:409-436). However, any method known in the art for introducing DNA into host cells can be used.

[0208] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0209] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, Oomycota, and all mitotic fungi (as defined by Hawksworth et al., in: Dictionary of the Fungi, Ainsworth and Bisby, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0210] The host cell for fungi can be a yeast cell. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeasts belonging to the class Fungi Imperfecti (Blastomycetes). Since yeast classification may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (Skinner, Passmore, and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0211] Yeast host cells can be cells of the genera *Candida*, *Hansenula*, *Kluyveromyces*, *Pichia pastoris*, *Saccharomyces*, *Saccharomyces*, or *Yerovia*, such as *Kluyveromyces lactis*, *Saccharomyces cerevisiae*, *Saccharomyces cerevisiae*, *Saccharomyces sacchariformis*, *Saccharomyces douglasii*, *Kluyveromyces kluyveromyces*, *Nordiya*, *Ovo*, or *Yerovia lipolytica*.

[0212] Fungal host cells can be filamentous fungal cells. "Filamentous fungi" includes all filamentous forms of the subphylum Eumycota and Oomycota (as defined above by Hawkesworth et al., 1995). Filamentous fungi are typically characterized by a hyphal wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal extension, and carbon metabolism is obligate aerobic. In contrast, yeast (such as Saccharomyces cerevisiae) grows vegetatively through budding of single-celled cells, and carbon metabolism can be fermentation.

[0213] The host cells of filamentous fungi can be cells from genera such as *Apertoire*, *Aspergillus*, *Bjerkandera*, *Pseudomonas*, *Aureospora*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Pyrophyllus*, *Pleurotus*, *Mucor*, *Pyrophyllus*, *Pleurotus ... or *Trichoderma*.

[0214] For example, the host cells of filamentous fungi can be *Aspergillus amblymorii*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidus*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsis saneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, *Chrysosporium inops*, *Chrysosporium lucknowense*, and *Chrysosporium foetida*. merdarium), rent spores, Queensland golden spores (Chrysosporium queenslandicum), tropical golden spores, brown golden spores (Chrysosporium zonatum), gray-capped coprinus (Coprinus cinereus), hairy-skinned spores (Coriolushirsutus), rod-shaped spores (Fusarium), cereal spores (Fusarium), kuweispore (Fusarium kuweiss), large scabra spores (Fusarium graminearum), grass spores (Fusarium graminearum), heterosporium (Fusarium graminearum), albinofuga spores (Fusarium graminearum), acuminata spores (Fusarium graminearum), multibranched spores (Fusarium graminearum), pink spores (Fusarium graminearum), elderberry spores (Fusarium graminearum), skin-colored spores (Fusarium graminearum), sulfidosa spores (Fusarium graminearum), round spores (Fusarium graminearum), patchy spores (Fusarium graminearum), specific humic molds (Fusarium graminearum), cottony humic molds (Fusarium graminearum), rice black mold (Mucor graminearum), thermophilic pyriformis (Mucor graminearum), rough spores (Nephrolepis commune), purpuric mold (Penicillium purpureum), Phanerochaete chrysosporium (Phanerochaete chrysosporium), Phlebiaradiata (Pleurotus eryngii) eryngii), terrestrial clostridium, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma corningensis, Trichoderma longibranchii, Trichoderma reesei, or green Trichoderma cells.

[0215] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP 238023; Yelton et al., 1984, Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA) 81:1470-1474; and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using procedures described in the following literature: Becker and Guarente, in Abelson, JN and Simon, MI (eds.), Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1920.

[0216] Generation method

[0217] The present invention also relates to methods for generating variants, the methods comprising: (a) culturing a host cell of the present invention under conditions suitable for the expression of the variant; and (b) recovering the variant.

[0218] These host cells are cultured in a nutrient medium suitable for producing the variant using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions that allow for the expression and / or isolation of the variant in a laboratory or industrial fermenter. The culture occurs using procedures known in the art in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the variant is secreted into the nutrient medium, it can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0219] These variants can be detected using methods specific to them known in the art. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, an enzyme assay can be used to determine the activity of the variant (e.g., those described in the examples).

[0220] The variant can be recovered using methods known in the art. For example, the variant can be recovered from the nutrient medium through a variety of routine procedures, including but not limited to collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0221] Variants can be purified to obtain substantially pure variants by a variety of procedures known in the art, including but not limited to chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0222] Alternatively, instead of recycling the variant, the host cell of the present invention expressing the variant is used as the source of the variant.

[0223] Composition

[0224] Compositions comprising the polypeptides of the present invention are considered. In some aspects, the present invention relates to detergent compositions comprising a variant of a parental lipase, wherein said variant has at least 60% but less than 100% sequence identity with SEQ ID NO:2, has lipase activity, and the variant comprises substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2; and remains unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2. In one aspect, the parental lipase comprises or consists of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

[0225] In one aspect, the present invention relates to compositions comprising lipase variants, which further comprise one or more (e.g., several) substitutions in the cap region corresponding to positions 81-99 of SEQ ID NO:2, preferably R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P; and K98D / Q. In another aspect, the present invention relates to compositions comprising lipase variants, which further comprise one or more (e.g., several) substitutions at positions corresponding to positions 51, 136, 211, 252, and 255 of SEQ ID NO:2, preferably F51V, P136H, F211L, I252T, and I255T.

[0226] In one aspect, the present invention relates to compositions comprising lipase variants, which further comprise one or more (e.g., several) substitutions corresponding to any one of the following positions: 4, 27, 33, 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 compositions comprising lipase variants, which further comprise one or more (e.g., several) substitutions corresponding to any one of the following positions: 4V, 27R, 33Q, 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.

[0227] In some respects, the variant exhibits increased Ca-independence compared to the parental lipase.

[0228] The non-limiting list of compositional components set forth below is suitable for use in these compositions, and the methods herein may be voluntarily incorporated into certain aspects of the invention, for example, to aid or enhance cleaning performance, to treat a substrate to be cleaned, or to enhance the aesthetic appeal of the composition, as in the case of fragrances, colorants, dyes, or the like. The level of any such component incorporated into any composition is in addition to any materials previously referenced for incorporation. The precise properties of these additional components and their incorporation levels will depend on the physical form of the composition and the nature of the cleaning operation in which the composition will be used. Although the components mentioned below are classified under a general heading according to their specific functionality, this is not to be construed as limiting, as it will be understood by one of ordinary skill in the art that a component may include additional functionality.

[0229] Unless otherwise stated, amounts expressed as percentages are by weight (wt%) of the composition. Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibitors, dispersants, enzymes and enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymeric dispersants, clay removers / anti-redeposition agents, brighteners, foam inhibitors, dyes, tinting dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble additives, processing aids, solvents, and / or pigments. Suitable examples and levels of use of other such components, in addition to those disclosed below, are found in US 5576282, US 6306812, and US 6326348, which are incorporated herein by reference.

[0230] Therefore, in some embodiments, the present invention does not contain or exclude one or more of the following auxiliary materials: surfactants, soaps, detergent builders, chelating agents, dye transfer inhibitors, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, polymer dispersants, clay removal / anti-redeposition agents, brightening agents, foam inhibitors, dyes, fragrances, fragrance delivery systems, structural elastic agents, fabric softeners, carriers, water-soluble additives, processing aids, solvents, and / or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

[0231] surfactants- The compositions according to the invention may include a surfactant or surfactant system, wherein the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, facultative zwitterionic surfactants, semipolar nonionic surfactants, and mixtures thereof. When present, the surfactant is typically present at levels ranging from 0.1 wt% to 60 wt%, from 0.2 wt% to 40 wt%, from 0.5 wt% to 30 wt%, from 1 wt% to 50 wt%, from 1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1 wt% to 20 wt%, from 3 wt% to 10 wt%, from 3 wt% to 5 wt%, from 5 wt% to 40 wt%, from 5 wt% to 30 wt%, from 5 wt% to 15 wt%, from 3 wt% to 20 wt%, from 3 wt% to 10 wt%, from 8 wt% to 12 wt%, from 10 wt% to 12 wt%, from 20 wt% to 25 wt%, or from 25 wt% to 60 wt%.

[0232] Suitable anionic detergency surfactants include sulfate and sulfonate detergency surfactants.

[0233] Suitable sulfonate detergency surfactants include alkylbenzene sulfonates, which in one aspect are C 10-13 Alkylbenzene sulfonates. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonation of commercially available linear alkylbenzenes (LABs). Suitable LABs include low-carbon 2-phenyl LABs, such as... or Other suitable LABs include high-carbon 2-phenyl LABs, such as... Suitable anionic detergency surfactants are alkylbenzene sulfonates obtained via DETAL catalytic processes, but other synthetic routes (such as HF) may also be suitable. In one approach, magnesium salts of LAS are used.

[0234] Suitable sulfate detergency surfactants include alkyl sulfates, in one respect, C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfates.

[0235] Another suitable sulfate detergency surfactant is alkylalkoxylated sulfate, which is alkylethoxylated sulfate on one hand and C on the other. 8-18 Alkyl alkoxylated sulfates, on the one hand, are C 8-18 Alkyl ethoxylated sulfates, typically alkyl alkoxylated sulfates have an average degree of alkoxylation from 0.5 to 20 or from 0.5 to 10, and are typically C10-2 ... 8-18 Alkyl ethoxylated sulfates having an average degree of ethoxylation of 0.5 to 10, 0.5 to 7, 0.5 to 5, or 0.5 to 3.

[0236] Alkyl sulfates, alkylalkoxylated sulfates, and alkylbenzene sulfonates can be linear or branched, substituted or unsubstituted.

[0237] The detergency surfactant can be a medium-chain branched detergency surfactant, on one hand a medium-chain branched anionic detergency surfactant, on the other hand a medium-chain branched alkyl sulfate and / or medium-chain branched alkylbenzene sulfonate, such as a medium-chain branched alkyl sulfate. On one hand, the medium chain branch is C 1-4 Alkyl groups, typically methyl and / or ethyl.

[0238] Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkyl-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates, and disulfonates, alkyl sulfates (AS) (such as sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES, AEOS, or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0239] Suitable nonionic detergency surfactants are selected from the following group, which consists of the following items: C8-C 18 Alkyl ethoxylates, such as C6-C 12 Alkylphenol alkoxylates, wherein the alkoxyl unit may be an ethyleneoxy unit, an propyleneoxy unit, or a mixture thereof; C 12 -C 18 Alcohols and C6-C 12 Condensations of alkylphenols with ethylene oxide / propylene oxide block polymers, such as Pluronic. C 14 -C 22 Medium-chain branched alcohols; C 14 -C 22 Medium-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation from 1 to 30; alkyl polysaccharides, in one respect as alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

[0240] Suitable nonionic detergency surfactants include alkyl polysaccharides and / or alkyl alkoxylated alcohols.

[0241] On the one hand, nonionic detergency surfactants include alkylalkoxylated alcohols, and on the other hand, they are C 8-18 Alkyl alkoxylated alcohols, such as C 8-18 Alkyl ethoxylated alcohols, wherein the alkyl alkoxylated alcohols may have an average degree of alkoxylation ranging from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C 8-18 Alkyl ethoxylated alcohols have an average degree of ethoxylation ranging from 1 to 10, from 1 to 7, more commonly from 1 to 5, or from 3 to 7. Alkyl alkoxylated alcohols can be straight-chain or branched, and substituted or unsubstituted. Suitable nonionic surfactants include...

[0242] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA), or fatty acid glucosamide (FAGA)), together with products available under the trade names SPAN and TWEEN, and combinations thereof.

[0243] Suitable cationic detergency surfactants include alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds, and mixtures thereof.

[0244] Suitable cationic detergency surfactants are quaternary ammonium compounds having the following general formula: (R)(R1)(R2)(R3)N + X - Where R is a straight or branched, substituted or unsubstituted C 6-18 The alkyl or alkenyl moiety, R1 and R2 are independently selected from the methyl or ethyl moiety, R3 is a hydroxy, hydroxymethyl, or hydroxyethyl moiety, and X is an anion that provides a neutral charge; suitable anions include halides, such as chlorides; sulfates; and sulfonates. Suitable cationic detergency surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. A highly suitable cationic detergency surfactant is mono-C...8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C 10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

[0245] Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium compounds, and combinations thereof.

[0246] Suitable amphoteric / zombie surfactants include amine oxides and betaines (such as alkyl dimethyl betaine, sulfobetaine), or combinations thereof.

[0247] The amine-neutralized anionic surfactants of the present invention—anionic surfactants and associated anionic cosurfactants—can be present in acidic form, and said acidic form can be neutralized to form surfactant salts desired for use in the detergent compositions of the present invention. Typical agents for neutralization include metal counterionic bases, such as hydroxides, like NaOH or KOH. Other preferred agents for neutralizing the anionic surfactants of the present invention and associated anionic surfactants or cosurfactants in their acidic form include ammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other straight-chain or branched alkanolamines known in the art; for example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization can be carried out to a complete or partial extent; for example, a portion of the anionic surfactant mixture can be neutralized by sodium or potassium, and a portion of the anionic surfactant mixture can be neutralized by an amine or alkanolamine.

[0248] Non-limiting examples of semi-polar surfactants include amine oxides (AO), such as alkyl dimethylamine oxides.

[0249] A surfactant system comprising one or more anionic surfactants and one or more other nonionic surfactants, and optionally a mixture with other surfactants such as cationic surfactants, is preferred. A preferred weight ratio of anionic to nonionic surfactants is at least 2:1, or at least 1:1 to 1:10.

[0250] In one aspect, the surfactant system may include a mixture of isoprene-like surfactants represented by chemical formulas A and B:

[0251]

[0252] Where Y is CH2 or empty, and Z can be selected such that the resulting surfactant is selected from the following surfactants: alkyl carboxyl ester surfactants, alkyl polyalkoxy surfactants, alkyl anionic polyalkoxy sulfate surfactants, alkyl glycerol ester sulfonate surfactants, alkyl dimethylamine oxide surfactants, alkyl polyhydroxy-based surfactants, alkyl phosphate ester surfactants, alkyl glycerol sulfonate surfactants, alkyl polyglucose ester surfactants, alkyl polyphosphate ester surfactants, alkyl phosphonate surfactants, alkyl polyglycoside surfactants, alkyl monosaccharide surfactants, alkyl disaccharide surfactants, alkyl sulfosuccinate surfactants, alkyl disulfate surfactants, alkyl disulfonate surfactants, alkyl sulfonated succinate surfactants, alkyl glucosamide surfactants. Surfactants, alkyl taurine ester surfactants, alkyl sarcosine ester 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 glycerol ester surfactants, alkyl glycerol ester sulfate surfactants, alkyl glycerol ether surfactants, alkyl glycerol ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglycerol ether surfactants, alkyl polyglycerol ether sulfate surfactants, alkyl sorbitan ester surfactants, alkyl aminoalkane sulfonate surfactants, alkylamidopropyl betaine surfactants, and alkyl-based allylated quaternary salts (alkyl Surfactants including allylated quat, alkylated quat, monoalkyl quat, alkylated quat, trimethylammonium quat, oxypropyl quat, alkyl glycerol ester quat, and alkyl glycolamine.The surfactants include alkyl monomethyl dihydroxyethyl quaternary ammonium surfactants, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactants, alkyl trimethylammonium 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, alkylphenol ethoxylate surfactants, alkylbenzene 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 benzyl dimethylammonium surfactants, alkyl aminopropionate surfactants, alkyl amide propyl dimethylamine surfactants, or mixtures thereof; and if Z is a charged portion, Z is charge-balanced by a suitable metal or organic counterion. Suitable counterions include metal counterions, amines, or alkanolamines, such as C1-C6 alkanolammonium. More specifically, suitable counterions include Na+, Ca+, Li+, K+, Mg+, such as monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, 1-amino-3-propanol, or mixtures thereof. In one aspect, the composition comprises from 5% to 97% of one or more non-isoprene surfactants; and one or more auxiliary cleaning additives; wherein the weight ratio of the surfactant having formula A to the surfactant having formula B is from 50:50 to 95:5.

[0253] Soap – The compositions herein may contain soap. Without being theoretically limited, it is desirable to include soap because it partially acts as a surfactant and partially as a detergent builder, and can be used to suppress foaming; furthermore, it can advantageously interact with a variety of cationic compounds in the composition to enhance the softness of textile fabrics treated with the compositions of the present invention. Any soap known in the art for use in laundry detergents can be utilized. In one aspect, these compositions contain from 0 wt% to 20 wt%, from 0.5 wt% to 20 wt%, from 4 wt% to 10 wt%, or from 4 wt% to 7 wt% soap.

[0254] Examples of soaps useful here include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps with different chain lengths and degrees of substitution. One such mixture is top-coated palm kernel fatty acid.

[0255] On one hand, the soap is selected from free fatty acids. Suitable fatty acids are saturated and / or unsaturated and can be obtained from natural sources such as plant or animal esters (e.g., palm kernel oil, palm oil, coconut oil, babassu oil, safflower oil, tallow, castor oil, tallow and fish oil, fats and oils, and mixtures thereof), or synthesized (e.g., by oxidation of petroleum or by hydrogenation of carbon monoxide via the Fisher Tropsch process).

[0256] Examples of suitable saturated fatty acids for use in the compositions of the present invention include decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and benzyl acid. Suitable unsaturated fatty acids include palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and ricinoleic acid. Preferred examples of fatty acids are saturated Cn fatty acids, saturated Ci2-Ci4 fatty acids, and saturated or unsaturated Cn to Ci8 fatty acids, and mixtures thereof.

[0257] When present, the weight ratio of the fabric-softening cationic auxiliary surfactant to the fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, and most preferably about 1:1.

[0258] The levels of soap and non-soap anionic surfactants mentioned herein are percentages by weight of the detergent composition specified on an acidic basis. However, as is generally understood in the art, in practice, sodium, potassium, or alkanol ammonium bases such as sodium hydroxide or monoethanolamine are used to neutralize the anionic surfactants and soap.

[0259] Water-soluble- The compositions of the present invention may include one or more co-hydrophiles. A co-hydrophile is a compound that dissolves a hydrophobic compound (or conversely, a polar substance in a nonpolar environment) in an aqueous solution. Typically, co-hydrophiles possess both hydrophilic and hydrophobic characteristics (such as the so-called amphiphilic properties known from surfactants); however, the molecular structure of co-hydrophiles generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12:121-128. Co-hydrophiles do not exhibit a critical concentration above which self-aggregation, as observed with surfactants, and lipid formation of micelles, thin layers, or other well-defined intermediate phases occur. Instead, many co-hydrophiles exhibit a continuous type of aggregation process, where the size of the aggregates increases with increasing concentration. However, many water-soluble solvents alter the phase behavior, stability, and colloidal properties of systems containing substances with both polar and nonpolar characteristics (including mixtures of water, oils, surfactants, and polymers). Water-soluble solvents are classically used across industries, from pharmaceuticals and personal care to food and technical applications. The use of water-soluble solvents in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0260] Detergents may contain 0 to 10 wt%, such as 0 to 5 wt%, 0.5 wt% to 5 wt%, or 3 wt% to 5 wt% of a water-soluble solvent. Any water-soluble solvent known in the art for use in detergents may be used. Non-limiting examples of water-soluble solvents include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthenate, sodium hydroxynaphthenate, sodium ethylhexylsulfonate, and combinations thereof.

[0261] builder - The compositions of the present invention may include one or more detergent builders, co-builders, detergent builder systems, or mixtures thereof. When a detergent builder is used, the cleaning composition will typically include 0 to 65 wt%, at least 1 wt%, 2 wt% to 60 wt%, or 5 wt% to 10 wt% of the detergent builder. In dishwashing cleaning compositions, the level of detergent builder is typically 40 wt% to 65 wt% or 50 wt% to 65 wt%. The composition may be substantially free of detergent builders; substantially free of zeolite and / or phosphates, meaning "no intentionally added". Typical zeolite detergent builders include zeolite A, zeolite P, and zeolite MAP. Typical phosphate detergent builders are sodium tripolyphosphate.

[0262] Builders and / or co-builders may specifically be chelating agents that form water-soluble complexes having Ca and Mg. Any builders and / or co-builders known in the art for use in detergents may be used. Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hearst Corporation), ethanolamines such as 2-aminoethanol (MEA), iminodiethanol (DEA), and 2,2',2'-triazine (TEA), and carboxymethyl inulin (CMI), and combinations thereof.

[0263] Cleaning compositions may include a co-agent alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of co-agents include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-dimethylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), and N-(2-sulfoethyl)aspartic acid (SEAS). N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), p-aminobenzenesulfonic acid-N,N-diacetic acid (SLDA), aminoethanesulfonic acid-N,N-diacetic acid (TUDA), and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolamine glycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations thereof and salts thereof. Further exemplary builders and / or co-builders are described in, for example, WO 09 / 102854, US 5977053.

[0264] In one aspect, the present invention relates to compositions comprising a variant of a parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with SEQ ID NO:2, has lipase activity, and the variant comprises substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2; and remains unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2. The composition comprises up to 10 wt% or 15 wt% aluminosilicate (anhydrous base) and / or phosphate builders, and the composition has a reserve alkalinity greater than 4 or 7.5. In another aspect, the parental lipase comprises or consists of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. On the one hand, lipase variants can be selected from the following: S83T E87K G91L N92D F95Y D96T L97P K98Q; R81Q S83T R84H S85T I86L E87TN88Q I90M G91A N92K L93F N94K F95A D96L K98Q; S83T E87A G91A N94D D96I L97PK98D; I86P E87A G91A N92D F95Y D96T L97P K98Q I252T I255T; I86W E87A G91A N92D F95Y D96T L97P K98Q I252TI255T; I86P E87A G91A N92D L93F F95Y D96T L97P K98Q I252T I255T; I86P E87T G91LN94D D96I L97F K98D; I90L F95L F211L I252T; S83T G91A N92D E96T; S83T G91A N92DF95Y E96L L97P K98Q; S83T I86P G91A N92D D96T K98Q; F51V I86P E87A G91A N92DF95Y D96T L97P K98Q T252I T255I; S83T E87T G91L N92D D96I L97F K98D; E87T G91AN92D N94D D96L K98Q; E87K G91A N92D N94D D96L K98Q;S83T I86P G91A N92D D96L L97P K98Q; S83T I86P G91A N92D F95Y D96L; S83T I86P G91L N92D D96L; S83T F95Y D96L; N92D D96L K98Q; S83T I86P G91L N92D D96L L97P; S83T I86P G91A N92D D96L K98Q; S83T I86W G91L N92D F95Y; S83T I86P G91L N92DF95Y D96T; S83T G91L N92D K98Q P136H; and I86P G91L N92K L97P. As used herein, the term "reserve alkalinity" is a measure of the buffering capacity of the composition (g / NaOH / 100g composition) determined by titrating a 1% (w / v) solution of the composition to pH 7.5 with hydrochloric acid, i.e., for calculating the reserve alkalinity. The reserve alkalinity can be calculated as disclosed on page 9 of WO 2006 / 090335. In another aspect, the present invention relates to compositions comprising lipase variants that further comprise one or more substitutions in the cap region corresponding to positions 81-99 of SEQ ID NO:2, preferably R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90L / M; G91A / L; L93F; N94D / K; F95A / L / Y; L97F / P;And K98D / Q. In one aspect, the present invention relates to compositions comprising a lipase variant, the variant further comprising one or more substitutions at positions corresponding to positions 51, 136, 211, 252 and 255 of SEQ ID NO:2, preferably F51V, P136H, F211L, I252T and I255T. In another aspect, the present invention relates to compositions comprising a lipase variant, the variant further comprising one or more (e.g., several) substitutions corresponding to any one of the following positions: 4, 27, 33, 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 compositions comprising lipase variants, the variants further comprising one or more (e.g., several) substitutions corresponding to any one of the following positions: 4V, 27R, 33Q, 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.

[0265] Chelating agents and crystal growth inhibitors - The composition herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, ionic, and / or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), disodium hydroxide of 1,2-dihydroxybenzene-3,5-disulfonic acid, ethylenediamine, diethylenetriamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin, and 2-phosphinocarboxybutane-1,2,4-tricarboxylic acid ( AM) and its derivatives. Typically, the composition may include chelating agents or crystal growth inhibitors from 0.005 wt% to 15 wt% or from 3.0 wt% to 10 wt%.

[0266] bleaching componentsSuitable bleaching components for incorporation into the methods and compositions of the present invention include one or a mixture of more than one bleaching component. Suitable bleaching components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide, hydrogen peroxide sources, pre-formed peracids, and mixtures thereof. Typically, when using bleaching components, the compositions of the present invention may include from 0 to 30 wt%, from 0.00001 wt% to 90 wt%, from 0.0001 wt% to 50 wt%, from 0.001 wt% to 25 wt%, or from 1 wt% to 20 wt%. Examples of suitable bleaching components include:

[0267] (1) Preformed peracid: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of preformed peroxyacids or their salts, typically peroxycarboxylic acids or their salts, or persulfate or their salts.

[0268] The pre-formed peroxyacid or its salt is preferably a peroxycarboxylic acid or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0269]

[0270] Where: R 14 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 14 The functional group can be straight-chain or branched, substituted or unsubstituted; and Y is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 14 Is it a linear or branched, substituted or unsubstituted C? 6-9 Alkyl group. Preferably, the peroxy acid or its salt is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and their salts, or any combination thereof. Particularly preferred peroxy acids are phthalimide-peroxy-alkanoic acids, especially ε-phthalimide-peroxyhexanoic acid (PAP). Preferably, the peroxy acid or its salt has a melting point in the range of 30°C to 60°C.

[0271] The pre-formed peroxyacid or its salt can also be persulfate or its salt, typically having a chemical structure corresponding to the following chemical formula:

[0272]

[0273] Where: R 15 Selected from alkyl, aralkyl, cycloalkyl, aryl, or heterocyclic groups; R 15 The group can be straight-chain or branched, substituted or unsubstituted; and Z is any suitable counterion that achieves charge neutrality, preferably selected from hydrogen, sodium, or potassium. Preferably, R... 15 Is it a linear or branched, substituted or unsubstituted C? 6-9Alkyl groups. Preferably, such bleaching components may be present in the compositions of the present invention in an amount from 0.01 wt% to 50 wt% or from 0.1 wt% to 20 wt%.

[0274] (2) Hydrogen peroxide sources include, for example, inorganic hydrogen peroxide salts, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, sodium salts of persilicates, and mixtures thereof. In one aspect of the invention, inorganic hydrogen peroxide salts are, for example, those selected from the group consisting of perborates, sodium salts of percarbonates, and mixtures thereof. When used, inorganic hydrogen peroxide salts are typically present in amounts from 0.05 wt% to 40 wt% or 1 wt% to 30 wt% of the total composition and are typically incorporated into such compositions as crystalline solids that can be coated. Suitable coatings include: inorganic salts, such as alkali metal silicates, carbonates, or borates, or mixtures thereof, or organic materials, such as water-soluble or water-dispersible polymers, waxes, oils, or fatty soaps. Preferably, such bleaching components may be present in the compositions of the invention in amounts from 0.01 wt% to 50 wt% or 0.1 wt% to 20 wt%.

[0275] (3) The term bleaching activator herein refers to a compound that reacts with hydrogen peroxide to form a peracid via a perhydrolysis reaction. The peracid formed in this manner constitutes an activated bleaching agent. Suitable bleaching activators to be used herein include those belonging to the classes of esters, amides, imides, or anhydrides. Suitable bleaching activators are those having R-(C=O)-L, wherein R is an alkyl group (preferably branched), having from 6 to 14 carbon atoms or from 8 to 12 carbon atoms when the bleaching activator is hydrophobic, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleaching activator is hydrophilic; and is an L leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives—especially benzenesulfonates. Suitable bleaching activators include dodecyloxybenzenesulfonate, decyloxybenzenesulfonate, decyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecyloxy)benzene-1-sulfonate (LOBS), 4-(decyloxy)benzene-1-sulfonate, 4-(decyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A family of bleaching activators disclosed in EP 624154, and particularly preferred in that family, is triethyl acetyl citrate (ATC). ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly. Furthermore, triethyl acetylacetic acid (ATC) and triacetin exhibit good hydrolytic stability in the product during storage and are effective bleaching activators. Finally, ATC is versatile because the citrate released during the hydrolysis reaction can act as a detergent builder. Alternatively, the bleaching system may include, for example, peroxy acids of the form of amides, imides, or sulfones. The bleaching system may also include peracids, such as 6-(phthalimide)percaptoic acid (PAP). Suitable bleaching activators are also disclosed in WO 98 / 17767. Although any suitable bleaching activator may be used, in one aspect of the invention, the subject cleaning composition may include NOBS, TAED, or mixtures thereof. When present, peracids and / or bleaching activators are typically present in the composition in amounts of 0.1 wt% to 60 wt%, 0.5 wt% to 40 wt%, or 0.6 wt% to 10 wt%, depending on the fabric and home care composition. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors. Preferably, such bleaching components may be present in the compositions of the present invention in an amount of 0.01 wt% to 50 wt% or 0.1 wt% to 20 wt%.

[0276] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0277] (4) Diacyl peroxides – Preferred diacyl peroxide bleaching agents include those selected from diacyl peroxides having the following general formula: R 1 -C(O)-OO-(O)CR 2 , where R 1 Represents a C6-C 18 Alkyl groups, preferably comprising a straight chain having at least 5 carbon atoms and optionally containing one or more substituents (e.g., -N). + (CH3)3, -COOH or -CN) and / or one or more interruptions inserted between adjacent carbon atoms of an alkyl group (e.g., -CONH- or -CH=CH-) at C6-C 12 Alkyl groups, and R 2 This represents an aliphatic group compatible with the peroxide moiety, such that R 1 and R 2 Together they contain a total of 8 to 30 carbon atoms. In a preferred aspect, R 1 and R 2 It is a straight-chain, unsubstituted C6-C 12 Alkyl chain. Most preferably, R 1 and R 2 They are the same. Diacyl peroxide (where R) 1 and R 2 All are C6-C 12 Alkyl groups are particularly preferred. Preferably, at least one of the R groups (R1 or R2) does not contain a branched or drooping ring at the α-position, or preferably does not contain a branched or drooping ring at either the α- or β-position, or most preferably does not contain a branched or drooping ring at either the α-, β-, or γ-position. In a further preferred embodiment, the DAP may be asymmetric, such that the R1 acyl group preferably hydrolyzes rapidly to produce a superacid, but the R2 acyl group hydrolyzes slowly.

[0278] Tetraacyl peroxide bleaching agents are preferably selected from tetraacyl peroxides having the following general formula: R 3 -C(O)-OO-C(O)-(CH2)nC(O)-OO-C(O)-R 3 , where R 3 Indicates C1-C9 alkyl, or C 3- The C7 group, and n represents an integer from 2 to 12 or from 4 to 10 (inclusive of the end value).

[0279] Preferably, the diacyl and / or tetraacyl peroxide bleaching agents are present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm of detergent solution by weight. In a preferred embodiment, these bleaching agents are present in an amount sufficient to provide detergent solution from 0.5 ppm to 300 ppm, or from 30 ppm to 150 ppm by weight.

[0280] Preferably, the bleaching component includes bleaching catalysts (5 and 6).

[0281] (5) Preferably, organic (non-metallic) bleaching catalysts are included, comprising bleaching catalysts capable of accepting oxygen atoms from peroxy acids and / or their salts and transferring those oxygen atoms to an oxidizable substrate. Suitable bleaching catalysts include, but are not limited to: imine-onium cations and polyions; imine-onium zwitterions; modified amines; modified amine oxides; N-sulfonylimides; N-phosphonylimides; N-acylimides; thiadiazole dioxides; perfluoroimides; cyclic glycoketones and mixtures thereof.

[0282] Suitable imine-onium cations and polyions include, but are not limited to, N-methyl-3,4-dihydroisoquinolineonium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (e.g., compound 4, page 433); N-methyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5360569 (e.g., column 11, example 1); and n-octyl-3,4-dihydroisoquinolineonium p-toluenesulfonate, prepared as described in US 5360568 (e.g., column 10, example 3).

[0283] Suitable imine-onium zwitterions include, but are not limited to, N-(3-sulfopropyl)-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5576282 (e.g., column 31, Example II); N-[2-(sulfonoxy)dodecyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in US 5817614 (e.g., column 32, Example V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt, prepared as described in WO 05 / 047264 (e.g., page 18, Example 8); and 2-[3-[(2-butyloctyl)oxy]-2-(sulfonoxy)propyl]-3,4-dihydroisoquinolineonium, an inner salt.

[0284] Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinoline alcohol, which can be prepared according to the method described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxygen transfer catalysts include, but are not limited to, sodium 1-hydroxy-N-oxy-N-[2-(sulfonoxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0285] Suitable N-sulfonylimide oxygen transfer catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide prepared according to the procedure described in the Journal of Organic Chemistry (1990), 55(4), 1254-61.

[0286] Suitable N-phosphonylimide oxygen transfer catalysts include, but are not limited to, [R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-p-phenyl-p-(2,4,6-trimethylphenyl)phosphonamide, which can be prepared according to the method described in the Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70.

[0287] Suitable N-acylimide oxygen transfer catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, which can be manufactured according to the procedure described in the Polish Journal of Chemistry (2003), 77(5), 577-590.

[0288] Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, which may be manufactured according to the procedure described in US 5753599 (Column 9, Example 2).

[0289] Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butyrylimide fluoride, which can be prepared according to the method described in Tetrahedron Letters (1994), 35(34), 6329-30.

[0290] Suitable cyclic glycoketone oxygen transfer catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexanedione-2,6-pyranose, as prepared in US 6649085 (column 12, Example 1).

[0291] Preferably, the bleaching catalyst comprises imine-onium and / or carbonyl functional groups, and is generally capable of forming oxaziridinium and / or diethylene oxide functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises oxaziridinium functional groups and / or is capable of forming oxaziridinium functional groups upon accepting oxygen atoms, particularly from peroxy acids and / or their salts. Preferably, the bleaching catalyst comprises cyclic imine-onium functional groups, preferably wherein the cyclic portion has a ring size of five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleaching catalyst comprises arylimine-onium functional groups, preferably bicyclic arylimine functional groups, and more preferably 3,4-dihydroisoquinoline-onium functional groups. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary peroxyimine cation functional group when accepting an oxygen atom, particularly when accepting an oxygen atom from a peroxy acid and / or its salt. In one aspect, the detergent composition comprises a logP having a value not greater than 0, not greater than -0.5, not greater than -1.0, not greater than -1.5, not greater than -2.0, not greater than -2.5, not greater than -3.0, or not greater than -3.5. o / w The bleaching component. The following describes in more detail the components used to determine logP. o / w The method.

[0292] Typically, bleaching agents can produce X values ​​ranging from 0.01 to 0.30, from 0.05 to 0.25, or from 0.10 to 0.20. SO The types of bleaching are described in more detail below for determining X. SO The method. For example, bleaching agents with an isoquinoline onion structure can produce bleaching species with a peroxyimine cation structure. In this example, X SO It is X, a type of peroxide imine cation bleaching. SO .

[0293] Preferably, the bleaching catalyst has a chemical structure corresponding to the following chemical formula:

[0294]

[0295] Where: n and m independently range from 0 to 4, preferably both n and m are 0; each R 1Independently selected, either substituted or unsubstituted, groups selected from the group consisting of: hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocyclic, fused heterocyclic, nitro, halogen, cyano, sulfonate, alkoxy, ketone, carboxyl, and alkoxycarbonyl; and any two R groups in the connected positions 1 Substituents can merge to form fused aryl, fused carbocyclic, or fused heterocyclic groups; each R 2 Independently selected, either substituted or unsubstituted, groups independently selected from the group consisting of: hydrogen, hydroxyl, alkyl, cycloalkyl, alkylaryl, aryl, aralkyl, alkylene, heterocyclic, alkoxy, arylcarbonyl, carboxyalkyl, and amide groups; any R 2 It can be used with any other R 2 They are combined to form part of a common ring; any harmonic R 2 They can merge to form a carbonyl group; and any two R groups can be combined to form a carbonyl group. 2 They can merge to form a substituted or unsubstituted fused unsaturated portion; R 3 It is C1 to C 20 Substituted or unsubstituted alkyl groups; R 4 Is it hydrogen or Q? t -A portion, where: Q is a branched or unbranched alkene, t = 0 or 1, and A is an anionic group selected from the group consisting of the following: OSO3 - SO3 - CO2 - OCO2 - OPO3 2- OPO3H - and OPO2 - ;R 5 Is it hydrogen or -CR? 11 R 12 -YG b -Y c -[(CR 9 R 10 ) y -O] k -R 8 Part, wherein: each Y is independently selected from the following group, which consists of: O, S, NH, or NR. 8 And each R 8 Independently selected from the group consisting of alkyl, aryl, and heteroaryl groups, wherein the moiety is substituted or unsubstituted, and wherein the substituted or unsubstituted moiety has less than 21 carbons; each G is independently selected from the group consisting of CO, SO2, SO, PO, and PO2; R 9 and R 10 Independently selected from the group consisting of: H and C1-C4 alkyl groups; R11 and R 12 Independently selected from the group consisting of: H and alkyl groups, or those that can combine to form a carbonyl group when placed together; b = 0 or 1; c can = 0 or 1, but if b = 0, c must = 0; y is an integer from 1 to 6; k is an integer from 0 to 20; R 6 It is an H, or alkyl, aryl, or heteroaryl moiety; said moiety is substituted or unsubstituted; and X, if present, is a suitable charge-balancing counterion, preferably when R 4 When it is hydrogen, X exists. Suitable X includes, but is not limited to: chlorides, bromides, sulfates, methoxysulfates, sulfonates, p-toluenesulfonates, borotetrafluorides, and phosphates.

[0296] In one aspect of the invention, the bleaching catalyst has a structure corresponding to the following general formula:

[0297]

[0298] Where R 13 It is a branched alkyl group (including branched carbon atoms) containing three to 24 carbon atoms or a straight-chain alkyl group containing one to 24 carbon atoms; preferably, R 13 It is a branched alkyl group containing eight to 18 carbon atoms or a straight-chain alkyl group containing eight to eighteen carbon atoms; preferably, R 13 Selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isothidecyl, and isopentetranyl; preferably, R 13 Selected from the group consisting of the following: 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, isotridecyl and isopentadecanyl.

[0299] Preferably, in addition to the bleaching catalyst, particularly an organic bleaching catalyst, the bleaching component also includes a peracid source. The peracid source may be selected from (a) pre-formed peracid; (b) percarbonate, perborate, or percarbonate (hydrogen peroxide source), preferably in combination with a bleaching activator; and (c) perhydrolase and ester, used to form the peracid in situ in the presence of water during textile or hard surface treatment steps.

[0300] When present, based on the composition, the peracid and / or bleaching activator are typically present in the composition in amounts from 0.1 wt% to 60 wt%, from 0.5 wt% to 40 wt%, or from 0.6 wt% to 10 wt%. One or more hydrophobic peracids or their precursors may be used in combination with one or more hydrophilic peracids or their precursors.

[0301] The amounts of hydrogen peroxide source and peracid or bleaching activator can be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

[0302] (6) Metal-containing bleaching catalysts – the bleaching component can be provided by a catalytic metal complex. One type of metal-containing bleaching catalyst is a catalytic system comprising a transition metal cation (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation) having defined bleaching catalytic activity, an auxiliary metal cation (e.g., zinc or aluminum cation) having little or no bleaching catalytic activity, and an insulator having defined stability constants for both the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and their water-soluble salts. Such catalysts are disclosed in US 4430243. Preferred catalysts are described in WO 09 / 839406, US 6218351, and WO 00 / 012667. Transition metal catalysts or ligands thus serving as cross-bridged multidentate N-donor ligands are particularly preferred.

[0303] If desired, the composition herein can be catalyzed using manganese compounds. Such compounds and the levels of use are well known in the art and include, for example, manganese-based catalysts disclosed in US 5576282.

[0304] Useful cobalt bleaching catalysts are known and described, for example, in US 5597936 and US 5595967. Such cobalt catalysts can be readily prepared by known procedures, such as those taught in US 5597936 and US 5595967.

[0305] The compositions herein may also suitably include transition metal complexes of ligands, such as bispidone (US 7501389) and / or multicyclic rigid ligands – abbreviated as “MRL”. As a practical problem and not for limitation, the compositions and methods herein may be adapted to provide approximately at least one part per hundred million of active MRL species in an aqueous washing medium, and will typically provide MRL levels from 0.005 ppm to 25 ppm, from 0.05 ppm to 10 ppm, or from 0.1 ppm to 5 ppm in the washing solution.

[0306] Suitable transition metals for readily available transition metal bleaching catalysts include, for example, manganese, iron, and chromium. Suitable MRLs include 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitable transition metal MRLs can be readily prepared by known procedures, such as those described in US 6225464 and WO 00 / 32601.

[0307] (7) Photobleaching agents – Suitable photobleaching agents include, for example, sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, zeatin dyes, and mixtures thereof. Preferred bleaching components used in these compositions of the invention include a hydrogen peroxide source, a bleaching activator, and / or an organic peroxyacid, optionally generated in situ by a reaction of the hydrogen peroxide source and the bleaching activator in combination with a bleaching catalyst. Preferred bleaching components include a bleaching catalyst, preferably an organic bleaching catalyst as described above.

[0308] The preferred bleaching component is a bleaching catalyst, especially an organic bleaching catalyst.

[0309] Exemplary bleaching systems are also described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242.

[0310] Fabric colorant - The composition may include a fabric colorant. Suitable fabric colorants include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes belonging to the following colorimetric index (CI) classifications: direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet, and basic red, or mixtures thereof.

[0311] On the one hand, suitable small molecule dyes include those selected from the group consisting of the following: colorimetric index (Society of Dyers and Colorists, Bradford, UK) number: Direct Violet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, Acid Violet 50 Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75, Basic Blue 159 and mixtures thereof. In one respect, suitable small molecule dyes include those selected from the group consisting of: colorimetric index (Institute of Dyers and Colorists, Bradford, UK) numbers Acid Violet 17, Acid Violet 43, Acid Red 52, Acid Red 73, Acid Red 88, Acid Red 150, Acid Blue 25, Acid Blue 29, Acid Blue 45, Acid Blue 113, Acid Black 1, Direct Blue 1, Direct Blue 71, Direct Violet 51, and mixtures thereof. In another respect, suitable small molecule dyes include those selected from the group consisting of: colorimetric index (Institute of Dyers and Colorists, Bradford, UK) numbers Acid Violet 17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113, and mixtures thereof.

[0312] Suitable polymer dyes include polymer dyes selected from the group consisting of polymers containing conjugated chromogens (dye-polymer conjugates) and polymers copolymerized with chromogens into the polymer backbone, and mixtures thereof.

[0313] On the one hand, suitable polymer dyes include polymer dyes selected from the group consisting of the following: Substantial fabric colorants under the name Milliken are dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of: polymers comprising portions of the group consisting of hydroxyl moieties, primary amine moieties, secondary amine moieties, thiols, and mixtures thereof. Still in one aspect, suitable polymer dyes include polymer dyes selected from the group consisting of: Purple CT, carboxymethyl cellulose (CMC) conjugated with reactive blue, reactive purple or reactive red dyes, such as CMC conjugated with CI reactive blue 19 (sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC), alkoxylated triphenylmethane polymer colorants, alkoxylated thiophene polymer colorants, and mixtures thereof.

[0314] Preferred tinting dyes include whitening agents found in WO 08 / 87497. These whitening agents can be characterized by the following structure (I):

[0315]

[0316] R1 and R2 can be independently selected from:

[0317] a)[(CH2CR'HO) x (CH2CR"HO) y H]

[0318] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

[0319] b) R1 = alkyl, aryl, or arylalkyl, and R2 = [(CH2CR'HO) x (CH2CR"HO) y H]

[0320] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

[0321] c) R1 = [CH2CH2(OR3)CH2OR4] and R2 = [CH2CH2(OR3)CH2OR4]

[0322] R3 is selected from the following group, which consists of the following items: H, (CH2CH2O). z H, and mixtures thereof; and wherein z = 0 to 10;

[0323] R4 is selected from the following group, which consists of the following items: (C1-C 16 )alkyl, aryl, and mixtures thereof; and

[0324] d) Wherein R1 and R2 may be independently selected from styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, tert-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and glycidyl hexadecyl ether, followed by an addition of 1 to 10 epoxide units.

[0325] The preferred whitening agent of the present invention can be characterized by the following structure (II):

[0326]

[0327] R' is selected from the following group, which consists of the following items: H, CH3, CH2O (CH2CH2O). z H and its mixtures; wherein R” is selected from the group consisting of the following items: H, CH2O (CH2CH2O). z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

[0328] Another preferred whitening agent of the present invention can be characterized by the following structure (III):

[0329]

[0330] Typically, this includes mixtures having a total of 5 EO groups. Suitable preferred molecules are those in structure I that have the side groups described in “part a” above.

[0331] Table 1

[0332] 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

[0333] Other whitening agents used include those described in US 2008 / 34511 (Unilever). The preferred agent is "Violet 13".

[0334] Suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of at least one cationic / basic dye and green clay, and mixtures thereof. In one aspect, suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of a cationic / basic dye and a clay, wherein the cationic / basic dye is selected from the group consisting of: CI Basic Yellow 1 to 108, CI Basic Orange 1 to 69, CI Basic Red 1 to 118, CI Basic Violet 1 to 51, CI Basic Blue 1 to 164, CI Basic Green 1 to 14, CI Basic Brown 1 to 23, CI Basic Black 1 to 11, and the clay is selected from the group consisting of: montmorillonite clay, hydrophoretic clay, soapstone clay, and mixtures thereof. On the other hand, suitable dye clay conjugates include those selected from the group consisting of: montmorillonite basic blue B7CI42595 conjugate, montmorillonite basic blue B9CI52015 conjugate, montmorillonite basic violet V3CI42555 conjugate, montmorillonite basic green G1CI42040 conjugate, montmorillonite basic red R1CI45160 conjugate, montmorillonite CI basic black 2 conjugate, lithium montmorillonite basic blue B7CI42595 conjugate, and lithium montmorillonite basic blue B9CI52015 conjugate. Compounds, including lithium montmorillonite basic violet V3CI42555 chalcogenide, lithium montmorillonite basic green G1CI42040 chalcogenide, lithium montmorillonite basic red R1CI45160 chalcogenide, lithium montmorillonite CI basic black 2 chalcogenide, soapstone basic blue B7CI42595 chalcogenide, soapstone basic blue B9CI52015 chalcogenide, soapstone basic violet V3CI42555 chalcogenide, soapstone basic green G1CI42040 chalcogenide, soapstone basic red R1CI45160 chalcogenide, soapstone CI basic black 2 chalcogenide, and mixtures thereof.

[0335] Suitable pigments include pigments selected from the group consisting of: flavonoids, indanone, chlorinated indanone containing 1 to 4 chlorine atoms, pinanthrone, dichloropinanthrone, monobromodichloropinanthrone, dibromodichloropinanthrone, tetrabromopinanthrone, dinaphthyl-3,4,9,10-tetracarboxylic acid diimide (wherein the imide group may be unsubstituted or substituted with C1-C3-alkyl or phenyl or heterocyclic groups, and wherein the phenyl and heterocyclic groups may additionally have substituents that do not impart solubility in water), anthraquinone pyrimidine carboxylic acid amide, anthrone violet, isoanthrone violet, dioxazine pigments, copper phthalocyanine per molecule that may contain up to 2 chlorine atoms, polychlorinated copper phthalocyanine or polybrominated chloro-copper phthalocyanine per molecule containing up to 14 bromine atoms, and mixtures thereof.

[0336] On the one hand, suitable pigments include those selected from the group consisting of ultramarine (CI Pigment Blue 29), ultramarine violet (CI Pigment Violet 15), and mixtures thereof.

[0337] The above-mentioned fabric toners can be used in combination (any mixture of fabric toners can be used). Suitable toners are described in more detail in US 7208459. The preferred level of dye in the compositions of the present invention is 0.00001 wt% to 0.5 wt%, or 0.0001 wt% to 0.25 wt%. The concentration of dye used in water for treatment and / or cleaning steps is preferably from 1 ppb to 5 ppm, 10 ppb to 5 ppm, or 20 ppb to 5 ppm. In preferred compositions, the concentration of surfactant will be from 0.2 to 3 g / L.

[0338] Encapsulation - The composition may include an encapsulated compound. In one aspect, the encapsulated compound includes a core and a shell having an inner surface and an outer surface, the shell encapsulating the core.

[0339] In one aspect of the encapsulated compound, the core may comprise a material selected from the group consisting of: fragrances; brighteners; dyes; insect repellents; silicones; waxes; flavorings; vitamins; fabric softeners; skin care agents, in one aspect, paraffin wax; enzymes; antibacterial agents; bleaching agents; sensates; and mixtures thereof; and the shell may comprise a material selected from the group consisting of: polyethylene; polyamides; polyvinyl alcohol, optionally including other comonomers; polystyrene; polyisoprene; polycarbonate; polyester; polyacrylate; amino plastics, in one aspect, the amino plastics may include polyurea, polyurethane and / or polyurea polyurethane, in one aspect, the polyurea may include polyoxymethylene urea and / or melamine formaldehyde; polyolefins; polysaccharides, in one aspect, the polysaccharides may include alginate and / or chitosan; gelatin; shellac; epoxy resins; water-insoluble inorganic vinyl polymers; silicones; and mixtures thereof.

[0340] In one aspect of the encapsulated compound, the core may include a fragrance.

[0341] In one aspect of the encapsulation, the shell may comprise melamine formaldehyde and / or cross-linked melamine formaldehyde.

[0342] In one aspect, it is disclosed that suitable encapsulations 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 encapsulation may have a tensile strength from 0.2 MPa to 10 MPa, from 0.4 MPa to 5 MPa, from 0.6 MPa to 3.5 MPa, or from 0.7 MPa to 3 MPa; and have a beneficial reagent leakage of 0% to 30%, 0% to 20%, or 0% to 5%.

[0343] In one respect, at least 75%, 85%, or 90% of the encapsulated material may have a particle size of 1 to 80 micrometers, 5 to 60 micrometers, 10 to 50 micrometers, or 15 to 40 micrometers.

[0344] In one respect, at least 75%, 85%, or 90% of the encapsulated material may have a particle wall thickness of 30 to 250 nm, 80 to 180 nm, or 100 to 160 nm.

[0345] In one aspect, the core material of the encapsulated compound may include a material selected from the group consisting of: a flavoring ingredient and / or optionally a material selected from the group consisting of: vegetable oils, including undiluted and / or blended vegetable oils (including castor oil, coconut oil, cottonseed oil, grape oil, rapeseed oil, soybean oil, corn oil, palm oil, flaxseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil, and mixtures thereof); vegetable oil esters Esters, including dibutyl adipate, dibutyl phthalate, benzyl butyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl phosphate, and mixtures thereof; straight-chain or branched hydrocarbons, including those with boiling points above about 80°C; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls, including monoisopropyl biphenyls, alkyl naphthalenes (including dipropyl naphthalene), gasoline (including kerosene), mineral oils, and mixtures thereof; aromatic solvents, including benzene, toluene, and mixtures thereof; silicone oils; and mixtures thereof.

[0346] In one aspect, the wall material of the encapsulation may include a suitable resin comprising a reaction product of an aldehyde and an amine, with suitable aldehydes including formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycourea, and mixtures thereof. Suitable melamines include hydroxymethyl melamine, methylated hydroxymethyl melamine, iminomelamine, and mixtures thereof. Suitable ureas include dihydroxymethyl urea, methylated dihydroxymethyl urea, urea-resorcinol, and mixtures thereof.

[0347] On one hand, before, during, or after the addition of the encapsulation compound to the composition, a suitable formaldehyde scavenger may be used with and / or added to such composition, for example, in a capsule slurry. Suitable capsules may be made according to the following teachings of US 2008 / 0305982; and / or US 2009 / 0247449.

[0348] In a preferred aspect, the composition may further comprise a deposition aid, preferably composed of a group consisting of cationic or nonionic polymers. Suitable polymers include cationic starch, cationic hydroxyethyl cellulose, polyethylene formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene terephthalate, and polymers comprising dimethylaminoethyl methacrylate and optionally having one or more monomers selected from the group consisting of acrylic acid and acrylamide.

[0349] spices- In one aspect, the composition comprises a fragrance containing one or more fragrance ingredients selected from the group consisting of: 1,1'-oxybis-2-propanol; 1,4-cyclohexanedicarboxylic acid, diethyl ester; (ethoxymethoxy)cyclododecane; 1,3-nonanediol, monoacetic acid ester; (3-methylbutoxy)acetic acid, 2-propenyl ester; β-methylcyclododecaneethanol; 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol; oxetane-2-one; α-methyl-benzyl alcohol acetate; trans-3-ethoxy-1,1,5-trimethylcyclohexane; 4-(1,1-dimethylethyl)cyclohexanol acetate; dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; β-methylphenylpropanal; β-methyl-3-(1-methylethyl)phenylpropanal; 4-phenyl-2-butanone; 2-methylbutyric acid, ethyl ester; benzaldehyde; 2-methylbutyric acid, 1-methylethyl ester; dihydro-5-pentyl-2(3H)furanone; (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecaaldehyde; undecanoal; 2-ethyl-α,α-dimethylphenylpropanal; decanal; α,α-dimethylphenylethanol acetate; 2-( 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoic acid, methyl ester; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one; 2-pentylcyclopentanone; 3-oxo-2-pentylcyclopentaneacetic acid, methyl ester; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde; 2-heptylcyclopentanone; 1-(4-methylphenyl)ethyl ketone; (3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one; (3E)-4-(2,6,6-trimethyl-3-cyclohexen-1-yl)-3-buten-2-one; (2H-1-benzopyran-2-one); phenethyl alcohol; 2H-1-benzopyran-2-one; 4-methoxybenzaldehyde; 10-undecenal; propionic acid, phenyl methyl ester; β-methylphenylpentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene; α,α-dimethylphenethyl alcohol; (2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; acetic acid, phenyl methyl ester; cyclohexanepropionic acid, 2-propenyl ester; hexanoic acid, 2-propenyl ester; 1,2-dimethoxy-4-(2-propenyl)benzene; 1,5-dimethyl-bicyclo[3.2].1] Oct-8-one oxime; 4-(4-hydroxy-4-methylpentyl)-3-cyclohexen-1-carboxaldehyde; 3-buten-2-ol; 2-[[[2,4(or 3,5)-dimethyl-3-cyclohexenyl-1-yl]methylene]amino]benzoic acid, methyl ester; 8-cyclohexadecyl-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol; 2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-octadien-1-ol; 2-hydroxy-benzoic acid, (3Z)-3-hexenyl ester; 2-tridecenoic acid; 4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; acetic acid, (2-methylbutoxy)-, 2-propenyl ester; benzoic acid, 2-hydroxy-, 3-methylbutyl ester; 2-buten-1-one, 1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, (Z)-; cyclopentanecarboxylic acid, 2-hexyl-3-oxo- methyl esters; phenylpropanal, 4-ethyl-α,α-dimethyl-; 3-cyclohexene-1-carboxaldehyde, 3-(4-hydroxy-4-methylpentyl)-; acetone, 1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanolchamomile-5-yl)-, [3R-(3.α.,3a.β.,7.β.,8a.α.)]-; undecaldehyde, 2-methyl-2H-pyran-2-one, 6-butyltetrahydro-; phenylpropanal, 4-(1,1-dimethylacetone) α-methyl-; 2(3H)-furanone, 5-heptyldihydro-; benzoic acid, 2-[(7-hydroxy-3,7-dimethyl-octyl)amino]-, methyl; benzoic acid, 2-hydroxy-, phenylmethyl ester; naphthalene, 2-methoxy-; 2-cyclopenten-1-one, 2-hexyl-; 2(3H)-furanone, 5-hexyldihydro-; oxacyclopropanecarboxylic acid, 3-methyl-3-phenyl-, ethyl ester; 2-oxabicyclo[2.2.2]octane, 1,3,3-trimethyl-; phenylpentanol, γ-.-Methyl-; 3-octanol, 3,7-dimethyl-; 3,7-dimethyl-2,6-octadienone; 3,7-dimethyl-6-octen-1-ol; terpineol acetate; 2-methyl-6-methylene-7-octen-2-ol, dihydro derivative; 3a,4,5,6,7,7a-hexahydro-4,7-methanol-1H-inden-6-phenol propionate; 3-methyl-2-buten-1-ol acetate; (Z)-3-hexen-1-ol acetate; 2-ethyl-4 -(2,2,3-trimethyl-3-cyclopenten-1-yl)-2-buten-1-ol; 4-(octahydro-4,7-methanol-5H-indenyne-5-yl)-butanal; 3-2,4-dimethyl-cyclohexen-1-carboxaldehyde; 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthyl)-acetone; 2-hydroxybenzoic acid, methyl ester; 2-hydroxybenzoic acid, hexyl ester; 2-phenoxy-ethanol; 2-hydroxy-benzene Formic acid, pentyl ester; 2,3-heptanedione; 2-hexen-1-ol; 6-octen-2-ol, 2,6-dimethyl-; daemonone (α, β, γ or δ or mixtures thereof), 4,7-methanol-1H-inden-6-phenol, 3a,4,5,6,7,7a-hexahydro-, acetate; 9-undecenal; 8-undecenal; heterocyclic citral; acetone, 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2-naphthyl) -; 3-cyclohexene-1-carboxaldehyde, 3,5-dimethyl-; 3-cyclohexene-1-carboxaldehyde, 2,4-dimethyl-; 1,6-octadien-3-ol, 3,7-dimethyl-; 1,6-octadien-3-ol, 3,7-dimethyl-, acetate; lily aldehyde (pt-Bucinal); and cyclopentanone, 2-[2-(4-methyl-3-cyclohexene-1-yl)propyl]- and 1-methyl-4-(1-methylvinyl)cyclohexene and mixtures thereof.

[0350] In one aspect, the composition may include encapsulated fragrance particles containing a water-soluble hydroxy compound or melamine-formaldehyde or modified polyvinyl alcohol. In another aspect, the encapsulation comprises (a) at least partially water-soluble solid matrix containing one or more water-soluble hydroxy compounds, preferably starch; and (b) fragrance oil encapsulated by the solid matrix.

[0351] On the other hand, the flavoring can be pre-complexed with a polyamine (preferably polyethyleneimine) to form a Schiff base.

[0352] polymer- The composition may include one or more polymers. Examples include carboxymethyl cellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazolium), polycarboxylate (such as polyacrylate), maleic acid / acrylic acid copolymer, and lauryl methacrylate / acrylic acid copolymer.

[0353] The composition may include one or more amphiphilic cleaning polymers, such as compounds having the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + -(CH3)-bis((C2H5O)(C2H4O)n), where n = from 20 to 30 and x = from 3 to 8, or its sulfonated or sulfonated variants.

[0354] The composition may include amphiphilic alkoxylated grease-removing polymers that have a balanced hydrophilic and hydrophobic property, enabling them to remove grease particles from fabrics and surfaces. Specific embodiments of the amphiphilic alkoxylated grease-removing polymers of the present invention include a core structure and a plurality of alkoxylated groups attached to that core structure. These may include alkoxylated polyalkylenimines, preferably having an inner ethylene oxide block and an outer propylene oxide block.

[0355] Here, alkoxylated polycarboxylate esters (such as those prepared from polyacrylates) can be used to provide additional grease-removing properties. Such materials are described in WO 91 / 08281 and PCT 90 / 01815. Chemically, these materials comprise polyacrylates having one ethoxylated side chain per 7-8 acrylate units. The side chain has the chemical formula -(CH2CH2O). m (CH2) n CH3, where m is 2-3 and n is 6-12. Side chains are esters attached to the polyacrylate "main chain" to provide a "comb-like" polymer-type structure. Molecular weights can vary, but are typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylic acid esters can be included in the compositions herein from 0.05 wt% to 10 wt%.

[0356] The isoprene-derived surfactants of the present invention, and mixtures thereof formed with other auxiliary surfactants and adjuvants, are particularly suitable for use with amphiphilic graft copolymers, preferably comprising (i) a polyethylene glycol backbone; and (ii) and at least one dangling portion selected from polyvinyl acetate, polyvinyl alcohol, and mixtures thereof. A preferred amphiphilic graft copolymer is Sokalan HP22 supplied by BASF. Suitable polymers include random graft copolymers, preferably polyvinyl acetate-grafted polyethylene oxide copolymers, having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000, and the weight ratio of polyethylene oxide to polyvinyl acetate is 40:60, with no more than one grafting point per 50 ethylene oxide units.

[0357] Carboxylic acid ester polymers - The compositions of the present invention further include one or more carboxylate polymers, such as maleate / acrylate random copolymers or polyacrylate homopolymers. In one aspect, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of 4,000 Da to 9,000 Da or 6,000 Da to 9,000 Da.

[0358] Fouling release polymers - The compositions of the present invention may further comprise one or more dirt-releasing polymers having a structure as defined by one of the following structures (I), (II) or (III):

[0359] (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d

[0360] (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e

[0361] (III)-[(OCHR 5 -CHR 6 ) c -OR 7 ] f

[0362] in:

[0363] a, b, and c are from 1 to 200;

[0364] d, e, and f are from 1 to 50;

[0365] Ar is a 1,4-substituted phenylene;

[0366] sAr is a 1,3-substituted phenylene with SO3Me substituted at the 5-position;

[0367] Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, mono-, di-, tri-, or tetraalkylammonium, wherein the alkyl group is C1-C. 18 Alkyl or C2-C 10 Hydroxyalkyl groups, or mixtures thereof;

[0368] R 1 R 2 R 3 R 4 R 5 and R 6 Independently selected from H or C1-C 18 n- or iso-alkyl; and

[0369] R 7 Is it a straight or branched C1-C? 18 Alkyl groups, or straight-chain or branched C2-C 30 Alkenyl, or cycloalkyl having 5 to 9 carbon atoms, or C8-C 30 Aryl, or C6-C 30 Arylalkyl.

[0370] Suitable detergency polymers are polyester detergency polymers, such as Repel-o-tex polymers, including Repel-o-tex, SF-2, and SRP6, supplied by Rhodia. Other suitable detergency polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300, and SRN325, supplied by Clariant. Other suitable detergency polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0371] Cellulose polymers The compositions of the present invention further include one or more cellulose polymers, including those selected from alkyl cellulose, alkylalkoxyalkyl cellulose, carboxyalkyl cellulose, and alkylcarboxyalkyl cellulose. In one aspect, the cellulose polymer is selected from the group consisting of carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose, and mixtures thereof. In one aspect, the carboxymethyl cellulose has a degree of carboxymethyl substitution from 0.5 to 0.9 and a molecular weight from 100,000 Da to 300,000 Da.

[0372] Enzymes – The composition may include one or more enzymes that provide cleaning properties and / or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tanninase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase, or mixtures thereof. A typical combination is an enzyme mixture that may contain, for example, proteases and lipases together with amylase. When present in the composition, the aforementioned additional enzymes may be present at a level of enzyme protein from 0.00001 wt% to 2 wt%, from 0.0001 wt% to 1 wt%, or from 0.001 wt% to 0.5 wt% by weight of the composition.

[0373] Generally, the properties of one or more selected enzymes should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymes and non-enzyme components, etc.), and the one or more enzymes should be present in an effective amount.

[0374] In one respect, the preferred enzyme will include a cellulase. Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified variants or protein-engineered variants. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humus, Fusarium, Clostridium, and Cladosporium, such as the fungal cellulases produced by specific Humus, Thermophilus, and Fusarium disclosed in US4435307, US 5648263, US 5691178, US 5776757, and WO 89 / 09259.

[0375] Particularly suitable cellulases are alkaline or neutral cellulases that offer color-care benefits. Examples of such cellulases are those described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0531315, US 5457046, US 5686593, US 5763254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0376] Commercially available cellulases include Celluzyme TM and Carezyme TM(Novozymes A / S), Clazinase TM and Puradax HA TM (Genencor International Inc.) and KAC-500(B) TM (Kao Corporation).

[0377] In one respect, preferred enzymes will include proteases. Suitable proteases include those of bacterial, fungal, plant, viral, or animal origin, such as those of plant or microbial origin. Microbial origin is preferred. This includes chemically modified variants or protein-engineered variants. It can be an alkaline protease, such as a serine protease or a metalloproteinase. Serine proteases can be, for example, from the S1 family (such as trypsin) or the S8 family (such as subtilisin). Metalloproteinases can be, for example, thermophilic bacterial proteases from, for example, family M4, or other metalloproteinases, such as those from the M5, M7, or M8 families.

[0378] The term "subtilase" refers to the serine protease subgroup according to Siezen et al., Protein Engineering 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by the presence of a serine residue at its active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subfamilies: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanathionine antibiotic peptidase family, the Kexin family, and the Pyrolysin family.

[0379] Examples of subtilisinases are those derived from the genus Bacillus, such as *Bacillus lentus*, *Bacillus alkalophilus*, *Bacillus subtilis*, *Bacillus amyloliquefaciens*, *Bacillus pumilus*, and *Bacillus giganteus* as described in US 7262042 and WO 09 / 021867; and *Lentus*, *Novo*, *Carlsberg*, *Bacillus licheniformis*, *BPN'*, *309*, *147*, and *168* as described in WO 89 / 06279, and *PD138* as described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024, and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g., from pigs or cattle) and Fusarium proteases (described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372), as well as chymotrypsin derived from Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0380] Further preferred proteases are alkaline proteases from Bacillus tarda DSM 5483 (as described in, for example, WO95 / 23221), and their variants (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0381] Examples of metalloproteinases are neutral metalloproteinases as described in WO 07 / 044993 (Genencor Int.), such as those derived from Bacillus amyloliquefaciens.

[0382] Examples of useful proteases are described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO Variants in 11 / 036264, especially those with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, use BPN' numbers. More preferably, these Bacillus subtilis enzyme variants may include the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).

[0383] Suitable commercially available proteases include those marketed under trade names. Duralase Tm Durazym Tm , Ultra Ultra Ultra Ultra as well as Those that are sold, all of them can be sold at or (Sold by Novozymes A / S); those sold under the following trade names: Purafect Preferenz Tm Purafect Purafect Purafect Effectenz Tm , as well as (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG) and KAP (Bacillus subtilis protease) from Kao Corporation.

[0384] In one respect, the preferred enzyme will include an amylase. Suitable amylases may be α-amylases or glucosylamylases and may be of bacterial or fungal origin. This includes chemically modified variants or protein-engineered variants. Amylases include, for example, α-amylases obtained from the genus *Bacillus*, such as the α-amylases of specific strains of *Bacillus licheniformis* described in more detail in GB 1296839.

[0385] Suitable amylases include the amylase having SEQ ID NO:3 in WO 95 / 10603 or a variant thereof having 90% sequence identity with SEQ ID NO:3. Preferred variants are described in SEQ ID NO:4 of WO 94 / 02597, WO 94 / 18314, WO 97 / 43424 and WO 99 / 019467, for example, variants having substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181, 188, 190, 197, 201, 202, 207, 208, 209, 211, 243, 264, 304, 305, 391, 408 and 444.

[0386] Suitable amylases include the amylase having SEQ ID NO:6 in WO 02 / 010355 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those with deletions at positions 181 and 182 and substitutions at position 193.

[0387] Other suitable amylases are hybrid α-amylases comprising residues 1-33 of the α-amylase derived from *Bacillus amyloliquefaciens* as shown in SEQ ID NO:6 of WO 2006 / 066594 and residues 36-483 of the α-amylase derived from *Bacillus licheniformis* as shown in SEQ ID NO:4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of this hybrid α-amylase are those having substitutions, deletions, or insertions at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209, and Q264. The most preferred variant of the hybrid α-amylase, including residues 1-33 of the α-amylase derived from Bacillus amyloliquefaciens shown in WO 2006 / 066594 and residues 36-483 of SEQ ID NO:4, is those having the following substitutions:

[0388] M197T;

[0389] H156Y+A181T+N190F+A209V+Q264S; or

[0390] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0391] Suitable additional amylases are those having SEQ ID NO:6 in WO 99 / 019467 or variants thereof having 90% sequence identity with SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having substitutions, deletions, or insertions at one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216, and K269. Particularly preferred amylases are those having deletions at positions R181 and G182 or positions H183 and G184.

[0392] Other amylases that can be used are those of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:2, or SEQ ID NO:7 having WO 96 / 023873, or variants thereof having 90% sequence identity with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7. Preferred variants of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:7 are those with substitutions, deletions, or insertions at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304, and 476. More preferred variants are those with deletions at positions 181 and 182 or positions 183 and 184. The most preferred amylase variants of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:7 are those with deletions in positions 183 and 184 and substitutions in one or more of positions 140, 195, 206, 243, 260, 304 and 476.

[0393] Other amylases that may be used are those having SEQ ID NO:2 in WO 08 / 153815, SEQ ID NO:10 in WO 01 / 66712, or variants thereof having 90% sequence identity with SEQ ID NO:2 in WO 08 / 153815 or 90% sequence identity with SEQ ID NO:10 in WO 01 / 66712. Preferred variants of SEQ ID NO:10 in WO 01 / 66712 are those having substitutions, deletions, or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0394] Other suitable amylases are the amylase having SEQ ID NO:2 in WO 09 / 061380 or a variant thereof having 90% sequence identity with SEQ ID NO:2. Preferred variants of SEQ ID NO:2 are those having C-terminal truncation and / or substitution, deletion, or insertion at one or more of the following positions: Q87, Q98, S125, N128, T131, T165, K178, R180, S181, T182, G183, M201, F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444, and G475. More preferred variants of SEQ ID NO:2 are those having substitutions at one or more of the following positions: Q87E,R, Q98R, S125A, N128C, T131I, T165I, K178L, T182G, M201L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E, and G475K, and / or the deletion of positions R180 and / or S181 or T182 and / or G183. The most preferred amylase variants of SEQ ID NO:2 are those having the following substitutions:

[0395] N128C+K178L+T182G+Y305R+G475K;

[0396] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0397] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0398] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein these variants are C-terminated and optionally further include a substitution at position 243 and / or a deletion at positions 180 and / or 181.

[0399] Other suitable amylases are those having SEQ ID NO:12 in WO 01 / 66712 or variants thereof having 90% sequence identity with SEQ ID NO:12. Preferred amylase variants are those having substitutions, deletions, or insertions at one or more of the following positions in SEQ ID NO:12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and substitutions of R118K, N195F, R320K, and R458K, as well as variants having substitutions at one or more positions selected from the group consisting of M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345, and A339, with the most preferred being variants having substitutions at all of these positions.

[0400] Other examples are amylase variants, such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0401] Commercially available amylase is Duramyl TM Terminyl TM Terminyl Ultra TM Fungaly TM Ban TM Stainzyme TM Stainzyme Plus TM , Supramyl TM Natalase TM Everest TM Liquozyme X and BAN TM (From Novozymes A / S) AT 9000BiozymBiotech Trading GmbH Wehlistrasse 27b A-1200Wien Austria, and Rapidase TM Purastar TM / EffectenzTM , Powerase, Preferenz S100, Preferenx S110, OPTISIZE HT and PURASTAR (Danisco / DuPont) and (Kao Co., Ltd. (Kao)).

[0402] Suitable lipases and keratins include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermophilic*, such as those from *Thermophilic Hypotherium latifolium* (formerly named *Pythium latifolium*) as described in EP 258068 and EP305216; cutinases from the genus *Pythium*, such as *Pythium salivarium* (WO 96 / 13580); lipases from strains of the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *Alcaligenes* or *Alcaligenes-like* (EP 218272), *Pseudomonas cepacia* (EP331376), *Pseudomonas* strain SD705 (WO 95 / 06720 & WO 96 / 27002), *Pseudomonas wisconsinensis* (WO 96 / 12012); GDSL-type *Streptomyces* lipase (WO 10 / 065455); and cutinases from *Bacillus oryzae* (WO 10 / 065455). 10 / 107560); cutinase from Pseudomonas mendoza (US 5,389,536); lipase from Thermobifida fusca (WO 11 / 084412, WO 13 / 033318); lipase from Bacillus stearothermophilus (WO 11 / 084417); lipase from Bacillus subtilis (WO 11 / 084599); and lipase from Streptomyces griseus (WO 11 / 150157) and Streptomyces pristinaespiralis (WO 12 / 137147).

[0403] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508, and WO 09 / 109500.

[0404] Preferred commercially available lipase products include Lipolase TM Lipex TM Lipex Evity TM Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0405] Other examples are lipases sometimes called acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarctica lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and variants of Mycobacterium smegmatis perhydrolases (particularly the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd) (WO 10 / 100028).

[0406] In one aspect, other preferred enzymes include microbial-derived endoglucanases exhibiting endo-β-1,4-glucanase activity (EC3.2.1.4), including endogenous bacterial polypeptides for members of the genus Bacillus, having a sequence having at least 90%, 94%, 97%, or 99% homology with the amino acid sequence SEQ ID NO:2 in US7141403, and mixtures thereof. Suitable endoglucanases are those listed under the trademark name. and Sold by (Novozymes).

[0407] Other preferred enzymes include those listed in the trademark name. The pectin lyase sold under the trademark, and the pectin lyase sold under the trademark. The mannanase sold by Novozymes, and (Danisco / DuPont).

[0408] These one or more detergent enzymes can be included in the detergent composition by adding a separate additive comprising one or more enzymes, or by adding a combination additive comprising all of these enzymes. The detergent additives of the present invention, i.e., individual or combined additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, particularly dust-free granules; liquids, particularly stabilized liquids; or slurries.

[0409] Non-dust particles can be manufactured, for example, as disclosed in US 4106991 and US 4661452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molar weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and has 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and monoglycerides, diglycerides, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugars or sugar alcohols, lactic acid, or boric acid according to established methods. Protected enzymes can be prepared according to the methods disclosed in EP238216.

[0410] Dye transfer inhibitors - The compositions of the present invention may further include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone, and polyvinylimidazole or mixtures thereof. When present in the composition, the dye transfer inhibitor may be present at levels from 0.0001 wt% to 10 wt%, from 0.01 wt% to 5 wt%, or from 0.1 wt% to 3 wt%.

[0411] Brightening agent - The compositions of the present invention may also contain or include additional components that can color the cleaned article, such as fluorescent brighteners.

[0412] The composition may include CI fluorescent brightener 260 in an α-crystalline form having the following structure:

[0413]

[0414] On one hand, the brightener is a cold water-soluble brightener, such as CI fluorescent brightener 260 in α-crystalline form. On the other hand, the brightener is primarily in α-crystalline form, meaning that typically at least 50 wt%, at least 75 wt%, at least 90 wt%, at least 99 wt%, or even substantially all of CI fluorescent brightener 260 is in α-crystalline form.

[0415] Brightening agents are typically in the form of microparticles with a weighted average primary particle size ranging from 3 to 30 micrometers, from 3 to 20 micrometers, or from 3 to 10 micrometers.

[0416] The composition may include CI fluorescent brightener 260 in β-crystalline form, and the weight ratio of (i) CI fluorescent brightener 260 in α-crystalline form to (ii) CI fluorescent brightener 260 in β-crystalline form may be at least 0.1 or at least 0.6. BE 680847 relates to a method for obtaining CI fluorescent brightener 260 in α-crystalline form.

[0417] Commercial optical brighteners that can be used in this invention can be classified into several subgroups, which include, but are not necessarily limited to, stilbene, pyrazoline, coumarin, carboxylic acids, methinecyanine, dibenzothiophene-5,5-dioxide, azoles, derivatives of 5- and 6-membered heterocyclic rings, and other hybrids. Examples of such brighteners are disclosed in “Production and Application of Fluorescent Brighteners,” M. Zahradnik, John Wiley & Sons, New York (1982). Specific, non-limiting examples of optical brighteners that can be used in the compositions of this invention are those identified in US4790856 and US3646015.

[0418] Other suitable brightening agents have the following structure:

[0419]

[0420] Suitable levels of fluorescent brighteners range from lower levels of 0.01 wt%, 0.05 wt%, 0.1 wt%, or 0.2 wt% to higher levels of 0.5 wt% or 0.75 wt%.

[0421] On the one hand, brightening agents can be loaded onto clay to form granules.

[0422] silicatesThe compositions of the present invention may also contain or comprise silicates, such as sodium silicate or potassium silicate. The compositions may comprise silicates from 0 wt% to less than 10 wt%, up to 9 wt%, or up to 8 wt%, or up to 7 wt%, or up to 6 wt%, or up to 5 wt%, or up to 4 wt%, or up to 3 wt%, or even up to 2 wt%, and from more than 0 wt%, or from 0.5 wt%, or from 1 wt%. A suitable silicate is sodium silicate.

[0423] dispersant - The compositions of the present invention may also contain or include a dispersant. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms.

[0424] Enzyme stabilizers - The enzymes used in the composition can be stabilized by various techniques. The enzymes used herein can be stabilized in the presence of a water-soluble source of calcium and / or magnesium ions. Examples of conventional stabilizers are, for example, polyols such as propylene glycol or glycerol, sugars or sugar alcohols, peptide aldehydes, lactic acid, boric acid or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid, and the composition can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708. In the case of aqueous compositions containing proteases, reversible protease inhibitors, such as boron compounds including borates, 4-formylphenylboronic acid, phenylboronic acid and their derivatives, or compounds such as calcium formate, sodium formate and 1,2-propanediol, can be added to further improve stability. Peptide aldehydes may have the formula B2-B1-B0-R, wherein: R is hydrogen, CH3, CX3, CHX2, or CH2X, where X is a halogen atom; B0 is a phenylalanine residue having an OH substituent at the para and / or meta positions. B1 is a single amino acid residue; and B2 consists of one or more amino acid residues, optionally including an N-terminal protecting group. Preferred peptide aldehydes include, but are not limited to: Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H, or Ac-WLVY-H, wherein Z is a benzyloxycarbonyl group and Ac is an acetyl group.

[0425] solvent Suitable solvents include water and other solvents, such as lipophilic fluids. Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerol derivatives (e.g., glycerol ethers), perfluorinated amines, perfluorinated and hydrofluoroether solvents, low-volatility nonfluorinated organic solvents, glycol solvents, other environmentally friendly solvents, and mixtures thereof.

[0426] Structurer / Thickener - Structured liquids can be internally structured, whereby the structure is formed by primary components (e.g., surfactant materials), and / or externally structured by providing a three-dimensional matrix structure through the use of secondary components (e.g., polymers, clays, and / or silicate materials). The composition may include 0.01 wt% to 5 wt%, or 0.1 wt% to 2.0 wt% of a structuring agent. The structuring agent is typically selected from the group consisting of: diglycerides and triglycerides, ethylene glycol stearate diester, microcrystalline cellulose, cellulose-based materials, microfibrillated cellulose, hydrophobically modified basic expandable emulsions (e.g., Polygel W30 (3VSigma)), biopolymers, xanthan gum, glucan gum, and mixtures thereof. Suitable structuring agents include hydrogenated castor oil and its non-ethoxylated derivatives. Suitable structuring agents are disclosed in US6855680. Such structuring agents have a thread-like structuring system with a range of aspect ratios. Other suitable structuring agents and methods for their preparation are described in WO 10 / 034736.

[0427] regulator - The compositions of the present invention may include high-melting-point fatty acid compounds. Useful high-melting-point fatty acid compounds herein have a melting point of 25°C or higher and are selected from the group consisting of fatty alcohols, fatty acids, fatty alcohol derivatives, fatty acid derivatives, and mixtures thereof. Such compounds having low melting points are not intended to be included in this section. Non-limiting examples of high-melting-point compounds are found in the International Dictionary of Cosmetic Ingredients, 5th Edition, 1993, and the CTFA Handbook of Cosmetic Ingredients, 2nd Edition, 1992.

[0428] In view of providing improved conditioning benefits (such as a smooth, soft feel during application to wet hair and a moisturizing feel on dry hair), high melting point fatty compounds are included in the composition at levels ranging from 0.1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1.5 wt% to 16 wt%, and from 1.5 wt% to 8 wt%.

[0429] The compositions of the present invention may comprise or include cationic polymers. The concentration of the cationic polymer in the composition typically ranges from 0.05 wt% to 3 wt%, from 0.075 wt% to 2.0 wt%, or from 0.1 wt% to 1.0 wt%. Suitable cationic polymers will have a cationic charge density of at least 0.5 meq / gm, at least 0.9 meq / gm, at least 1.2 meq / gm, at least 1.5 meq / gm, or less than 7 meq / gm, and less than 5 meq / gm at the pH at which the composition is intended to be used, in a pH range generally from pH 3 to pH 9, or between pH 4 and pH 8. Here, the “cationic charge density” of the polymer refers to the ratio of the number of positive charges on the polymer to the molecular weight of the polymer. Such suitable cationic polymers will have an average molecular weight generally between 10,000 and 10 million, between 50,000 and 5 million, or between 100,000 and 3 million.

[0430] Suitable cationic polymers used in the compositions of the present invention comprise or contain a cationic nitrogen-containing moiety, such as a quaternary ammonium or a cationic protonated amino moiety. Any anionic counterion may be used in association with the cationic polymer, provided that the polymer remains dissolved in water, in the composition, or in the condensed phase of the composition, and provided that the counterion is physically and chemically compatible with the main component of the composition or otherwise does not unduly impair the properties, stability, or aesthetics of the composition. Non-limiting examples of such counterions include halides (e.g., chlorides, fluorides, bromides, iodides), sulfates, and methyl sulfates.

[0431] Non-limiting examples of such polymers are described in the CTFA Dictionary of Cosmetic Ingredients, Third Edition, by Estrin, Crosley, and Haynes (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, 1982).

[0432] Other suitable cationic polymers for use in this composition include polysaccharide polymers, cationic guar gum derivatives, tetravalent nitrogen-containing cellulose ethers, synthetic polymers, and copolymers of etherified cellulose, guar gum, and starch. When used, the cationic polymer herein is soluble in the composition or in a soluble complex condensed phase formed from the cationic polymer described above and anionic, amphoteric, and / or facultative zwitterionic surfactant components. The complex condensate of the cationic polymer may also form with other charged materials in the composition. Suitable cationic polymers are described in US 3962418; US 3958581; and US 2007 / 0207109.

[0433] The compositions of the present invention may include nonionic polymers as modifiers. Polyalkylene glycols having a molecular weight greater than 1000 are useful here. Those having the following general formulas are useful:

[0434]

[0435] Where R 95 The following group is selected, which consists of: H, methyl groups, and mixtures thereof. Modifiers, and in particular silicones, may be included in the composition. Modifiers used in the compositions of the present invention typically include water-insoluble, water-dispersible, non-volatile liquids that form emulsion liquid particles. Suitable modifiers for use in this composition are those generally characterized as: silicones (e.g., silicone oils, cationic silicones, silicone gels, high-refractive silicones, and silicone resins), organic modifier oils (e.g., hydrocarbon oils, polyolefins, and fatty esters) or combinations thereof, or those modifiers that otherwise form liquid dispersion particles in an aqueous surfactant matrix herein. Such modifiers should be physically and chemically compatible with the main components of the composition and should not otherwise unduly impair the stability, aesthetics, or performance of the composition.

[0436] The concentration of the modifier in the composition should be sufficient to provide the desired moderating benefits. This concentration can vary depending on the modifier, the desired moderating performance, the average size of the modifier particles, the type and concentration of other components, and other similar factors.

[0437] The concentration range of silicone modifiers is typically from 0.01 wt% to 10 wt%. Non-limiting examples of suitable silicone modifiers and optional suspending agents for silicones are described in U.S. Republication Patent Nos. 34,584; US 5104646; US5106609; US 4152416; US 2826551; US ​​3964500; US 4364837; US 6607717; US 6482969; US5807956; US 5981681; US ​​6207782; US 7465439; US 7041767; US 7217777; US 2007 / 0286837 A1; US ​​2005 / 0048549 A1; US ​​2007 / 0041929 A1; GB 849433; DE All references are cited in 10036533; Chemistry and Technology of Silicones, New York: Academic Press (1968); GE Silicone Rubber Product Data Sheets SE 30, SE 33, SE54 and SE 76; Silicon Compounds, Petrarch Systems, Inc. (1984); and Encyclopedia of Polymer Science and Engineering, Volume 15, 2nd Edition, pp. 204-308, John Wiley & Sons, Inc. (1989).

[0438] The compositions of the present invention may further comprise from 0.05 wt% to 3 wt% of at least one organic conditioning oil as a conditioning agent, alone or in combination with other conditioning agents such as silicone (described herein). Suitable conditioning oils include hydrocarbon oils, polyolefins, and fatty esters. Also suitable for use in the compositions herein are the conditioning agents described in US 5674478 and US 5750122 or in US 4529586; US 4507280; US 4663158; US 4197865; US 4217914; US 4381919; and US 4422853.

[0439] Hygiene and Odor -The compositions of the present invention may further include zinc ricinoleate, thymol, quaternary ammonium salts (e.g., zinc ricinoleate, thymol, quaternary ammonium salts). ), polyethyleneimine (e.g., from BASF) ) and its zinc complexes, silver and silver compounds (especially those designed to slowly release Ag) +One or more of the following: (or those nano-silver dispersions).

[0440] Probiotics These compositions may include prebiotics, such as those described in WO 09 / 043709.

[0441] foaming agent - If high foaming is desired, use a foaming agent (e.g., C). 10 -C 16 Alkylamide or C 10 -C 14 Alkyl sulfates can typically be incorporated into the composition at levels ranging from 1 wt% to 10 wt%. 10 -C 14 Monoethanol and diethanolamides illustrate typical categories of such foaming agents. These foaming agents are also advantageous when used with high levels of foaming adjuvants and surfactants (e.g., the aforementioned amine oxides, betaine, and sulfobetaine). If desired, water-soluble magnesium and / or calcium salts (e.g., MgCl2, MgSO4, CaCl2, CaSO4, etc.) can typically be added at levels from 0.1 wt% to 2 wt% to provide additional foam and enhance grease removal performance.

[0442] Foam inhibitors - Compounds for reducing or inhibiting foam formation may be incorporated into the compositions of the present invention. Foam inhibition may be particularly important in so-called “high-concentration cleaning processes” as described in US 4489455 and US 4489574, and in front-loading-style washing machines. A wide variety of materials can be used as foam inhibitors, and foam inhibitors are well known to those skilled in the art. See, for example, Kirk Othmer Encyclopedia of Chemical Technology, 3rd Edition, Volume 7, pp. 430-447 (John Willie & Sons, 1979). Examples of foam inhibitors include monocarboxylic fatty acids and their soluble salts, high molecular weight hydrocarbons such as paraffin, fatty acid esters (e.g., fatty acid triglycerides), fatty acid esters of monovalent alcohols, aliphatic C 18 -C 40Ketones (e.g., stearone), N-alkylated aminotriazines, preferably wax hydrocarbons having a melting point below about 100°C, silicone foam inhibitors, and secondary alcohols. Foam inhibitors are described in US 2954347; US 4265779; US 4265779; US3455839; US 3933672; US 4652392; US 4978471; US ​​4983316; US 5288431; US ​​4639489; US4749740; US 4798679; US 4075118; EP 89307851.9; EP 150872; and DOS 2,124,526.

[0443] For any detergent composition intended for use in an automatic washing machine, foam should not form to the extent that it overflows the washing machine. When used, the foam inhibitor is preferably present in a "foam-inhibiting amount." "Foam-inhibiting amount" refers to the amount of this foam control agent that the formulator of the composition can choose to adequately control foam to result in a low-foaming laundry detergent for use in an automatic washing machine.

[0444] The compositions herein will typically include 0 to 10 wt% of a foam inhibitor. When used as a foam inhibitor, monocarboxylic fatty acids and their salts will typically be present in amounts up to 5 wt%. Preferably, a fatty monocarboxylic acid ester foam inhibitor is used in amounts from 0.5 wt% to 3 wt%. Silicone foam inhibitors are typically used in amounts up to 2.0 wt%, although higher amounts may be used. Monostearyl phosphate foam inhibitors are typically used in amounts ranging from 0.1 wt% to 2 wt%. Hydrocarbon foam inhibitors are typically used in amounts ranging from 0.01 wt% to 5 wt%, although higher levels may be used. Alcohol foam inhibitors are typically used in amounts from 0.2 wt% to 3 wt%.

[0445] The compositions herein can exhibit cleaning activity over a wide pH range. In some embodiments, these compositions have cleaning activity from pH 4 to pH 11.5. In other embodiments, these compositions are active from pH 6 to pH 11, from pH 7 to pH 11, from pH 8 to pH 11, from pH 9 to pH 11, or from pH 10 to pH 11.5.

[0446] The compositions herein can exhibit cleaning activity over a wide temperature range (e.g., from 10°C or lower to 90°C). Preferably, this temperature will be below 50°C, 40°C, or even 30°C. In some embodiments, the optimal temperature range for these compositions is from 10°C to 20°C, from 15°C to 25°C, from 15°C to 30°C, from 20°C to 30°C, from 25°C to 35°C, from 30°C to 40°C, from 35°C to 45°C, or from 40°C to 50°C.

[0447] Forms of the composition

[0448] The compositions described herein are advantageously used in applications such as laundry, hard surface cleaning, dishwashing, and cosmetic applications (e.g., dentures, teeth, hair, and skin). The compositions of the invention are specifically solid or liquid cleaning and / or treatment compositions. In one aspect, the invention relates to a composition selected from the group consisting of: regular, compressed, or concentrated liquids; gels; ointments; soap bars; regular or compressed powders; granular solids; homogeneous or multilayer tablets having two or more layers (same or different phases); bags having one or more chambers; single or multiple chamber unit dosage forms; or any combination thereof.

[0449] The composition is designed to physically separate the components from each other in multiple compartments (e.g., like a water-soluble pouch) or different layers of a tablet. This avoids negative storage interactions between the components. Furthermore, the different dissolution profiles of each compartment in the washing solution can also cause delayed dissolution of selected components.

[0450] The bag can be configured as a single or multiple chambers. It can have any form, shape, and material suitable for containing the composition, for example, preventing the composition from being released from the bag before contact with water. The bag is made of a water-soluble membrane encapsulating an inner volume. The inner volume can be divided into chambers containing the bag. Preferred membranes are polymeric materials that form membranes or sheets, preferably polymers. Preferred polymers, copolymers, or derivatives thereof are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylates, most preferably polyvinyl alcohol copolymers, and hydroxypropyl methylcellulose (HPMC). Preferably, the polymer level in the membrane, such as PVA, is at least about 60%. Preferred average molecular weights will typically be from about 20,000 to about 150,000. The membrane can also be a blend composition comprising a hydrolyzable and water-soluble polymer blend, such as polylactic acid and polyvinyl alcohol (known under Trade Reference M8630, such as those sold by MonoSol LLC, Indiana, USA), plus plasticizers, such as glycerin, ethylene glycol, propylene glycol, sorbitol, and mixtures thereof. These bags may include solid laundry cleaning compositions or portions thereof and / or liquid cleaning compositions or portions thereof separated by a water-soluble membrane. Chambers for liquid components may differ in composition from chambers comprising solid components (US 2009 / 0011970 A1).

[0451] Lipase granules

[0452] The lipase variants contained in the water-soluble membrane of the present invention can be present as lipase particles. These lipase particles may even contain one or more additional enzymes, as described below.

[0453] Lipase particles are any form of lipase variant in solid particulate form. Lipase particles can be lipase crystals, lipase precipitates, spray-dried or lyophilized lipase, or any form of granular lipase, as a powder or suspension in a liquid. Typically, the particle size of lipase particles, measured as equivalent spherical diameter (average particle size based on volume), is less than 2 mm, preferably less than 1 mm, less than 0.5 mm, less than 0.25 mm, or less than 0.1 mm; and greater than 0.05 μm, preferably greater than 0.1 μm, greater than 0.5 μm, greater than 1 μm, greater than 5 μm, or greater than 10 μm.

[0454] In a preferred embodiment, the particle size of the lipase particles ranges from 0.5 μm to 100 μm.

[0455] The lipase particles contain at least 1% w / w lipase protein, preferably at least 5% w / w lipase protein, at least 10% w / w lipase protein, at least 20% w / w lipase protein, at least 30% w / w lipase protein, at least 40% w / w lipase protein, at least 50% w / w lipase protein, at least 60% w / w lipase protein, at least 70% w / w lipase protein, at least 80% w / w lipase protein, or at least 90% w / w lipase protein.

[0456] In a preferred embodiment, the lipase particles are lipase crystals, or the lipase protein is in a crystal form.

[0457] Enzyme crystallization can be performed in a variety of ways as known in the art (e.g., as described in WO 91 / 09943 or WO94 / 22903).

[0458] Lipases can be formulated in lipase particles as known in the art for use in solid enzyme formulations, such as formulations for reducing dust, improving stability, and / or altering enzyme release rates. Lipase particles can also be formulated in a matrix or coated with reagents that inhibit the dissolution of the enzyme particles in PVOH / membrane solutions used to prepare water-soluble membranes.

[0459] The lipase molecules on the surface of lipase particles can also be cross-linked, such as CLEC (cross-linked enzyme crystals) or CLEA (cross-linked enzyme aggregates).

[0460] Water-soluble membrane

[0461] Water-soluble films, optional components therein, and methods for preparing them are well known in the art. In one type of embodiment, the water-soluble film comprises PVOH. PVOH is a synthetic resin typically prepared by alcoholysis (commonly referred to as hydrolysis or saponification) of polyvinyl acetate. Fully hydrolyzed PVOH, in which almost all acetate groups have been converted to alcohol groups, is a highly crystalline polymer with strong hydrogen bonds that dissolves only in hot water (above about 140°F (60°C)). If a sufficient number of acetate groups are allowed to remain after the hydrolysis of polyvinyl acetate, the PVOH polymer is called partially hydrolyzed, with weaker hydrogen bonds and lower crystallinity, and is soluble in cold water (below about 50°F (10°C)). An intermediate cold / hot water-soluble film may comprise, for example, intermediately partially hydrolyzed PVOH (e.g., having a degree of hydrolysis of about 94% to about 98%), and is readily soluble only in warm water (e.g., rapidly dissolving at temperatures of about 40°C and above). Both fully and partially hydrolyzed PVOH types are commonly referred to as PVOH homopolymers, although the partially hydrolyzed type is technically a vinyl alcohol-vinyl acetate copolymer.

[0462] The degree of hydrolysis of PVOH contained in the water-soluble membrane disclosed herein can be from about 75% to about 99%. When the degree of hydrolysis decreases, the membrane made from the resin will have reduced mechanical strength but will dissolve more quickly at temperatures below about 20°C. When the degree of hydrolysis increases, the membrane made from the resin will tend to have higher mechanical strength and its thermoformability will tend to decrease. The degree of hydrolysis of PVOH can be selected such that the water solubility of the resin is temperature-dependent, and thus the solubility of the membrane made from the resin, compatibility reagents, and other components is also affected. In one type of embodiment, the membrane is cold-water soluble. A cold-water soluble membrane (soluble in water at temperatures below 10°C) may include PVOH with a degree of hydrolysis in the range of about 75% to about 90%, or in the range of about 80% to about 90%, or in the range of about 85% to about 90%. In another type of embodiment, the membrane is hot-water soluble. A hot-water soluble membrane (soluble in water at temperatures of at least about 60°C) may include PVOH with a degree of hydrolysis of at least about 98%.

[0463] In addition to or as an alternative to PVOH, other film-forming resins used may include, but are not limited to, modified polyvinyl alcohol, polyacrylates, water-soluble acrylate copolymers, polyacrylates, polyacrylamide, polyvinylpyrrolidone, pullulan, water-soluble natural polymers including but not limited to guar gum, xanthan gum, carrageenan, and starch, and water-soluble polymer derivatives including but not limited to ethoxylated starch and hydroxypropylated starch, poly(acrylamido-2-methylpropanesulfonate), polymethyl maleate, copolymers thereof, and combinations thereof. In one embodiment, the film-forming resin is a terpolymer composed of vinyl alcohol, vinyl acetate, and acrylamide-2-methylpropanesulfonate. Surprisingly, water-soluble films based on the terpolymer of vinyl alcohol, vinyl acetate, and acrylamide-2-methylpropanesulfonate have shown high percentages of enzyme recovery.

[0464] The water-soluble resin can be included in the water-soluble film in any suitable amount, for example, in the range of about 35 wt% to about 90 wt%. The preferred weight ratio of the amount of the water-soluble resin to the combined amount of all enzymes, enzyme stabilizers and auxiliary additives can be any suitable ratio, for example, in the range of about 0.5 to about 5, or about 1 to about 3, or about 1 to about 2.

[0465] The water-soluble resins (including, but not limited to, PVOH resins) used in the membranes described herein can be characterized by any viscosity suitable for the desired membrane properties, optionally in the range of about 5.0 to about 30.0 cP, or about 10.0 cP to about 25 cP. The viscosity of the PVOH resin is determined by measuring the freshly prepared solution using a Brookfield LV viscometer with a UL adapter, as described in Annex E of British Standard EN ISO 15023-2:2006, Brookfield Test Method. It is international practice to describe the viscosity of a 4% aqueous polyvinyl alcohol solution at 20°C. All PVOH viscosities specified herein in cP should be understood to refer to the viscosity of a 4% aqueous polyvinyl alcohol solution at 20°C, unless otherwise stated.

[0466] It is well known in the art that the viscosity of a PVOH resin is related to its weight-average molecular weight. Related, and this viscosity is often used as Therefore, the weight-average molecular weight of the water-soluble resin may optionally be in the range of about 35,000 to about 190,000, or about 80,000 to about 160,000. The molecular weight of the resin only needs to be sufficient for it to be molded into a plastic film using suitable techniques.

[0467] The water-soluble film disclosed herein may include, for example, other optional additive components in an amount suitable for its intended purpose, including but not limited to, plasticizers, surfactants, defoamers, film-forming agents, anti-blocking agents, internal release agents, anti-yellowing agents, and other functional components.

[0468] Water is considered a very effective plasticizer for PVOH and other polymers; however, its volatility limits its effectiveness because polymer films need to have at least some tolerance (robustness) to a variety of environmental conditions, including low and high relative humidity. Glycerin is much less volatile than water and has been well established as an effective plasticizer for PVOH and other polymers. If the levels used in film formulations are too high, glycerin or other such liquid plasticizers can themselves cause surface “sweating” and greasiness. This can lead to problems in films such as giving consumers an unacceptable tactile experience and, if not mitigated in some way (e.g., surface dusting), can even cause the film to become stuck on rollers or in stacks of sheets. This can be characterized as overplasticization. However, if too little plasticizer is added to the film, the film may lack sufficient stretch and flexibility for many end uses, such as when being converted to end-use types, such as bags.

[0469] Plasticizers used in the water-soluble membranes disclosed herein include, but are not limited to, sorbitol, glycerol, diglycerol, propylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, polyethylene glycol (up to 400 MW), 2-methyl-1,3-propanediol, lactic acid, glyceryl monoacetate, triacetyl ester, triethyl citrate, 1,3-butanediol, trimethylolpropane (TMP), polyether triols, and combinations thereof. As mentioned above, polyols are generally used as plasticizers. The less plasticizer used, the more brittle the membrane may become, while the more plasticizer used, the more likely the membrane may lose tensile strength. Plasticizers may be included in the water-soluble membrane in amounts ranging from, for example, from about 25 phr (parts per 100 parts of resin) to about 50 phr, or from about 30 phr to about 45 phr, or from about 32 phr to about 42 phr.

[0470] Surfactants used in water-soluble membranes are well known in the art. Optionally, surfactants are included to assist in the dispersion of the resin solution during casting. Suitable surfactants for the water-soluble membranes disclosed herein include, but are not limited to, dialkyl sulfosuccinates, lactated fatty acid esters of glycerol and propylene glycol, fatty acid lactyl esters, sodium alkyl sulfate, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80, alkyl polyoxyethylene ethers, lecithin, acetylated fatty acid esters of glycerol and propylene glycol, sodium lauryl sulfate, acetylated fatty acid esters, myristyl dimethylamine oxide, trimethyl tallow alkyl ammonium chloride, quaternary ammonium compounds, their salts, and combinations thereof. Thus, surfactants may be included in the water-soluble membrane in amounts, for example, less than about 2 phr, such as less than about 1 phr, or less than about 0.5 phr.

[0471] One type of auxiliary ingredient to be considered is a defoamer. Defoamers can help coalesce foam bubbles. Suitable defoamers for use in water-soluble films according to this disclosure include, but are not limited to, hydrophobic silica, such as fine-grained silica or calcined silica, including Foam. Defoamers (available from Emerald Performance Materials), including Foam 327, Foam UVD, Foam 163. Foam 269. Foam 338, Foam 290, Foam 332, Foam 349. Foam 550 and Foam 339, these are proprietary non-mineral oil defoamers. In embodiments, the defoamer may be used in amounts of 0.5 phr or less, such as 0.05 phr, 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr. Preferably, significant amounts of silica will be avoided to prevent stress whitening.

[0472] Methods for preparing water-soluble articles (including films) include casting, blow molding, extrusion, or blow extrusion, as known in the art. One class of considered embodiments is characterized by the formation of water-soluble films described herein by casting, for example by applying the mixture to a surface by mixing the components described herein with water to produce an aqueous mixture (e.g., a solution having optionally dispersed solids), and drying to remove the water to produce a film. Similarly, other compositions can be formed by drying the mixture while confining it to a desired shape.

[0473] In one considered embodiment, the water-soluble film is formed by casting a water-soluble mixture, wherein the water-soluble mixture is prepared according to the following steps:

[0474] (a) Provide a mixture of water-soluble resin, water, and any optional additives (excluding plasticizers);

[0475] (b) Boil the mixture for 30 minutes;

[0476] (c) Degas the mixture in an oven at a temperature of at least 40°C; optionally in the range of 40°C to 70°C, for example about 65°C;

[0477] (d) Add one or more enzymes, plasticizers, and additional water to the mixture at a temperature of 65°C or lower; and

[0478] (e) Stir the mixture without vortexing until the mixture appears substantially uniform in color and consistency; optionally for a period ranging from 30 to 90 minutes, optionally for at least 1 hour; and

[0479] (f) Pour the mixture quickly after the mixing period (e.g., within 4 hours, 2 hours, or 1 hour).

[0480] If enzymes are added to a mixture too early (e.g., along with auxiliary additives or resins), enzyme activity may decrease. Without intending to be bound by any specific theory, it is believed that boiling a mixture containing enzymes causes enzyme denaturation, and prolonged storage in solution also leads to decreased enzyme activity.

[0481] In one embodiment, the water-soluble membrane according to this disclosure is maintained at high enzyme activity by rapid drying under moderate to mild conditions. As used herein, rapid drying means a drying time of less than 24 hours, optionally less than 12 hours, optionally less than 8 hours, optionally less than 2 hours, optionally less than 1 hour, optionally less than 45 minutes, optionally less than 30 minutes, optionally less than 20 minutes, optionally less than 10 minutes, for example, in the range of about 6 minutes to about 10 minutes or 8 minutes. As used herein, moderate to mild conditions mean a drying temperature below 170°F (77°C), optionally in the range of about 150°F to about 170°F (about 66°C to about 77°C), for example, 165°F (74°C). As the drying temperature increases, the enzyme tends to denature more quickly, while as the drying temperature decreases, the drying time increases, thereby exposing the enzyme to solution for an extended period of time.

[0482] This membrane is used to produce a package containing a composition, such as a laundry or dishwashing composition, thereby forming a bag. The membrane described herein can also be used to prepare a package having two or more compartments, made from the same membrane or a membrane combined with other polymeric materials. Additional membranes can be obtained, for example, by casting, blow molding, extrusion, or blow extrusion of the same or different polymeric materials, as known in the art. In one type of embodiment, polymers, copolymers, or derivatives thereof suitable for use as additional membranes are selected from polyvinyl alcohol, polyvinylpyrrolidone, polyepoxides, polyacrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetate, polycarboxylic acids and salts, polyamino acids or peptides, polyamides, polyacrylamide, maleic acid / acrylic acid copolymers, polysaccharides (including starch and gelatin), and natural gums (e.g., xanthan gum and carrageenan). For example, the polymer may be selected from polyacrylate and water-soluble acrylate copolymers, methylcellulose, sodium carboxymethylcellulose, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, maltodextrin, polymethyl acrylate and combinations thereof, or from polyvinyl alcohol, polyvinyl alcohol copolymers and hydroxypropyl methylcellulose (HPMC) and combinations thereof.

[0483] The bags and / or packages disclosed herein include at least one sealed compartment. Therefore, these bags may include a single compartment or multiple compartments. These bags may have enzyme-containing and enzyme-free areas. In embodiments including multiple compartments, each compartment may contain the same and / or different compositions. Furthermore, these compositions may take any suitable form, including but not limited to liquids, solids, and combinations thereof (e.g., solids suspended in a liquid). In some embodiments, these bags include first, second, and third compartments, wherein each compartment contains a different first, second, and third composition, respectively. In some embodiments, as described in EP 2258820, these compositions may be visually different.

[0484] The compartments of a multi-compartment bag and / or pack may have one or more identical or different sizes and / or volumes. The compartments of the multi-compartment bag of the present invention may be separate or combined in any suitable manner. In some embodiments, the second and / or third and / or subsequent compartments are stacked on top of the first compartment. In one aspect, the third compartment may be stacked on top of the second compartment, which in turn is stacked on top of the first compartment in a sandwich configuration. Alternatively, the second and third compartments may be stacked on top of the first compartment. However, it is also contemplated that the first, second, and optionally third and subsequent compartments may be attached to each other in a side-by-side relationship. These compartments may be packaged in a string, each compartment being separable individually by perforated lines. Thus, each compartment can be individually torn off by the end user from the remainder of the string.

[0485] In some embodiments, a multi-compartment bag and / or package comprises three compartments, consisting of a large first compartment and two smaller compartments. The smaller second and third compartments are stacked on top of the large first compartment. The dimensions and geometry of these compartments are chosen to make this arrangement feasible. The geometries of the compartments may be the same or different. In some embodiments, the second and optionally third compartments each have a different geometry and shape compared to the first compartment. In these embodiments, the second and optionally third compartments are arranged on top of the first compartment in a design. This design may be decorative, didactic, or illustrative, for example, to illustrate a concept or guide, and / or to indicate the origin of the product. In some embodiments, the first compartment is the largest compartment, having two large, perimeter-sealed surfaces, while the second compartment is smaller, covering less than about 75%, or less than about 50%, of the surface area of ​​one surface of the first compartment. In embodiments where a third compartment exists, the above structure may be the same, but the second and third compartments cover less than about 60%, or less than about 50%, or less than about 45% of the surface area of ​​one side of the first compartment.

[0486] The bags and / or pouches disclosed herein may include one or more different films. For example, in a single-compartment embodiment, the pouch may be made of a wall folded onto itself and sealed at the edges, or alternatively, of two walls sealed together at the edges. In a multi-compartment embodiment, the pouch may be made of one or more films such that any given pouch compartment may include walls made of a single film or multiple films with different compositions. In one aspect, a multi-compartment bag includes at least three walls: an outer upper wall; an outer lower wall; and a partition wall. The outer upper wall and the outer lower wall are generally opposite each other and form the exterior of the bag. The partition wall is inside the bag and is secured to these generally opposite outer walls along a sealing line. The partition wall divides the interior of the multi-compartment bag into at least one first compartment and one second compartment. In one type of embodiment, the partition wall may be the only membrane containing the enzymes, thereby minimizing consumer exposure to these enzymes.

[0487] Bags and pouches can be made using any suitable equipment and methods. For example, individual compartment bags can be made using vertical filling, horizontal filling, or drum filling techniques known in the art. Such processes can be continuous or intermittent. The membrane can be wetted and / or heated to improve its extensibility. The method may also involve using a vacuum to draw the membrane into a suitable mold. The vacuum drawing the membrane into the mold can be applied for about 0.2 to about 5 seconds, or about 0.3 to about 3 seconds, or about 0.5 to about 1.5 seconds, once the membrane is on the horizontal portion of the surface. This vacuum can be such that it provides a pressure, for example, in the range of 10 mbar to 1000 mbar, or in the range of 100 mbar to 600 mbar.

[0488] These molds (in which bags can be made) can have any shape, length, width, and depth, depending on the dimensions required for these bags. These molds can also differ from one another in size and shape, if desired. For example, the final bag volume can be from about 5 ml to about 300 ml, or from about 10 to 150 ml, or from about 20 to about 100 ml, and the size of these molds can be adjusted accordingly.

[0489] In one aspect, the package includes a first and a second sealed compartment. Typically, the second compartment is superimposed on the first sealed compartment, such that the second sealed compartment and the first sealed compartment share a partition wall inside the bag.

[0490] In one aspect, the package, which includes a first and a second compartment, further includes a third sealed compartment. Typically, the third sealed compartment is superimposed on the first sealed compartment, such that the third sealed compartment and the first sealed compartment share a partition wall inside the bag.

[0491] In various respects, the first composition and the second composition are selected from one of the following combinations: liquid, liquid; liquid and powder; powder, powder; and powder and liquid.

[0492] In different respects, the first, second and third compositions are selected from one of the following combinations: solid, liquid, liquid and liquid, liquid, liquid.

[0493] In one aspect, the single compartment or multiple sealed compartments comprise a composition. The multiple compartments may each contain the same or different compositions. The composition is selected from a liquid, a solid, or a combination thereof.

[0494] In this method, heat can be applied to the film, commonly referred to as thermoforming. Heat can be applied by any suitable method. For example, the film can be directly heated before or once it is supplied to a surface by placing it under a heating element or by hot air. Alternatively, for example, it can be indirectly heated by heating the surface or by applying a hot article to the film. Infrared light can be used to heat the film. The film can be heated to at least 50°C, for example, about 50°C to about 150°C, about 50°C to about 120°C, about 60°C to about 130°C, about 70°C to about 120°C, or about 60°C to about 90°C.

[0495] Alternatively, the membrane can be wetted by any suitable means, for example, by spraying a wetting agent (including water, a solution of the membrane composition, a plasticizer for the membrane composition, or any combination thereof) directly onto the membrane before or once it is supplied to a surface, or indirectly by wetting the surface or by applying a wetting article to the membrane.

[0496] Once the film has been heated and / or wetted, it can be drawn into a suitable mold, preferably using a vacuum. For example, the film can be thermoformed at a stretch ratio of at least about 1.5, and optionally up to a stretch ratio of 2. The molding film can be filled using any suitable means. In some embodiments, the most preferred method will depend on the product form and the required filling speed. In some embodiments, the molding film is filled using an in-line filling technique. The filled open bags are then closed using any suitable method with a second film to form a pouch. This can be done while in a horizontal position and in continuous, uniform motion. The closure can be achieved by continuously supplying a second film (preferably a water-soluble film) above and over these open bags, and then preferably sealing the first and second films together, typically in the area between the molds and thus between the bags.

[0497] Any suitable sealing package and / or its individual compartments can be used. Non-limiting examples of such means include heat sealing, solvent welding, solvent sealing, or liquid sealing, and combinations thereof. The water-soluble package and / or its individual compartments can be heat-sealed at a temperature of at least 200°F (93°C), for example, in the range of about 220°F (about 105°C) to about 290°F (about 145°C), or about 230°F (about 110°C) to about 280°F (about 140°C). Typically, only the area to be sealed is treated with heat or solvent. Typically, heat or solvent can be applied to the sealing material by any method, and typically only to the area to be sealed. If solvent sealing, liquid sealing, or welding is used, it is preferable to also apply heat. Preferred liquid sealing or solvent sealing / welding methods involve selectively applying a solvent to the area between molds or to the sealing material, by spraying or printing it onto these areas, and then applying pressure to these areas to form a seal. For example, sealing rollers and belts as described above can be used (optionally, heat is also provided).

[0498] The formed bag can then be cut using a cutting device. Any known method can be used to perform the cutting. Preferably, the cutting can be performed continuously, and preferably at a constant speed and preferably when in a horizontal position. The cutting device can be, for example, a sharp object, a hot object, or a laser, whereby, in the latter case, the hot object or laser “burns” through the film / seal area.

[0499] The different compartments of a multi-compartment bag can be made together in a side-by-side pattern, wherein the resulting one-piece bag can be separated by cutting or can remain together. Alternatively, the compartments can be made separately.

[0500] In some embodiments, the bag may be manufactured according to a method including the following steps:

[0501] a) Forming a first compartment (as described above);

[0502] b) Forming notches in some or all of the closed compartments formed in step (a) to create a second molded compartment superimposed on the first compartment;

[0503] c) The second compartment is filled and sealed by means of a third membrane;

[0504] d) Seal the first, second, and third membranes; and

[0505] e) Cut these membranes to create a multi-compartment bag.

[0506] The notch formed in step (b) can be achieved by applying a vacuum to the compartment prepared in step (a).

[0507] In some embodiments, the second and / or third compartments may be made in separate steps and then combined with the first compartment, as described in EP 2088187 or WO 2009 / 152031.

[0508] In other embodiments, the bag can be made according to a method including the following steps:

[0509] a) Optionally using heat and / or vacuum, a first compartment is formed on a first forming machine using a first membrane;

[0510] b) Fill the first compartment with the first composition;

[0511] c) On a second molding machine, heat and vacuum are optionally used to deform the second film to form a second and optionally a third molded compartment;

[0512] d) Fill the second and optionally third compartment;

[0513] e) Seal the second and optionally third compartments using a third membrane;

[0514] f) Place the sealed second and optionally third compartments onto the first compartment;

[0515] g) Seal the first, second, and optionally third compartments; and

[0516] h) Cut these membranes to create a multi-compartment bag.

[0517] The suitability of the above methods can be selected based on the first and second molding machines. In some embodiments, the first molding machine is preferably a horizontal molding machine, and the second molding machine is preferably a rotary drum molding machine, preferably located above the first molding machine.

[0518] It should be understood that, by using appropriate feed stations, it is possible to manufacture multi-compartment bags incorporating a variety of different or unique compositions and / or different or unique liquid, gel, or paste compositions.

[0519] Method for manufacturing the composition

[0520] The compositions of the present invention can be formulated in any suitable form and can be prepared by any method of choice of the formulation party, non-limiting examples of which are described in the applicant’s examples and in US 4990280; US20030087791 A1; US ​​20030087790 A1; US ​​20050003983 A1; US ​​20040048764 A1; US4762636; US 6291412; US 20050227891 A1; EP 1070115 A2; US 5879584; US 5691297; US5574005; US 5569645; US 5565422; US 5516448; US 5489392; US 5486303, all of which are incorporated herein by reference. The compositions of the present invention, or compositions prepared according to the present invention, comprise cleaning and / or treatment compositions, including but not limited to compositions for treating fabrics, hard surfaces, and any other surfaces in the fields of fabric and home care, including: air care (including air fresheners and odor delivery systems), car care, dishwashing, fabric conditioning (including softening and / or freshening), laundry detergents, laundry and rinsing additives and / or care, hard surface cleaning and / or treatments (including floor and toilet cleaners), general-purpose or “heavy-duty” detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, gel, or paste form, especially so-called heavy-duty liquid types; liquid fine-fabric detergents; hand or light-duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including various tablet, granule, liquid, and rinsing aid types for use in homes and public institutions: car or carpet shampoos, bathroom cleaners (including toilet cleaners); and cleaning aids, such as bleaching additives and “stain-stick” or pretreatment types, matrix-loaded compositions (e.g., sheets with added desiccants). Preferred are compositions and methods for cleaning and / or treating textiles and / or hard surfaces (most preferably textiles). The compositions are preferably used in the pretreatment step or the main washing step of the washing process (most preferably for the textile washing step).

[0521] As used herein, the term "fabric and / or hard surface cleaning and / or treatment composition" is a subset of cleaning and treatment compositions, which, unless otherwise indicated, includes general-purpose or "heavy-duty" detergents in granular or powder form, especially cleaning detergents; general-purpose detergents in liquid, glue, or paste form, especially so-called heavy-duty liquid types; liquid fine fabric detergents; hand or light-duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including various tablet, granular, liquid, and rinsing aid types for use in homes and public institutions; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners (including toilet cleaners); fabric conditioning compositions (including softeners and / or fresheners), which may be in liquid, solid, and / or desiccant sheet form; and matrix-loaded compositions (e.g., desiccant sheets) together with cleaning aids, such as bleach additives and "stain remover sticks" or pretreatment types. All applicable such compositions may be in standard, concentrated, or even highly concentrated forms, even to the point that such compositions may be non-aqueous in some respects.

[0522] How to use

[0523] This invention includes methods for cleaning any surface (including treating textiles or hard or other surfaces) in the fields of fabrics and / or home care. The cleaning described herein can be considered on a small scale (e.g., family household) as well as on a large scale (e.g., in industrial and professional settings). In one aspect of the invention, the method includes a step of contacting the surface to be treated in a pretreatment step or a main washing step of the washing process (most preferably used in textile washing steps or alternatively used in dishwashing (including both manual and automatic / mechanical dishwashing)). In one aspect of the invention, a lipase variant and other components are sequentially added to the method for cleaning and / or treating the surface. Alternatively, the lipase variant and other components are added simultaneously.

[0524] As used herein, washing includes, but is not limited to, scrubbing and mechanical agitation. Washing can be carried out with foam compositions (as described in WO 08 / 101958) and / or by applying alternating pressure (pressure / vacuum) as an additional or alternative method to scrubbing and mechanical agitation. Drying such surfaces or fabrics can be accomplished by any of the common means employed in domestic or industrial environments. The cleaning compositions of the present invention are ideally suited for use in laundry and dishwashing applications. Therefore, the present invention includes a method for cleaning objects (including, but not limited to, fabrics, tableware, knives, and kitchen utensils). The method includes the step of contacting the object to be cleaned with the cleaning composition, which includes at least one aspect of the applicant's cleaning composition, cleaning additives, or mixtures thereof. Fabrics can include virtually any fabric capable of being washed under normal consumer or public institution use conditions. The solution can have a pH from 8 to 10.5. The composition can be used in the solution at concentrations from 500 ppm to 15,000 ppm. The water temperature range is typically from 5°C to 90°C. The water-to-fabric ratio is typically from 1:1 to 30:1.

[0525] In one aspect, the present invention relates to a method using a lipase variant of a parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with SEQ ID NO:2 and has lipase activity, the variant comprising substitutions at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2; and remaining unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2. In another aspect, the present invention relates to the use of this composition for cleaning objects.

[0526] In one aspect, the present invention relates to a method of producing a composition comprising adding a variant of a parental lipase, wherein the variant has at least 60% but less than 100% sequence identity with SEQ ID NO:2 and has lipase activity, the variant comprising substitution at positions corresponding to positions 92 and / or 96 of SEQ ID NO:2; and remaining unchanged at positions corresponding to positions 231, 233, and 254 of SEQ ID NO:2. In one aspect, the present invention relates to a method for cleaning a surface, comprising contacting a lipid stain present on the surface to be cleaned with the cleaning composition. In one aspect, the present invention relates to a method for hydrolyzing stains and / or lipids present on a surface, comprising contacting the stains and / or stains with a cleaning composition. In one aspect, the present invention relates to the use of the composition in the hydrolysis of carboxylic 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 in the manufacture of stable formulations.

[0527] plant

[0528] This invention also relates to plants, such as transgenic plants, plant parts, or plant cells, which include the polynucleotides of this invention to express and produce the variant in a recyclable amount. The variant can be recovered from the plant or plant part. Alternatively, the plant or plant part containing the variant can be used as is to improve the quality of food or feed, for example, to improve nutritional value, palatability, and rheological properties, or to neutralize anti-nutritional factors.

[0529] Genetically modified plants can be dicotyledonous (dicotyledonous plants) or monocotyledonous (monocotyledonous plants). Examples of monocotyledonous plants are grasses, such as meadow grass (bluegrass, Kentucky bluegrass); forage grasses, such as fescue (Festuca) and ryegrass (Lolium); temperate grasses, such as creeping bentgrass (Agrostis); and cereals, such as wheat, oats, rye, barley, rice, sorghum, and corn.

[0530] Examples of dicotyledonous plants include tobacco, legumes (such as lupins, potatoes, sugar beets, peas, beans, and soybeans), and cruciferous plants (such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana).

[0531] Examples of plant parts include stems, callus, leaves, roots, fruits, seeds, and tubers, as well as individual tissues comprising these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, and meristem. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm, are also considered plant parts. Furthermore, any plant cell, regardless of its tissue origin, is considered a plant part. Similarly, plant parts, such as specific tissues and cells isolated to facilitate the use of this invention, are also considered plant parts, such as embryo, endosperm, aleurone, and seed coat.

[0532] Also included within the scope of this invention are such plants, plant parts, and offspring of plant cells.

[0533] Transgenic plants or plant cells expressing variants can be constructed according to methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more expression constructs encoding the variant into the plant host genome or chloroplast genome, and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

[0534] The expression construct is preferably a nucleic acid construct comprising a polynucleotide encoding a variant, which is operatively linked to an appropriate regulatory sequence required for expression of the polynucleotide in a selected plant or plant part. Furthermore, the expression construct may include a selectable marker for identifying plant cells incorporating the expression construct, and the DNA sequence necessary for introducing the construct into the plant in question (the latter depending on the method used to introduce the DNA).

[0535] For example, the selection of regulatory sequences such as promoter and terminator sequences and optional signaling or transport sequences is determined based on when, where, and how the variant is desired to be expressed. For instance, the expression of a gene encoding a variant can be constitutive or inducible, or developmentally, stage-, or tissue-specific, and can target the gene product to a specific tissue or plant part, such as a seed or leaf. Regulatory sequences are described, for example, by Tague et al., 1988, Plant Physiology 86:506.

[0536] For constitutive expression, the 35S-CaMV, maize ubiquitin-1, or rice actin-1 promoters can be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Molecular Biology 18:675-689; Zhang et al., 1991, Plant Cell 3:1155-1165). Organ-specific promoters can be promoters from the following sources: those from storage bank tissues (e.g., seeds, potato tubers, and fruits) (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303); those from metabolic bank tissues (e.g., meristems) (Ito et al., 1994, Plant Molecular Biology 24:863-878); seed-specific promoters, such as glutenin, prolysin, globulin, or albumin promoters from rice (Wu et al., 1998, Plant Cell Physiology 39:885-889); fava bean promoters from ginsenoglobulin B4; and unknown seed protein genes from fava bean (Conrad et al., 1998, Journal of Plant Physiology). Physiol. 152:708-711), promoters from seed oil body proteins (Chen et al., 1998, Plant Cell Physiol. 39:935-941), promoters from the napA storage protein from rapeseed, or any other seed-specific promoters known in the art, for example, as described in WO 91 / 14772. In addition, promoters can be leaf-specific promoters, such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102:991-1000), the Chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26:85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Molecular Genetics and Genomics 248:668-674), or wound-inducible promoters (such as the potato pin2 promoter) (Xu et al., 1993, Plant Molecular Biology 22:573-588).Similarly, the promoter can be induced by abiotic treatments such as temperature, drought or salinity changes, or by exogenous application of substances that activate the promoter (e.g., ethanol; estrogens; plant hormones such as ethylene, abscisic acid and gibberellic acid; and heavy metals).

[0537] Promoter enhancer elements can also be used to achieve higher expression of variants in plants. For example, a promoter enhancer element can be an intron positioned between the promoter and the multinucleotide encoding the variant. For example, Xu et al., 1993, see above, disclosed the use of the first intron of the rice actin 1 gene to enhance expression.

[0538] The selective marker gene and any other part of the expression construct may be selected from those available in the art.

[0539] Nucleic acid constructs can be incorporated into plant genomes using conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, bioprojectile transformation, and electroporation (Gasser et al., 1990, Science 244:1293; Potrykus, 1990, Bio / Technology 8:535; Shimamoto et al., 1989, Nature 338:274).

[0540] Currently, *Agrobacterium tumefaciens*-mediated gene transfer is used to produce transgenic dicotyledonous plants (for a review, see Hooykas and Schilperoort, 1992, *Plant Molecular Biology* 19:15-38) and to transform monocotyledonous plants, although other transformation methods can be used for these plants. Methods for producing transgenic monocotyledonous plants involve bombarding embryonic callus or developing embryos with particles (microscopic gold or tungsten particles coated with transforming DNA) (Christou, 1992, *Plant J.* 2:275-281; ​​Shimamoto, 1994, *Curr. Opin. Biotechnol.* 5:158-162; Vasil et al., 1992, *Bio / Technology* 10:667-674). Alternative methods for transforming monocotyledons are based on protoplast transformation, as described by Omirulleh et al., 1993, Plant Molecular Biology 21:415-428. Other transformation methods include those described in U.S. Patent Nos. 6,395,966 and 7,151,204 (both incorporated herein by reference in their entirety).

[0541] Following transformation, transformants incorporating the expression construct are selected using methods well-known in the art and regenerated into complete plants. Transformation programs are typically designed to selectively eliminate the selection gene during regeneration or in subsequent generations by methods such as co-transformation with two independent T-DNA constructs or by specifically excising the selection gene using specific recombinase sites.

[0542] In addition to directly transforming a specific plant genotype using the constructs of the present invention, transgenic plants can also be produced by hybridizing a plant with the construct with a second plant lacking the construct. For example, a construct encoding a variant can be introduced into a specific plant variety through hybridization, without always directly transforming the plant of that given variety. Therefore, the present invention covers not only plants directly regenerated from cells transformed according to the present invention, but also the offspring of such plants. As used herein, offspring can refer to the offspring of any generation of the parent plant prepared according to the present invention. Such offspring may include the DNA constructs prepared according to the present invention. Hybridization results in the introduction of transgenes into a plant line through cross-pollination between the donor plant line and the starting line. Non-limiting examples of such steps are described in U.S. Patent No. 7,151,204.

[0543] Plants can be generated through backcross transformation. For example, plants include genotypes, lineages, inbreds, or hybrids that have undergone backcross transformation.

[0544] Genetic markers can be used to assist the introgression of one or more transgenes of the present invention from one genetic background to another. Marker-assisted selection offers advantages over conventional breeding in that it can be used to avoid errors caused by phenotypic variation. Furthermore, genetic markers can provide data on the relative degree of superior germplasm in individual offspring of a specific hybrid. For example, when a plant with the desired trait and an additional genetic background not agronomically desired is crossed with a superior parent, genetic markers can be used to select offspring that not only possess the trait of interest but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required for the introgression of one or more traits into a specific genetic background is minimized.

[0545] The present invention also relates to methods for producing variants of the invention, the methods comprising: (a) culturing a transgenic plant or a plant cell comprising a polynucleotide encoding the variant under conditions favorable to producing the variant; and (b) recovering the variant.

[0546] The invention described and claimed herein is not limited to the specific aspects disclosed herein, as these aspects are intended to illustrate several aspects of the invention. Any equivalent aspects are contemplated within the scope of the invention. In fact, various modifications to the invention, other than 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 case of conflict, the disclosure including the definition shall prevail.

[0547] Example

[0548] Example 1: Measurement

[0549] Determination of p-nitrophenyl (pNP):

[0550] The hydrolytic activity of lipases was determined by kinetic assays using p-nitrophenyl acyl ester as a substrate.

[0551] Dilute the following substrates in 100 mM stock solutions in DMSO to a final concentration of 1 mM 25 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 laurylate (C12), and p-nitrophenyl palmitate (C16) (all from Sigma-Aldrich Danmark A / S, Kirkebjerg Allé 84, 2605 Brøndby). Catalog numbers: C3: N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752.

[0552] The lipase of the present invention, the parental lipase, and appropriate controls, such as buffer (negative), Lipolase™ & Lipex™ (positive), were added to the substrate solution in a 96-well NUNC plate (catalog number: 260836, Kamstrupvej 90, DK-4000, Roskilde) at the following final concentrations: 0.01 mg / ml; 5 x 10⁻³ mg / ml; 2.5 x 10⁻⁴ mg / ml; and 1.25 x 10⁻⁴ mg / ml. On a Spectra max190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberlach am Ries, Germany), the release of p-nitrophenol from the hydrolysis of p-nitrophenylyl can be monitored at 405 nm for 5 minutes at 10-second intervals. The hydrolytic activity of the variants against one or more substrates can be compared with the hydrolytic activity of the parent lipase.

[0553] Standard determination:

[0554] The specific activities of reference lipase and lipase variants were measured in a 96-well microtiter plate (MTP) at 25°C using 4 μg / mL of enzyme in the final assay volume. The assay plate was prepared by coating the bottom of each well with 100 nmol olive oil (CAS No. 8001-25-0) and 100 nmol pNP-decanoate (CAS No. 1956-09-8) dissolved in 99% hexane and evaporating in a fume hood with the lights off for 2 hours. 180 μL of buffer, 100 mM Tris pH 8.5, and 2 mM CalCl2 or 2 mM EDTA were transferred to the assay plate. The reaction was initiated by adding 20 μL of each enzyme dilution to the assay plate. Immediately place the plate in a Spectra max 190 (Molecular Devices GmbH, Bismarckring 39, 88400 Biberlach am Ries, Germany), shake for 5 seconds, and absorb at 405 nm for the following 10 minutes. Using readily available Spectra max 190 plates from Molecular Devices, activity is given as the change in absorbance (mAU / min) over 5 minutes. Calculate the mean and standard deviation using triplicate.

[0555] Example 2: Lipase activity and Ca-dependence

[0556] The activities of the lipase variant of the present invention, the reference lipase, and the prior art lipase were determined according to the standard assays described in Example 1. The activities of the lipase in the presence of CalCl2 (Ca) and EDTA (EDTA) are shown in the table below along with their respective standard deviations (Ca SD & EDTA SD). In the presence of EDTA, the concentration of Ca is reduced, i.e., low.

[0557] The ratio of lipase activity in the presence of EDTA and Ca (EDTA / Ca) is calculated and presented as a percentage (%). This ratio reflects the percentage of lipase activity at reduced / low levels of Ca in the presence of Ca. A 100% EDTA / Ca ratio indicates that lipase activity is independent of the presence of Ca, while a low percentage indicates that lipase activity is dependent on the presence of Ca.

[0558] Table 1: Ca-dependent lipase

[0559]

[0560]

[0561] Table 2: Ca-dependence of lipases

[0562]

[0563] sequence list <110> Novozymes A / S <120> Lipase variants and the polynucleotides that encode them <130> 13015-WO-PCT <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 807 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> CDS <222> (1)..(807) <400> 1 gag gtc tcg cag gat ctg ttt aac cag ttc aat ctc ttt gca cag tat 48 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 tct gca gcc gca tac tgc gga aaa aac aat agg gcc cca gct ggt aca 96 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Arg Ala Pro Ala Gly Thr 20 25 30 aac att acg tgc acg gcc aat gcc tgc ccc gag gta gag aag gcg gat 144 Asn Ile Thr Cys Thr Ala Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 gca acg ttt ctc tac tcg ttt gaa gac tct gga gtg ggc gat gtc acc 192 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 ggc ttc ctt gct ctc gac aac acg aac aaa ttg atc gtc ctc tct ttc 240 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 cgt ggc tct cgt tcc ata gag aac tgg atc ggg aat ctt aac ttc gag 288 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 ttg aaa gaa ata aat gac att tgc tcc ggc tgc agg gga cat gcc ggc 336 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 ttc act tcg tcc tgg agg tct gta gcc gat acg tta agg cag aag gtg 384 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 gag gat gct gtg agg gag cat ccc gac tat cgc gtg gtg ttt acc gga 432 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 cat agc ttg ggt ggt gca ttg gca act gtt gcc gga gca gac ctg cgt 480 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 gga aat aag tat gat atc gac gtg ttt tca tat ggc gcc ccc cga gtc 528 Gly Asn Lys Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 gga aac agg gct ttt gca gaa ttc ctg acc gta cag acc ggc gga aca 576 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 ctc tac cgc att acc cac acc aat gat att gtc cct aga ctc ccg ccg 624 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 cgc gaa ttc ggt tac agc cat tct agc cca gaa tac tgg atc aaa tct 672 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 gga acc ctt gtc ccc gtc cgg cga cga gac atc gtg aag ata gaa ggc 720 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 atc gat gcc acc ggc ggc aat aac cag cct aac att ccg tcc atc acc 768 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Thr 245 250 255 gcg cac cta tgg tac ttc ggg tta att ggg aca tgt ctt 807 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 2 <211> 269 <212> PRT <213> artificial sequence <220> <223> Synthesis structure <400> 2 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Arg Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Ala Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Lys Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Thr 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 3 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> CDS <222> (1)..(807) <400> 3 gag gtc tcg cag gat ctg ttt aac cag ttc aat ctc ttt gca cag tat 48 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 tct gca gcc gca tac tgc gga aaa aac aat gat gcc cca gct ggt aca 96 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 aac att acg tgc acg gga aat gcc tgc ccc gag gta gag aag gcg gat 144 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 gca acg ttt ctc tac tcg ttt gaa gac tct gga gtg ggc gat gtc acc 192 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 ggc ttc ctt gct ctc gac aac acg aac aaa ttg atc gtc ctc tct ttc 240 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 cgt ggc tct cgt tcc ata gag aac tgg atc ggg aat ctt aac ttc gag 288 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 ttg aaa gaa ata aat gac att tgc tcc ggc tgc agg gga cat gcc ggc 336 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 ttc act tcg tcc tgg agg tct gta gcc gat acg tta agg cag aag gtg 384 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 gag gat gct gtg agg gag cat ccc gac tat cgc gtg gtg ttt acc gga 432 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 cat agc ttg ggt ggt gca ttg gca act gtt gcc gga gca gac ctg cgt 480 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 gga aat ggg tat gat atc gac gtg ttt tca tat ggc gcc ccc cga gtc 528 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 gga aac agg gct ttt gca gaa ttc ctg acc gta cag acc ggc gga aca 576 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 ctc tac cgc att acc cac acc aat gat att gtc cct aga ctc ccg ccg 624 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 cgc gaa ttc ggt tac agc cat tct agc cca gaa tac tgg atc aaa tct 672 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 gga acc ctt gtc ccc gtc cgg cga cga gac atc gtg aag ata gaa ggc 720 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 atc gat gcc acc ggc ggc aat aac cag cct aac att ccg tcc atc cct 768 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Pro 245 250 255 gcg cac cta tgg tac ttc ggg tta att ggg aca tgt ctt 807 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 4 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 4 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Ala Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 5 <211> 807 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <220> <221> CDS <222> (1)..(807) <400> 5 gag gtc tcg cag gat ctg ttt aac cag ttc aat ctc ttt gca cag tat 48 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 tct gca gcc gca tac tgc gga aaa aac aat gat gcc cca gct ggt aca 96 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 aac att acg tgc acg gga aat gcc tgc ccc gag gta gag aag gcg gat 144 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 gca acg ttt ctc tac tcg ttt gaa gac tct gga gtg ggc gat gtc acc 192 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 ggc ttc ctt gct ctc gac aac acg aac aaa ttg atc gtc ctc tct ttc 240 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 cgt ggc tct cgt tcc ata gag aac tgg atc ggg aat ctt aac ttc gag 288 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 ttg aaa gaa ata aat gac att tgc tcc ggc tgc agg gga cat gac ggc 336 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 ttc act tcg tcc tgg agg tct gta gcc gat acg tta agg cag aag gtg 384 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 gag gat gct gtg agg gag cat ccc gac tat cgc gtg gtg ttt acc gga 432 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 cat agc ttg ggt ggt gca ttg gca act gtt gcc gga gca gac ctg cgt 480 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 gga aat ggg tat gat atc gac gtg ttt tca tat ggc gcc ccc cga gtc 528 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 gga aac agg gct ttt gca gaa ttc ctg acc gta cag acc ggc gga aca 576 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 ctc tac cgc att acc cac acc aat gat att gtc cct aga ctc ccg ccg 624 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 cgc gaa ttc ggt tac agc cat tct agc cca gaa tac tgg atc aaa tct 672 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 gga acc ctt gtc ccc gtc cgg cga cga gac atc gtg aag ata gaa ggc 720 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 atc gat gcc acc ggc ggc aat aac cag cct aac att ccg tcg atc cct 768 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Pro 245 250 255 gcg cac cta tgg tac ttc ggg tta att ggg aca tgt ctt 807 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265 <210> 6 <211> 269 <212> PRT <213> artificial sequence <220> <223> Synthetic construct <400> 6 Glu Val Ser Gln Asp Leu Phe Asn Gln Phe Asn Leu Phe Ala Gln Tyr 1 5 10 15 Ser Ala Ala Ala Tyr Cys Gly Lys Asn Asn Asp Ala Pro Ala Gly Thr 20 25 30 Asn Ile Thr Cys Thr Gly Asn Ala Cys Pro Glu Val Glu Lys Ala Asp 35 40 45 Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr 50 55 60 Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe 65 70 75 80 Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Glu 85 90 95 Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly 100 105 110 Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val 115 120 125 Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly 130 135 140 His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg 145 150 155 160 Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val 165 170 175 Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr 180 185 190 Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro 195 200 205 Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser 210 215 220 Gly Thr Leu Val Pro Val Arg Arg Arg Asp Ile Val Lys Ile Glu Gly 225 230 235 240 Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Ser Ile Pro 245 250 255 Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu 260 265

Claims

1. A variant of a parental lipase, wherein the variant has lipase activity; and remains unchanged at positions 231, 233, and 254 of SEQ ID NO: 2; wherein the amino acid residues at positions 231, 233, and 254 of SEQ ID NO: 2 are Arg, Arg, and Ser, respectively; and wherein the following substitutions are made based on SEQ ID NO: 2: a. S83T E87K G91L N92D F95Y E96T L97P K98Q; b. R81Q S83T R84H S85T I86L E87T N88Q I90M G91A N92K L93F N94K F95A E96LK98Q; c. S83T E87A G91A N94D E96I L97P K98D; d. I86P E87A G91A N92D F95Y E96T L97P K98Q I252T I255T; e. I86W E87A G91A N92D F95Y E96T K98Q I252T I255T; f. I86W E87A G91A N92D F95Y E96T L97P K98Q I252T I255T; g. I86P E87A G91A N92D L93F F95Y E96T L97P K98Q I252T I255T; h. I86P E87T G91L N94D E96I L97F K98D; j. S83T G91A N92D E96T; k. S83T G91A N92D F95Y E96L L97P K98Q; l. S83T I86P G91A N92D E96T K98Q; m. F51V I86P E87A G91A N92D F95Y E96T L97P K98Q T252I T255I; o. E87T G91A N92D N94D E96L K98Q; p. E87K G91A N92D N94D E96L K98Q; q. S83T E87T G91A N92D F95Y E96T L97P K98Q; r. S83T I86W G91L N92D F95Y; s. S83T I86P G91A N92D E96L L97P K98Q; v. S83T F95Y E96L; w. S83T I86W G91L N92D F95Y E96T; z. S83T I86P G91A N92D E96L K98Q; bb. S83T I86P G91L N92D F95Y E96T; cc. S83T G91L N92D K98Q P136H; dd. I86P G91L N92K L97P; ee. N33K F51V S83T I86P G91T N92D N94D F95A E96L K98Q; ff. S83T I86P E87T G91A N92D F95Y E96T K98Q; ii. I86P E87T G91A N92D E96L L97P K98Q; jj. E87A G91L N92D L97P K98E E96D; mm. S83T I86P G91A N92D F95Y E96T; oo. N33K F51V S83T R84H G91A N92D L93F F95A E96L K98E; uu. N33K F51V S83T I86P I90F G91L N92D L93F E96L L259F; vv. N33K F51V L93I N94R F95V E96P E99T; ww. N33K F51V L93I N94R F95V E96H; xx. N33K F51V L93W N94R F95V E96P; and yy. E56K S83T G91A N92D N94D E96L K98Q E239P K163G.

2. The variant of claim 1, wherein the variant has at least one improved property compared to the parental lipase, said property being selected from the group consisting of: Increased lipase activity; Increased lipase activity in reduced / low Ca levels; increased lipase activity in the presence of EDTA; and increased Ca-independence.

3. A composition comprising any one of the variants described in claims 1-2.

4. The composition of claim 3, wherein the composition is a detergent composition.

5. The detergent composition of claim 4, further comprising a surfactant or surfactant system, wherein the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, facultative zwitterionic surfactants, semipolar nonionic surfactants, and mixtures thereof.

6. The detergent composition of claim 5, wherein the surfactant is present at a level ranging from 0.1 wt% to 60 wt%, from 0.2 wt% to 40 wt%, from 0.5 wt% to 30 wt%, from 1 wt% to 50 wt%, from 1 wt% to 40 wt%, from 1 wt% to 30 wt%, from 1 wt% to 20 wt%, from 3 wt% to 10 wt%, from 3 wt% to 5 wt%, from 5 wt% to 40 wt%, from 5 wt% to 30 wt%, from 5 wt% to 15 wt%, from 3 wt% to 20 wt%, from 3 wt% to 10 wt%, from 8 wt% to 12 wt%, from 10 wt% to 12 wt%, from 20 wt% to 25 wt%, or from 25 wt% to 60 wt%.

7. The detergent composition of claim 5, comprising anionic surfactant.

8. The detergent composition of claim 5, wherein the anionic surfactant is a sulfate or sulfonate surfactant.

9. The detergent composition of claim 8, wherein the sulfonate surfactant is selected from the group consisting of alkylbenzene sulfonates.

10. The detergent composition of claim 9, wherein the sulfonate surfactant is C 10-13 Alkylbenzene sulfonates, including alkylbenzene sulfonates (LAS).

11. The detergent composition of claim 8, wherein the sulfate surfactant is selected from the group consisting of alkyl sulfates.

12. The detergent composition of claim 11, wherein the alkyl sulfate is C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfates.

13. The detergent composition of claim 8, wherein the sulfate surfactant is selected from the group consisting of alkylalkoxylated sulfates.

14. The detergent composition of claim 13, wherein the alkylalkoxylated sulfate is an alkylethoxylated sulfate.

15. The detergent composition of claim 14, wherein the alkylalkoxylated sulfate is C 8-18 Alkyl alkoxylated sulfates.

16. The detergent composition of claim 14, wherein the alkylalkoxylated sulfate is C 8-18 Alkyl ethoxylated sulfates.

17. The detergent composition of claim 14, wherein the alkylalkoxylated sulfate has an average degree of alkoxylation from 0.5 to 20 or from 0.5 to 10, or wherein the alkylalkoxylated sulfate is a C44 having an average degree of ethoxylation from 0.5 to 10, from 0.5 to 7, from 0.5 to 5, or from 0.5 to 3. 8-18 Alkyl ethoxylated sulfates.

18. The detergent composition of any one of claims 7-17, wherein the composition comprises linear or branched, substituted or unsubstituted alkyl sulfates, alkylalkoxylated sulfates, and alkylbenzene sulfonates.

19. The detergent composition of claim 5, wherein the surfactant is a medium-chain branched surfactant.

20. The detergent composition of claim 19, wherein the surfactant is a medium-chain branched anionic surfactant.

21. The detergent composition of claim 20, wherein the surfactant is a medium-chain branched alkyl sulfate and / or a medium-chain branched alkylbenzene sulfonate.

22. The detergent composition of claim 21, wherein the surfactant is a medium-chain branched alkyl sulfate.

23. The detergent composition of claim 22, wherein the medium-chain branch is C 1-4 alkyl.

24. The detergent composition of claim 23, wherein the medium-chain branch is methyl and / or ethyl.

25. The detergent composition of claim 5, wherein the anionic surfactant is selected from sulfates and sulfonates, particularly linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, and alkene sulfonates. sulfonates), alkanes-2,3-dimethylbis(sulfates), hydroxyalkane sulfonates and disulfonates, alkyl sulfates (AS), 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), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

26. The detergent composition of claim 25, wherein the alkyl sulfate (AS) is sodium dodecyl sulfate (SDS).

27. The detergent composition of claim 5, wherein the nonionic surfactant is selected from the group consisting of C8-C6. 18 Alkyl ethoxylates; C6-C 12 Alkylphenol alkoxylates, wherein the alkoxyl unit may be an ethyleneoxy unit, an propyleneoxy unit, or a mixture thereof; C 12 -C 18 Alcohols and C6-C 12 Condensation products of alkylphenols and ethylene oxide / propylene oxide block polymers; C 14 -C 22 Medium-chain branched alcohols; C 14 -C 22 Medium-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation from 1 to 30; alkyl polysaccharides, in one respect as alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

28. The detergent composition of claim 27, wherein the nonionic surfactant is NEODOL®.

29. The detergent composition of claim 27, wherein the nonionic surfactant is Pluronic®.

30. The detergent composition of claim 27, wherein the nonionic surfactant comprises alkyl polysaccharide and / or alkyl alkoxylated alcohol.

31. The detergent composition of claim 5, wherein the nonionic surfactant is selected from the group consisting of alkylalkoxylated alcohols.

32. The detergent composition of claim 31, wherein the alkylalkoxylated alcohol is C 8-18 Alkyl alkoxylated alcohols.

33. The detergent composition of claim 32, wherein the alkylalkoxylated alcohol is C 8-18 Alkyl ethoxylated alcohols.

34. The detergent composition of claim 32, wherein the alkylalkoxylated alcohol may have an average degree of alkoxylation ranging from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10.

35. The detergent composition of claim 32, wherein the alkylalkoxylated alcohol may be a C10 having an average degree of ethoxylation of 1 to 10, 1 to 7, more often 1 to 5, or 3 to 7. 8-18 Alkyl ethoxylated alcohols.

36. The detergent composition of claim 32, wherein the alkylalkoxylated alcohol may be linear or branched, and may be substituted or unsubstituted.

37. The detergent composition of claim 32, wherein the nonionic surfactant comprises Lutensol®.

38. The detergent composition of claim 5, wherein the nonionic surfactant is selected from the group consisting of alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA) or fatty acid glucosamide (FAGA)), together with products available under the trade names SPAN and TWEEN, and combinations thereof.

39. The detergent composition of claim 38, wherein the alkoxylated fatty acid alkyl ester is an ethoxylated and / or propoxylated fatty acid alkyl ester.

40. The detergent composition of claim 5, wherein the cationic surfactant is selected from the group consisting of alkylpyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds, and mixtures thereof.

41. The detergent composition of claim 5, wherein the cationic surfactant is a quaternary ammonium compound having the following general formula: (R)(R1)(R2)(R3)N + X - Where R is a straight or branched, substituted or unsubstituted C 6-18 The alkyl or alkenyl moiety, R1 and R2 are independently selected from the methyl or ethyl moiety, R3 is a hydroxy, hydroxymethyl, or hydroxyethyl moiety, and X is an anion that provides charge neutrality. Suitable anions include halides; sulfates; and sulfonates. Suitable cationic detergency surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, a highly suitable cationic detergency surfactant is mono-C 8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C 10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride.

42. The detergent composition of claim 41, wherein the halide is a chloride.

43. The detergent composition of claim 5, wherein the cationic surfactant is selected from the group consisting of alkyldimethylethanolamine quaternary ammonium salt (ADMEAQ), hexadecyltrimethylammonium bromide (CTAB), dimethyldistearate ammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium compounds, and combinations thereof.

44. The detergent composition of claim 5, wherein the amphoteric / zombie surfactant is selected from the group consisting of amine oxides and betaine, or combinations thereof.

45. The detergent composition of claim 44, wherein the betaine is alkyl dimethyl betaine, sulfobetaine, or a combination thereof.

46. ​​The detergent composition of claim 5, wherein the semi-polar surfactant is selected from the group consisting of amine oxides (AO).

47. The detergent composition of claim 46, wherein the semi-polar surfactant is an alkyl dimethylamine oxide.

48. The detergent composition of claim 5, wherein the surfactant system comprises one or more anionic surfactants and one or more nonionic surfactants, and optionally a mixture with other surfactants.

49. The detergent composition of claim 48, wherein the additional surfactant is a cationic surfactant.

50. The detergent composition of claim 48, wherein the weight ratio of the anionic surfactant to the nonionic surfactant is at least 2:1, or at least 1:1 to 1:

10.

51. The detergent composition of claim 4 or 5, comprising linear alkylbenzene sulfonic acid (LAS), sodium lauryl ether sulfate (SLES), and alcohol ethoxylate (AEO).

52. The detergent composition of claim 4, further comprising one or more enzymes that provide cleaning performance and / or fabric care benefits.

53. The detergent composition of claim 52, wherein the one or more enzymes are selected from the group consisting of hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, keratinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenol oxidase, lipoxygenase, ligninase, amylopectinase, tannic acidase, pentosanase, malanase, β-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase, or mixtures thereof.

54. The detergent composition of claim 52, wherein the one or more enzymes include protease, lipase, and amylase.

55. The detergent composition of any one of claims 52-54, wherein the one or more enzymes are present at a level of enzyme protein from 0.00001 wt% to 2 wt%, from 0.0001 wt% to 1 wt%, or from 0.001 wt% to 0.5 wt% based on the weight of the composition.

56. Use of the variants of any one of claims 1-2 for hydrolyzing lipase substrates.

57. A method for cleaning a surface, the method comprising the step of contacting the surface with a variant as described in any one of claims 1-2.

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

59. A nucleic acid construct comprising the polynucleotide as described in claim 58.

60. An expression vector comprising the polynucleotide as described in claim 58.

61. A host cell comprising the polynucleotide of claim 58; the nucleic acid construct of claim 59; or the expression vector of claim 60, wherein the host cell is not a plant cell.

62. A method for producing a lipase variant, the method comprising: (a) Culture the host cells as described in claim 61 under conditions suitable for the expression of the variant; And (b) recycle the variant.

63. A method for obtaining a lipase variant as claimed in any one of claims 1-2, the method comprising introducing a substitution into the parental lipase at one or more positions corresponding to position 92 and / or 96 of SEQ ID NO: 2, wherein the variant has lipase activity; and recovering the variant.

64. The method of claim 63, wherein the substitution at position 92 is performed using Asp or Lys.

65. The method of claim 63, wherein the substitution at position 96 is performed using Ile, Leu, or Thr.

66. The method of any one of claims 63-65, wherein the substitution for the parental lipase is selected from the group consisting of: N92D, N92K, E96I, E96L, E96T, N92D+E96I, N92D+E96L, N92D+E96T, N92K+E96I, N92K+E96L, and N92K+E96T.

67. The method of claim 63, wherein the variant further comprises one or more substitutions corresponding to positions 81-99 of SEQ ID NO: 2, and wherein the substitutions are selected from the following: R81Q; S83T; R84H; S85T; I86L / P / W; E87A / K / T; N88Q; I90M; G91A / L; L93F; N94D / K; F95A / Y; L97F / P; and K98D / Q.

68. The method of claim 63, wherein the variant further comprises one or more substitutions at positions corresponding to positions 51, 136, 252 and 255 of SEQ ID NO: 2, and wherein the substitutions are selected from the following: F51V, P136H, I252T and I255T.

69. The method of any one of claims 63-68, wherein the following substituents are introduced into the parental lipase: a.S83T E87K G91L N92D F95Y E96T L97P K98Q; b.R81Q S83T R84H S85T I86L E87T N88Q I90M G91A N92K L93F N94K F95A E96LK98Q; c.S83T E87A G91A N94D E96I L97P K98D; d.I86P E87A G91A N92D F95Y E96T L97P K98Q I252T I255T; e.I86W E87A G91A N92D F95Y E96T K98Q I252T I255T; f.I86W E87A G91A N92D F95Y E96T L97P K98Q I252T I255T; g.I86P E87A G91A N92D L93F F95Y E96T L97P K98Q I252T I255T; h.I86P E87T G91L N94D E96I L97F K98D; j.S83T G91A N92D E96T; k.S83T G91A N92D F95Y E96L L97P K98Q; l.S83T I86P G91A N92D E96T K98Q; m.F51V I86P E87A G91A N92D F95Y E96T L97P K98Q T252I T255I; o.E87T G91A N92D N94D E96L K98Q; p.E87K G91A N92D N94D E96L K98Q; q.S83T E87T G91A N92D F95Y E96T L97P K98Q; r.S83T I86W G91L N92D F95Y; s.S83T I86P G91A N92D E96L L97P K98Q; v.S83T F95Y E96L; w.S83T I86W G91L N92D F95Y E96T; z.S83T I86P G91A N92D E96L K98Q; bb.S83T I86P G91L N92D F95Y E96T; cc.S83T G91L N92D K98Q P136H; dd.I86P G91L N92K L97P; ee.N33K F51V S83T I86P G91T N92D N94D F95A E96L K98Q; ff.S83T I86P E87T G91A N92D F95Y E96T K98Q; ii.I86P E87T G91A N92D E96L L97P K98Q; jj.E87A G91L N92D L97P K98E E96D; mm.S83T I86P G91A N92D F95Y E96T; oo.N33K F51V S83T R84H G91A N92D L93F F95A E96L K98E; uu.N33K F51V S83T I86P I90F G91L N92D L93F E96L L259F; vv.N33K F51V L93I N94R F95V E96P E99T; ww.N33K F51V L93I N94R F95V E96H; xx.N33K F51V L93W N94R F95V E96P; and yy.E56K S83T G91A N92D N94D E96L K98Q E239P K163G.

70. The method of claim 63, wherein the variant has at least one improved property compared to the parental lipase, said property being selected from the group consisting of: Increased lipase activity; Increased lipase activity in reduced / low Ca levels; increased lipase activity in the presence of EDTA; and increased Ca-independence.

Images