Lipase variants and compositions comprising such lipase variants

Abstract

The present invention relates to lipase variants having reduced activity at approximately neutral pH compared to the parent. The invention also relates to compositions comprising the lipase variants of the invention; a polynucleotide encoding the lipase variant of the invention; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing and using the variants for cleaning.

Description

[0001] References to sequence listings

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Technical Field

[0004] The present invention relates to lipase variants, compositions comprising the lipase variants of the present invention, polynucleotides encoding the variants of the present invention, nucleic acid constructs comprising the polynucleotides of the present invention, expression vectors comprising the polynucleotides or nucleic acid constructs of the present invention, host cells comprising the nucleic acid constructs or expression vectors of the present invention. Finally, the present invention relates to methods for cleaning surfaces using the variants or compositions of the present invention, methods for hydrolyzing lipase substrates using the lipase variants or compositions of the present invention, methods for washing laundry using the variants or compositions of the present invention, and methods for producing the variants of the present invention. Background Art

[0005] Lipases are important biocatalysts that have been shown to be useful for various applications. Variants of wild-type Thermomyces lanuginosus lipase (synonymous with Humicola lanuginosa) have been commercialized as active ingredients in detergent compositions for the removal of lipid stains by hydrolyzing triglycerides to produce fatty acids.

[0006] Detergent, cleaning and / or fabric care compositions contain active ingredients that interfere with the ability of lipases to remove lipid stains. Many known Thermomyces lanuginosus lipase variants with good wash performance form odor-producing short-chain fatty acids during the wash and / or have short storage stability.

[0007] WO 2016 / 050661 (Novozymes) relates to Thermomyces lanuginosus lipase variants with reduced odor production, wherein the lipase variants comprise substitutions at positions corresponding to position 210 (which is not a negatively charged amino acid) and position 255 (which is not I), and wherein position 256 is not K.

[0008] WO 2017 / 001673 (Novozymes) discloses Thermomyces lanuginosus lipase variants with reduced odor production, wherein the lipase variants comprise one or more substitutions selected from the group consisting of F7H / K / R, F51A / I / L / V / Y, T143A / G / S / V, A150G / V, H198A / D / E / F / G / I / L / N / Q / S / T / V / Y, N200H / K / Q / R, I202G / L / V, S224C / F / H / I / L / P / Y, L2 27D / E / K / R, V228P, P229H / K / R, V230H / K / L / R, I255A / G / N / P / S / T / V / Y, P256A / K / N / Q / R / S / T / W, A257F / H / I / L / V / Y, L259F / Y, and W260D / E / F / H / I / L / N / Q / S / T / Y, or selected from H198A / D / E / F / G / I / L / N / Q / S / T / V / Y, F7H / K / R, F51A / I / L / V / Y, T143A / G / S / V, A150G / V, N200H / K / Q / R, I202G / L / V, S224C / F / H / I / L / P / Y, L227D / E / Substitutions of: K / R, V228P, P229H / K / R, V230H / K / L / R, I255A / G / N / P / S / T / V / Y, T256A / K / N / Q / R / S / P / W, A257F / H / I / L / V / Y, L259F / Y, and W260D / E / F / H / I / L / N / Q / S / T / Y.

[0009] Despite the progress made, there remains a need and desire for lipases with improved properties. Summary of the Invention

[0010] An important object of the present invention is to provide a lipase variant with reduced lipase activity at a pH around neutral (i.e., about pH 6-8, particularly about pH 7). The reduced activity results in a reduction in the odor produced by the lipase variant that hydrolyzes short-chain lipid substrates. At a higher pH, i.e., about pH 8-11, the odor produced by the lipase variant is higher than that at a pH around neutral. Such a lipase variant can be advantageously used, for example, to clean clothes. During the washing of clothes, the pH of the washing solution is higher (e.g., pH 8-11), while the pH of the rinse water during the subsequent rinse cycle is about neutral, i.e., pH 6-8. Therefore, the lipase variant of the present invention alleviates the odor generation problem that occurs during the wash cycle at high pH by reducing the odor generation during the rinse cycle when the pH is relatively low (i.e., about neutral).

[0011] The present invention relates to an isolated lipase variant selected from one or more of the groups (i), (ii) and (iii) comprising:

[0012] (i) substitutions at one or more of positions 202, 252, and 269 of the polypeptide corresponding to SEQ ID NO: 8;

[0013] (ii) substitutions at one or more of positions 40, 56, 57, 91, 98, 108, 118, 210, 244, and 254 of the polypeptide corresponding to SEQ ID NO: 8; and

[0014] (iii) substitutions at one or more of positions 23, 27, 40, 51, 56, 60, 118, 244, and 256 of the polypeptide corresponding to SEQ ID NO: 8;

[0015] wherein the variant has lipase activity, and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 8, wherein the variant optionally comprises one or more amino acid extensions at the N-terminus and / or C-terminus, or one or more amino acid truncations at the N-terminus and / or C-terminus, and wherein the variant has lipase activity.

[0016] In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i). In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (ii). In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i), and one or more substitutions, particularly all substitutions, from group (ii). In another preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i), and one or more substitutions, particularly all substitutions, from group (iii).

[0017] In one aspect, the present invention relates to particles comprising:

[0018] (a) a core comprising a variant of the invention, and, optionally

[0019] (b) A coating consisting of one or more layers surrounding the core.

[0020] In another aspect, the invention relates to liquid compositions comprising a variant of the invention and an enzyme stabilizer, e.g. a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boronic acid or a boronic acid derivative, e.g. an aromatic boronic ester or a phenylboronic acid derivative such as 4-formylphenylboronic acid.

[0021] The present invention also relates to a composition comprising a variant of the invention, a particle of the invention, or a liquid composition of the invention.In a preferred embodiment, the composition comprises one or more surfactants.

[0022] The present invention also relates to a polynucleotide encoding a variant of the present invention.

[0023] In one aspect, the present invention relates to a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The present invention also relates to a recombinant host cell transformed with a polynucleotide of the present invention. In one aspect, the present invention relates to a method for producing a lipase variant of the present invention, the method comprising:

[0024] a. culturing the recombinant host cell of the present invention under conditions suitable for expressing the variant; and

[0025] b. Recover the variant.

[0026] The present invention also relates to a method for hydrolyzing a lipase substrate, the method comprising admixing the substrate with a lipase variant of the present invention or a composition of the present invention under conditions conducive for hydrolysis of the substrate by the lipase variant of the present invention.

[0027] In another aspect, the present invention relates to methods for removing lipid staining materials from surfaces, the methods comprising contacting the lipid staining material with a lipase variant of the present invention or a composition of the present invention under conditions conducive for the lipase variant of the present invention to hydrolyze the lipid staining material.

[0028] The present invention also relates to methods for removing lipid stains from surfaces, these methods comprising contacting the stain with a lipase variant of the present invention or a composition of the present invention, followed by rinsing the surface and optionally drying.

[0029] The present invention also relates to methods for removing lipid stains from surfaces, which methods comprise contacting the stain with a lipase variant of the present invention or a composition of the present invention, followed by rinsing and optionally drying, in which method, odor development is reduced when compared to a method wherein a parent lipase (in particular one of SEQ ID NO: 2, 4, 6 or 8, respectively) is contacted with the stain.

[0030] Finally, the present invention also relates to the use of a lipase variant according to the invention or a composition according to the invention for cleaning a surface, which use comprises applying the lipase variant to the surface to be cleaned, followed by rinsing and optionally drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 An alignment of the parent lipases of SEQ ID NO: 2 (wild-type Thermomyces lanuginosus lipase), SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8 using the Clustal Omega (1.2.4) multiple sequence alignment software available on the European Bioinformatics Institute webpage of EMBL (www.ebi.ac.uk).

[0032] definition

[0033] In light of this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0035] Lipase: The terms "lipase," "lipase enzyme," "lipolytic enzyme," "lipid esterase," "lipolytic polypeptide," and "lipolytic protein" refer to an enzyme in class EC 3.1.1 as defined in enzyme nomenclature. It may have lipase activity (triacylglycerol lipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74), sterol esterase activity (EC 3.1.1.13), and / or wax ester hydrolase activity (EC 3.1.1.50).

[0036] Lipase activity: For the purposes of the present invention, lipase activity (ie, the hydrolytic activity of the lipase) can be determined with a pNP assay using substrates with different chain lengths as described in the Examples.

[0037] In one aspect, the variants of the invention have at least 20%, e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the lipase activity of the parent lipase. In one aspect, the parent lipase is the polypeptide of SEQ ID NO: 8, or a fragment thereof having lipase activity. SEQ ID NO: 8 is identical to the wild-type Thermomyces lanuginosus lipase set forth in SEQ ID NO: 2 having T231R+N233R substitutions.

[0038] Benefit-Risk Factor: The benefit-risk factor (BRF) describes the wash performance (benefit) compared to the odor release (risk) and is defined as RP(wash) / RP(odor). If the benefit-risk factor of a lipase variant is greater than 1.0, the lipase has better wash performance with respect to odor release than the reference lipase (particularly the parent lipase in SEQ ID NO: 2, 4, 6 or 8, respectively).

[0039] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA, which is processed through a series of steps, including splicing, before appearing as mature, spliced ​​mRNA.

[0040] Coding sequence: The term "coding sequence" means a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of a coding sequence are generally determined by an open reading frame, which begins with a start codon (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0041] Control sequence: the term "control sequence" means a nucleic acid sequence that is directed to regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence can be native (i.e., from the same gene) or heterologous (i.e., from different genes) for the polynucleotide encoding the variant, and is native or heterologous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, prepropeptides, propeptides, signal peptides, promoters, terminators, enhancers, and transcription or translation initiator and terminator sequences. At a minimum, these control sequences include promoters, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that promote the connection of the control sequence to the coding region of the polynucleotide encoding the variant, the control sequence can be provided with a joint.

[0042] Expression: The term "expression" includes any step involved in the production of the variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant operably linked to appropriate control sequences capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers, and sequences that control termination of transcription and translation.

[0044] Extension: The term "extension" means the addition of one or more amino acids to the amino and / or carboxyl terminus of a variant, wherein the "extended" variant has lipase activity.

[0045] Fragment: The term "fragment" means a variant having one or more amino acids deleted from the amino and / or carboxyl terminus of the variant; wherein the fragment has lipase activity.

[0046] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which one polypeptide is fused to the N-terminus and / or C-terminus of a variant of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides can also be constructed using intein technology, in which fusion polypeptides are produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12:2575-2583; Dawson et al., 1994, Science [Science] 266:776-779). The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 199 5, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0047] Heterologous: With respect to a host cell, the term "heterologous" means that the polypeptide or nucleic acid is not naturally present in the host cell. With respect to a polypeptide or nucleic acid, the term "heterologous" means that the control sequence (e.g., the promoter of the polypeptide or nucleic acid) is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0048] Host strain or host cell: "Host strain" or "host cell" refers to an organism into which an expression vector, phage, virus, or other DNA construct (including a polynucleotide encoding a variant) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by a cell.

[0049] Improved properties: The term "improved properties" refers to characteristics associated with a variant that are improved relative to a parent. Such improved properties include, but are not limited to, reduced lipase activity and / or reduced odor production at around neutral pH, i.e., about pH 6-8, preferably about pH 7, and / or an increased benefit-risk factor (BRF), compared to a parent lipase, particularly SEQ ID NO: 2, 4, 6 or 8, respectively.

[0050] Introduced: In the context of inserting a nucleic acid sequence into a cell, the term "introduced" means "transfection," "transformation," or "transduction," as known in the art.

[0051] Isolated: The term "isolated" means a variant, nucleic acid, cell, or other designated material or component that is separated from at least one other material or component, including but not limited to other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other material is in a form not found in nature. An isolated polypeptide includes, but is not limited to, a culture medium containing a secreted variant expressed in a host cell.

[0052] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its mature form following N-terminal processing and / or C-terminal processing (eg, removal of a signal peptide).

[0053] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having lipase activity.

[0054] Mutant: The term "mutant" means a polynucleotide encoding a variant.

[0055] Native: The term "native" means a nucleic acid or polypeptide that occurs naturally in a host cell.

[0056] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present compositions and methods encompass nucleotide sequences that encode a specific amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5' to 3' orientation.

[0057] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified in a manner not originally found in nature to contain a segment of nucleic acid or is synthetic and comprises one or more control sequences operably linked to the nucleic acid sequence.

[0058] Operably linked: "Operably linked" means that the specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under the control of the regulatory sequence.

[0059] Parent or parent lipase: The term "parent" or "parent lipase" means the lipase that is altered to produce the lipase variant of the present invention.

[0060] Purified: The term "purified" means a nucleic acid, variant, or cell that is substantially free of other components, as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form discrete bands in an electrophoretic gel, chromatography eluate, and / or culture medium subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, typically at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., by weight or by mole). In a related sense, a composition is enriched for a molecule when the concentration of the molecule is substantially increased following application of a purification or enrichment technique. The term "enriched" refers to the presence of a compound, variant, cell, nucleic acid, amino acid or other designated material or component in a composition at a relative or absolute concentration greater than that of the starting composition.

[0061] In one aspect, the term "purified" as used herein refers to a variant or cell that is substantially free of components (especially insoluble components) from the producing organism. In other aspects, the term "purified" refers to a variant that is substantially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some soluble components of the organism and culture medium from which it is recovered. The variant can be purified (i.e., isolated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0062] Accordingly, variants can be purified so that only small amounts of other proteins, particularly other polypeptides, are present. As used herein, the term "purified" can refer to the removal of other components present in the cell of the polypeptide source, particularly other proteins and most particularly other enzymes. Variants can be "substantially pure," i.e., free of other components from the organism from which they were produced (e.g., the host organism used to recombinantly produce the variant). In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation containing at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide materials with which the polypeptide is naturally or recombinantly associated.

[0063] Thus, it is preferred that a substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, and most preferably at least 99.5% pure, based on the weight of the total polypeptide material present in the preparation. The variants of the present invention are preferably in substantially pure form (i.e., the preparation is substantially free of other polypeptide material with which it is naturally or recombinantly associated). This can be accomplished, for example, by preparing the variant via well-known recombinant methods or via classical purification methods.

[0064] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation (e.g., cleavage and rejoining) of a nucleic acid sequence to form a sequence population that is different from that found in nature. The term recombinant refers to a cell, nucleic acid, variant, or vector that has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes at different levels or under different conditions than found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0065] Recovery: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, e.g., recovery of the polypeptide from a whole fermentation broth or from a cell-free fermentation broth by harvesting the polypeptide crystals, by filtration (e.g., depth filtration (by using filter aids or packed filter media, cloth filtration in box filters, drum filtration, drum filtration, rotary vacuum drum filtration, candle filters, horizontal leaf filters, or the like, filtration using sheets or pads in frame or modular units), or membrane filtration (using plate filtration, module filtration, candle filtration, microfiltration, ultrafiltration in crossflow, dynamic crossflow, or dead-end operation)), or by centrifugation (using a horizontal centrifuge, a disk stack centrifuge, a hyrdo cyclone, or the like), or by precipitating the polypeptide and using related solid-liquid separation methods to harvest the polypeptide from a broth medium by using size fractionation. Recovery encompasses isolation and / or purification of the polypeptide.

[0066] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0067] For the purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of "longest identity" using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) as implemented 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 6.6.0 or later). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -non-simplified (nobrief) option must be specified in the command line. The output of the "longest identity" labeled Needle is calculated as follows:

[0068] (Identical residues x 100) / (Alignment length - Total number of gaps in the alignment)

[0069] For purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, the same) implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, the same) (preferred 6.6.0 version or later) is used to determine the output of the sequence identity between two polynucleotide sequences as "longest identity". The parameters used are gap open penalty 10, gap extension penalty 0.5 and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. In order to make the Needle program report the longest identity, it is necessary to specify the non-simplified option in the command line. The output calculation of the "longest identity" of the Needle mark is as follows:

[0070] (number of identical deoxyribonucleotides x 100) / (length of alignment – ​​total number of gaps in the alignment)

[0071] Signal peptide: A "signal peptide" is an amino acid sequence attached to the N-terminal portion of a protein that promotes secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

[0072] Subsequence: The term "subsequence" means a polynucleotide having one or more nucleotides deleted from the 5' end and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity.

[0073] Variant: The term "variant" means a polypeptide having lipase activity comprising a substitution, insertion (including extension) and / or deletion (e.g., truncation) at one or more positions. Substitution means replacing the amino acid occupying a position with a different amino acid; deletion means removing the amino acid occupying a position; and insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0074] Wild-type: The term "wild-type" when referring to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., a protein, amino acid, or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in a laboratory, or modifications of a wild-type sequence).

[0075] Variant naming conventions

[0076] For the purposes of the present invention, the mature polypeptide disclosed in SEQ ID NO: 4 is used to determine the corresponding amino acid position in another lipase. The amino acid sequence of another lipase is aligned with the lipase disclosed in SEQ ID NO: 4, and based on the alignment, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needleman program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, 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: 4. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0077] In describing the variants of the present invention, the nomenclature described below has been adapted for ease of reference. Accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0078] replace For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 by alanine is represented as "Thr226Ala" or "T226A." Multiple mutations are separated by a plus sign ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represents the substitution of glycine (G) at position 205 and serine (S) at position 411 by arginine (R) and phenylalanine (F), respectively.

[0079] Missing For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Accordingly, a deletion of glycine at position 195 is indicated as "Gly195*" or "G195*." Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*."

[0080] insert.For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK." Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA."

[0081] In such cases, the inserted amino acid residue(s) are numbered by adding lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would therefore be:

[0082] <![CDATA[ Parents: ]]> <![CDATA[ Variants: ]]> 195 195 195a 195b G GKA

[0083] Multiple changes Variants containing multiple alterations are separated by plus signs ("+"), for example, "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine at position 170 and glycine at position 195 by tyrosine and glutamic acid, respectively.

[0084] Different changes Where different changes can be introduced at one position, the different changes are separated by commas, e.g., "Arg170Tyr,Glu" represents a substitution of arginine at position 170 by tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants:

[0085] “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”. DETAILED DESCRIPTION

[0086] Variants of the invention

[0087] The present invention relates to a lipase variant selected from one or more of the groups (i), (ii) and (iii) comprising:

[0088] (i) substitutions at one or more of positions 202, 252, and 269 of the polypeptide corresponding to SEQ ID NO: 8;

[0089] (ii) substitutions at one or more of positions 40, 56, 57, 91, 98, 108, 118, 210, 244, and 254 of the polypeptide corresponding to SEQ ID NO: 8; and

[0090] (iii) substitutions at one or more of positions 23, 27, 40, 51, 56, 60, 118, 244, and 256 of the polypeptide corresponding to SEQ ID NO: 8;

[0091] wherein the variant has lipase activity, and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 8.

[0092] In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i). In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (ii). In a preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i), and one or more substitutions, particularly all substitutions, from group (ii). In another preferred embodiment, the lipase variant has one or more substitutions, particularly all substitutions, from group (i), and one or more substitutions, particularly all substitutions, from group (iii).

[0093] The variants may further comprise an extension of one or more amino acids at the N-terminus and / or C-terminus (i.e., a peptide addition). In a preferred embodiment, the extension is a SPIRR-peptide (or one or more amino acids thereof) located at the N-terminus of the lipase. Suitable lipase extensions are disclosed in WO 1997 / 004079 (incorporated herein by reference). Examples of C-terminal extensions are disclosed in WO 2000 / 060063 (incorporated herein by reference). Alternatively, the variants may further comprise a truncation of one or more amino acids at the N-terminus and / or C-terminus.

[0094] In embodiments, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, but less than 100% sequence identity to the amino acid sequence of the parent lipase.

[0095] In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 2.

[0096] In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to the polypeptide of SEQ ID NO: 4.

[0097] In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6.

[0098] In another embodiment, the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 8.

[0099] In one aspect, the number of alterations (particularly substitutions) in a variant of the invention is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations (particularly substitutions).

[0100] In an embodiment, the variant of the invention comprises or consists of one or more substitutions (particularly all substitutions) corresponding to positions in SEQ ID NO: 8, which substitutions are selected from the group consisting of:

[0101] H substitution of the amino acid residue at position 202;

[0102] H substitution of the amino acid residue at position 252; and

[0103] H substitution of the amino acid residue at position 269.

[0104] In embodiments, a variant of the invention comprises or consists of one of the following substitution groups corresponding to: 202H+252H; 202H+269H; 252H+269H; or 202H+252H+269H (using SEQ ID NO: 8 for numbering).

[0105] In a preferred embodiment, the variant of the invention comprises or consists of one or more substitutions (particularly all substitutions) corresponding to the positions in SEQ ID NO: 8, which substitutions are selected from the group consisting of:

[0106] E substitution of the amino acid residue at position 40;

[0107] R substitution of the amino acid residue at position 56;

[0108] N substitution of the amino acid residue at position 57;

[0109] T substitution of the amino acid residue at position 91;

[0110] E substitution of the amino acid residue at position 98;

[0111] K substitution of the amino acid residue at position 108;

[0112] F substitution of the amino acid residue at position 118;

[0113] K substitution of the amino acid residue at position 210;

[0114] E substitution of the amino acid residue at position 244; and

[0115] S substitution of the amino acid residue at position 254.

[0116] In another preferred embodiment, the variant of the present invention comprises or consists of one or more substitutions (in particular all substitutions) corresponding to the positions in SEQ ID NO: 8, which substitutions are selected from the group consisting of:

[0117] S substitution of the amino acid residue at position 23;

[0118] N substitution of the amino acid residue at position 27;

[0119] 1. Substitution of the amino acid residue at position 40;

[0120] 1. Substitution of the amino acid residue at position 51;

[0121] R substitution of the amino acid residue at position 56;

[0122] K substitution of the amino acid residue at position 60;

[0123] F substitution of the amino acid residue at position 118;

[0124] E substitution of the amino acid residue at position 244; and

[0125] T substitution of the amino acid residue at position 256.

[0126] In embodiments, a variant of the invention has a substitution corresponding to I202H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to: A40E, E56R, D57N, G91T, K98E, R108K, R118F, E210K, T244E, A40E+E56R, A40E+D57N, A40E+G91T, A40E+K98E, A40E+R108K, A40E+R118F, A40E+E210K, A40E+T244E, A40E+D254S, E56R+D57N, E56R+G91T, E56R+K98E, E56R+R108K, E56R+R118F, E210K, +E210K, E56R+T244E, E56R+D254S, D57N+G91T, D57N+K98E, D57N+R108K, D57N+R118F, D57N+E210K, D57N+T244E, D57N+D254S, G91T+K98E, G91T+R 108K, G91T+R118F, G91T+E210K, G91T+T244E, G91T+D254S, K98E+R108K, K98E+R118F, K98E+E210K, K98E+T244E, K98E+D254S, R108K+R118F, R108K +E210K, R108K+T244E, R108K+D254S, R118F+E210K, R118F+T244E, R118F +D254S, E210K+T244E, E210K+D254S, T244E+D254S, A40E+E56R+D57N, A4 0E+E56R+G91T, A40E+E56R+K98E, A40E+E56R+R108K, A40E+E56R+R118F, A40E+E56R+E210K, A40E+E56R+T244E, A40E+E56R+D254S, A40E+D57N+G9 1T, A40E+D57N+K98E, A40E+D57N+R108K, A40E+D57N+R118F, A40E+D57N+ E210K, A40E+D57N+T244E, A40E+D57N+D254S, A40E+G91T+K98E, A40E+G9 1T+R108K, A40E+G91T+R118F, A40E+G91T+E210K, A40E+G91T+T244E, A40 E+G91T+D254S, A40E+K98E+R108K, A40E+K98E+R118F, A40E+K98E+E210K,A40E+K98E+T244E、A40E+K98E+D254S、A40E+R108K+R118F、A40E+R108K+E210K、A40E+R108K+T244E、A40E+R108K+D254S、A40E+R118F+E210K、A40E+R118F+T244E、A40E+R118F+D254S、A40E+E210K+T244E、A40E+E210K+D254S、A40E+T244E+D254S、E56R+D57N+G91T、E56R+D57N+K98E、E56R+D57N+R108K、E56R+D57N+R118F、E56R+D57N+E210K、E56R+D57N+T244E、E56R+D57N+D254S、E56R+G91T+K98E、E56R+G91T+R108K、E56R+G91T+R118F、E56R+G91T+E210K、E56R+G91T+T244E、E56R+G91T+D254S、E56R+K98E+R108K、E56R+K98E+R118F、E56R+K98E+E210K、E56R+K98E+T244E、E56R+K98E+D254S、E56R+R108K+R118F、E56R+R108K+E210K、E56R+R108K+T244E、E56R+R108K+D254S、E56R+R118F+E210K、E56R+R118F+T244E、E56R+R118F+D254S、E56R+E210K+T244E、E56R+E210K+D254S、E56R+T244E+D254S、D57N+G91T+K98E、D57N+G91T+R108K、D57N+G91T+R118F、D57N+G91T+E210K、D57N+G91T+T244E、D57N+G91T+D254S、D57N+K98E+R108K、D57N+K98E+R118F、D57N+K98E+E210K、D57N+K98E+T244E、D57N+K98E+D254S、D57N+R108K+R118F、D57N+R108K+E210K、D57N+R108K+T244E、D57N+R108K+D254S、D57N+R118F+E210K、D57N+R118F+T244E、D57N+R118F+D254S、D57N+E210K+T244E、D57N+E210K+D254S、D57N+T244E+D254S、G91T+K98E+R108K、G91T+K98E+R118F、G91T+K98E+E210K、G91T+K98E+T244E、G91T+K98E+D254S、G91T+R108K+R118F、G91T+R108K+E210K、G91T+R108K+T244E、G91T+R108K+D254S、G91T+R118F+E210K、G91T+R118F+T244E、G91T+R118F+D254S、G91T+E210K+T244E、G91T+E210K+D254S、G91T+T244E+D254S、K98E+R108K+R118F、K98E+R108K+E210K、K98E+R108K+T244E、K98E+R108K+D254S、K98E+R118F+E210K、K98E+R118F+T244E、K98E+R118F+D254S、K98E+E210K+T244E、K98E+E210K+D254S、K98E+T244E+D254S、R108K+R118F+E210K、R108K+R118F+T244E、R108K+R118F+D254S、R108K+E210K+T244E、R108K+E210K+D254S、R108K+T244E+D254S、R118F+E210K+T244E、R118F+E210K+D254S、R118F+T244E+D254S、E210K+T244E+D254S、A40E+E56R+D57N+G91T、A40E+E56R+D57N+K98E、A40E+E56R+D57N+R108K、A40E+E56R+D57N+R118F、A40E+E56R+D57N+E210K、A40E+E56R+D57N+T244E、A40E+E56R+D57N+D254S、A40E+E56R+G91T+K98E、A40E+E56R+G91T+R108K、A40E+E56R+G91T+R118F、A40E+E56R+G91T+E210K、A40E+E56R+G91T+T244E、A40E+E56R+G91T+D254S、A40E+E56R+K98E+R108K、A40E+E56R+K98E+R118F、A40E+E56R+K98E+E210K、A40E+E56R+K98E+T244E、A40E+E56R+K98E+D254S、A40E+E56R+R108K+R118F、A40E+E56R+R108K+E210K、A40E+E56R+R108K+T244E、A40E+E56R+R108K+D254S、A40E+E56R+R118F+E210K、A40E+E56R+R118F+T244E、A40E+E56R+R118F+D254S、A40E+E56R+E210K+T244E、A40E+E56R+E210K+D254S、A40E+E56R+T244E+D254S、A40E+D57N+G91T+K98E、A40E+D57N+G91T+R108K、A40E+D57N+G91T+R118F、A40E+D57N+G91T+E210K、A40E+D57N+G91T+T244E、A40E+D57N+G91T+D254S、A40E+D57N+K98E+R108K、A40E+D57N+K98E+R118F、A40E+D57N+K98E+E210K、A40E+D57N+K98E+T244E、A40E+D57N+K98E+D254S、A40E+D57N+R108K+R118F、A40E+D57N+R108K+E210K、A40E+D57N+R108K+T244E、A40E+D57N+R108K+D254S、A40E+D57N+R118F+E210K、A40E+D57N+R118F+T244E、A40E+D57N+R118F+D254S、A40E+D57N+E210K+T244E、A40E+D57N+E210K+D254S、A40E+D57N+T244E+D254S、A40E+G91T+K98E+R108K、A40E+G91T+K98E+R118F、A40E+G91T+K98E+E210K、A40E+G91T+K98E+T244E、A40E+G91T+K98E+D254S、A40E+G91T+R108K+R118F、A40E+G91T+R108K+E210K、A40E+G91T+R108K+T244E、A40E+G91T+R108K+D254S、A40E+G91T+R118F+E210K、A40E+G91T+R118F+T244E、A40E+G91T+R118F+D254S、A40E+G91T+E210K+T244E、A40E+G91T+E210K+D254S、A40E+G91T+T244E+D254S、A40E+K98E+R108K+R118F、A40E+K98E+R108K+E210K、A40E+K98E+R108K+T244E、A40E+K98E+R108K+D254S、A40E+K98E+R118F+E210K、A40E+K98E+R118F+T244E、A40E+K98E+R118F+D254S、A40E+K98E+E210K+T244E、A40E+K98E+E210K+D254S、A40E+K98E+T244E+D254S、A40E+R108K+R118F+E210K、A40E+R108K+R118F+T244E、A40E+R108K+R118F+D254S、A40E+R108K+E210K+T244E、A40E+R108K+E210K+D254S、A40E+R108K+T244E+D254S、A40E+R118F+E210K+T244E、A40E+R118F+E210K+D254S、A40E+R118F+T244E+D254S、A40E+E210K+T244E+D254S、E56R+D57N+G91T+K98E、E56R+D57N+G91T+R108K、E56R+D57N+G91T+R118F、E56R+D57N+G91T+E210K、E56R+D57N+G91T+T244E、E56R+D57N+G91T+D254S、E56R+D57N+K98E+R108K、E56R+D57N+K98E+R118F、E56R+D57N+K98E+E210K、E56R+D57N+K98E+T244E、E56R+D57N+K98E+D254S、E56R+D57N+R108K+R118F、E56R+D57N+R108K+E210K、E56R+D57N+R108K+T244E、E56R+D57N+R108K+D254S、E56R+D57N+R118F+E210K、E56R+D57N+R118F+T244E、E56R+D57N+R118F+D254S、E56R+D57N+E210K+T244E、E56R+D57N+E210K+D254S、E56R+D57N+T244E+D254S、E56R+G91T+K98E+R108K、E56R+G91T+K98E+R118F、E56R+G91T+K98E+E210K、E56R+G91T+K98E+T244E、E56R+G91T+K98E+D254S、E56R+G91T+R108K+R118F、E56R+G91T+R108K+E210K、E56R+G91T+R108K+T244E、E56R+G91T+R108K+D254S、E56R+G91T+R118F+E210K、E56R+G91T+R118F+T244E、E56R+G91T+R118F+D254S、E56R+G91T+E210K+T244E、E56R+G91T+E210K+D254S、E56R+G91T+T244E+D254S、E56R+K98E+R108K+R118F、E56R+K98E+R108K+E210K、E56R+K98E+R108K+T244E、E56R+K98E+R108K+D254S、E56R+K98E+R118F+E210K、E56R+K98E+R118F+T244E、E56R+K98E+R118F+D254S、E56R+K98E+E210K+T244E、E56R+K98E+E210K+D254S、E56R+K98E+T244E+D254S、E56R+R108K+R118F+E210K、E56R+R108K+R118F+T244E、E56R+R108K+R118F+D254S、E56R+R108K+E210K+T244E、E56R+R108K+E210K+D254S、E56R+R108K+T244E+D254S、E56R+R118F+E210K+T244E、E56R+R118F+E210K+D254S、E56R+R118F+T244E+D254S、E56R+E210K+T244E+D254S、D57N+G91T+K98E+R108K、D57N+G91T+K98E+R118F、D57N+G91T+K98E+E210K、D57N+G91T+K98E+T244E、D57N+G91T+K98E+D254S、D57N+G91T+R108K+R118F、D57N+G91T+R108K+E210K、D57N+G91T+R108K+T244E、D57N+G91T+R108K+D254S、D57N+G91T+R118F+E210K、D57N+G91T+R118F+T244E、D57N+G91T+R118F+D254S、D57N+G91T+E210K+T244E、D57N+G91T+E210K+D254S、D57N+G91T+T244E+D254S、D57N+K98E+R1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98E+R118F+E210K+T244E+D254S、G91T+R108K+R118F+E210K+T244E+D254S、K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F、A40E+E56R+D57N+G91T+K98E+R108K+E210K、A40E+E56R+D57N+G91T+K98E+R108K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R118F+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E、A40E+E56R+D57N+G91T+K98E+E210K+D254S、A40E+E56R+D57N+G91T+K98E+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K、A40E+E56R+D57N+G91T+R108K+R118F+T244E、A40E+E56R+D57N+G91T+R108K+R118F+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E、A40E+E56R+D57N+G91T+R108K+E210K+D254S、A40E+E56R+D57N+G91T+R108K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R118F+T244E+D254S、A40E+E56R+D57N+G91T+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K、A40E+E56R+D57N+K98E+R108K+R118F+T244E、A40E+E56R+D57N+K98E+R108K+R118F+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E、A40E+E56R+D57N+K98E+R108K+E210K+D254S、A40E+E56R+D57N+K98E+R108K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R118F+T244E+D254S、A40E+E56R+D57N+K98E+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E、A40E+E56R+D57N+R108K+R118F+E210K+D254S、A40E+E56R+D57N+R108K+R118F+T244E+D254S、A40E+E56R+D57N+R108K+E210K+T244E+D254S、A40E+E56R+D57N+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K、A40E+E56R+G91T+K98E+R108K+R118F+T244E、A40E+E56R+G91T+K98E+R108K+R118F+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E、A40E+E56R+G91T+K98E+R108K+E210K+D254S、A40E+E56R+G91T+K98E+R108K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R118F+T244E+D254S、A40E+E56R+G91T+K98E+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E、A40E+E56R+G91T+R108K+R118F+E210K+D254S、A40E+E56R+G91T+R108K+R118F+T244E+D254S、A40E+E56R+G91T+R108K+E210K+T244E+D254S、A40E+E56R+G91T+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E、A40E+E56R+K98E+R108K+R118F+E210K+D254S、A40E+E56R+K98E+R108K+R118F+T244E+D254S、A40E+E56R+K98E+R108K+E210K+T244E+D254S、A40E+E56R+K98E+R118F+E210K+T244E+D254S、A40E+E56R+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K、A40E+D57N+G91T+K98E+R108K+R118F+T244E、A40E+D57N+G91T+K98E+R108K+R118F+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E、A40E+D57N+G91T+K98E+R108K+E210K+D254S、A40E+D57N+G91T+K98E+R108K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R118F+T244E+D254S、A40E+D57N+G91T+K98E+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E、A40E+D57N+G91T+R108K+R118F+E210K+D254S、A40E+D57N+G91T+R108K+R118F+T244E+D254S、A40E+D57N+G91T+R108K+E210K+T244E+D254S、A40E+D57N+G91T+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E、A40E+D57N+K98E+R108K+R118F+E210K+D254S、A40E+D57N+K98E+R108K+R118F+T244E+D254S、A40E+D57N+K98E+R108K+E210K+T244E+D254S、A40E+D57N+K98E+R118F+E210K+T244E+D254S、A40E+D57N+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E、A40E+G91T+K98E+R108K+R118F+E210K+D254S、A40E+G91T+K98E+R108K+R118F+T244E+D254S、A40E+G91T+K98E+R108K+E210K+T244E+D254S、A40E+G91T+K98E+R118F+E210K+T244E+D254S、A40E+G91T+R108K+R118F+E210K+T244E+D254S、A40E+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K、E56R+D57N+G91T+K98E+R108K+R118F+T244E、E56R+D57N+G91T+K98E+R108K+R118F+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E、E56R+D57N+G91T+K98E+R108K+E210K+D254S、E56R+D57N+G91T+K98E+R108K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R118F+T244E+D254S、E56R+D57N+G91T+K98E+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E、E56R+D57N+G91T+R108K+R118F+E210K+D254S、E56R+D57N+G91T+R108K+R118F+T244E+D254S、E56R+D57N+G91T+R108K+E210K+T244E+D254S、E56R+D57N+G91T+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E、E56R+D57N+K98E+R108K+R118F+E210K+D254S、E56R+D57N+K98E+R108K+R118F+T244E+D254S、E56R+D57N+K98E+R108K+E210K+T244E+D254S、E56R+D57N+K98E+R118F+E210K+T244E+D254S、E56R+D57N+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E、E56R+G91T+K98E+R108K+R118F+E210K+D254S、E56R+G91T+K98E+R108K+R118F+T244E+D254S、E56R+G91T+K98E+R108K+E210K+T244E+D254S、E56R+G91T+K98E+R118F+E210K+T244E+D254S、E56R+G91T+R108K+R118F+E210K+T244E+D254S、E56R+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E、D57N+G91T+K98E+R108K+R118F+E210K+D254S、D57N+G91T+K98E+R108K+R118F+T244E+D254S、D57N+G91T+K98E+R108K+E210K+T244E+D254S、D57N+G91T+K98E+R118F+E210K+T244E+D254S、D57N+G91T+R108K+R118F+E210K+T244E+D254S、D57N+K98E+R108K+R118F+E210K+T244E+D254S、G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S。、

[0127] In embodiments, a variant of the invention has a substitution corresponding to I252H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to: A40E, E56R, D57N, G91T, K98E, R108K, R118F, E210K, T244E, A40E+E56R, A40E+D57N, A40E+G91T, A40E+K98E, A40E+R108K, A40E+R118F, A40E+E210K, A40E+T244E, A40E+D254S, E56R+D57N, E56R+G91T, E56R+K98E, E56R+R108K, E56R+R118F, E210K, +E210K, E56R+T244E, E56R+D254S, D57N+G91T, D57N+K98E, D57N+R108K, D57N+R118F, D57N+E210K, D57N+T244E, D57N+D254S, G91T+K98E, G91T+R 108K, G91T+R118F, G91T+E210K, G91T+T244E, G91T+D254S, K98E+R108K, K98E+R118F, K98E+E210K, K98E+T244E, K98E+D254S, R108K+R118F, R108K +E210K, R108K+T244E, R108K+D254S, R118F+E210K, R118F+T244E, R118F +D254S, E210K+T244E, E210K+D254S, T244E+D254S, A40E+E56R+D57N, A4 0E+E56R+G91T, A40E+E56R+K98E, A40E+E56R+R108K, A40E+E56R+R118F, A40E+E56R+E210K, A40E+E56R+T244E, A40E+E56R+D254S, A40E+D57N+G9 1T, A40E+D57N+K98E, A40E+D57N+R108K, A40E+D57N+R118F, A40E+D57N+ E210K, A40E+D57N+T244E, A40E+D57N+D254S, A40E+G91T+K98E, A40E+G9 1T+R108K, A40E+G91T+R118F, A40E+G91T+E210K, A40E+G91T+T244E, A40 E+G91T+D254S, A40E+K98E+R108K, A40E+K98E+R118F, A40E+K98E+E210K,A40E+K98E+T244E、A40E+K98E+D254S、A40E+R108K+R118F、A40E+R108K+E210K、A40E+R108K+T244E、A40E+R108K+D254S、A40E+R118F+E210K、A40E+R118F+T244E、A40E+R118F+D254S、A40E+E210K+T244E、A40E+E210K+D254S、A40E+T244E+D254S、E56R+D57N+G91T、E56R+D57N+K98E、E56R+D57N+R108K、E56R+D57N+R118F、E56R+D57N+E210K、E56R+D57N+T244E、E56R+D57N+D254S、E56R+G91T+K98E、E56R+G91T+R108K、E56R+G91T+R118F、E56R+G91T+E210K、E56R+G91T+T244E、E56R+G91T+D254S、E56R+K98E+R108K、E56R+K98E+R118F、E56R+K98E+E210K、E56R+K98E+T244E、E56R+K98E+D254S、E56R+R108K+R118F、E56R+R108K+E210K、E56R+R108K+T244E、E56R+R108K+D254S、E56R+R118F+E210K、E56R+R118F+T244E、E56R+R118F+D254S、E56R+E210K+T244E、E56R+E210K+D254S、E56R+T244E+D254S、D57N+G91T+K98E、D57N+G91T+R108K、D57N+G91T+R118F、D57N+G91T+E210K、D57N+G91T+T244E、D57N+G91T+D254S、D57N+K98E+R108K、D57N+K98E+R118F、D57N+K98E+E210K、D57N+K98E+T244E、D57N+K98E+D254S、D57N+R108K+R118F、D57N+R108K+E210K、D57N+R108K+T244E、D57N+R108K+D254S、D57N+R118F+E210K、D57N+R118F+T244E、D57N+R118F+D254S、D57N+E210K+T244E、D57N+E210K+D254S、D57N+T244E+D254S、G91T+K98E+R108K、G91T+K98E+R118F、G91T+K98E+E210K、G91T+K98E+T244E、G91T+K98E+D254S、G91T+R108K+R118F、G91T+R108K+E210K、G91T+R108K+T244E、G91T+R108K+D254S、G91T+R118F+E210K、G91T+R118F+T244E、G91T+R118F+D254S、G91T+E210K+T244E、G91T+E210K+D254S、G91T+T244E+D254S、K98E+R108K+R118F、K98E+R108K+E210K、K98E+R108K+T244E、K98E+R108K+D254S、K98E+R118F+E210K、K98E+R118F+T244E、K98E+R118F+D254S、K98E+E210K+T244E、K98E+E210K+D254S、K98E+T244E+D254S、R108K+R118F+E210K、R108K+R118F+T244E、R108K+R118F+D254S、R108K+E210K+T244E、R108K+E210K+D254S、R108K+T244E+D254S、R118F+E210K+T244E、R118F+E210K+D254S、R118F+T244E+D254S、E210K+T244E+D254S、A40E+E56R+D57N+G91T、A40E+E56R+D57N+K98E、A40E+E56R+D57N+R108K、A40E+E56R+D57N+R118F、A40E+E56R+D57N+E210K、A40E+E56R+D57N+T244E、A40E+E56R+D57N+D254S、A40E+E56R+G91T+K98E、A40E+E56R+G91T+R108K、A40E+E56R+G91T+R118F、A40E+E56R+G91T+E210K、A40E+E56R+G91T+T244E、A40E+E56R+G91T+D254S、A40E+E56R+K98E+R108K、A40E+E56R+K98E+R118F、A40E+E56R+K98E+E210K、A40E+E56R+K98E+T244E、A40E+E56R+K98E+D254S、A40E+E56R+R108K+R118F、A40E+E56R+R108K+E210K、A40E+E56R+R108K+T244E、A40E+E56R+R108K+D254S、A40E+E56R+R118F+E210K、A40E+E56R+R118F+T244E、A40E+E56R+R118F+D254S、A40E+E56R+E210K+T244E、A40E+E56R+E210K+D254S、A40E+E56R+T244E+D254S、A40E+D57N+G91T+K98E、A40E+D57N+G91T+R108K、A40E+D57N+G91T+R118F、A40E+D57N+G91T+E210K、A40E+D57N+G91T+T244E、A40E+D57N+G91T+D254S、A40E+D57N+K98E+R108K、A40E+D57N+K98E+R118F、A40E+D57N+K98E+E210K、A40E+D57N+K98E+T244E、A40E+D57N+K98E+D254S、A40E+D57N+R108K+R118F、A40E+D57N+R108K+E210K、A40E+D57N+R108K+T244E、A40E+D57N+R108K+D254S、A40E+D57N+R118F+E210K、A40E+D57N+R118F+T244E、A40E+D57N+R118F+D254S、A40E+D57N+E210K+T244E、A40E+D57N+E210K+D254S、A40E+D57N+T244E+D254S、A40E+G91T+K98E+R108K、A40E+G91T+K98E+R118F、A40E+G91T+K98E+E210K、A40E+G91T+K98E+T244E、A40E+G91T+K98E+D254S、A40E+G91T+R108K+R118F、A40E+G91T+R108K+E210K、A40E+G91T+R108K+T244E、A40E+G91T+R108K+D254S、A40E+G91T+R118F+E210K、A40E+G91T+R118F+T244E、A40E+G91T+R118F+D254S、A40E+G91T+E210K+T244E、A40E+G91T+E210K+D254S、A40E+G91T+T244E+D254S、A40E+K98E+R108K+R118F、A40E+K98E+R108K+E210K、A40E+K98E+R108K+T244E、A40E+K98E+R108K+D254S、A40E+K98E+R118F+E210K、A40E+K98E+R118F+T244E、A40E+K98E+R118F+D254S、A40E+K98E+E210K+T244E、A40E+K98E+E210K+D254S、A40E+K98E+T244E+D254S、A40E+R108K+R118F+E210K、A40E+R108K+R118F+T244E、A40E+R108K+R118F+D254S、A40E+R108K+E210K+T244E、A40E+R108K+E210K+D254S、A40E+R108K+T244E+D254S、A40E+R118F+E210K+T244E、A40E+R118F+E210K+D254S、A40E+R118F+T244E+D254S、A40E+E210K+T244E+D254S、E56R+D57N+G91T+K98E、E56R+D57N+G91T+R108K、E56R+D57N+G91T+R118F、E56R+D57N+G91T+E210K、E56R+D57N+G91T+T244E、E56R+D57N+G91T+D254S、E56R+D57N+K98E+R108K、E56R+D57N+K98E+R118F、E56R+D57N+K98E+E210K、E56R+D57N+K98E+T244E、E56R+D57N+K98E+D254S、E56R+D57N+R108K+R118F、E56R+D57N+R108K+E210K、E56R+D57N+R108K+T244E、E56R+D57N+R108K+D254S、E56R+D57N+R118F+E210K、E56R+D57N+R118F+T244E、E56R+D57N+R118F+D254S、E56R+D57N+E210K+T244E、E56R+D57N+E210K+D254S、E56R+D57N+T244E+D254S、E56R+G91T+K98E+R108K、E56R+G91T+K98E+R118F、E56R+G91T+K98E+E210K、E56R+G91T+K98E+T244E、E56R+G91T+K98E+D254S、E56R+G91T+R108K+R118F、E56R+G91T+R108K+E210K、E56R+G91T+R108K+T244E、E56R+G91T+R108K+D254S、E56R+G91T+R118F+E210K、E56R+G91T+R118F+T244E、E56R+G91T+R118F+D254S、E56R+G91T+E210K+T244E、E56R+G91T+E210K+D254S、E56R+G91T+T244E+D254S、E56R+K98E+R108K+R118F、E56R+K98E+R108K+E210K、E56R+K98E+R108K+T244E、E56R+K98E+R108K+D254S、E56R+K98E+R118F+E210K、E56R+K98E+R118F+T244E、E56R+K98E+R118F+D254S、E56R+K98E+E210K+T244E、E56R+K98E+E210K+D254S、E56R+K98E+T244E+D254S、E56R+R108K+R118F+E210K、E56R+R108K+R118F+T244E、E56R+R108K+R118F+D254S、E56R+R108K+E210K+T244E、E56R+R108K+E210K+D254S、E56R+R108K+T244E+D254S、E56R+R118F+E210K+T244E、E56R+R118F+E210K+D254S、E56R+R118F+T244E+D254S、E56R+E210K+T244E+D254S、D57N+G91T+K98E+R108K、D57N+G91T+K98E+R118F、D57N+G91T+K98E+E210K、D57N+G91T+K98E+T244E、D57N+G91T+K98E+D254S、D57N+G91T+R108K+R118F、D57N+G91T+R108K+E210K、D57N+G91T+R108K+T244E、D57N+G91T+R108K+D254S、D57N+G91T+R118F+E210K、D57N+G91T+R118F+T244E、D57N+G91T+R118F+D254S、D57N+G91T+E210K+T244E、D57N+G91T+E210K+D254S、D57N+G91T+T244E+D254S、D57N+K98E+R108K+R118F、D57N+K98E+R108K+E210K、D57N+K98E+R108K+T244E、D57N+K98E+R108K+D254S、D57N+K98E+R118F+E210K、D57N+K98E+R118F+T244E、D57N+K98E+R118F+D254S、D57N+K98E+E210K+T244E、D57N+K98E+E210K+D254S、D57N+K98E+T244E+D254S、D57N+R108K+R118F+E210K、D57N+R108K+R118F+T244E、D57N+R108K+R118F+D254S、D57N+R108K+E210K+T244E、D57N+R108K+E210K+D254S、D57N+R108K+T244E+D254S、D57N+R118F+E210K+T244E、D57N+R118F+E210K+D254S、D57N+R118F+T244E+D254S、D57N+E210K+T244E+D254S、G91T+K98E+R108K+R118F、G91T+K98E+R108K+E210K、G91T+K98E+R108K+T244E、G91T+K98E+R108K+D254S、G91T+K98E+R118F+E210K、G91T+K98E+R118F+T244E、G91T+K98E+R118F+D254S、G91T+K98E+E210K+T244E、G91T+K98E+E210K+D254S、G91T+K98E+T244E+D254S、G91T+R108K+R118F+E210K、G91T+R108K+R118F+T244E、G91T+R108K+R118F+D254S、G91T+R108K+E210K+T244E、G91T+R108K+E210K+D254S、G91T+R108K+T244E+D254S、G91T+R118F+E210K+T244E、G91T+R118F+E210K+D254S、G91T+R118F+T244E+D254S、G91T+E210K+T244E+D254S、K98E+R108K+R118F+E210K、K98E+R108K+R118F+T244E、K98E+R108K+R118F+D254S、K98E+R108K+E210K+T244E、K98E+R108K+E210K+D254S、K98E+R108K+T244E+D254S、K98E+R118F+E210K+T244E、K98E+R118F+E210K+D254S、K98E+R118F+T244E+D254S、K98E+E210K+T244E+D254S、R108K+R118F+E210K+T244E、R108K+R118F+E210K+D254S、R108K+R118F+T244E+D254S、R108K+E210K+T244E+D254S、R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E、A40E+E56R+D57N+G91T+R108K、A40E+E56R+D57N+G91T+R118F、A40E+E56R+D57N+G91T+E210K、A40E+E56R+D57N+G91T+T244E、A40E+E56R+D57N+G91T+D254S、A40E+E56R+D57N+K98E+R108K、A40E+E56R+D57N+K98E+R118F、A40E+E56R+D57N+K98E+E210K、A40E+E56R+D57N+K98E+T244E、A40E+E56R+D57N+K98E+D254S、A40E+E56R+D57N+R108K+R118F、A40E+E56R+D57N+R108K+E210K、A40E+E56R+D57N+R108K+T244E、A40E+E56R+D57N+R108K+D254S、A40E+E56R+D57N+R118F+E210K、A40E+E56R+D57N+R118F+T244E、A40E+E56R+D57N+R118F+D254S、A40E+E56R+D57N+E210K+T244E、A40E+E56R+D57N+E210K+D254S、A40E+E56R+D57N+T244E+D254S、A40E+E56R+G91T+K98E+R108K、A40E+E56R+G91T+K98E+R118F、A40E+E56R+G91T+K98E+E210K、A40E+E56R+G91T+K98E+T244E、A40E+E56R+G91T+K98E+D254S、A40E+E56R+G91T+R108K+R118F、A40E+E56R+G91T+R108K+E210K、A40E+E56R+G91T+R108K+T244E、A40E+E56R+G91T+R108K+D254S、A40E+E56R+G91T+R118F+E210K、A40E+E56R+G91T+R118F+T244E、A40E+E56R+G91T+R118F+D254S、A40E+E56R+G91T+E210K+T244E、A40E+E56R+G91T+E210K+D254S、A40E+E56R+G91T+T244E+D254S、A40E+E56R+K98E+R108K+R118F、A40E+E56R+K98E+R108K+E210K、A40E+E56R+K98E+R108K+T244E、A40E+E56R+K98E+R108K+D254S、A40E+E56R+K98E+R118F+E210K、A40E+E56R+K98E+R118F+T244E、A40E+E56R+K98E+R118F+D254S、A40E+E56R+K98E+E210K+T244E、A40E+E56R+K98E+E210K+D254S、A40E+E56R+K98E+T244E+D254S、A40E+E56R+R108K+R118F+E210K、A40E+E56R+R108K+R118F+T244E、A40E+E56R+R108K+R118F+D254S、A40E+E56R+R108K+E210K+T244E、A40E+E56R+R108K+E210K+D254S、A40E+E56R+R108K+T244E+D254S、A40E+E56R+R118F+E210K+T244E、A40E+E56R+R118F+E210K+D254S、A40E+E56R+R118F+T244E+D254S、A40E+E56R+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K、A40E+D57N+G91T+K98E+R118F、A40E+D57N+G91T+K98E+E210K、A40E+D57N+G91T+K98E+T244E、A40E+D57N+G91T+K98E+D254S、A40E+D57N+G91T+R108K+R118F、A40E+D57N+G91T+R108K+E210K、A40E+D57N+G91T+R108K+T244E、A40E+D57N+G91T+R108K+D254S、A40E+D57N+G91T+R118F+E210K、A40E+D57N+G91T+R118F+T244E、A40E+D57N+G91T+R118F+D254S、A40E+D57N+G91T+E210K+T244E、A40E+D57N+G91T+E210K+D254S、A40E+D57N+G91T+T244E+D254S、A40E+D57N+K98E+R108K+R118F、A40E+D57N+K98E+R108K+E210K、A40E+D57N+K98E+R108K+T244E、A40E+D57N+K98E+R108K+D254S、A40E+D57N+K98E+R118F+E210K、A40E+D57N+K98E+R118F+T244E、A40E+D57N+K98E+R118F+D254S、A40E+D57N+K98E+E210K+T244E、A40E+D57N+K98E+E210K+D254S、A40E+D57N+K98E+T244E+D254S、A40E+D57N+R108K+R118F+E210K、A40E+D57N+R108K+R118F+T244E、A40E+D57N+R108K+R118F+D254S、A40E+D57N+R108K+E210K+T244E、A40E+D57N+R108K+E210K+D254S、A40E+D57N+R108K+T244E+D254S、A40E+D57N+R118F+E210K+T244E、A40E+D57N+R118F+E210K+D254S、A40E+D57N+R118F+T244E+D254S、A40E+D57N+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F、A40E+G91T+K98E+R108K+E210K、A40E+G91T+K98E+R108K+T244E、A40E+G91T+K98E+R108K+D254S、A40E+G91T+K98E+R118F+E210K、A40E+G91T+K98E+R118F+T244E、A40E+G91T+K98E+R118F+D254S、A40E+G91T+K98E+E210K+T244E、A40E+G91T+K98E+E210K+D254S、A40E+G91T+K98E+T244E+D254S、A40E+G91T+R108K+R118F+E210K、A40E+G91T+R108K+R118F+T244E、A40E+G91T+R108K+R118F+D254S、A40E+G91T+R108K+E210K+T244E、A40E+G91T+R108K+E210K+D254S、A40E+G91T+R108K+T244E+D254S、A40E+G91T+R118F+E210K+T244E、A40E+G91T+R118F+E210K+D254S、A40E+G91T+R118F+T244E+D254S、A40E+G91T+E210K+T244E+D254S、A40E+K98E+R108K+R118F+E210K、A40E+K98E+R108K+R118F+T244E、A40E+K98E+R108K+R118F+D254S、A40E+K98E+R108K+E210K+T244E、A40E+K98E+R108K+E210K+D254S、A40E+K98E+R108K+T244E+D254S、A40E+K98E+R118F+E210K+T244E、A40E+K98E+R118F+E210K+D254S、A40E+K98E+R118F+T244E+D254S、A40E+K98E+E210K+T244E+D254S、A40E+R108K+R118F+E210K+T244E、A40E+R108K+R118F+E210K+D254S、A40E+R108K+R118F+T244E+D254S、A40E+R108K+E210K+T244E+D254S、A40E+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K、E56R+D57N+G91T+K98E+R118F、E56R+D57N+G91T+K98E+E210K、E56R+D57N+G91T+K98E+T244E、E56R+D57N+G91T+K98E+D254S、E56R+D57N+G91T+R108K+R118F、E56R+D57N+G91T+R108K+E210K、E56R+D57N+G91T+R108K+T244E、E56R+D57N+G91T+R108K+D254S、E56R+D57N+G91T+R118F+E210K、E56R+D57N+G91T+R118F+T244E、E56R+D57N+G91T+R118F+D254S、E56R+D57N+G91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98E+R118F+E210K+T244E+D254S、G91T+R108K+R118F+E210K+T244E+D254S、K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F、A40E+E56R+D57N+G91T+K98E+R108K+E210K、A40E+E56R+D57N+G91T+K98E+R108K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R118F+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E、A40E+E56R+D57N+G91T+K98E+E210K+D254S、A40E+E56R+D57N+G91T+K98E+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K、A40E+E56R+D57N+G91T+R108K+R118F+T244E、A40E+E56R+D57N+G91T+R108K+R118F+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E、A40E+E56R+D57N+G91T+R108K+E210K+D254S、A40E+E56R+D57N+G91T+R108K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R118F+T244E+D254S、A40E+E56R+D57N+G91T+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K、A40E+E56R+D57N+K98E+R108K+R118F+T244E、A40E+E56R+D57N+K98E+R108K+R118F+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E、A40E+E56R+D57N+K98E+R108K+E210K+D254S、A40E+E56R+D57N+K98E+R108K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R118F+T244E+D254S、A40E+E56R+D57N+K98E+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E、A40E+E56R+D57N+R108K+R118F+E210K+D254S、A40E+E56R+D57N+R108K+R118F+T244E+D254S、A40E+E56R+D57N+R108K+E210K+T244E+D254S、A40E+E56R+D57N+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K、A40E+E56R+G91T+K98E+R108K+R118F+T244E、A40E+E56R+G91T+K98E+R108K+R118F+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E、A40E+E56R+G91T+K98E+R108K+E210K+D254S、A40E+E56R+G91T+K98E+R108K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R118F+T244E+D254S、A40E+E56R+G91T+K98E+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E、A40E+E56R+G91T+R108K+R118F+E210K+D254S、A40E+E56R+G91T+R108K+R118F+T244E+D254S、A40E+E56R+G91T+R108K+E210K+T244E+D254S、A40E+E56R+G91T+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E、A40E+E56R+K98E+R108K+R118F+E210K+D254S、A40E+E56R+K98E+R108K+R118F+T244E+D254S、A40E+E56R+K98E+R108K+E210K+T244E+D254S、A40E+E56R+K98E+R118F+E210K+T244E+D254S、A40E+E56R+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K、A40E+D57N+G91T+K98E+R108K+R118F+T244E、A40E+D57N+G91T+K98E+R108K+R118F+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E、A40E+D57N+G91T+K98E+R108K+E210K+D254S、A40E+D57N+G91T+K98E+R108K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R118F+T244E+D254S、A40E+D57N+G91T+K98E+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E、A40E+D57N+G91T+R108K+R118F+E210K+D254S、A40E+D57N+G91T+R108K+R118F+T244E+D254S、A40E+D57N+G91T+R108K+E210K+T244E+D254S、A40E+D57N+G91T+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E、A40E+D57N+K98E+R108K+R118F+E210K+D254S、A40E+D57N+K98E+R108K+R118F+T244E+D254S、A40E+D57N+K98E+R108K+E210K+T244E+D254S、A40E+D57N+K98E+R118F+E210K+T244E+D254S、A40E+D57N+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E、A40E+G91T+K98E+R108K+R118F+E210K+D254S、A40E+G91T+K98E+R108K+R118F+T244E+D254S、A40E+G91T+K98E+R108K+E210K+T244E+D254S、A40E+G91T+K98E+R118F+E210K+T244E+D254S、A40E+G91T+R108K+R118F+E210K+T244E+D254S、A40E+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K、E56R+D57N+G91T+K98E+R108K+R118F+T244E、E56R+D57N+G91T+K98E+R108K+R118F+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E、E56R+D57N+G91T+K98E+R108K+E210K+D254S、E56R+D57N+G91T+K98E+R108K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R118F+T244E+D254S、E56R+D57N+G91T+K98E+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E、E56R+D57N+G91T+R108K+R118F+E210K+D254S、E56R+D57N+G91T+R108K+R118F+T244E+D254S、E56R+D57N+G91T+R108K+E210K+T244E+D254S、E56R+D57N+G91T+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E、E56R+D57N+K98E+R108K+R118F+E210K+D254S、E56R+D57N+K98E+R108K+R118F+T244E+D254S、E56R+D57N+K98E+R108K+E210K+T244E+D254S、E56R+D57N+K98E+R118F+E210K+T244E+D254S、E56R+D57N+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E、E56R+G91T+K98E+R108K+R118F+E210K+D254S、E56R+G91T+K98E+R108K+R118F+T244E+D254S、E56R+G91T+K98E+R108K+E210K+T244E+D254S、E56R+G91T+K98E+R118F+E210K+T244E+D254S、E56R+G91T+R108K+R118F+E210K+T244E+D254S、E56R+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E、D57N+G91T+K98E+R108K+R118F+E210K+D254S、D57N+G91T+K98E+R108K+R118F+T244E+D254S、D57N+G91T+K98E+R108K+E210K+T244E+D254S、D57N+G91T+K98E+R118F+E210K+T244E+D254S、D57N+G91T+R108K+R118F+E210K+T244E+D254S、D57N+K98E+R108K+R118F+E210K+T244E+D254S、G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S。、

[0128] In embodiments, a variant of the invention has a substitution corresponding to L269H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to: A40E, E56R, D57N, G91T, K98E, R108K, R118F, E210K, T244E, A40E+E56R, A40E+D57N, A40E+G91T, A40E+K98E, A40E+R108K, A40E+R118F, A40E+E210K, A40E+T244E, A40E+D254S, E56R+D57N, E56R+G91T, E56R+K98E, E56R+R108K, E56R+R118F, E210K, +E210K, E56R+T244E, E56R+D254S, D57N+G91T, D57N+K98E, D57N+R108K, D57N+R118F, D57N+E210K, D57N+T244E, D57N+D254S, G91T+K98E, G91T+R 108K, G91T+R118F, G91T+E210K, G91T+T244E, G91T+D254S, K98E+R108K, K98E+R118F, K98E+E210K, K98E+T244E, K98E+D254S, R108K+R118F, R108K +E210K, R108K+T244E, R108K+D254S, R118F+E210K, R118F+T244E, R118F +D254S, E210K+T244E, E210K+D254S, T244E+D254S, A40E+E56R+D57N, A4 0E+E56R+G91T, A40E+E56R+K98E, A40E+E56R+R108K, A40E+E56R+R118F, A40E+E56R+E210K, A40E+E56R+T244E, A40E+E56R+D254S, A40E+D57N+G9 1T, A40E+D57N+K98E, A40E+D57N+R108K, A40E+D57N+R118F, A40E+D57N+ E210K, A40E+D57N+T244E, A40E+D57N+D254S, A40E+G91T+K98E, A40E+G9 1T+R108K, A40E+G91T+R118F, A40E+G91T+E210K, A40E+G91T+T244E, A40 E+G91T+D254S, A40E+K98E+R108K, A40E+K98E+R118F, A40E+K98E+E210K,A40E+K98E+T244E、A40E+K98E+D254S、A40E+R108K+R118F、A40E+R108K+E210K、A40E+R108K+T244E、A40E+R108K+D254S、A40E+R118F+E210K、A40E+R118F+T244E、A40E+R118F+D254S、A40E+E210K+T244E、A40E+E210K+D254S、A40E+T244E+D254S、E56R+D57N+G91T、E56R+D57N+K98E、E56R+D57N+R108K、E56R+D57N+R118F、E56R+D57N+E210K、E56R+D57N+T244E、E56R+D57N+D254S、E56R+G91T+K98E、E56R+G91T+R108K、E56R+G91T+R118F、E56R+G91T+E210K、E56R+G91T+T244E、E56R+G91T+D254S、E56R+K98E+R108K、E56R+K98E+R118F、E56R+K98E+E210K、E56R+K98E+T244E、E56R+K98E+D254S、E56R+R108K+R118F、E56R+R108K+E210K、E56R+R108K+T244E、E56R+R108K+D254S、E56R+R118F+E210K、E56R+R118F+T244E、E56R+R118F+D254S、E56R+E210K+T244E、E56R+E210K+D254S、E56R+T244E+D254S、D57N+G91T+K98E、D57N+G91T+R108K、D57N+G91T+R118F、D57N+G91T+E210K、D57N+G91T+T244E、D57N+G91T+D254S、D57N+K98E+R108K、D57N+K98E+R118F、D57N+K98E+E210K、D57N+K98E+T244E、D57N+K98E+D254S、D57N+R108K+R118F、D57N+R108K+E210K、D57N+R108K+T244E、D57N+R108K+D254S、D57N+R118F+E210K、D57N+R118F+T244E、D57N+R118F+D254S、D57N+E210K+T244E、D57N+E210K+D254S、D57N+T244E+D254S、G91T+K98E+R108K、G91T+K98E+R118F、G91T+K98E+E210K、G91T+K98E+T244E、G91T+K98E+D254S、G91T+R108K+R118F、G91T+R108K+E210K、G91T+R108K+T244E、G91T+R108K+D254S、G91T+R118F+E210K、G91T+R118F+T244E、G91T+R118F+D254S、G91T+E210K+T244E、G91T+E210K+D254S、G91T+T244E+D254S、K98E+R108K+R118F、K98E+R108K+E210K、K98E+R108K+T244E、K98E+R108K+D254S、K98E+R118F+E210K、K98E+R118F+T244E、K98E+R118F+D254S、K98E+E210K+T244E、K98E+E210K+D254S、K98E+T244E+D254S、R108K+R118F+E210K、R108K+R118F+T244E、R108K+R118F+D254S、R108K+E210K+T244E、R108K+E210K+D254S、R108K+T244E+D254S、R118F+E210K+T244E、R118F+E210K+D254S、R118F+T244E+D254S、E210K+T244E+D254S、A40E+E56R+D57N+G91T、A40E+E56R+D57N+K98E、A40E+E56R+D57N+R108K、A40E+E56R+D57N+R118F、A40E+E56R+D57N+E210K、A40E+E56R+D57N+T244E、A40E+E56R+D57N+D254S、A40E+E56R+G91T+K98E、A40E+E56R+G91T+R108K、A40E+E56R+G91T+R118F、A40E+E56R+G91T+E210K、A40E+E56R+G91T+T244E、A40E+E56R+G91T+D254S、A40E+E56R+K98E+R108K、A40E+E56R+K98E+R118F、A40E+E56R+K98E+E210K、A40E+E56R+K98E+T244E、A40E+E56R+K98E+D254S、A40E+E56R+R108K+R118F、A40E+E56R+R108K+E210K、A40E+E56R+R108K+T244E、A40E+E56R+R108K+D254S、A40E+E56R+R118F+E210K、A40E+E56R+R118F+T244E、A40E+E56R+R118F+D254S、A40E+E56R+E210K+T244E、A40E+E56R+E210K+D254S、A40E+E56R+T244E+D254S、A40E+D57N+G91T+K98E、A40E+D57N+G91T+R108K、A40E+D57N+G91T+R118F、A40E+D57N+G91T+E210K、A40E+D57N+G91T+T244E、A40E+D57N+G91T+D254S、A40E+D57N+K98E+R108K、A40E+D57N+K98E+R118F、A40E+D57N+K98E+E210K、A40E+D57N+K98E+T244E、A40E+D57N+K98E+D254S、A40E+D57N+R108K+R118F、A40E+D57N+R108K+E210K、A40E+D57N+R108K+T244E、A40E+D57N+R108K+D254S、A40E+D57N+R118F+E210K、A40E+D57N+R118F+T244E、A40E+D57N+R118F+D254S、A40E+D57N+E210K+T244E、A40E+D57N+E210K+D254S、A40E+D57N+T244E+D254S、A40E+G91T+K98E+R108K、A40E+G91T+K98E+R118F、A40E+G91T+K98E+E210K、A40E+G91T+K98E+T244E、A40E+G91T+K98E+D254S、A40E+G91T+R108K+R118F、A40E+G91T+R108K+E210K、A40E+G91T+R108K+T244E、A40E+G91T+R108K+D254S、A40E+G91T+R118F+E210K、A40E+G91T+R118F+T244E、A40E+G91T+R118F+D254S、A40E+G91T+E210K+T244E、A40E+G91T+E210K+D254S、A40E+G91T+T244E+D254S、A40E+K98E+R108K+R118F、A40E+K98E+R108K+E210K、A40E+K98E+R108K+T244E、A40E+K98E+R108K+D254S、A40E+K98E+R118F+E210K、A40E+K98E+R118F+T244E、A40E+K98E+R118F+D254S、A40E+K98E+E210K+T244E、A40E+K98E+E210K+D254S、A40E+K98E+T244E+D254S、A40E+R108K+R118F+E210K、A40E+R108K+R118F+T244E、A40E+R108K+R118F+D254S、A40E+R108K+E210K+T244E、A40E+R108K+E210K+D254S、A40E+R108K+T244E+D254S、A40E+R118F+E210K+T244E、A40E+R118F+E210K+D254S、A40E+R118F+T244E+D254S、A40E+E210K+T244E+D254S、E56R+D57N+G91T+K98E、E56R+D57N+G91T+R108K、E56R+D57N+G91T+R118F、E56R+D57N+G91T+E210K、E56R+D57N+G91T+T244E、E56R+D57N+G91T+D254S、E56R+D57N+K98E+R108K、E56R+D57N+K98E+R118F、E56R+D57N+K98E+E210K、E56R+D57N+K98E+T244E、E56R+D57N+K98E+D254S、E56R+D57N+R108K+R118F、E56R+D57N+R108K+E210K、E56R+D57N+R108K+T244E、E56R+D57N+R108K+D254S、E56R+D57N+R118F+E210K、E56R+D57N+R118F+T244E、E56R+D57N+R118F+D254S、E56R+D57N+E210K+T244E、E56R+D57N+E210K+D254S、E56R+D57N+T244E+D254S、E56R+G91T+K98E+R108K、E56R+G91T+K98E+R118F、E56R+G91T+K98E+E210K、E56R+G91T+K98E+T244E、E56R+G91T+K98E+D254S、E56R+G91T+R108K+R118F、E56R+G91T+R108K+E210K、E56R+G91T+R108K+T244E、E56R+G91T+R108K+D254S、E56R+G91T+R118F+E210K、E56R+G91T+R118F+T244E、E56R+G91T+R118F+D254S、E56R+G91T+E210K+T244E、E56R+G91T+E210K+D254S、E56R+G91T+T244E+D254S、E56R+K98E+R108K+R118F、E56R+K98E+R108K+E210K、E56R+K98E+R108K+T244E、E56R+K98E+R108K+D254S、E56R+K98E+R118F+E210K、E56R+K98E+R118F+T244E、E56R+K98E+R118F+D254S、E56R+K98E+E210K+T244E、E56R+K98E+E210K+D254S、E56R+K98E+T244E+D254S、E56R+R108K+R118F+E210K、E56R+R108K+R118F+T244E、E56R+R108K+R118F+D254S、E56R+R108K+E210K+T244E、E56R+R108K+E210K+D254S、E56R+R108K+T244E+D254S、E56R+R118F+E210K+T244E、E56R+R118F+E210K+D254S、E56R+R118F+T244E+D254S、E56R+E210K+T244E+D254S、D57N+G91T+K98E+R108K、D57N+G91T+K98E+R118F、D57N+G91T+K98E+E210K、D57N+G91T+K98E+T244E、D57N+G91T+K98E+D254S、D57N+G91T+R108K+R118F、D57N+G91T+R108K+E210K、D57N+G91T+R108K+T244E、D57N+G91T+R108K+D254S、D57N+G91T+R118F+E210K、D57N+G91T+R118F+T244E、D57N+G91T+R118F+D254S、D57N+G91T+E210K+T244E、D57N+G91T+E210K+D254S、D57N+G91T+T244E+D254S、D57N+K98E+R108K+R118F、D57N+K98E+R108K+E210K、D57N+K98E+R108K+T244E、D57N+K98E+R108K+D254S、D57N+K98E+R118F+E210K、D57N+K98E+R118F+T244E、D57N+K98E+R118F+D254S、D57N+K98E+E210K+T244E、D57N+K98E+E210K+D254S、D57N+K98E+T244E+D254S、D57N+R108K+R118F+E210K、D57N+R108K+R118F+T244E、D57N+R108K+R118F+D254S、D57N+R108K+E210K+T244E、D57N+R108K+E210K+D254S、D57N+R108K+T244E+D254S、D57N+R118F+E210K+T244E、D57N+R118F+E210K+D254S、D57N+R118F+T244E+D254S、D57N+E210K+T244E+D254S、G91T+K98E+R108K+R118F、G91T+K98E+R108K+E210K、G91T+K98E+R108K+T244E、G91T+K98E+R108K+D254S、G91T+K98E+R118F+E210K、G91T+K98E+R118F+T244E、G91T+K98E+R118F+D254S、G91T+K98E+E210K+T244E、G91T+K98E+E210K+D254S、G91T+K98E+T244E+D254S、G91T+R108K+R118F+E210K、G91T+R108K+R118F+T244E、G91T+R108K+R118F+D254S、G91T+R108K+E210K+T244E、G91T+R108K+E210K+D254S、G91T+R108K+T244E+D254S、G91T+R118F+E210K+T244E、G91T+R118F+E210K+D254S、G91T+R118F+T244E+D254S、G91T+E210K+T244E+D254S、K98E+R108K+R118F+E210K、K98E+R108K+R118F+T244E、K98E+R108K+R118F+D254S、K98E+R108K+E210K+T244E、K98E+R108K+E210K+D254S、K98E+R108K+T244E+D254S、K98E+R118F+E210K+T244E、K98E+R118F+E210K+D254S、K98E+R118F+T244E+D254S、K98E+E210K+T244E+D254S、R108K+R118F+E210K+T244E、R108K+R118F+E210K+D254S、R108K+R118F+T244E+D254S、R108K+E210K+T244E+D254S、R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E、A40E+E56R+D57N+G91T+R108K、A40E+E56R+D57N+G91T+R118F、A40E+E56R+D57N+G91T+E210K、A40E+E56R+D57N+G91T+T244E、A40E+E56R+D57N+G91T+D254S、A40E+E56R+D57N+K98E+R108K、A40E+E56R+D57N+K98E+R118F、A40E+E56R+D57N+K98E+E210K、A40E+E56R+D57N+K98E+T244E、A40E+E56R+D57N+K98E+D254S、A40E+E56R+D57N+R108K+R118F、A40E+E56R+D57N+R108K+E210K、A40E+E56R+D57N+R108K+T244E、A40E+E56R+D57N+R108K+D254S、A40E+E56R+D57N+R118F+E210K、A40E+E56R+D57N+R118F+T244E、A40E+E56R+D57N+R118F+D254S、A40E+E56R+D57N+E210K+T244E、A40E+E56R+D57N+E210K+D254S、A40E+E56R+D57N+T244E+D254S、A40E+E56R+G91T+K98E+R108K、A40E+E56R+G91T+K98E+R118F、A40E+E56R+G91T+K98E+E210K、A40E+E56R+G91T+K98E+T244E、A40E+E56R+G91T+K98E+D254S、A40E+E56R+G91T+R108K+R118F、A40E+E56R+G91T+R108K+E210K、A40E+E56R+G91T+R108K+T244E、A40E+E56R+G91T+R108K+D254S、A40E+E56R+G91T+R118F+E210K、A40E+E56R+G91T+R118F+T244E、A40E+E56R+G91T+R118F+D254S、A40E+E56R+G91T+E210K+T244E、A40E+E56R+G91T+E210K+D254S、A40E+E56R+G91T+T244E+D254S、A40E+E56R+K98E+R108K+R118F、A40E+E56R+K98E+R108K+E210K、A40E+E56R+K98E+R108K+T244E、A40E+E56R+K98E+R108K+D254S、A40E+E56R+K98E+R118F+E210K、A40E+E56R+K98E+R118F+T244E、A40E+E56R+K98E+R118F+D254S、A40E+E56R+K98E+E210K+T244E、A40E+E56R+K98E+E210K+D254S、A40E+E56R+K98E+T244E+D254S、A40E+E56R+R108K+R118F+E210K、A40E+E56R+R108K+R118F+T244E、A40E+E56R+R108K+R118F+D254S、A40E+E56R+R108K+E210K+T244E、A40E+E56R+R108K+E210K+D254S、A40E+E56R+R108K+T244E+D254S、A40E+E56R+R118F+E210K+T244E、A40E+E56R+R118F+E210K+D254S、A40E+E56R+R118F+T244E+D254S、A40E+E56R+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K、A40E+D57N+G91T+K98E+R118F、A40E+D57N+G91T+K98E+E210K、A40E+D57N+G91T+K98E+T244E、A40E+D57N+G91T+K98E+D254S、A40E+D57N+G91T+R108K+R118F、A40E+D57N+G91T+R108K+E210K、A40E+D57N+G91T+R108K+T244E、A40E+D57N+G91T+R108K+D254S、A40E+D57N+G91T+R118F+E210K、A40E+D57N+G91T+R118F+T244E、A40E+D57N+G91T+R118F+D254S、A40E+D57N+G91T+E210K+T244E、A40E+D57N+G91T+E210K+D254S、A40E+D57N+G91T+T244E+D254S、A40E+D57N+K98E+R108K+R118F、A40E+D57N+K98E+R108K+E210K、A40E+D57N+K98E+R108K+T244E、A40E+D57N+K98E+R108K+D254S、A40E+D57N+K98E+R118F+E210K、A40E+D57N+K98E+R118F+T244E、A40E+D57N+K98E+R118F+D254S、A40E+D57N+K98E+E210K+T244E、A40E+D57N+K98E+E210K+D254S、A40E+D57N+K98E+T244E+D254S、A40E+D57N+R108K+R118F+E210K、A40E+D57N+R108K+R118F+T244E、A40E+D57N+R108K+R118F+D254S、A40E+D57N+R108K+E210K+T244E、A40E+D57N+R108K+E210K+D254S、A40E+D57N+R108K+T244E+D254S、A40E+D57N+R118F+E210K+T244E、A40E+D57N+R118F+E210K+D254S、A40E+D57N+R118F+T244E+D254S、A40E+D57N+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F、A40E+G91T+K98E+R108K+E210K、A40E+G91T+K98E+R108K+T244E、A40E+G91T+K98E+R108K+D254S、A40E+G91T+K98E+R118F+E210K、A40E+G91T+K98E+R118F+T244E、A40E+G91T+K98E+R118F+D254S、A40E+G91T+K98E+E210K+T244E、A40E+G91T+K98E+E210K+D254S、A40E+G91T+K98E+T244E+D254S、A40E+G91T+R108K+R118F+E210K、A40E+G91T+R108K+R118F+T244E、A40E+G91T+R108K+R118F+D254S、A40E+G91T+R108K+E210K+T244E、A40E+G91T+R108K+E210K+D254S、A40E+G91T+R108K+T244E+D254S、A40E+G91T+R118F+E210K+T244E、A40E+G91T+R118F+E210K+D254S、A40E+G91T+R118F+T244E+D254S、A40E+G91T+E210K+T244E+D254S、A40E+K98E+R108K+R118F+E210K、A40E+K98E+R108K+R118F+T244E、A40E+K98E+R108K+R118F+D254S、A40E+K98E+R108K+E210K+T244E、A40E+K98E+R108K+E210K+D254S、A40E+K98E+R108K+T244E+D254S、A40E+K98E+R118F+E210K+T244E、A40E+K98E+R118F+E210K+D254S、A40E+K98E+R118F+T244E+D254S、A40E+K98E+E210K+T244E+D254S、A40E+R108K+R118F+E210K+T244E、A40E+R108K+R118F+E210K+D254S、A40E+R108K+R118F+T244E+D254S、A40E+R108K+E210K+T244E+D254S、A40E+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K、E56R+D57N+G91T+K98E+R118F、E56R+D57N+G91T+K98E+E210K、E56R+D57N+G91T+K98E+T244E、E56R+D57N+G91T+K98E+D254S、E56R+D57N+G91T+R108K+R118F、E56R+D57N+G91T+R108K+E210K、E56R+D57N+G91T+R108K+T244E、E56R+D57N+G91T+R108K+D254S、E56R+D57N+G91T+R118F+E210K、E56R+D57N+G91T+R118F+T244E、E56R+D57N+G91T+R118F+D254S、E56R+D57N+G91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98E+R118F+E210K+T244E+D254S、G91T+R108K+R118F+E210K+T244E+D254S、K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F、A40E+E56R+D57N+G91T+K98E+R108K+E210K、A40E+E56R+D57N+G91T+K98E+R108K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R118F+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E、A40E+E56R+D57N+G91T+K98E+E210K+D254S、A40E+E56R+D57N+G91T+K98E+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K、A40E+E56R+D57N+G91T+R108K+R118F+T244E、A40E+E56R+D57N+G91T+R108K+R118F+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E、A40E+E56R+D57N+G91T+R108K+E210K+D254S、A40E+E56R+D57N+G91T+R108K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R118F+T244E+D254S、A40E+E56R+D57N+G91T+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K、A40E+E56R+D57N+K98E+R108K+R118F+T244E、A40E+E56R+D57N+K98E+R108K+R118F+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E、A40E+E56R+D57N+K98E+R108K+E210K+D254S、A40E+E56R+D57N+K98E+R108K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R118F+T244E+D254S、A40E+E56R+D57N+K98E+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E、A40E+E56R+D57N+R108K+R118F+E210K+D254S、A40E+E56R+D57N+R108K+R118F+T244E+D254S、A40E+E56R+D57N+R108K+E210K+T244E+D254S、A40E+E56R+D57N+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K、A40E+E56R+G91T+K98E+R108K+R118F+T244E、A40E+E56R+G91T+K98E+R108K+R118F+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E、A40E+E56R+G91T+K98E+R108K+E210K+D254S、A40E+E56R+G91T+K98E+R108K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R118F+T244E+D254S、A40E+E56R+G91T+K98E+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E、A40E+E56R+G91T+R108K+R118F+E210K+D254S、A40E+E56R+G91T+R108K+R118F+T244E+D254S、A40E+E56R+G91T+R108K+E210K+T244E+D254S、A40E+E56R+G91T+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E、A40E+E56R+K98E+R108K+R118F+E210K+D254S、A40E+E56R+K98E+R108K+R118F+T244E+D254S、A40E+E56R+K98E+R108K+E210K+T244E+D254S、A40E+E56R+K98E+R118F+E210K+T244E+D254S、A40E+E56R+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K、A40E+D57N+G91T+K98E+R108K+R118F+T244E、A40E+D57N+G91T+K98E+R108K+R118F+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E、A40E+D57N+G91T+K98E+R108K+E210K+D254S、A40E+D57N+G91T+K98E+R108K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R118F+T244E+D254S、A40E+D57N+G91T+K98E+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E、A40E+D57N+G91T+R108K+R118F+E210K+D254S、A40E+D57N+G91T+R108K+R118F+T244E+D254S、A40E+D57N+G91T+R108K+E210K+T244E+D254S、A40E+D57N+G91T+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E、A40E+D57N+K98E+R108K+R118F+E210K+D254S、A40E+D57N+K98E+R108K+R118F+T244E+D254S、A40E+D57N+K98E+R108K+E210K+T244E+D254S、A40E+D57N+K98E+R118F+E210K+T244E+D254S、A40E+D57N+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E、A40E+G91T+K98E+R108K+R118F+E210K+D254S、A40E+G91T+K98E+R108K+R118F+T244E+D254S、A40E+G91T+K98E+R108K+E210K+T244E+D254S、A40E+G91T+K98E+R118F+E210K+T244E+D254S、A40E+G91T+R108K+R118F+E210K+T244E+D254S、A40E+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K、E56R+D57N+G91T+K98E+R108K+R118F+T244E、E56R+D57N+G91T+K98E+R108K+R118F+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E、E56R+D57N+G91T+K98E+R108K+E210K+D254S、E56R+D57N+G91T+K98E+R108K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R118F+T244E+D254S、E56R+D57N+G91T+K98E+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E、E56R+D57N+G91T+R108K+R118F+E210K+D254S、E56R+D57N+G91T+R108K+R118F+T244E+D254S、E56R+D57N+G91T+R108K+E210K+T244E+D254S、E56R+D57N+G91T+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E、E56R+D57N+K98E+R108K+R118F+E210K+D254S、E56R+D57N+K98E+R108K+R118F+T244E+D254S、E56R+D57N+K98E+R108K+E210K+T244E+D254S、E56R+D57N+K98E+R118F+E210K+T244E+D254S、E56R+D57N+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E、E56R+G91T+K98E+R108K+R118F+E210K+D254S、E56R+G91T+K98E+R108K+R118F+T244E+D254S、E56R+G91T+K98E+R108K+E210K+T244E+D254S、E56R+G91T+K98E+R118F+E210K+T244E+D254S、E56R+G91T+R108K+R118F+E210K+T244E+D254S、E56R+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E、D57N+G91T+K98E+R108K+R118F+E210K+D254S、D57N+G91T+K98E+R108K+R118F+T244E+D254S、D57N+G91T+K98E+R108K+E210K+T244E+D254S、D57N+G91T+K98E+R118F+E210K+T244E+D254S、D57N+G91T+R108K+R118F+E210K+T244E+D254S、D57N+K98E+R108K+R118F+E210K+T244E+D254S、G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S、A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S。、

[0129] In embodiments, a variant of the invention has a substitution corresponding to I202H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to G23S, D27N, A40I, F51I, E56R, V60K, R118F, T244E, P256T, G23S+D27N, G23S+A40I, G23S+F51I, G23S+E56R, G23S+V60K, G23S+R118F, G23S+T244E, G23S+P256T, D27N+A40I, D27N+F51I, D27N+E56R, D27N+V60K, D27N+R118F, D27N+T244E, , A40I+F51I, A40I+E56R, A40I+V60K, A40I+R118F, A40I+T244E, A40I+P25 6T, F51I+E56R, F51I+V60K, F51I+R118F, F51I+T244E, F51I+P256T, E56R+ V60K, E56R+R118F, E56R+T244E, E56R+P256T, V60K+R118F, V60K+T244E, V 60K+P256T, R118F+T244E, R118F+P256T, T244E+P256T, G23S+D27N+A40I, G23S+D27N+F51I, G23S+D27N+E56R, G23S+D27N+V60K, G23S+D27N+R118F , G23S+D27N+T244E, G23S+D27N+P256T, G23S+A40I+F51I, G23S+A40I+E56 R、G23S+A40I+V60K、G23S+A40I+R118F、G23S+A40I+T244E、G23S+A40I+P2 56T, G23S+F51I+E56R, G23S+F51I+V60K, G23S+F51I+R118F, G23S+F51I+T 244E, G23S+F51I+P256T, G23S+E56R+V60K, G23S+E56R+R118F, G23S+E56R +T244E, G23S+E56R+P256T, G23S+V60K+R118F, G23S+V60K+T244E, G23S+V 60K+P256T, G23S+R118F+T244E, G23S+R118F+P256T, G23S+T244E+P256T, D27N+A40I+F51I, D27N+A40I+E56R, D27N+A40I+V60K, 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23S+D27N+A40I+E56R+V60K+R118F+P256T、G23S+D27N+A40I+E56R+V60K+T244E+P256T、G23S+D27N+A40I+E56R+R118F+T244E+P256T、G23S+D27N+A40I+V60K+R118F+T244E+P256T、G23S+D27N+F51I+E56R+V60K+R118F+T244E、G23S+D27N+F51I+E56R+V60K+R118F+P256T、G23S+D27N+F51I+E56R+V60K+T244E+P256T、G23S+D27N+F51I+E56R+R118F+T244E+P256T、G23S+D27N+F51I+V60K+R118F+T244E+P256T、G23S+D27N+E56R+V60K+R118F+T244E+P256T、G23S+A40I+F51I+E56R+V60K+R118F+T244E、G23S+A40I+F51I+E56R+V60K+R118F+P256T、G23S+A40I+F51I+E56R+V60K+T244E+P256T、G23S+A40I+F51I+E56R+R118F+T244E+P256T、G23S+A40I+F51I+V60K+R118F+T244E+P256T、G23S+A40I+E56R+V60K+R118F+T244E+P256T、G23S+F51I+E56R+V60K+R118F+T244E+P256T、D27N+A40I+F51I+E56R+V60K+R118F+T244E、D27N+A40I+F51I+E56R+V60K+R118F+P256T、D27N+A40I+F51I+E56R+V60K+T244E+P256T、D27N+A40I+F51I+E56R+R118F+T244E+P256T、D27N+A40I+F51I+V60K+R118F+T244E+P256T、D27N+A40I+E56R+V60K+R118F+T244E+P256T、D27N+F51I+E56R+V60K+R118F+T244E+P256T、A40I+F51I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E、G23S+D27N+A40I+F51I+E56R+V60K+R118F+P256T、G23S+D27N+A40I+F51I+E56R+V60K+T244E+P256T、G23S+D27N+A40I+F51I+E56R+R118F+T244E+P256T、G23S+D27N+A40I+F51I+V60K+R118F+T244E+P256T、G23S+D27N+A40I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+F51I+E56R+V60K+R118F+T244E+P256T、G23S+A40I+F51I+E56R+V60K+R118F+T244E+P256T、D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T。、

[0130] In embodiments, a variant of the invention has a substitution corresponding to I252H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to G23S, D27N, A40I, F51I, E56R, V60K, R118F, T244E, P256T, G23S+D27N, G23S+A40I, G23S+F51I, G23S+E56R, G23S+V60K, G23S+R118F, G23S+T244E, G23S+P256T, D27N+A40I, D27N+F51I, D27N+E56R, D27N+V60K, D27N+R118F, D27N+T244E, , A40I+F51I, A40I+E56R, A40I+V60K, A40I+R118F, A40I+T244E, A40I+P25 6T, F51I+E56R, F51I+V60K, F51I+R118F, F51I+T244E, F51I+P256T, E56R+ V60K, E56R+R118F, E56R+T244E, E56R+P256T, V60K+R118F, V60K+T244E, V 60K+P256T, R118F+T244E, R118F+P256T, T244E+P256T, G23S+D27N+A40I, G23S+D27N+F51I, G23S+D27N+E56R, G23S+D27N+V60K, G23S+D27N+R118F , G23S+D27N+T244E, G23S+D27N+P256T, G23S+A40I+F51I, G23S+A40I+E56 R、G23S+A40I+V60K、G23S+A40I+R118F、G23S+A40I+T244E、G23S+A40I+P2 56T, G23S+F51I+E56R, G23S+F51I+V60K, G23S+F51I+R118F, G23S+F51I+T 244E, G23S+F51I+P256T, G23S+E56R+V60K, G23S+E56R+R118F, G23S+E56R +T244E, G23S+E56R+P256T, G23S+V60K+R118F, G23S+V60K+T244E, G23S+V 60K+P256T, G23S+R118F+T244E, G23S+R118F+P256T, G23S+T244E+P256T, D27N+A40I+F51I, D27N+A40I+E56R, D27N+A40I+V60K, 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23S+D27N+A40I+E56R+V60K+R118F+P256T、G23S+D27N+A40I+E56R+V60K+T244E+P256T、G23S+D27N+A40I+E56R+R118F+T244E+P256T、G23S+D27N+A40I+V60K+R118F+T244E+P256T、G23S+D27N+F51I+E56R+V60K+R118F+T244E、G23S+D27N+F51I+E56R+V60K+R118F+P256T、G23S+D27N+F51I+E56R+V60K+T244E+P256T、G23S+D27N+F51I+E56R+R118F+T244E+P256T、G23S+D27N+F51I+V60K+R118F+T244E+P256T、G23S+D27N+E56R+V60K+R118F+T244E+P256T、G23S+A40I+F51I+E56R+V60K+R118F+T244E、G23S+A40I+F51I+E56R+V60K+R118F+P256T、G23S+A40I+F51I+E56R+V60K+T244E+P256T、G23S+A40I+F51I+E56R+R118F+T244E+P256T、G23S+A40I+F51I+V60K+R118F+T244E+P256T、G23S+A40I+E56R+V60K+R118F+T244E+P256T、G23S+F51I+E56R+V60K+R118F+T244E+P256T、D27N+A40I+F51I+E56R+V60K+R118F+T244E、D27N+A40I+F51I+E56R+V60K+R118F+P256T、D27N+A40I+F51I+E56R+V60K+T244E+P256T、D27N+A40I+F51I+E56R+R118F+T244E+P256T、D27N+A40I+F51I+V60K+R118F+T244E+P256T、D27N+A40I+E56R+V60K+R118F+T244E+P256T、D27N+F51I+E56R+V60K+R118F+T244E+P256T、A40I+F51I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E、G23S+D27N+A40I+F51I+E56R+V60K+R118F+P256T、G23S+D27N+A40I+F51I+E56R+V60K+T244E+P256T、G23S+D27N+A40I+F51I+E56R+R118F+T244E+P256T、G23S+D27N+A40I+F51I+V60K+R118F+T244E+P256T、G23S+D27N+A40I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+F51I+E56R+V60K+R118F+T244E+P256T、G23S+A40I+F51I+E56R+V60K+R118F+T244E+P256T、D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T。、

[0131] In embodiments, a variant of the invention has a substitution corresponding to L269H of SEQ ID NO: 8 and further comprises one of the following substitutions or groups of substitutions corresponding to G23S, D27N, A40I, F51I, E56R, V60K, R118F, T244E, P256T, G23S+D27N, G23S+A40I, G23S+F51I, G23S+E56R, G23S+V60K, G23S+R118F, G23S+T244E, G23S+P256T, D27N+A40I, D27N+F51I, D27N+E56R, D27N+V60K, D27N+R118F, D27N+T244E, , A40I+F51I, A40I+E56R, A40I+V60K, A40I+R118F, A40I+T244E, A40I+P25 6T, F51I+E56R, F51I+V60K, F51I+R118F, F51I+T244E, F51I+P256T, E56R+ V60K, E56R+R118F, E56R+T244E, E56R+P256T, V60K+R118F, V60K+T244E, V 60K+P256T, R118F+T244E, R118F+P256T, T244E+P256T, G23S+D27N+A40I, G23S+D27N+F51I, G23S+D27N+E56R, G23S+D27N+V60K, G23S+D27N+R118F , G23S+D27N+T244E, G23S+D27N+P256T, G23S+A40I+F51I, G23S+A40I+E56 R、G23S+A40I+V60K、G23S+A40I+R118F、G23S+A40I+T244E、G23S+A40I+P2 56T, G23S+F51I+E56R, G23S+F51I+V60K, G23S+F51I+R118F, G23S+F51I+T 244E, G23S+F51I+P256T, G23S+E56R+V60K, G23S+E56R+R118F, G23S+E56R +T244E, G23S+E56R+P256T, G23S+V60K+R118F, G23S+V60K+T244E, G23S+V 60K+P256T, G23S+R118F+T244E, G23S+R118F+P256T, G23S+T244E+P256T, D27N+A40I+F51I, D27N+A40I+E56R, D27N+A40I+V60K, 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23S+D27N+A40I+E56R+V60K+R118F+P256T、G23S+D27N+A40I+E56R+V60K+T244E+P256T、G23S+D27N+A40I+E56R+R118F+T244E+P256T、G23S+D27N+A40I+V60K+R118F+T244E+P256T、G23S+D27N+F51I+E56R+V60K+R118F+T244E、G23S+D27N+F51I+E56R+V60K+R118F+P256T、G23S+D27N+F51I+E56R+V60K+T244E+P256T、G23S+D27N+F51I+E56R+R118F+T244E+P256T、G23S+D27N+F51I+V60K+R118F+T244E+P256T、G23S+D27N+E56R+V60K+R118F+T244E+P256T、G23S+A40I+F51I+E56R+V60K+R118F+T244E、G23S+A40I+F51I+E56R+V60K+R118F+P256T、G23S+A40I+F51I+E56R+V60K+T244E+P256T、G23S+A40I+F51I+E56R+R118F+T244E+P256T、G23S+A40I+F51I+V60K+R118F+T244E+P256T、G23S+A40I+E56R+V60K+R118F+T244E+P256T、G23S+F51I+E56R+V60K+R118F+T244E+P256T、D27N+A40I+F51I+E56R+V60K+R118F+T244E、D27N+A40I+F51I+E56R+V60K+R118F+P256T、D27N+A40I+F51I+E56R+V60K+T244E+P256T、D27N+A40I+F51I+E56R+R118F+T244E+P256T、D27N+A40I+F51I+V60K+R118F+T244E+P256T、D27N+A40I+E56R+V60K+R118F+T244E+P256T, D27N+F51I+E56R+V60K+R118F+T244 E+P256T, A40I+F51I+E56R+V60K+R118F+T244E+P256T, G23S+D27N+A40I+F51I+E5 6R+V60K+R118F+T244E, G23S+D27N+A40I+F51I+E56R+V60K+R118F+P256T, G23S+D 27N+A40I+F51I+E56R+V60K+T244E+P256T, G23S+D27N+A40I+F51I+E56R+R118F+T2 44E+P256T, G23S+D27N+A40I+F51I+V60K+R118F+T244E+P256T, G23S+D27N+A40I+ E56R+V60K+R118F+T244E+P256T, G23S+D27N+F51I+E56R+V60K+R118F+T244E+P25 6T, G23S+A40I+F51I+E56R+V60K+R118F+T244E+P256T, D27N+A40I+F51I+E56R+V6 0K+R118F+T244E+P256T, G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T. ,

[0132] In a preferred embodiment, the variant of the present invention comprises or consists of one of the following substitution groups corresponding to:

[0133] A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S+I202H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254 S+I252H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S+L269H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T2 44E+D254S+I202H+I252H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S+I202H+L269H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S+I252H+L269H, A40E+E56R+D57N+G91T+K98E+R108K+R118F+E210K+T244E+D254S+I252H+L269H (using SEQ ID NO: 8 for numbering).

[0134] In a preferred embodiment, the variant of the present invention comprises or consists of one of the following substitution groups corresponding to:

[0135] G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+I202H, G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+I 252H、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+L269H、G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P2 56T+I202H+I252H, G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+I202H+L269H, G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+I252H+L269H, G23S+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T+I202H+H252H+L269H (using SEQ ID NO: 8 for numbering).

[0136] In a preferred embodiment, the variant of the present invention may further comprise one or more of the substitutions corresponding to F7K, F51I, F51L, F51V, F51Y, H198D, H198G, H198F, H198I, H198L, H198N, H198S, H198T, H198Y, N200Q, S224F, S224P, L227D, L227E, L227R, V228P, V230R, I255G, I255N, A257F and A257I (using SEQ ID NO: 8 for numbering).

[0137] The amino acid changes may be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0138] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL Hill, 1979, in The Proteins, Academic Press, New York. Common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0139] Alternatively, these amino acid changes have such a property that the physicochemical properties of the polypeptide are altered. For example, amino acid changes can improve the thermal stability of the polypeptide, change substrate specificity, change the optimal pH, etc.

[0140] Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for lipase activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutating putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a family of polypeptides or proteins derived from a common ancestor (typically having similar three-dimensional structure, function, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596:583-589.

[0141] The variants of the present invention preferably consist of 269 amino acids, but may also contain peptide extensions / additions at the N-terminus and / or C-terminus. The peptide extensions may be 1-20 amino acids long. At the N-terminus, the peptide extensions / additions may preferably contain 1-5 amino acid SPIRRs.

[0142] The polypeptide may be a fusion polypeptide comprising a variant of the invention.In one aspect, the variant is isolated.

[0143] The variants of the present invention have improved properties relative to the parent in that the lipase variants of the present invention have reduced lipase activity and / or reduced odor production at around neutral pH (i.e., about pH 6-8, preferably about pH 7), and / or have an increased benefit-risk factor (BRF) compared to the parent lipase (in particular SEQ ID NO: 2, 4, 6 or 8, respectively).

[0144] According to the present invention, the wash performance may be measured as relative wash performance (RP(wash)) compared to a parent lipase (particularly SEQ ID NO: 2, 4, 6 or 8, respectively).

[0145] In an embodiment, the relative wash performance (RP(wash)) is greater than 1.0, preferably greater than 1.1, preferably greater than 1.2, preferably greater than 1.3, preferably greater than 1.4, preferably greater than 1.5, preferably greater than 1.6, preferably greater than 1.7, preferably greater than 1.8, preferably greater than 1.9, preferably greater than 2.0, preferably greater than 2.5, preferably greater than 3.0, preferably greater than 3.5, preferably greater than 4.0, preferably greater than 5.0, preferably greater than 6.0, preferably greater than 7.0, preferably greater than 8.0, preferably greater than 9.0, preferably greater than 10.0.

[0146] According to the present invention, odor production may be measured as relative odor production (RP(odor)) compared to a parent lipase (particularly SEQ ID NO: 2, 4, 6 or 8, respectively).

[0147] In an embodiment, the odor generation is less than 1.0, preferably less than 0.9, preferably less than 0.8, preferably less than 0.7, preferably less than 0.6, preferably less than 0.5, preferably less than 0.4, preferably less than 0.3, preferably less than 0.2, preferably less than 0.1.

[0148] According to the present invention, the benefit-risk factor (BRF) is the relative wash performance (benefit) compared to the relative odor generation (risk) and is calculated as RP(wash) / RP(odor). If the benefit-risk factor of a lipase variant is greater than 1.0, the lipase has a better wash performance with respect to the odor released than the reference lipase (particularly the parent lipase in SEQ ID NO: 2, 4, 6 or 8, respectively).

[0149] In an embodiment, the BRF is greater than 1.0, preferably greater than 1.1, preferably greater than 1.2, preferably greater than 1.3, preferably greater than 1.4, preferably greater than 1.5, preferably greater than 1.6, preferably greater than 1.7, preferably greater than 1.8, preferably greater than 1.9, preferably greater than 2.0, preferably greater than 2.5, preferably greater than 3.0, preferably greater than 3.5, preferably greater than 4.0, preferably greater than 5.0, preferably greater than 6.0, preferably greater than 7.0, preferably greater than 8.0, preferably greater than 9.0, preferably greater than 10.0.

[0150] In preferred embodiments, the relative wash performance (RP(wash)) and BRF are both greater than 1.0, preferably 1.1, preferably greater than 1.2, preferably greater than 1.3, preferably greater than 1.4, preferably greater than 1.5, preferably greater than 1.6, preferably greater than 1.7, preferably greater than 1.8, preferably greater than 1.9, preferably greater than 2.0, preferably greater than 2.5, preferably greater than 3.0, preferably greater than 3.5, preferably greater than 4.0, preferably greater than 5.0, preferably greater than 6.0, preferably greater than 7.0, preferably greater than 8.0, preferably greater than 9.0, preferably greater than 10.0.

[0151] Parent lipase

[0152] The parent lipase has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO: 2, 4, 6 or 8, respectively (having lipase activity).

[0153] SEQ ID NO: 2 is the mature wild-type Thermomyces lanuginosus lipase (TLL).

[0154] EVSQDLFNQF NLFAQYSAAA YCGKNNDAPA GTNITCTGNA CPEVEKADAT

[0155] FLYSFEDSGV GDVTGFLALD NTNKLIVLSF RGSRSIENWI GNLNFDLKEI

[0156] NDICSGCRGH DGFTSSWRSV ADTLRQKVED AVREHPDYRV VFTGHSLGGA

[0157] LATVAGADLR GNGYDIDVFS YGAPRVGNRA FAEFLTVQTG GTLYRITHTN

[0158] DIVPRLPPRE FGYSHSSPEY WIKSGTLVPV TRNDIVKIEG IDATGGNNQP

[0159] NIPDIPAHLW YFGLIGTCL

[0160] SEQ ID NO:4 is a variant of the lipase in SEQ ID NO:8.

[0161] EVSQDLFNQF NLFAQYSAAA YCGKNNDAPA GTNITCTGNE CPEVEKADAT

[0162] FLYSFRNSGV GDVTGFLALD NTNKLIVLSF RGSRSIENWI TNLNFDLEEI

[0163] NDICSGCKGH DGFTSSWFSV ADTLRQKVED AVREHPDYRV VFTGHSLGGA

[0164] LATVAGADLR GNGYDIDVFS YGAPRVGNRA FAEFLTVQTG GTLYRITHTN

[0165] DIVPRLPPRK FGYSHSSPEY WIKSGTLVPV RRRDIVKIEG IDAEGGNNQP

[0166] NIPSIPAHLW YFGLIGTCL

[0167] SEQ ID NO:6 is a variant of the lipase shown in SEQ ID NO:8.

[0168] EVSQDLFNQF NLFAQYSAAA YCSKNNNAPA GTNITCTGNI CPEVEKADAT

[0169] ILYSFRDSGK GDVTGFLALD NTNKLIVLSF RGSRSIENWI GNLNFDLKEI

[0170] NDICSGCRGH DGFTSSWFSV ADTLRQKVED AVREHPDYRV VFTGHSLGGA

[0171] LATVAGADLR GNGYDIDVFS YGAPRVGNRA FAEFLTVQTG GTLYRITHTN

[0172] DIVPRLPPRE FGYSHSSPEY WIKSGTLVPV RRRDIVKIEG IDAEGGNNQP

[0173] NIPDITAHLW YFGLIGTCL

[0174] SEQ ID NO:8 is a variant of the wild-type Thermomyces lanuginosus lipase (TLL) shown in SEQ ID NO:2.

[0175] EVSQDLFNQF NLFAQYSAAA YCGKNNDAPA GTNITCTGNA CPEVEKADAT

[0176] FLYSFEDSGV GDVTGFLALD NTNKLIVLSF RGSRSIENWI GNLNFDLKEI

[0177] NDICSGCRGH DGFTSSWRSV ADTLRQKVED AVREHPDYRV VFTGHSLGGA

[0178] LATVAGADLR GNGYDIDVFS YGAPRVGNRA FAEFLTVQTG GTLYRITHTN

[0179] DIVPRLPPRE FGYSHSSPEY WIKSGTLVPV RRRDIVKIEG IDATGGNNQP

[0180] NIPDIPAHLW YFGLIGTCL

[0181] In a preferred aspect, any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8, respectively, may be a parent lipase.

[0182] In one aspect, the amino acid sequence of the parent differs from the polypeptide of SEQ ID NO: 2, 4, 6 or 8 by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, respectively. In another aspect, the parent comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, 4, 6 or 8. In another aspect, the parent is a fragment of the polypeptide of SEQ ID NO: 2, 4, 6 or 8 containing at least 200 amino acid residues, e.g., at least 250 amino acid residues and at least 260 amino acid residues.

[0183] The parent can be a fusion polypeptide or a cleavable fusion polypeptide. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include connecting the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of one or more identical promoters and terminators. Fusion polypeptides can also be constructed using intein technology, wherein the fusion polypeptide is produced after translation (Cooper et al., 1993, EMBO J. [Journal of the European Molecular Biology Association] 12: 2575-2583; Dawson et al., 1994, Science [Science] 266: 776-779).

[0184] The fusion polypeptide may further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63:3488-3493; Ward et al., 199 5, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0185] The parent can be obtained from any genus of microorganism. For the purposes of the present invention, as used herein in conjunction with a given source, the term "obtained from" shall mean that the parent encoded by the polynucleotide is produced by that source or by a strain into which a polynucleotide from that source has been inserted. In one aspect, the parent is secreted extracellularly.

[0186] In a preferred embodiment, the parent is a Thermomyces lipase, particularly a wild-type Thermomyces lanuginosus lipase, especially the lipase of SEQ ID NO: 2 (trade name LIPOLASE TM In another preferred aspect, the parent lipase is a variant of the Thermomyces lanuginosus lipase of SEQ ID NO: 2. In a preferred embodiment, the parent lipase is the lipase shown in SEQ ID NO: 8 (available under the trade name LIPEX TM18N+D27N+A40I+F51I+E56R+V60K+R118F+T244E+P256T, and is also disclosed in WO 2019 / 063499 (incorporated herein by reference).

[0187] Preparation of variants

[0188] The present invention also relates to methods for obtaining variants of the present invention having lipase activity, comprising:

[0189] (a) introducing one or more substitutions into a parent lipase selected from one or more of the groups (i), (ii) and (iii) comprising:

[0190] (i) substitutions at one or more of positions 202, 252, and 269 of the polypeptide corresponding to SEQ ID NO: 8;

[0191] (ii) substitutions at one or more of positions 40, 56, 57, 91, 98, 108, 118, 210, 244, and 254 of the polypeptide corresponding to SEQ ID NO: 8; and

[0192] (iii) substitutions at one or more of positions 23, 27, 40, 51, 56, 60, 118, 244, and 256 of the polypeptide corresponding to SEQ ID NO: 8;

[0193] wherein the variant has lipase activity, and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 8, wherein the variant optionally comprises one or more amino acid extensions at the N-terminus and / or C-terminus, or one or more amino acid truncations at the N-terminus and / or C-terminus, and wherein the variant has lipase activity; and

[0194] (b) recovering the variant of the invention.

[0195] Any variation of the present invention may be produced as described above.

[0196] In a preferred embodiment, the variant generated is selected from the variants of the invention comprising or consisting of one or more substitutions (in particular all substitutions) corresponding to the positions in SEQ ID NO: 8, which are selected from the group consisting of:

[0197] H substitution of the amino acid residue at position 202;

[0198] H substitution of the amino acid residue at position 252; and

[0199] H substitution of the amino acid residue at position 269.

[0200] In a preferred embodiment, the variant generated is selected from variants of the invention comprising or consisting of one of the following substitution groups corresponding to: 202H+252H; 202H+269H; 252H+269H; or 202H+252H+269H (using SEQ ID NO: 8 for numbering).

[0201] In another preferred embodiment, the variant produced is selected from the variants of the present invention, which comprise or consist of one or more substitutions (in particular all substitutions) corresponding to the positions in SEQ ID NO: 8, which substitutions are selected from the group consisting of:

[0202] E substitution of the amino acid residue at position 40;

[0203] R substitution of the amino acid residue at position 56;

[0204] N substitution of the amino acid residue at position 57;

[0205] T substitution of the amino acid residue at position 91;

[0206] E substitution of the amino acid residue at position 98;

[0207] K substitution of the amino acid residue at position 108;

[0208] F substitution of the amino acid residue at position 118;

[0209] K substitution of the amino acid residue at position 210;

[0210] E substitution of the amino acid residue at position 244; and

[0211] S substitution of the amino acid residue at position 254.

[0212] In another embodiment, the variant generated is selected from the variants of the invention comprising or consisting of one or more substitutions (particularly all substitutions) corresponding to the positions in SEQ ID NO: 8, these substitutions being selected from the group consisting of:

[0213] S substitution of the amino acid residue at position 23;

[0214] N substitution of the amino acid residue at position 27;

[0215] 1. Substitution of the amino acid residue at position 40;

[0216] 1. Substitution of the amino acid residue at position 51;

[0217] R substitution of the amino acid residue at position 56;

[0218] K substitution of the amino acid residue at position 60;

[0219] F substitution of the amino acid residue at position 118;

[0220] E substitution of the amino acid residue at position 244; and

[0221] T substitution of the amino acid residue at position 256.

[0222] 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, and the like.

[0223] Site-directed mutagenesis is a technique that introduces one or more mutations at one or more defined sites in a polynucleotide encoding a parent.

[0224] Site-directed mutagenesis can be achieved in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. In vitro site-directed mutagenesis can also be performed by cassette mutagenesis, which involves cutting at a site in a plasmid containing a polynucleotide encoding the parent by a restriction enzyme and then connecting the oligonucleotide containing the mutation to the polynucleotide. Typically, the restriction enzyme that digests the plasmid and the oligonucleotide is the same, allowing the sticky ends of the plasmid and the insert to connect to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. [Nucleic Acids Research] 18: 7349-4966.

[0225] Site-directed mutagenesis can also be achieved in vivo by methods known in the art. See, for example, US 2004 / 0171154; Storici et al., 2001, Nature Biotechnol. 19:773-776; Kren et al., 1998, Nat. Med. 4:285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43:15-16.

[0226] Any site-directed mutagenesis procedure may be used in the present invention.There are many commercially available kits that can be used to prepare variants.

[0227] Synthetic gene construction requires the in vitro synthesis of designed polynucleotide molecules to encode the desired polypeptide. Gene synthesis can be performed using a variety of techniques, such as the multiplexed microchip-based technology described by Tian et al., 2004, Nature 432:1050-1054, and similar techniques in which oligonucleotides are synthesized and assembled on optically programmable microfluidic chips.

[0228] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0229] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

[0230] Many aspects realize semi-synthetic gene construction through combination synthetic gene construction and / or site-directed mutagenesis and / or random mutagenesis and / or reorganization.The semi-synthetic construction typically utilizes the process of synthetic polynucleotide fragments in conjunction with PCR technology.Therefore, the restricted region of gene can be synthesized from the beginning, and other zones can use site-specific mutagenic primers to increase, and also have other zones to carry out fallibility PCR or non-fallibility PCR amplification.Then can polynucleotide subsequence be reorganized.

[0231] particles

[0232] The present invention also relates to enzyme granules / particles comprising the lipase variant of the invention.In an embodiment, the granule comprises a core and optionally one or more coatings (outer layers) surrounding the core.

[0233] The diameter of the core (measured as an equivalent spherical diameter (average particle size based on volume)) can be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm. The core diameter measured as an equivalent spherical diameter can be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0234] In an embodiment, the core comprises a lipase variant of the invention.

[0235] The core may include additional materials such as fillers, fibrous materials (cellulose or synthetic fibers), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants, and fragrances.

[0236] The core may include a binder such as a synthetic polymer, wax, fat or carbohydrate.

[0237] The core may include, typically as a homogeneous blend, a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposition catalyst, and / or an acidic buffer component.

[0238] The core may comprise an inert particle into which the variant is adsorbed, or applied (eg, by fluidized bed coating) to a surface.

[0239] The diameter of the core may be 20-2000 μm, in particular 50-1500 μm, 100-1500 μm or 250-1200 μm.

[0240] The core may be surrounded by at least one coating, for example to improve storage stability, reduce dust formation during handling or for coloring the granules. Optional coating(s) may include salt coatings or other suitable coating materials such as polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

[0241] The coating may be applied in an amount of at least 0.1% (e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%) by weight of the core. The amount may be up to 100%, 70%, 50%, 40%, or 30%.

[0242] The coating is preferably at least 0.1 μm thick, in particular at least 0.5 μm, at least 1 μm or at least 5 μm thick.In some embodiments, the coating has a thickness of less than 100 μm, such as less than 60 μm or less than 40 μm.

[0243] The coating should seal the core unit by forming a substantially continuous layer. A substantially continuous layer is understood to be a coating with few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should in particular be uniform in thickness.

[0244] The coating may further comprise other materials as known in the art, such as fillers, anti-sticking agents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0245] The salt coating may comprise at least 60% by weight salt, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% by weight.

[0246] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, such as at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm or at least 8 μm. In particular embodiments, the salt coating has a thickness of less than 100 μm, such as less than 60 μm or less than 40 μm.

[0247] The salt may be added from a salt solution (wherein the salt is completely dissolved) or from a salt suspension (wherein the fine particles are smaller than 50 μm, such as smaller than 10 μm or smaller than 5 μm).

[0248] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water-soluble, in particular having a solubility in 100 g of water at 20°C of at least 0.1 g, preferably at least 0.5 g / 100 g of water, such as at least 1 g / 100 g of water, for example at least 5 g / 100 g of water.

[0249] The salt can be an inorganic salt, for example a sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or a salt of a simple organic acid (less than 10 carbon atoms, for example 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or alkaline earth metal ions, ammonium ions or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromine, iodine, sulfate, sulfite, bisulfite, thiosulfate, phosphate, dihydrogen phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, silicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali or alkaline earth metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrates, malonates or acetates can be used.

[0250] The salt in the coating may have a constant humidity of 60% or more, in particular 70% or more, 80% or more or 85% or more at 20° C., or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0251] A specific example of a suitable salt is NaCl(CH 20℃ =76%), Na2CO3(CH 20℃ =92%), NaNO3(CH 20℃ =73%), Na2HPO4(CH 20℃ =95%), Na3PO4(CH 25℃ =92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93,0%), NH4H2PO4(CH 20℃ =93.1%), (NH4)2SO4(CH 20℃ =81.1%), KCl(CH 20℃ =85%), K2HPO4(CH 20℃ =92%), KH2PO4(CH 20℃=96.5%), KNO3(CH 20℃ =93.5%), Na2SO4(CH 20℃ =93%), K2SO4(CH 20℃ =98%), KHSO4(CH 20℃ =86%), MgSO4(CH 20℃ =90%), ZnSO4(CH 20℃ =90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0252] The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate having one or more waters of crystallization, as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO4.7H2O), zinc sulfate heptahydrate (ZnSO4.7H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4.7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0253] Preferably, the salt is used as a salt solution, for example using a fluidized bed.

[0254] The coating material may be a waxy coating material and a film-forming coating material. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) having an average molecular weight of 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein there are 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 by fluidized bed technology are given in GB 1483591.

[0255] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93 / 07263 and WO 97 / 23606.

[0256] The core may be prepared by granulating a blend of ingredients, for example by methods including granulation techniques such as crystallization, precipitation, pan-coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, particle size reduction, drum granulation and / or high shear granulation.

[0257] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CE Capes; Volume 1; 1980; Elsevier. Preparation methods include known feed and pellet formulation techniques, such as:

[0258] (a) Spray-dried products, wherein a liquid enzyme-containing solution is atomized in a spray-drying tower to form small droplets which dry as they descend along the drying tower to form a particulate material containing the enzyme. Very small particles can be produced in this way (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0259] (b) layered products, wherein the enzyme is coated in layers around preformed inert core particles, wherein the enzyme solution is typically atomized in a fluidized bed apparatus, in which the preformed core particles are fluidized and the enzyme solution is attached to the core particles and dried until the enzyme layer is left on the surface of the core particles. If a useful core particle with a desired size can be found, the particle with the desired size can be obtained in this way. This type of product is described in, for example, WO 97 / 23606.

[0260] (c) Adsorbed core particles, wherein instead of the variant being coated as a layer around the core, the enzyme is adsorbed on and / or in the surface of the core. Such a method is described in WO 97 / 39116.

[0261] (d) Extruded or pelletized products, in which a paste containing the variant is pressed into pellets or extruded under pressure through a small opening and cut into particles, and the pellets are subsequently dried. Such particles are generally of considerable size because the material through which the extrusion openings are formed (usually a flat plate with drilled holes) limits the permissible pressure drop across the extrusion openings. Furthermore, when small openings are used, very high extrusion pressures increase the heat generation in the enzyme paste, which is detrimental to the enzyme (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0262] (e) Spray-granulated products, in which a powder containing the modification is suspended in molten wax and the suspension is sprayed (e.g., by a rotating disk atomizer) into a cooling chamber where the droplets rapidly solidify (Michael S. Showell (ed.); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the modification is uniformly distributed throughout the inert material rather than concentrated on its surface. U.S. Pat. No. 4,016,040 and U.S. Pat. No. 4,713,245 describe this technique.

[0263] (f) mixer granulation product, wherein the liquid containing variant is added to the dry powder composition of conventional granulation components.Liquid and powder are mixed in a suitable ratio, and because the moisture of the liquid is absorbed by the dry powder, the components of the dry powder begin to adhere and agglomerate, and particles will accumulate to form particles comprising enzymes. Such methods are described in US 4,106,991, EP 170360, EP 304332, EP 304331, WO 90 / 09440 and WO 90 / 09428. In the particular aspects of this method, various high shear mixers can be used as granulators. The particles composed of variant, filler and adhesive etc. are mixed with cellulose fibers to strengthen particles, thereby producing so-called T-particles. The strengthened particles are more solid and release less enzyme dust.

[0264] (g) Particle size reduction, wherein cores are produced by milling or crushing larger enzyme-containing particles, pellets, platelets, briquettes, etc. The desired core particle fraction is obtained by sieving the milled or crushed product. Oversized and undersized particles can be recovered. Particle size reduction is described in Martin Rhodes (ed.); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0265] (h) Fluidized bed granulation. Fluidized bed granulation involves suspending fine particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. The particles struck by the sprayed liquid droplets become wet and sticky. The sticky particles collide with other particles and adhere to them to form granules.

[0266] (i) The cores can be dried, for example, in a fluidized bed dryer. Those skilled in the art can use other known methods for drying granules in the feed or enzyme industry. Drying is preferably carried out at a product temperature of 25°C to 90°C. For some enzymes, it is important that the cores containing the variant contain a small amount of water before coating with salt. If water-sensitive enzymes are coated with salt before the excess water is removed, the excess water will be trapped in the cores and may negatively affect the activity of the enzyme. After drying, the cores preferably contain 0.1% to 10% w / w water.

[0267] Non-dusting granules may be produced, for example, as disclosed in US 4,106,991 and US 4,661,452, and may optionally be coated by methods known in the art.

[0268] The granules may further comprise one or more additional enzymes. Each enzyme will then be present in more granules, ensuring a more even distribution of the enzymes and also reducing the physical separation of the different enzymes due to the different particle sizes. A method for producing multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0269] Another example of formulating enzymes by using co-granules is disclosed in WO 2013 / 188331.

[0270] The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.

[0271] In an embodiment, the particle further comprises one or more additional enzymes, e.g., hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0272] Liquid composition

[0273] The present invention also relates to a liquid composition comprising a variant of the present invention. The composition may comprise an enzyme stabilizer (examples of such enzyme stabilizers include polyols (such as propylene glycol or glycerol), sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives such as aromatic borate esters, or phenylboronic acid derivatives such as 4-formylphenylboronic acid).

[0274] In certain embodiments, one or more fillers or one or more carrier materials are included to increase the volume of such compositions. Suitable fillers or carrier materials include, but are not limited to, various salts of sulfates, carbonates, and silicates, as well as talc, clay, etc. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols (including polyols and diols). Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In certain embodiments, these compositions contain about 5% to about 90% of such materials.

[0275] In one aspect, the liquid formulation comprises 20%-80% w / w of a polyol.In one embodiment, the liquid formulation comprises 0.001%-2% w / w of a preservative.

[0276] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0277] (A) 0.001%-25% w / w of a variant of the invention;

[0278] (B) 20% to 80% w / w of a polyol;

[0279] (C) optionally 0.001%-2% w / w preservative; and

[0280] (D) Water.

[0281] In another embodiment, the present invention is directed to a liquid formulation comprising:

[0282] (A) 0.001%-25% w / w of a variant of the invention;

[0283] (B) 0.001%-2% w / w preservative;

[0284] (C) optionally 20% to 80% w / w of a polyol; and

[0285] (D) Water.

[0286] In another embodiment, the liquid formulation comprises one or more formulating agents, for example, a formulating agent selected from the group consisting of: polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of: sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of: glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600 and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably selected from the group consisting of: glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0287] In another embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol. In one embodiment, the liquid formulation comprises 20%-80% polyol, such as 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20%-80% polyol (i.e., the total amount of polyol), e.g., 25%-75% polyol, 30%-70% polyol, 35%-65% polyol, or 40%-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0288] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02%-1.5% w / w preservative, such as 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001%-2% w / w preservative (i.e., the total amount of preservative), such as 0.02%-1.5% w / w preservative, 0.05%-1% w / w preservative or 0.1%-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0289] In another embodiment, the liquid formulation further comprises one or more additional enzymes, e.g., hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. The one or more additional enzymes are preferably selected from the group consisting of: acetyl xylan esterase, acylglycerol lipase, amylase, α-amylase, β-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, α-galactosidase, β-galactosidase, β-glucanase, β-glucosidase, lysophospholipase, lysozyme, α-mannosidase, β-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, β-xylosidase, or any combination thereof.

[0290] Composition

[0291] The present invention further relates to a composition comprising the lipase variant of the present invention, the granules of the present invention or the liquid composition of the present invention.

[0292] The compositions of the present invention have reduced odor development and / or increased benefit risk factor (BRF) compared to the same composition comprising a parent lipase (particularly SEQ ID NO: 2, 4, 6, 8, respectively).

[0293] In preferred embodiments, the composition comprises one or more surfactants.

[0294] The non-limiting list of composition components set forth below is applicable to use in composition, and the method herein can be ideally incorporated in certain embodiments of the present invention, for example, in order to assist or enhance cleaning performance, for processing substrate to be cleaned, or in order to modify the aesthetic feeling of composition when together with spices, coloring agents, dyes etc. The level of any such component incorporated in any composition is except any material previously cited for incorporation. The precise nature of these other components and their incorporation level will depend on the physical form of composition and the property of the cleaning operation in which composition will be used therein. Although the components mentioned below are classified by general titles according to specific functionality, this is not to be construed as restriction, because as will be understood by those of ordinary skill, component can comprise other functionality.

[0295] Unless otherwise indicated, amounts in percentages are by weight of the composition (wt %). Suitable component materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay removal / anti-redeposition agents, brighteners, foam inhibitors, dyes, hueing dyes, perfumes, perfume delivery systems, structural elastic agents, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments. In addition to the disclosure below, suitable examples of such other components and usage levels are found in US Pat. No. 5,576,282, US Pat. No. 6,306,812, and US Pat. No. 6,326,348, which are hereby incorporated by reference.

[0296] Thus, in certain embodiments, the present invention does not contain one or more of the following adjunct materials: surfactants, soaps, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersants, clay soil removal / anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structural stretch agents, fabric softeners, carriers, hydrotropes, processing aids, solvents, and / or pigments. However, when one or more components are present, such one or more components may be present as detailed below:

[0297] surfactants- The composition according to the present invention may comprise a surfactant or surfactant system, wherein the surfactant may be selected from nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof. When present, the surfactant is typically present at a level of from 0.1 to 60 wt%, from 0.2 to 40 wt%, from 0.5 to 30 wt%, from 1 to 50 wt%, from 1 to 40 wt%, from 1 to 30 wt%, from 1 to 20 wt%, from 3 to 10 wt%, from 3 to 5 wt%, from 5 to 40 wt%, from 5 to 30 wt%, from 5 to 15 wt%, from 3 to 20 wt%, from 3 to 10 wt%, from 8 to 12 wt%, from 10 to 12 wt%, from 20 to 25 wt% or from 25 to 60 wt%.

[0298] Suitable anionic detersive surfactants include sulfate and sulfonate detersive surfactants.

[0299] Suitable sulfonate detersive surfactants include alkylbenzene sulfonates, in one aspect C 10-13 Alkylbenzene sulfonates. Suitable alkylbenzene sulfonates (LAS) can be obtained by sulfonating commercially available linear alkylbenzenes (LAB); suitable LABs include low 2-phenyl LABs, such as or Other suitable LABs include high 2-phenyl LABs, such as Suitable anionic detersive surfactants are alkylbenzene sulfonates obtained by the DETAL catalysed process, but other synthesis routes (eg HF) may also be suitable. In one aspect, the magnesium salt of LAS is used.

[0300] Suitable sulfate detersive surfactants include alkyl sulfates, in one aspect C 8-18 Alkyl sulfates, or mainly C 12 Alkyl sulfate.

[0301] Another suitable sulfate detersive surfactant is an alkyl alkoxylated sulfate, in one aspect an alkyl ethoxylated sulfate, in one aspect C 8-18 Alkyl alkoxylated sulfate, in another aspect C 8-18 Alkyl ethoxylated sulfate, typically the alkyl alkoxylated sulfate has an average degree of alkoxylation of from 0.5 to 20 or from 0.5 to 10, typically the alkyl alkoxylated sulfate is C 8-18 Alkyl ethoxylated sulfates having an average degree of ethoxylation of from 0.5 to 10, from 0.5 to 7, from 0.5 to 5, or from 0.5 to 3.

[0302] The alkyl sulfates, alkyl alkoxylated sulfates and alkyl benzene sulfonates may be linear or branched, substituted or unsubstituted.

[0303] The detersive surfactant can be a mid-chain branched detersive surfactant, in one aspect a mid-chain branched anionic detersive surfactant, in one aspect a mid-chain branched alkyl sulfate and / or a mid-chain branched alkylbenzene sulfonate, such as a mid-chain branched alkyl sulfate. In one aspect, the mid-chain branch is C 1-4 Alkyl groups are typically methyl and / or ethyl groups.

[0304] Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenylalkanesulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS) , alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkane sulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic fatty acid methyl esters (α-SFMe or SES) (including methyl sulfonate (MES)), alkyl succinic acid or alkenyl succinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfosuccinic acid or soap, and combinations thereof.

[0305] Suitable nonionic detersive surfactants are selected from the group consisting of: C8-C 18 Alkyl ethoxylates, e.g. C6-C 12 Alkylphenol alkoxylates, wherein the alkoxylate units may be ethyleneoxy units, propyleneoxy units or mixtures thereof; C 12 -C 18 Alcohol and C6-C 12 Condensates of alkylphenols with ethylene oxide / propylene oxide block polymers, e.g. C 14 -C 22 Medium chain branched alcohol; C 14 -C 22 mid-chain branched alkyl alkoxylates, typically having an average degree of alkoxylation of from 1 to 30; alkyl polysaccharides, in one aspect alkyl polyglycosides; polyhydroxy fatty acid amides; ether-terminated poly(alkoxylated) alcohol surfactants; and mixtures thereof.

[0306] Suitable nonionic detersive surfactants include alkyl polyglycosides and / or alkyl alkoxylated alcohols.

[0307] In one aspect, the nonionic detersive surfactant comprises an alkyl alkoxylated alcohol, in one aspect C 8-18 Alkyl alkoxylated alcohols, such as C 8-18 The alkyl ethoxylated alcohol may have an average degree of alkoxylation of from 1 to 50, from 1 to 30, from 1 to 20, or from 1 to 10. In one aspect, the alkyl alkoxylated alcohol may be C 8-18 Alkyl ethoxylated alcohols having an average degree of ethoxylation of from 1 to 10, from 1 to 7, more preferably from 1 to 5 or from 3 to 7. The alkyl alkoxylated alcohols may be linear or branched, and substituted or unsubstituted. Suitable nonionic surfactants include

[0308] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)), and products available under the SPAN and TWEEN trade names, and combinations thereof.

[0309] Suitable cationic detersive surfactants include alkyl pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl trisulfonium compounds, and mixtures thereof.

[0310] Suitable cationic detersive surfactants are quaternary ammonium compounds having the general formula: (R)(R1)(R2)(R3)N + X - , wherein R is a linear or branched, substituted or unsubstituted C 6-18 An alkyl or alkenyl moiety, R1 and R2 are independently selected from methyl or ethyl moieties, R3 is a hydroxy, hydroxymethyl or hydroxyethyl moiety, and X is an anion providing charge neutrality, suitable anions include halides, such as chlorides; sulfates; and sulfonates. Suitable cationic detersive surfactants are mono-C 6-18 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride. Highly suitable cationic detersive surfactants are mono-C 8-10 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride, mono-C10-12 Alkyl mono-hydroxyethyl dimethyl quaternary ammonium chloride and mono-C 10 Alkyl mono-hydroxyethyldimethyl quaternary ammonium chloride.

[0311] Non-limiting examples of cationic surfactants include alkyl dimethylethanol quaternary ammonium (ADMEAQ), cetyl trimethyl ammonium bromide (CTAB), dimethyl distearylammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quaternary ammonium, and combinations thereof.

[0312] Suitable amphoteric / zwitterionic surfactants include amine oxides and betaines (e.g., alkyl dimethyl betaines, sulfobetaines), or combinations thereof. Amine-neutralized anionic surfactants - The anionic surfactants of the present invention and the auxiliary anionic co-surfactants can be present in acid form, and the acid form can be neutralized to form the surfactant salts desired for use in the detergent compositions of the present invention. Typical agents for neutralization include metal counterion bases, such as hydroxides, such as NaOH or KOH. Other preferred agents for neutralizing the anionic surfactants of the present invention and the auxiliary anionic surfactants or co-surfactants in their acid form include ammonia, amines, or alkanolamines. Alkanolamines are preferred. Suitable non-limiting examples include monoethanolamine, diethanolamine, triethanolamine, and other linear or branched alkanolamines known in the art; for example, highly preferred alkanolamines include 2-amino-1-propanol, 1-aminopropanol, monoisopropanolamine, or 1-amino-3-propanol. Amine neutralization can be performed to a complete or partial extent, for example, part of the anionic surfactant mixture can be neutralized with sodium or potassium, and part of the anionic surfactant mixture can be neutralized with an amine or alkanolamine.

[0313] Non-limiting examples of semi-polar surfactants include amine oxides (AO), such as alkyl dimethylamine oxide

[0314] Surfactant systems comprising a mixture of one or more anionic surfactants, and additional one or more nonionic surfactants, and optionally additional surfactants such as cationic surfactants, may be preferred. Preferred weight ratios of anionic to nonionic surfactants are at least 2:1, or at least 1:1 to 1:10.

[0315] In one aspect, the surfactant system can include a mixture of isoprenoid surfactants represented by Formula A and Formula B:

[0316]

[0317] wherein Y is CH2 or absent, and Z may be selected such that the resulting surfactant is a surfactant selected from the group consisting of alkyl carboxylate surfactants, alkyl polyalkoxy surfactants, alkyl anionic polyalkoxy sulfate surfactants, alkyl glyceride sulfonate surfactants, alkyl dimethyl amine oxide surfactants, alkyl polyhydroxy-based surfactants, alkyl phosphate surfactants, alkyl glycerol sulfonate surfactants, alkyl polygluconate surfactants, alkyl polyphosphate surfactants, alkyl phosphonate surfactants, alkyl polyglycoside surfactants, alkyl monoglycoside surfactants, alkyl diglycoside surfactants, alkyl sulfosuccinate surfactants, alkyl disulfate surfactants, alkyl disulfonic acid surfactants. Ester surfactants, alkyl sulfonated succinate surfactants, alkyl glucose amide surfactants, alkyl taurate surfactants, alkyl sarcosinate surfactants, alkyl glycine ester surfactants, alkyl isethionate surfactants, alkyl dialkanolamide surfactants, alkyl monoalkanolamide surfactants, alkyl monoalkanolamide sulfate surfactants, alkyl dihydroxyacetamide surfactants, alkyl dihydroxyacetamide sulfate surfactants, alkyl glyceride surfactants, alkyl glyceride sulfate surfactants, alkyl glyceryl ether surfactants, alkyl glyceryl ether sulfate surfactants, alkyl methyl ester sulfonate surfactants, alkyl polyglyceryl ether surfactants, alkyl polyglyceryl ether sulfate surfactants Surfactants, alkyl sorbitan ester surfactants, alkyl aminoalkane sulfonate surfactants, alkyl amidopropyl betaine surfactants, surfactants based on alkyl allylated quaternary ammonium salts, surfactants based on alkyl monohydroxyalkyl-di-alkylated quaternary ammonium salts, surfactants based on alkyl di-hydroxyalkyl monoalkyl quaternary ammonium salts, alkylated quaternary ammonium salt surfactants, alkyl trimethyl ammonium quaternary ammonium salt surfactants, surfactants based on alkyl polyhydroxyalkyloxypropyl quaternary ammonium salts, alkyl glyceride quaternary ammonium salt surfactants, alkyl glycol amine quaternary ammonium salt surfactants, alkyl monomethyl dihydroxyethyl quaternary ammonium surfactants, alkyl dimethyl monohydroxyethyl quaternary ammonium surfactants, alkyl trimethyl ammonium surfactants, based on alkyl imidazoline surfactants, alkene-2-yl-succinate surfactants, alkyl α-sulfonated carboxylic acid surfactants, alkyl α-sulfonated carboxylic acid alkyl ester surfactants, alpha olefin sulfonate surfactants, alkyl phenol ethoxylate surfactants, alkyl benzene sulfonate surfactants, alkyl sulfobetaine surfactants, alkyl hydroxysulfobetaine surfactants, alkylammonium carboxylic acid betaine surfactants, alkyl sucrose ester surfactants, alkyl alkanolamide surfactants, alkyl di(polyoxyethylene)monoalkylammonium surfactants, alkyl mono(polyoxyethylene)dialkylammonium surfactants, alkyl benzyldimethylammonium surfactants, alkylaminopropionate surfactants, alkylamidopropyldimethylamine surfactants, or mixtures thereof;And if Z is a charged moiety, Z is charge balanced by a suitable metal or organic counterion. Suitable counterions include metal counterions, amines, or alkanolamines, such as C1-C6 alkanol ammonium. More specifically, suitable counterions include Na+, Ca+, Li+, K+, Mg+, such as monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-1-propanol, 1-aminopropanol, methyldiethanolamine, dimethylethanolamine, monoisopropanolamine, triisopropanolamine, 1-amino-3-propanol, or mixtures thereof. In one embodiment, the composition contains from 5% to 97% of one or more non-isoprenoid surfactants; and one or more auxiliary cleaning additives; wherein the weight ratio of the surfactant having formula A to the surfactant having formula B is 50:50 to 95:5. ;

[0318] soap The compositions herein may contain soap. Without being limited by theory, it may be desirable to include soap because it acts partly as a surfactant and partly as a builder and can be used to suppress foam and, in addition, can advantageously interact with the various cationic compounds of 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 embodiment, the composition contains 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.

[0319] Examples of soaps useful herein include oleic acid soaps, palmitic acid soaps, palm kernel fatty acid soaps, and mixtures thereof. Typical soaps are in the form of mixtures of fatty acid soaps having varying chain lengths and degrees of substitution. One such mixture is topped palm kernel fatty acid.

[0320] In one embodiment, 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, tall oil, castor oil, tallow and fish oils, greases, and mixtures thereof), or synthetically prepared (e.g., via oxidation of petroleum or via hydrogenation of carbon monoxide by the Fisher Tropsch process).

[0321] The example of the applicable saturated fatty acid for using in the present composition comprises capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. The unsaturated fatty acid kind that is applicable comprises: palmitoleic acid, oleic acid, linoleic acid, linolenic acid and ricinoleic acid. The example of preferred fatty acid is saturated Cn fatty acid, saturated C1-C14 fatty acid and saturated or undersaturated Cn to C18 fatty acid and mixture thereof.

[0322] When present, the weight ratio of fabric softening cationic co-surfactant to fatty acid is preferably from about 1:3 to about 3:1, more preferably from about 1:1.5 to about 1.5:1, most preferably about 1:1.

[0323] The levels of soap and non-soap anionic surfactants herein are percentages by weight of the detergent composition specified on an acid basis. However, as is generally understood in the art, in practice sodium, potassium or alkanolammonium bases such as sodium hydroxide or monoethanolamine are used to neutralize the anionic surfactants and soaps.

[0324] hydrotrope The compositions of the present invention may include one or more hydrotropes. A hydrotrope is a compound that dissolves hydrophobic compounds in aqueous solution (or, conversely, polar substances in a non-polar environment). Typically, a hydrotrope has both hydrophilic and hydrophobic properties (so-called amphiphilic properties, as known from surfactants); however, the molecular structure of a hydrotrope is generally not conducive to spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science 12:121-128. Hydrotropes do not exhibit a critical concentration above which self-aggregation occurs, as found for surfactants, and lipids form micelles, lamellae, or other well-defined mesophases. Instead, many hydrotropes exhibit a continuous type of aggregation process, in which the size of the aggregates increases with increasing concentration. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances with polar and non-polar characteristics, including mixtures of water, oils, surfactants, and polymers. Hydrotropes are routinely used in various industries ranging from pharmaceuticals, personal care, food to technical applications. The use of hydrotropes in detergent compositions allows, for example, more concentrated surfactant formulations (such as in the process of compacting liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0325] The detergent may contain from 0 to 10 wt %, e.g., from 0 to 5 wt %, 0.5 to 5 wt %, or from 3 to 5 wt % of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymenesulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalenesulfonate, sodium ethylhexylsulfonate, and combinations thereof.

[0326] detergentThe compositions of the present invention may comprise one or more builders, co-builders, builder systems, or mixtures thereof. When a builder is used, the cleaning composition will typically comprise from 0 to 65 wt%, at least 1 wt%, from 2 to 60 wt%, or from 5 to 10 wt% builder. In dishwashing cleaning compositions, the builder level is typically 40 to 65 wt%, or 50 to 65 wt%. The composition may be substantially free of builder; substantially free means "no intentionally added" zeolite and / or phosphate. Typical zeolite builders include zeolite A, zeolite P, and zeolite MAP. A typical phosphate builder is sodium tripolyphosphate.

[0327] Builder and / or co-builder can particularly form the chelating agent of water-soluble complex with Ca and Mg.Any builder and / or co-builder known in the art for use in detergents can be used.The limiting examples of builder comprises zeolite, diphosphate (pyrophosphate), triphosphate such as sodium triphosphate (STP or STPP), carbonate such as sodium carbonate, soluble silicate such as sodium metasilicate, layered silicate (for example from the SKS-6 of Hurst Company (Hoechst)), ethanolamine (for example 2-amino 2-alcohol (MEA), iminodiethanol (DEA) and 2,2 ', 2 "-nitrilotriethanol (TEA)) and carboxymethyl inulin (CMI) and combination thereof.

[0328] The cleaning compositions can include co-builders individually or in combination with builders (e.g., zeolite builders). Non-limiting examples of co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or co-poly(acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents (e.g., aminocarboxylates, aminopolycarboxylates, and phosphonates), and alkyl succinic acids or alkenyl succinic acids. Further specific examples include 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonic acid) (HEDP), ethylenediaminetetra(methylene)tetra(phosphonic acid) (EDTMPA), diethylenetriaminepenta(methylene)penta(phosphonic acid) (DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SE N,N-diacetic acid (SDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta(methylenephosphonic acid) (DTPMP), aminotri(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Additional exemplary builders and / or co-builders are described, for example, in WO 09 / 102854, US 5977053.

[0329] Chelating agents and crystal growth inhibitors- The compositions herein may contain chelating agents and / or crystal growth inhibitors. Suitable molecules include copper, ion and / or manganese chelating agents and mixtures thereof. Suitable molecules include DTPA (diethylenetriaminepentaacetic acid), HEDP (hydroxyethanediphosphonic acid), DTPMP (diethylenetriaminepenta(methylenephosphonic acid)), 1,2-dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylenetriamine, ethylenediamine disuccinic acid (EDDS), N-hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), N-hydroxyethyliminodiacetic acid (HEIDA), dihydroxyethylglycine (DHEG), ethylenediaminetetrapropionic acid (EDTP), carboxymethyl inulin and 2-phosphonobutane 1,2,4-tricarboxylic acid ( Typically, the composition may comprise from 0.005 to 15 wt % or from 3.0 to 10 wt % of a chelating agent or crystal growth inhibitor.

[0330] bleaching components - Suitable bleaching components for incorporation into the methods and compositions of the present invention include one or a mixture of more than one bleaching components. Suitable bleaching components include bleach catalysts, photobleaches, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, and mixtures thereof. Generally, when a bleaching component is used, the compositions of the present invention may comprise from 0 to 30 wt%, from 0.00001 to 90 wt%, from 0.0001 to 50 wt%, from 0.001 to 25 wt%, or from 1 to 20 wt%. Examples of suitable bleaching components include:

[0331] (1) Preformed Peracids: Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of: preformed peroxyacids or salts thereof, typically peroxycarboxylic acids or salts thereof, or peroxysulfuric acid or salts thereof.

[0332] The preformed peroxyacid or salt thereof is preferably a peroxycarboxylic acid or salt thereof, typically having a chemical structure corresponding to the following formula:

[0333]

[0334] Where: R 14 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; R 14 The group may be linear or branched, substituted or unsubstituted; and Y is any suitable counterion to achieve charge neutrality, preferably, Y is selected from hydrogen, sodium or potassium. Preferably, R 14 is a linear or branched, substituted or unsubstituted C 6-9alkyl. Preferably, the peroxy acid or its salt is selected from peroxyhexanoic acid, peroxyheptanoic acid, peroxyoctanoic acid, peroxynonanoic acid, peroxydecanoic acid, and salts thereof, or any combination thereof. Particularly preferred peroxy acids are phthalimido-peroxy-alkanoic acids, in particular ε-phthalimidoperoxyhexanoic acid (PAP). Preferably, the peroxy acid or its salt has a melting point in the range of 30°C to 60°C.

[0335] The preformed peroxyacid or salt thereof may also be peroxysulfuric acid or a salt thereof, typically having a chemical structure corresponding to the following formula:

[0336]

[0337] Where: R 15 is selected from alkyl, aralkyl, cycloalkyl, aryl or heterocyclic groups; R 15 The group may be linear or branched, substituted or unsubstituted; and Z is any suitable counterion to achieve charge neutrality, preferably, Z is selected from hydrogen, sodium or potassium. Preferably, R 15 is a linear or branched, substituted or unsubstituted C 6-9 Preferably, such bleaching components may be present in the compositions of the present invention in an amount of from 0.01 to 50 wt% or from 0.1 to 20 wt%.

[0338] (2) Hydrogen peroxide sources include, for example, inorganic perhydrate salts, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, perphosphates, sodium persilicates, and mixtures thereof. In one aspect of the invention, the inorganic perhydrate salts are, for example, those selected from the group consisting of perborates, sodium percarbonates, and mixtures thereof. When used, the inorganic perhydrate salts are typically present in an amount of 0.05 to 40 wt % or 1 to 30 wt % of the overall composition and are typically incorporated into such compositions as crystalline solids that can be coated. Suitable coatings include inorganic salts such as alkali metal silicates, carbonates or borates, or mixtures thereof, or organic materials such as water-soluble or water-dispersible polymers, waxes, oils, or fatty soaps. Preferably, such bleaching components may be present in the compositions of the invention in an amount of 0.01 to 50 wt % or 0.1 to 20 wt %.

[0339] (3) The term bleach activator is intended herein to mean a compound which reacts with hydrogen peroxide to form a peracid via a perhydrolysis reaction. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters, amides, imides or anhydrides. Suitable bleach activators are those having R-(C=O)-L, wherein R is an alkyl group (optionally branched) having from 6 to 14 carbon atoms, or from 8 to 12 carbon atoms when the bleach activator is hydrophobic, and having less than 6 carbon atoms or less than 4 carbon atoms when the bleach activator is hydrophilic; and L is a leaving group. Examples of suitable leaving groups are benzoic acid and its derivatives - in particular benzenesulfonate. Suitable bleach activators include dodecanoyloxybenzenesulfonate, decanoyloxybenzenesulfonate, decanoyloxybenzoic acid or its salts, 3,5,5-trimethylhexanoyloxybenzenesulfonate, tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene-1-sulfonate (ISONOBS), 4-(dodecanoyloxy)benzene-1-sulfonate (LOBS), 4-(decanoyloxy)benzene-1-sulfonate, 4-(decanoyloxy)benzoate (DOBS or DOBA), 4-(nonanoyloxy)benzene-1-sulfonate (NOBS), and / or those disclosed in WO 98 / 17767. A family of bleach activators is disclosed in EP 624154, and particularly preferred within that family is acetyl triethyl citrate (ATC). ATC or short-chain triglycerides (such as triacetin) have the advantage that they are environmentally friendly. In addition, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product during storage and are effective bleach activators. Finally, ATC is multifunctional because the citrate released in the perhydrolysis reaction can act as a builder. Alternatively, the bleaching system can include peroxy acids of the amide, imide or sulfone types. The bleaching system can also include peracids, such as 6-(phthalimido)perhexanoic acid (PAP). Suitable bleach activators are also disclosed in WO98 / 17767. Although any suitable bleach activator can be used, in one aspect of the present invention, the subject cleaning composition can include NOBS, TAED or a mixture thereof. When present, the peracid and / or bleach activator is typically present in the composition in an amount of 0.1 to 60 wt%, 0.5 to 40 wt% or 0.6 to 10 wt%, based on the fabric and home care composition. One or more hydrophobic peracids or their precursors can be used in combination with one or more hydrophilic peracids or their precursors. Preferably, such bleaching components may be present in the compositions of the invention in amounts of from 0.01 to 50 wt% or from 0.1 to 20 wt%.

[0340] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.

[0341] (4) Diacyl Peroxides - Preferred diacyl peroxide bleaching species include those selected from the group consisting of diacyl peroxides having the general formula: R 1 -C(O)-OO-(O)CR 2 , where R 1 Indicates C6-C 18 An alkyl group, preferably a straight chain having at least 5 carbon atoms and optionally containing one or more substituents (e.g., —N + (CH3)3, -COOH or -CN) and / or one or more interrupting moieties (e.g., -CONH- or -CH=CH-) inserted between adjacent carbon atoms of the alkyl group. 12 alkyl group, and R 2 represents an aliphatic group that is partially compatible with the peroxide so that R 1 and R 2 Together, they contain a total of 8 to 30 carbon atoms. In a preferred aspect, R 1 and R 2 It is a straight chain unsubstituted C6-C 12 Most preferably, R 1 and R 2 are the same. 1 and R 2 All are C6-C 12 alkyl groups) are particularly preferred. Preferably, at least one, and most preferably only one, of the R groups (R1 or R2) does not contain a branched ring or a pendant ring at the α position, or preferably does not contain a branched ring or a pendant ring at either the α or β position, or most preferably does not contain a branched ring or a pendant ring at either the α, β, or γ position. In another preferred embodiment, the DAP may be asymmetric such that the R1 acyl group preferably hydrolyzes rapidly to produce a peracid, but the R2 acyl group hydrolyzes slowly.

[0342] The tetraacyl peroxide bleaching species is preferably selected from tetraacyl peroxides of the general formula: R 3 -C(O)-OO-C(O)-(CH2)nC(O)-OO-C(O)-R 3 , where R 3 represents a C1-C9 alkyl group or a C3-C7 group, and n represents an integer from 2 to 12 or 4 to 10 both inclusive.

[0343] Preferably, the diacyl and / or tetraacyl peroxide bleaching species is present in an amount sufficient to provide at least 0.5 ppm, at least 10 ppm, or at least 50 ppm by weight of the wash liquor. In a preferred embodiment, the bleaching species is present in an amount sufficient to provide from 0.5 ppm to 300 ppm, from 30 ppm to 150 ppm by weight of the wash liquor.

[0344] Preferably, the bleach component comprises a bleach catalyst (5 and 6).

[0345] (5) Preferred are organic (non-metallic) bleach catalysts, including bleach catalysts that are capable of accepting an oxygen atom from a peroxyacid and / or its salt and transferring the oxygen atom to an oxidizable substrate. Suitable bleach catalysts include, but are not limited to, iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulfonylimines; N-phosphorylimides; N-acylimides; thiadiazole dioxides; perfluoroimines; cyclic sugar ketones, and mixtures thereof.

[0346] Suitable iminium cations and polyions include, but are not limited to, N-methyl-3,4-dihydroisoquinolinium tetrafluoroborate, prepared as described in Tetrahedron (1992), 49(2), 423-38 (e.g., Compound 4, page 433); N-methyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in US 5360569 (e.g., column 11, Example 1); and N-octyl-3,4-dihydroisoquinolinium p-toluenesulfonate, prepared as described in US 5360568 (e.g., column 10, Example 3).

[0347] Suitable iminium zwitterions include, but are not limited to, N-(3-sulfopropyl)-3,4-dihydroisoquinolinium, inner salt, prepared as described in US 5576282 (e.g., column 31, Example II); N-[2-(sulfooxy)dodecyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in US 5817614 (e.g., column 32, Example V); 2-[3-[(2-ethylhexyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, inner salt, prepared as described in WO 05 / 047264 (e.g., Example 8 on page 18), and 2-[3-[(2-butyloctyl)oxy]-2-(sulfooxy)propyl]-3,4-dihydroisoquinolinium, inner salt.

[0348] Suitable modified amine oxygen transfer catalysts include, but are not limited to, 1,2,3,4-tetrahydro-2-methyl-1-isoquinolinol, which can be prepared according to the procedures described in Tetrahedron Letters (1987), 28(48), 6061-6064. Suitable modified amine oxide oxygen transfer catalysts include, but are not limited to, sodium 1-hydroxy-N-oxy-N-[2-(sulfooxy)decyl]-1,2,3,4-tetrahydroisoquinoline.

[0349] Suitable N-sulfonylimine oxygen transfer catalysts include, but are not limited to, 3-methyl-1,2-benzisothiazole 1,1-dioxide, which can be prepared according to the procedures described in Journal of Organic Chemistry (1990), 55(4), 1254-61.

[0350] Suitable N-phosphonoimide oxygen transfer catalysts include, but are not limited to, [R-(E)]-N-[(2-chloro-5-nitrophenyl)methylene]-p-phenyl-p-(2,4,6-trimethylphenyl)phosphinic acid amide, which can be prepared according to the procedures described in Journal of the Chemical Society, Chemical Communications (1994), (22), 2569-70.

[0351] Suitable N-acylimine oxygen transfer catalysts include, but are not limited to, [N(E)]-N-(phenylmethylene)acetamide, which can be prepared according to the procedure described in Polish Journal of Chemistry (2003), 77(5), 577-590.

[0352] Suitable thiadiazole dioxide oxygen transfer catalysts include, but are not limited to, 3-methyl-4-phenyl-1,2,5-thiadiazole 1,1-dioxide, which can be prepared according to the procedure described in US Pat. No. 5,753,599 (column 9, Example 2).

[0353] Suitable perfluoroimine oxygen transfer catalysts include, but are not limited to, (Z)-2,2,3,3,4,4,4-heptafluoro-N-(nonafluorobutyl)butyrimidinium fluoride, which can be prepared according to the procedure described in Tetrahedron Letters (1994), 35(34), 6329-30.

[0354] Suitable cyclic sugar ketone oxygen transfer catalysts include, but are not limited to, 1,2:4,5-di-O-isopropylidene-D-erythro-2,3-hexodiuro-2,6-pyranose as prepared in US 6649085 (column 12, Example 1).

[0355] Preferably, the bleaching catalyst comprises an iminium ion and / or a carbonyl functional group and is typically capable of forming an oxaziridinium and / or a dioxirane functional group upon accepting an oxygen atom, in particular from a peroxyacid and / or its salt. Preferably, the bleaching catalyst comprises an oxaziridinium functional group and / or is capable of forming an oxaziridinium functional group upon accepting an oxygen atom, in particular from a peroxyacid and / or its salt. Preferably, the bleaching catalyst comprises a cyclic iminium functional group, preferably wherein the cyclic moiety has a ring size of from five to eight atoms (including nitrogen atoms), preferably six atoms. Preferably, the bleaching catalyst comprises an aryliminium functional group, preferably a dicyclic aryliminium functional group, preferably a 3,4-dihydroisoquinolinium functional group. Typically, the imine functional group is a quaternary imine functional group and is typically capable of forming a quaternary oxaziridinium functional group upon accepting an oxygen atom, particularly upon accepting an oxygen atom from a peroxyacid and / or a salt thereof. In another aspect, the detergent composition comprises a detergent having a logP of no greater than 0, no greater than -0.5, no greater than -1.0, no greater than -1.5, no greater than -2.0, no greater than -2.5, no greater than -3.0, or no greater than -3.5. o / w The following describes in more detail the method for determining logP o / w method.

[0356] Typically, the bleaching component is capable of producing an X having an X of from 0.01 to 0.30, from 0.05 to 0.25 or from 0.10 to 0.20. SO The following describes in more detail the methods used to determine X SO For example, a bleaching component having an isoquinolinium structure can generate a bleaching species having an oxaziridinium structure. In this example, X SO It is a type of oxazide ion bleaching X SO .

[0357] Preferably, the bleach catalyst has a chemical structure corresponding to the following formula:

[0358]

[0359] wherein: n and m are independently 0 to 4, preferably n and m are both 0; each R 1are independently selected from substituted or unsubstituted groups selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, fused aryl, heterocycle, fused heterocycle, nitro, halide, cyano, sulfonate, alkoxy, keto, carboxyl and alkoxycarbonyl groups; and any two vicinal R 1 The substituents may be combined to form a fused aryl, a fused carbocyclic ring, or a fused heterocyclic ring; each R 2 independently selected from substituted or unsubstituted groups independently selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, alkaryl, aryl, aralkyl, alkylene, heterocycle, alkoxy, arylcarbonyl, carboxyalkyl and amide groups; any R 2 Can be used with any other R 2 joined together to form part of a common ring; any geminal R 2 can combine to form a carbonyl group; and any two R 2 May be combined to form a substituted or unsubstituted fused unsaturated moiety; R 3 C1 to C 20 Substituted or unsubstituted alkyl; R 4 is hydrogen or Q t -A moiety, wherein: Q is a branched or unbranched alkylene, t=0 or 1, and A is an anionic group selected from the group consisting of: OSO3 - 、SO3 - 、CO2 - 、OCO2 - 、OPO3 2- 、OPO3H - and OPO2 - ; R 5 is hydrogen or part of -CR 11 R 12 -YG b -Y c -[(CR 9 R 10 ) y -O] k -R 8 , wherein: each Y is independently selected from the group consisting of: O, S, NH or NR 8 ; and each R 8 independently selected from the group consisting of alkyl, aryl, and heteroaryl, said moiety being substituted or unsubstituted, and whether substituted or unsubstituted, said moiety having less than 21 carbons; each G being independently selected from the group consisting of CO, SO2, SO, PO, and PO2; R 9 and R 10 are independently selected from the group consisting of: H and C1-C4 alkyl; R 11 and R 12independently selected from the group consisting of: H and alkyl, or when taken together they may combine to form a carbonyl; b = 0 or 1; c may be 0 or 1, but if b = 0, c must be 0; y is an integer from 1 to 6; k is an integer from 0 to 20; R 6 is H, or an alkyl, aryl or heteroaryl moiety; said moiety being substituted or unsubstituted; and X, if present, is a suitable charge-balancing counterion, when R 4 X is preferably present when it is hydrogen. Suitable X include, but are not limited to, chloride, bromide, sulfate, methylsulfate, sulfonate, p-toluenesulfonate, boron tetrafluoride, and phosphate.

[0360] In one embodiment of the present invention, the bleach catalyst has a structure corresponding to the following general formula:

[0361]

[0362] where R 13 is a branched chain alkyl group containing from three to 24 carbon atoms (including branched carbon atoms) or a straight chain alkyl group containing from one to 24 carbon atoms; preferably, R 13 is a branched alkyl group containing from eight to 18 carbon atoms or a straight chain alkyl group containing from eight to eighteen carbon atoms; preferably, R 13 is selected from the group consisting of 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isononyl, isodecyl, isotridecyl and isopentadecyl; preferably, R 13 Selected from the group consisting of 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, iso-tridecyl and iso-pentadecyl.

[0363] Preferably, the bleaching component comprises, in addition to a bleaching catalyst, in particular an organic bleaching catalyst, a peracid source. The peracid source may be selected from (a) preformed peracids; (b) percarbonates, perborates or persulfates (sources of hydrogen peroxide), preferably in combination with a bleach activator; and (c) perhydrolases and esters for in situ formation of peracids in the presence of water during the textile or hard surface treatment step.

[0364] When present, the peracid and / or bleach activator is typically present in the composition in an amount of from 0.1 to 60 wt%, from 0.5 to 40 wt% or from 0.6 to 10 wt%, based on the composition. One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracids or precursors thereof.

[0365] The amounts of hydrogen peroxide source and peracid or bleach activator may be selected so that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or 2:1 to 10:1.

[0366] (6) Metal-containing bleach catalysts - the bleaching component can be provided by a catalytic metal complex. One type of metal-containing bleach catalyst is a catalytic system comprising a transition metal cation having a defined bleach catalytic activity (e.g., a copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cation), an auxiliary metal cation having little or no bleach catalytic activity (e.g., a zinc or aluminum cation), and a spacer having defined stability constants for the catalytic and auxiliary metal cations, in particular ethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid), and water-soluble salts thereof. Such catalysts are disclosed in US 4430243. Preferred catalysts are described in WO 09 / 839406, US 6218351, and WO 00 / 012667. Particularly preferred are transition metal catalysts or ligands thereof as cross-bridged multidentate N-donor ligands.

[0367] If desired, the compositions herein may be catalyzed with the aid of manganese compounds. Such compounds and usage levels are well known in the art and include, for example, the manganese-based catalysts disclosed in US Pat. No. 5,576,282.

[0368] Cobalt bleach catalysts useful herein are known and are described, for example, in US 5597936; US 5595967. Such cobalt catalysts may be readily prepared by known procedures, such as those taught in US 5597936 and US 5595967.

[0369] The compositions herein may also suitably include transition metal complexes of ligands, such as bispidones (US 7501389) and / or polycyclic rigid ligands - abbreviated as "MRLs." As a practical matter and not by way of limitation, the compositions and methods herein may be adjusted to provide about at least 1 part per 100 million of active MRL species in the aqueous wash medium, and will typically provide from 0.005 ppm to 25 ppm, from 0.05 ppm to 10 ppm, or from 0.1 ppm to 5 ppm of MRL in the wash liquor.

[0370] Suitable transition metals in the transition metal bleach catalysts of the present invention include, for example, manganese, iron, and chromium. Suitable MRLs include 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane. Suitable transition metal MRLs can be readily prepared by known procedures, such as those taught in US Pat. No. 6,225,464 and WO 00 / 32601.

[0371] (7) Photobleaches - Suitable photobleaches include, for example, sulfonated zinc phthalocyanine, sulfonated aluminum phthalocyanine, xanthene dyes, and mixtures thereof. Preferred bleaching components for use in the compositions of the present invention comprise a hydrogen peroxide source, a bleach activator, and / or an organic peroxyacid, optionally generated in situ by the reaction of a hydrogen peroxide source and a bleach activator in combination with a bleach catalyst. Preferred bleaching components comprise a bleach catalyst, preferably an organic bleach catalyst as described above.

[0372] Particularly preferred bleaching components are bleach catalysts, especially organic bleach catalysts.

[0373] Exemplary bleaching systems are also described in, for example, WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259 and WO 2007 / 087242.

[0374] fabric toner -The composition may comprise a fabric hueing agent. Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes classified by the following Color Index (CI): direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet, and basic red, or mixtures thereof.

[0375] In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of Color Index (Society of Dyers and Colorists, Bradford, UK) numbers Direct Violet 9, Direct Violet 35, Direct Violet 48, Direct Violet 51, Direct Violet 66, Direct Violet 99, Direct Blue 1, Direct Blue 71, Direct Blue 80, Direct Blue 279, Acid Red 17, Acid Red 73, Acid Red 88, Acid Red 150, Acid Violet 15, Acid Violet 17, Acid Violet 24, Acid Violet 43, Acid Red 52, Acid Violet 49, Acid Violet 50 , Acid Blue 15, Acid Blue 17, Acid Blue 25, Acid Blue 29, Acid Blue 40, Acid Blue 45, Acid Blue 75, Acid Blue 80, Acid Blue 83, Acid Blue 90 and Acid Blue 113, Acid Black 1, Basic Violet 1, Basic Violet 3, Basic Violet 4, Basic Violet 10, Basic Violet 35, Basic Blue 3, Basic Blue 16, Basic Blue 22, Basic Blue 47, Basic Blue 66, Basic Blue 75, Basic Blue 159 and mixtures thereof. In another aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: Color Index (Institute of Dyers and Colourists, 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 aspect, suitable small molecule dyes include small molecule dyes selected from the group consisting of: Color Index (Institute of Dyers and Colourists, Bradford, UK) numbers Acid Violet 17, Direct Blue 71, Direct Violet 51, Direct Blue 1, Acid Red 88, Acid Red 150, Acid Blue 29, Acid Blue 113, or mixtures thereof.

[0376] Suitable polymeric dyes include polymeric dyes selected from the group consisting of polymers containing a conjugated chromogen (dye-polymer conjugate) and polymers in which the chromogen is copolymerized into the polymer backbone, and mixtures thereof.

[0377] In another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of: A fabric substantial colorant sold under the name of Milliken, a dye-polymer conjugate formed from at least one reactive dye and a polymer selected from the group consisting of a polymer comprising a moiety selected from the group consisting of a hydroxyl moiety, a primary amine moiety, a secondary amine moiety, a thiol moiety, and mixtures thereof. In yet another aspect, suitable polymeric dyes include polymeric dyes selected from the group consisting of: Violet CT, carboxymethyl cellulose (CMC) conjugated with reactive blue, reactive violet or reactive red dye (such as CMC conjugated with CI Reactive Blue 19 (sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE production code S-ACMC)), alkoxylated triphenyl-methane polymer colorants, alkoxylated thiophene polymer colorants, and mixtures thereof.

[0378] Preferred hueing dyes include the brighteners found in WO 08 / 87497. These brighteners can be characterized by the following structure (I):

[0379]

[0380] Wherein R1 and R2 can be independently selected from:

[0381] a)[(CH2CR'HO) x (CH2CR"HO) y H]

[0382] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5;

[0383] b) R1 = alkyl, aryl or arylalkyl, and R2 = [(CH2CR'HO) x (CH2CR"HO) y H]

[0384] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤10; wherein y≥1; and wherein z=0 to 5;

[0385] c) R1=[CH2CH2(OR3)CH2OR4] and R2=[CH2CH2(OR3)CH2OR4]

[0386] wherein R3 is selected from the group consisting of: H, (CH2CH2O) z H and mixtures thereof; and wherein z = 0 to 10;

[0387] wherein R4 is selected from the group consisting of: (C1-C 16) alkyl groups, aryl groups, and mixtures thereof; and

[0388] d) wherein R1 and R2 may be independently selected from the group consisting of styrene oxide, glycidyl methyl ether, isobutyl glycidyl ether, isopropyl glycidyl ether, tert-butyl glycidyl ether, 2-ethylhexyl glycidyl ether, and amino addition products of glycidyl hexadecyl ether, followed by the addition of from 1 to 10 alkylene oxide units.

[0389] The preferred brighteners of the present invention can be characterized by the following structure (II):

[0390]

[0391] Wherein R' is selected from the group consisting of: H, CH3, CH2O (CH2CH2O) z H, and mixtures thereof; wherein R" is selected from the group consisting of: H, CH2O (CH2CH2O) z H, and mixtures thereof; wherein x+y≤5; wherein y≥1; and wherein z=0 to 5.

[0392] Another preferred brightener of the present invention can be characterized by the following structure (III):

[0393]

[0394] Typically a mixture having a total of 5 EO groups is included. Suitable preferred molecules are those in structure I having the following side groups in "part a" above.

[0395] Table 1

[0396] 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

[0397] Additional whitening agents used include those described in US 2008 / 34511 (Unilever).A preferred agent is "Violet 13".

[0398] Suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of at least one cationic / basic dye and a smectite clay and mixtures thereof. In another aspect, suitable dye-clay conjugates include dye-clay conjugates selected from the group consisting of a cationic / basic dye and a clay, the cationic / basic dye selected from the group consisting of CI Basic Yellow 1 to 108, CI Basic Orange 1 to 69, CI Basic Red 1 to 118, CI Basic Violet 1 to 51, CI Basic Blue 1 to 164, CI Basic Green 1 to 14, CI Basic Brown 1 to 23, CI Basic Black 1 to 11, and the clay selected from the group consisting of montmorillonite clay, hectorite clay, saponite clay and mixtures thereof. In still another aspect, suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: montmorillonite basic blue B7 CI42595 conjugate, montmorillonite basic blue B9 CI52015 conjugate, montmorillonite basic violet V3 CI42555 conjugate, montmorillonite basic green G1 CI 42040 conjugate, montmorillonite basic red R1 CI 45160 conjugate, montmorillonite CI basic black 2 conjugate, hectorite basic blue B7 CI 42595 conjugate, hectorite basic blue B9 CI 52015 conjugate, hectorite basic violet V3 CI 42555 conjugate, hectorite basic green G1 CI 42040 conjugate, hectorite basic red R1 CI 45160 conjugate. CI45160 conjugate, hectorite CI basic black 2 conjugate, saponite basic blue B7 CI 42595 conjugate, saponite basic blue B9 CI 52015 conjugate, saponite basic violet V3 CI 42555 conjugate, saponite basic green G1 CI 42040 conjugate, saponite basic red R1 CI 45160 conjugate, saponite CI basic black 2 conjugate and mixtures thereof.

[0399] Suitable pigments include pigments selected from the group consisting of flavonol, indanthrone, chloroindanthrone containing from 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide (wherein the imide groups may be unsubstituted or substituted by C1-C3-alkyl or phenyl or heterocyclic groups, and wherein the phenyl and heterocyclic groups may additionally carry substituents which do not impart solubility in water), anthrapyrimidinecarboxylic acid amides, anthrone violet, isoanthrone violet, dioxazine pigments, copper phthalocyanine which may contain up to 2 chlorine atoms per molecule, polychloro-copper phthalocyanine or polybrominated chloro-copper phthalocyanine containing up to 14 bromine atoms per molecule, and mixtures thereof.

[0400] In another aspect, suitable pigments include pigments selected from the group consisting of Ultramarine Blue (CI Pigment Blue 29), Ultramarine Violet (CI Pigment Violet 15), and mixtures thereof.

[0401] The above-mentioned fabric hueing agents can be used in combination (any mixture of fabric hueing agents can be used). Suitable hueing agents are described in more detail in US 7208459. The preferred level of dye in the composition of the present invention is 0.00001 to 0.5wt% or 0.0001 to 0.25wt%. Preferably, the concentration of dye in water for treatment and / or cleaning steps is from 1ppb to 5ppm, 10ppb to 5ppm or 20ppb to 5ppm. In preferred compositions, the concentration of surfactant will be from 0.2 to 3g / l.

[0402] Encapsulated - The composition may comprise an encapsulate. In one aspect, the encapsulate comprises a core, a shell having an inner surface and an outer surface, said shell encapsulating said core.

[0403] In one aspect of the encapsulate, 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 waxes; 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 containing other comonomers; polystyrene; polyisoprene; polycarbonates; polyesters; polyacrylates; aminoplasts, in one aspect, the aminoplasts may comprise polyureas, polyurethanes and / or polyurea polyurethanes, in one aspect, the polyureas may comprise polyoxymethylene ureas and / or melamine formaldehyde; polyolefins; polysaccharides, in one aspect, the polysaccharides may comprise alginate and / or chitosan; gelatin; shellac; epoxy resins; vinyl polymers; water-insoluble inorganics; silicones; and mixtures thereof.

[0404] In one aspect of the encapsulate, the core may comprise a fragrance.

[0405] In one aspect of the encapsulate, the shell may comprise melamine formaldehyde and / or cross-linked melamine formaldehyde.

[0406] In one aspect, suitable encapsulates are disclosed that can comprise a core material and a shell that at least partially surrounds the core material. At least 75%, 85%, or 90% of the encapsulates can have a breaking strength of from 0.2 to 10 MPa, from 0.4 to 5 MPa, from 0.6 to 3.5 MPa, or from 0.7 to 3 MPa; and have a beneficial agent leakage of from 0% to 30%, from 0% to 20%, or from 0% to 5%.

[0407] In one aspect, at least 75%, 85%, or 90% of the encapsulates may have a particle size of from 1 to 80 microns, from 5 to 60 microns, from 10 to 50 microns, or from 15 to 40 microns.

[0408] In one aspect, at least 75%, 85%, or 90% of the encapsulates may have a particle wall thickness of from 30 to 250 nm, from 80 to 180 nm, or from 100 to 160 nm.

[0409] In one aspect, the core material of the encapsulate may include a material selected from the group consisting of fragrance raw materials, and / or optionally includes a material selected from the group consisting of: vegetable oils, including pure vegetable oils and / or blended vegetable oils, including castor oil, coconut oil, cottonseed oil, grapeseed oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil and mixtures thereof; esters of vegetable oils, esters including dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, octyl benzyl adipate, tricresyl phosphate, trioctyl phosphate and mixtures thereof; straight or branched chain hydrocarbons, including those having a boiling point greater than about 80°C; partially hydrogenated terphenyls, dialkyl phthalates, alkyl biphenyls (including monoisopropyl biphenyl), alkylated naphthalenes (including dipropyl naphthalene), petroleum spirits (including kerosene), mineral oils and mixtures thereof; aromatic solvents including benzene, toluene and mixtures thereof; silicone oils; and mixtures thereof.

[0410] In one aspect, the wall material of the encapsulated object can include a suitable resin, the resin including the reaction product of an aldehyde and an amine, suitable aldehyde including formaldehyde. Suitable amines include melamine, urea, benzoguanamine, glycoluril and mixtures thereof. Suitable melamines include methylol melamine, methylated methylol melamine, imino melamine and mixtures thereof. Suitable ureas include dimethylol urea, methylated dimethylol urea, urea-resorcinol and mixtures thereof.

[0411] In one aspect, a suitable formaldehyde scavenger can be used with the encapsulated material, for example in a capsule slurry, and / or added to such a composition before, during, or after the encapsulated material is added to the composition. Suitable capsules can be made by following the teachings of US2008 / 0305982, and / or US2009 / 0247449.

[0412] In a preferred aspect, the composition may further comprise a deposition aid, preferably consisting of the group comprising cationic or nonionic polymers. Suitable polymers include cationic starch, cationic hydroxyethylcellulose, polyvinyl formaldehyde, locust bean gum, mannan, xyloglucan, tamarind gum, polyethylene glycol terephthalate, and polymers containing dimethylaminoethyl methacrylate, optionally with one or more monomers selected from the group comprising acrylic acid and acrylamide.

[0413] spices- In one aspect, the composition comprises a perfume comprising one or more perfume raw materials selected from the group consisting of: 1,1'-oxybis-2-propanol; diethyl 1,4-cyclohexanedicarboxylate; (ethoxymethoxy)cyclododecane; 1,3-nonanediol monoacetate; 2-propenyl (3-methylbutoxy)acetate; β-methylcyclododecaneethanol; 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-1-propanol; oxahexadecan-2-one; α-methyl-benzyl alcohol ester; trans-3-ethoxy-1,1,5-trimethylcyclohexane; 4-[ ...]oxy]-1-propanol; -(1,1-dimethylethyl)cyclohexanol acetate; dodecahydro-3a,6,6,9a-tetramethylnaphtho[2,1-b]furan; β-methylphenylpropionaldehyde; β-methyl-3-(1-methylethyl)phenylpropionaldehyde; 4-phenyl-2-butanone; ethyl 2-methylbutyrate; benzaldehyde; 1-methylethyl 2-methylbutyrate; dihydro-5-pentyl-2(3H)furanone; (2E)-1-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2-buten-1-one; dodecanal; undecanal; 2-ethyl-α,α-dimethylphenylpropionaldehyde; decanal; α,α-dimethylphenylethanol acetate; 2- (Phenylidene)octanal; methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate; 1-(2,6,6-trimethyl-3-cyclohexen-1-yl)-2-buten-1-one; 2-pentylcyclopentanone; methyl 3-oxo-2-pentylcyclopentaneacetate; 4-hydroxy-3-methoxybenzaldehyde; 3-ethoxy-4-hydroxybenzaldehyde; 2-heptylcyclopentanone; 1-(4-methylphenyl)ethanone; (3E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one; (3E)-4-(2,6,6- (trimethyl-2-cyclohexen-1-yl)-3-buten-2-one; phenylethyl alcohol; 2H-1-chromen-2-one; 4-methoxybenzaldehyde; 10-undecanal; benzyl propionate; β-methylphenylpentanol; 1,1-diethoxy-3,7-dimethyl-2,6-octadiene; α,α-dimethylphenylethyl alcohol; (2E)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; benzyl acetate; 2-propenyl cyclohexylpropionate; 2-propenyl hexanoate; 1,2-dimethoxy-4-(2-propenyl)benzene; 1,5-dimethyl-bicyclo[3.2.1] Octane-8-one oxime; 4-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carbaldehyde; 3-buten-2-ol; methyl 2-[[[2,4(or 3,5)-dimethyl-3-cyclohexen-1-yl]methylene]amino]benzoate; 8-cyclohexadecene-1-one; methyl ionone; 2,6-dimethyl-7-octen-2-ol; 2-methoxy-4-(2-propenyl)phenol; (2E)-3,7-dimethyl-2,6-octadien-1-ol ; (3Z)-3-hexenyl-2-hydroxybenzoate; 2-tridecenenitrile; 4-(2,2-dimethyl-6-methylenecyclohexyl)-3-methyl-3-buten-2-one; tetrahydro-4-methyl-2-(2-methyl-1-propenyl)-2H-pyran; (2-methylbutoxy)-2-propenyl acetate; 2-hydroxy-3-methylbutyl benzoate; (Z)-1-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-buten-1-one; 2- Methyl hexyl-3-oxocyclopentanecarboxylate; 4-ethyl-α,α-dimethyl-phenylpropanal; 3-(4-hydroxy-4-methylpentyl)-3-cyclohexene-1-carbaldehyde; 1-(2,3,4,7,8,8a-hexahydro-3,6,8,8-tetramethyl-1H-3a,7-methanoazulen-5-yl)-[3R-(3α,3aβ,7β,8aα)]-ethanone; 2-methyl-2H-pyran-2-one 6-butyltetrahydro-undecanal; 4-(1,1- dimethylethyl)-α-methyl-phenylpropanal; 5-heptyldihydro-2(3H)-furanone; methyl 2-[(7-hydroxy-3,7-dimethyloctylene)amino]benzoate; 2-hydroxy-benzyl benzoate; 2-methoxynaphthalene; 2-hexyl-2-cyclopenten-1-one; 5-hexyldihydro-2(3H)-furanone; ethyl 3-methyl-3-phenyl-oxiranecarboxylate; 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane; phenylpentanol, γ.-methyl-; 3,7-dimethyl-3-octanol; 3,7-dimethyl-2,6-octadienenitrile; 3,7-dimethyl-6-octen-1-ol; terpineol acetate; 2-methyl-6-methylene-7-octen-2-ol dihydro derivative; 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-ol 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-methano-5H-inden-5-ylidene)-butanal; 3-2,4-dimethyl-cyclohexene-1-carbaldehyde; 1-(1,2,3,4,5,6,7,8-octahydro-2,3,8,8-tetramethyl-2-naphthalene)-ethanone; 2-hydroxy-benzoic acid methyl ester; 2-hydroxy-benzoic acid hexyl ester; 2-phenoxy-ethanol; 2 1-Hydroxy-pentyl benzoate; 2,3-heptanedione; 2-hexen-1-ol; 2,6-dimethyl-6-octen-2-ol; damascenone (α, β, γ or δ or a mixture thereof), 3a,4,5,6,7,7a-hexahydro-4,7-methano-1H-inden-6-ol acetate; 9-undecenal; 8-undecenal; isocyclic citral; 1-(1,2,3,5,6,7,8,8a-octahydro-2,3,8,8-tetramethyl-2- naphthalene)-ethanone; 3,5-dimethyl-3-cyclohexene-1-carbaldehyde; 2,4-dimethyl-3-cyclohexene-1-carbaldehyde; 3,7-dimethyl-1,6-octadien-3-ol; 3,7-dimethyl-1,6-octadien-3-ol acetate; pt-Bucinal, and 2-[2-(4-methyl-3-cyclohexen-1-yl)propyl]-cyclopentanone and 1-methyl-4-(1-methylvinyl)cyclohexene and mixtures thereof.

[0414] In one aspect, the composition may comprise encapsulated fragrance particles comprising a water-soluble hydroxyl compound or melamine-formaldehyde or a modified polyvinyl alcohol. In one aspect, the encapsulate comprises (a) an at least partially water-soluble solid matrix comprising one or more water-soluble hydroxyl compounds, preferably starch; and (b) a fragrance oil encapsulated by the solid matrix.

[0415] In another aspect, the fragrance may be pre-complexed with a polyamine, preferably polyethyleneimine, to form a Schiff base.

[0416] polymerThe composition may comprise one or more polymers. Examples are carboxymethylcellulose, poly(vinyl-pyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates (e.g. polyacrylates), maleic acid / acrylic acid copolymers, and lauryl methacrylate / acrylic acid copolymers.

[0417] The composition may include one or more amphiphilic cleaning polymers, such as compounds having the following general structure: bis((C2H5O)(C2H4O)n)(CH3)-N + -C x H 2x -N + -(CH3)-bis((C2H5O)(C2H4O)n), wherein n = from 20 to 30, and x = from 3 to 8, or sulfated or sulfonated variants thereof.

[0418] The composition may comprise amphiphilic alkoxylated grease cleaning polymers having balanced hydrophilic and hydrophobic properties such that they remove grease particles from fabrics and surfaces. Specific embodiments of the amphiphilic alkoxylated grease cleaning polymers of the present invention comprise a core structure and a plurality of alkoxylated groups attached to that core structure. These may comprise alkoxylated polyalkylenimines, preferably having inner polyethylene oxide blocks and outer polypropylene oxide blocks.

[0419] Alkoxylated polycarboxylates (such as those prepared from polyacrylates) can be used herein to provide additional grease removal properties. Such materials are described in WO 91 / 08281 and PCT 90 / 01815. Chemically, these materials comprise polyacrylates having one ethoxy side chain per every 7-8 acrylate units. The side chains have the formula -(CH2CH2O) m (CH2) n CH3, wherein m is 2-3 and n is 6-12. The side chains are ester-linked to the polyacrylate "backbone" to provide a "comb-like" polymer type structure. The molecular weight can vary but is typically in the range of 2000 to 50,000. Such alkoxylated polycarboxylates may comprise from 0.05 wt % to 10 wt % of the composition herein.

[0420] The isoprenoid-derived surfactants of the present invention, and mixtures thereof with other co-surfactants and other auxiliary ingredients, are particularly suitable for use with amphiphilic graft copolymers, preferably comprising (i) a polyethylene glycol backbone; and (ii) at least one pendant moiety selected from polyvinyl acetate, polyvinyl alcohol, and mixtures thereof. A preferred amphiphilic graft copolymer is Sokalan HP22 supplied by BASF. Suitable polymers include random graft copolymers, preferably polyvinyl acetate grafted polyethylene oxide copolymers, having a polyethylene oxide backbone and multiple polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is preferably 6000, and the weight ratio of polyethylene oxide to polyvinyl acetate is 40 to 60, with no more than one grafting site per 50 ethylene oxide units.

[0421] Carboxylate polymers - The composition of the present invention may also include one or more carboxylate polymers, such as maleate / acrylate random copolymers or polyacrylate homopolymers. In one aspect, the carboxylate polymer is a polyacrylate homopolymer having a molecular weight of from 4,000 to 9,000 Da or from 6,000 to 9,000 Da.

[0422] Soil release polymers - The compositions of the present invention may also include one or more soil release polymers having a structure as defined by one of the following structures (I), (II) or (III):

[0423] (I)-[(OCHR 1 -CHR 2 ) a -O-OC-Ar-CO-] d

[0424] (II)-[(OCHR 3 -CHR 4 ) b -O-OC-sAr-CO-] e

[0425] (III)-[(OCHR 5 -CHR 6 ) c -OR 7 ] f

[0426] in:

[0427] a, b and c are from 1 to 200;

[0428] d, e, and f are from 1 to 50;

[0429] Ar is 1,4-substituted phenylene;

[0430] sAr is a 1,3-substituted phenylene group substituted at the 5-position by SO3Me;

[0431] Me is Li, K, Mg / 2, Ca / 2, Al / 3, ammonium, monoalkylammonium, dialkylammonium, trialkylammonium or tetraalkylammonium, wherein the alkyl group is C1-C 18 Alkyl or C2-C 10 hydroxyalkyl, or a mixture thereof;

[0432] R 1 、R 2 、R 3 、R 4 、R 5 and R 6 Independently selected from H or C1-C 18 n-alkyl or isoalkyl; and

[0433] R 7 Is a straight or branched C1-C 18 Alkyl, or linear or branched C2-C 30 alkenyl, or cycloalkyl having 5 to 9 carbon atoms, or C8-C 30 Aryl, or C6-C 30 Arylalkyl.

[0434] Suitable soil release polymers are polyester soil release polymers, such as Repel-o-tex polymers, including Repel-o-tex, SF-2 and SRP6, supplied by Rhodia. Other suitable soil release polymers include Texcare polymers, including Texcare SRA100, SRA300, SRN100, SRN170, SRN240, SRN300 and SRN325, supplied by Clariant. Other suitable soil release polymers are Marloquest polymers, such as Marloquest SL, supplied by Sasol.

[0435] Cellulose polymers The compositions of the present invention may also comprise one or more cellulosic polymers, including those selected from the group consisting of alkylcelluloses, alkylalkoxyalkylcelluloses, carboxyalkylcelluloses, and alkylcarboxyalkylcelluloses. In one aspect, the cellulosic polymer is selected from the group consisting of carboxymethylcellulose, methylcellulose, methylhydroxyethylcellulose, methylcarboxymethylcellulose, and mixtures thereof. In one aspect, the carboxymethylcellulose has a degree of carboxymethyl substitution of from 0.5 to 0.9 and a molecular weight of from 100,000 to 300,000 Da.

[0436] Enzyme - except lipase variants of the present invention, composition can also comprise one or more enzymes that provide cleaning performance and / or fabric care benefit.The example of applicable enzyme includes but is not limited to hemicellulase, peroxidase, protease, cellulase, xylanase, other lipolytic enzymes, phospholipases, esterases, cutinase, pectinase, mannanase, pectin lyase, keratinase, reductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosaccharase, Marana enzyme (malanase), beta-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, chlorophyllase, amylase or its mixture.Typical combination is enzyme mixture, can comprise for example protease and lipase together with amylase.When being present in composition, aforementioned other enzyme can exist with the level of from 0.00001wt% to 2wt%, from 0.0001wt% to 1wt% or from 0.001wt% to 0.5wt% enzyme protein by the weight of composition.

[0437] Generally, the enzyme(s) selected should have properties compatible with the chosen detergent (ie, pH optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in an effective amount.

[0438] On the one hand, preferred enzyme will include cellulase.Suitable cellulase includes those of bacterial origin or fungal origin.Including chemically modified mutants or protein engineered mutants.Suitable cellulase includes cellulase from the following genus: Bacillus (Bacillus), Pseudomonas (Pseudomonas), Humicola (Humicola), Fusarium (Fusarium), Thielavia (Thielavia), Acremonium (Acremonium), such as the fungal cellulase produced by Humicola insolens (Humicola insolens), Myceliophthora thermophila (Myceliophthora thermophila) and Fusarium oxysporum (Fusarium oxysporum) disclosed in US 4435307, US5648263, US 5691178, US 5776757 and WO 89 / 09259.

[0439] Particularly suitable cellulases are alkaline or neutral cellulases having color care benefits. Examples of such cellulases are the cellulases described in EP 0495257, EP 0531372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are cellulase variants such as those described in WO 94 / 07998, EP 0531315, US 5457046, US 5686593, US 5763254, WO 95 / 24471, WO 98 / 12307, and PCT / DK98 / 00299.

[0440] 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).

[0441] On the one hand, preferred enzymes will include proteases. Suitable proteases include those of bacterial, fungal, plant, viral or animal origin, such as plant or microbial origin. Preferably, microbial origin. Include chemically modified mutants or protein engineered mutants. It can be an alkaline protease, such as a serine protease or a metalloprotease. The serine protease can be, for example, an S1 family (such as trypsin) or an S8 family (such as subtilisin). The metalloprotease protease can be, for example, a thermolysin or other metalloprotease from, for example, an M4 family, such as those from an M5, M7 or M8 family.

[0442] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Engng. 4 (1991) 719-737 and Siezen et al., Protein Science 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subclasses, namely, the subtilisin family, the thermophilic protease family, the proteinase K family, the lanthionine antibiotic peptidase family, the Kexin family, and the pyrolysin family.

[0443] Examples of subtilases are those derived from Bacillus, such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US 7262042 and WO 09 / 021867; and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO 89 / 06279 and protease PD138 described in (WO 93 / 18140). Other useful proteases may be those described in WO 92 / 175177, WO 01 / 016285, WO 02 / 026024 and WO 02 / 016547. Examples of trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease (described in WO 89 / 06270, WO 94 / 25583 and WO 05 / 040372), and chymotrypsin from Cellumonas (described in WO 05 / 052161 and WO 05 / 052146).

[0444] Further preferred proteases are the alkaline protease from Bacillus lentus DSM 5483 (as described in, for example, WO 95 / 23221), and variants thereof (described in WO 92 / 21760, WO 95 / 23221, EP 1921147 and EP 1921148).

[0445] Examples of metalloproteases are neutral metalloproteases as described in WO 07 / 044993 (Genencor International), such as those derived from Bacillus amyloliquefaciens.

[0446] Examples of useful proteases are the variants described in WO 92 / 19729, WO 96 / 034946, WO 98 / 20115, WO 98 / 20116, WO 99 / 011768, WO 01 / 44452, WO 03 / 006602, WO 04 / 03186, WO 04 / 041979, WO 07 / 006305, WO 11 / 036263, WO 11 / 036264, particularly variants having substitutions at one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252, and 274, using BPN' for numbering. More preferably, the subtilase variant may comprise the following mutations: S3T, V4I, S9R, A15T, K27R, *36D, V68A, N76D, N87S,R, *97E, A98S, S99G,D,A, S99AD, S101G,M,R S103A, V104I,Y,N, S106A, G118V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (use BPN' for numbering).

[0447] Suitable commercially available protease enzymes include those sold under the following trade names: Duralase Tm 、Durazym Tm 、 Ultra, Ultra, Ultra, Ultra, and All of these can be or (Novozymes); those sold under the following trade names: Purafect Preferenz Tm 、Purafect Purafect Purafect Effectenz Tm 、 and (Danisco / DuPont), Axapem TM (Gist-Brocases NV), BLAP (sequence shown in Figure 29 of US 5352604) and its variants (Henkel AG), and KAP (alkalophilic Bacillus subtilisin) from Kao Corporation (Kao).

[0448] In one aspect, preferred enzymes will include amylases. Suitable amylases may be alpha-amylases or glucoamylases and may be of bacterial or fungal origin. Chemically modified mutants or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, such as the alpha-amylase of a particular strain of Bacillus licheniformis described in more detail in GB 1296839.

[0449] Suitable amylases include those having SEQ ID NO: 3 in WO 95 / 10603 or variants thereof having 90% sequence identity to SEQ ID NO: 3. Preferred variants are described in 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.

[0450] Various suitable amylases include those having SEQ ID NO: 6 in WO 02 / 010355 or variants thereof having 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having deletions in positions 181 and 182 and a substitution in position 193.

[0451] Other suitable amylases are hybrid alpha-amylases comprising residues 1-33 of an alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of an alpha-amylase from Bacillus licheniformis shown in SEQ ID NO: 4 of WO 2006 / 066594, or variants thereof having 90% sequence identity. Preferred variants of the hybrid alpha-amylase are those having a substitution, deletion, or insertion at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, 1201, A209, and Q264. Most preferred variants of the hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from Bacillus amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006 / 066594 and residues 36-483 of SEQ ID NO: 4 are those having the following substitutions:

[0452] M197T;

[0453] H156Y+A181T+N190F+A209V+Q264S; or

[0454] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

[0455] Suitable additional amylases are those having SEQ ID NO: 6 in WO 99 / 019467 or variants thereof having 90% sequence identity thereto. 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.

[0456] Additional amylases that can be used are those having SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96 / 023873 or variants thereof having 90% sequence identity thereto. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, deletion or insertion at one or more of the following positions: 140, 181, 182, 183, 184, 195, 206, 212, 243, 260, 269, 304 and 476. More preferred variants are those having a deletion at positions 181 and 182 or positions 183 and 184. Most preferred amylase variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are those having deletions at positions 183 and 184 and substitutions at one or more of positions 140, 195, 206, 243, 260, 304, and 476.

[0457] Other amylases that can be used are those having SEQ ID NO: 2 of WO 08 / 153815, SEQ ID NO: 10 in WO 01 / 66712, or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08 / 153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01 / 66712. Preferred variants of SEQ ID NO: 10 in WO 01 / 66712 are those having substitutions, deletions or insertions at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

[0458] Additional suitable amylases are those having SEQ ID NO: 2 of WO 09 / 061380 or a variant thereof having 90% sequence identity to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those having a 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 deletions at 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:

[0459] N128C+K178L+T182G+Y305R+G475K;

[0460] N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

[0461] S125A+N128C+K178L+T182G+Y305R+G475K; or

[0462] S125A+N128C+T131I+T165I+K178L+T182G+Y305R+G475K, wherein the variants are C-terminally truncated and optionally further comprise a substitution at position 243 and / or a deletion at position 180 and / or position 181.

[0463] Other suitable amylases are alpha-amylases having SEQ ID NO: 12 in WO 01 / 66712, or variants having at least 90% sequence identity to SEQ ID NO: 12. Preferred amylase variants are those having substitutions, deletions or insertions at one or more of the following positions of SEQ ID NO: 12 in WO 01 / 66712: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471, N484. Particularly preferred amylases include variants having deletions of D183 and G184 and having substitutions R118K, N195F, R320K, and R458K, as well as variants additionally 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 variants additionally having substitutions in all of these positions being most preferred.

[0464] Further examples are amylase variants such as those described in WO 2011 / 098531, WO 2013 / 001078 and WO 2013 / 001087.

[0465] Commercially available amylase is Duramyl TM Termamyl TM Termamyl Ultra TM、 Fungamyl TM 、Ban TM 、Stainzyme TM 、Stainzyme Plus TM 、 Prime, Supramyl TM 、Natalase TM , Liquozyme X and BAN TM (from Novozymes), AT 9000 (Biozym Biotech Trading GmbH, Wehlistrasse 27b A-1200 Wien Austria), and Rapidase TM 、Purastar TM / EffectenzTM , Powerase, Preferenz S100, Preferenx S110, OPTISIZE and PURASTAR (Danisco / DuPont) and (Kao Corporation).

[0466] Additional suitable lipases and cutinases include those of bacterial or fungal origin, including chemically modified mutant enzymes or protein engineered mutant enzymes. Examples include lipases from thermophilic fungi, e.g., from Thermomyces lanuginosa (formerly named Humicola lanuginosa) as described in EP 258068 and EP 305216; cutinases from Humicola, e.g., Humicola insolens (WO 96 / 13580); lipases from strains of Pseudomonas (some of these now renamed Burkholderia), e.g., Pseudomonas alcaligenes or Pseudomonas pseudoalcaligenes (EP 218272), Pseudomonas cepacia (EP 331376), Pseudomonas strain SD705 (WO 95 / 06720 & WO 96 / 27002), Pseudomonas wisconsinensis (WO 96 / 12012); GDSL-type Streptomyces lipase (WO 10 / 065455); a cutinase from Magnaporthe grisea (WO 10 / 107560); a cutinase from Pseudomonas mendocina (US 5,389,536); a lipase from Thermobifida fusca (WO 11 / 084412, WO 13 / 033318); a Geobacillus stearothermophilus lipase (WO 11 / 084417); a lipase from Bacillus subtilis (WO 11 / 084599); and lipases from Streptomyces griseus (WO 11 / 150157) and Streptomyces pristinaespiralis (WO 12 / 137147).

[0467] Other examples are further lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO 94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508 and WO 09 / 109500.

[0468] Preferred additional commercial lipase products include Lipolase TM 、Lipex TM ; Lipex evitry100L, Lipex Evity 200L, Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), Lipomax (from Gist-Brocades), and Preferenz L100 (from Danisco US Inc).

[0469] Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, such as the acyltransferase with homology to Candida antarctica lipase A (WO 10 / 111143), the acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), the perhydrolases from the CE 7 family (WO 09 / 67279), and variants of the M. smegmatis perhydrolase, in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 10 / 100028).

[0470] In one aspect, other preferred enzymes include endoglucanases (EC 3.2.1.4) of microbial origin exhibiting endo-β-1,4-glucanase activity, including bacterial polypeptides endogenous to members of the genus Bacillus (the polypeptide having a sequence having at least 90%, 94%, 97% or 99% identity to the amino acid sequence SEQ ID NO: 2 in US 7141403) and mixtures thereof. Suitable endoglucanases are available under the trade name and Sold under the name Novozymes.

[0471] Other preferred enzymes include those under the trade names Pectate lyases sold under the trade names Mannanase sold under the name Novozymes, and (Danisco / DuPont).

[0472] One or more detergent enzymes can be included in the detergent composition by adding a single additive containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additives of the present invention, i.e., the additives alone or in combination, can be formulated, for example, as granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, particularly non-dusting granules; liquids, particularly stabilized liquids; or slurries.

[0473] Dust-free particles can be manufactured as disclosed in, for example, US 4106991 and US 4661452, and can optionally be coated by methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with an average molecular weight of 1000 to 20000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms, and wherein there are 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 use by fluidized bed technology are provided in GB 1483591. Liquid enzyme preparations can be stabilized, for example, by adding polyols (such as propylene glycol), sugar or sugar alcohols, lactic acid or boric acid according to established methods. Protected enzymes can be prepared according to the method disclosed in EP 238216.

[0474] Dye transfer inhibition reagents The compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones, and polyvinylimidazoles, or mixtures thereof. When present in the composition, the dye transfer inhibiting agent may be present at a level of from 0.0001 to 10 wt%, from 0.01 to 5 wt%, or from 0.1 to 3 wt%.

[0475] Brightener - The compositions of the present invention may also contain additional ingredients which may impart colour to the items being cleaned, such as optical brighteners.

[0476] The composition may include a CI fluorescent brightener 260 in α-crystal form having the following structure:

[0477]

[0478] In one aspect, the brightener is a cold water soluble brightener, such as a CI fluorescent brightener in α-crystalline form 260. In one aspect, the brightener is predominantly 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 the CI fluorescent brightener 260 is in α-crystalline form.

[0479] The brightener is typically in the form of micronized particles having a weighted average primary particle size of from 3 to 30 microns, from 3 to 20 microns, or from 3 to 10 microns.

[0480] The composition may include a 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. BE680847 relates to a method for preparing a CI fluorescent brightener 260 in α-crystalline form.

[0481] Commercial optical brighteners that can be used in the present invention can be divided into several subgroups, including but not necessarily limited to: stilbenes, pyrazolines, coumarins, carboxylic acids, methine cyanines, dibenzothiophene-5,5-dioxide, azoles, derivatives of 5- and 6-membered heterocycles, and other miscellaneous agents. Examples of such brighteners are disclosed in "The Production and Application of Fluorescent Brightening Agents", M. Zahradnik, published by John Wiley & Sons, New York (1982). Specific non-limiting examples of optical brighteners that can be used in the compositions of the present invention are those identified in US Pat. No. 4,790,856 and US Pat. No. 3,646,015.

[0482] Another suitable brightener has the following structure:

[0483]

[0484] Suitable fluorescent brightener levels include lower levels of from 0.01 wt%, from 0.05 wt%, from 0.1 wt%, or from 0.2 wt%, to higher levels of 0.5 wt% or 0.75 wt%.

[0485] In one aspect, the brightener can be loaded onto the clay to form particles. Silicate - The composition of the present invention can also contain a silicate, such as sodium silicate or potassium silicate. The composition can contain from 0 wt% to less than 10 wt% silicate, to 9 wt%, or to 8 wt%, or to 7 wt%, or to 6 wt%, or to 5 wt%, or to 4 wt%, or to 3 wt%, or even to 2 wt%, and from above 0 wt%, or from 0.5 wt%, or from 1 wt% silicate. A suitable silicate is sodium silicate.

[0486] dispersants - The compositions of the invention may also contain a dispersant. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.

[0487] Enzyme stabilizers - The enzymes used in the composition can be stabilized by various techniques. The enzymes used herein can be stabilized by the presence of a water-soluble source of calcium and / or magnesium ions. Examples of conventional stabilizing agents 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 compositions can be formulated as described in, for example, WO 92 / 19709 and WO 92 / 19708. In the case of aqueous compositions comprising proteases, reversible protease inhibitors can be added to further improve stability, such as boron compounds including borates, 4-formylphenylboronic acid, phenylboronic acid and derivatives thereof; or compounds such as calcium formate, sodium formate and 1,2-propylene glycol. The peptide aldehyde may have the formula B2-B1-BO-R, wherein: R is hydrogen, CH3, CX3, CHX2 or CH2X, wherein X is a halogen atom; B0 is a phenylalanine residue having an OH substituent at the para position and / or at the meta position; B1 is a single amino acid residue; and B2 consists of one or more amino acid residues, optionally containing an N-terminal protecting group. Preferred peptide aldehydes include, but are not limited to, Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H or Ac-WLVY-H, wherein Z is benzyloxycarbonyl and Ac is acetyl.

[0488] solventSuitable solvents include water and other solvents such as lipophilic fluids. Examples of suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerol derivatives (e.g., glycerol ethers), perfluorinated amines, perfluorinated and hydrofluoroether solvents, low volatility non-fluorinated organic solvents, glycol solvents, other environmentally friendly solvents, and mixtures thereof.

[0489] Structuring agent / thickener -Structured liquids can be structured from the inside, whereby the structure is formed by a primary component (e.g., a surfactant material), and / or structured from the outside by providing a three-dimensional matrix structure using a secondary component (e.g., a polymer, clay, and / or silicate material). The composition can comprise from 0.01 to 5wt% or from 0.1 to 2.0wt% of a structuring agent. The structurin...

Claims

1. A lipase variant selected from one or more of the groups (i), (ii) and (iii) comprising: (i) substitutions at one or more of positions 252, 202 and 269 of the polypeptide corresponding to SEQ ID NO: 8; (ii) substitutions at one or more of positions 40, 56, 57, 91, 98, 108, 118, 210, 244 and 254 of the polypeptide corresponding to SEQ ID NO: 8; and (iii) substitutions at one or more of positions 23, 27, 40, 51, 56, 60, 118, 244, and 256 of the polypeptide corresponding to SEQ ID NO: 8; wherein the variant has lipase activity, and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 8, wherein the variant optionally comprises one or more amino acid extensions at the N-terminus and / or the C-terminus, or comprises one or more amino acid truncations at the N-terminus and / or the C-terminus, and wherein the variant has lipase activity.

2. The variant of claim 1, comprising or consisting of one or more substitutions corresponding to the positions in SEQ ID NO: 8, in particular all substitutions, selected from the group consisting of: H substitution of the amino acid residue at position 202; H substitution of the amino acid residue at position 252; and H substitution of the amino acid residue at position 269; or The variant comprises or consists of one of the following substitution groups: 202H+252H; 202H+269H; 252H+269H; or 202H+252H+269H.

3. The variant according to any one of claims 1 to 2, comprising or consisting of one or more substitutions corresponding to the positions in SEQ ID NO: 8, in particular all substitutions, selected from the group consisting of: E substitution of the amino acid residue at position 40; R substitution of the amino acid residue at position 56; Substitution of the amino acid residue at position 57 by N; T substitution of the amino acid residue at position 91; E substitution of the amino acid residue at position 98; Substitution of the amino acid residue at position 108 by K; F substitution of the amino acid residue at position 118; K substitution of the amino acid residue at position 210; E substitution of the amino acid residue at position 244; and S substitution of the amino acid residue at position 254.

4. The variant according to any one of claims 1 to 3, comprising or consisting of one or more substitutions corresponding to the positions in SEQ ID NO: 8, in particular all substitutions, which are selected from the group consisting of: S substitution of the amino acid residue at position 23; N substitution of the amino acid residue at position 27; 1. Substitution of the amino acid residue at position 40; 1. Substitution of the amino acid residue at position 51; R substitution of the amino acid residue at position 56; K substitution of the amino acid residue at position 60; F substitution of the amino acid residue at position 118; E substitution of the amino acid residue at position 244; and T is used to substitute the amino acid residue at position 256.

5. The variant of any one of claims 1 to 4, wherein the lipase variant has reduced lipase activity and / or reduced odor production at around neutral pH, i.e. about pH 6-8, preferably about pH 7, and / or has an increased benefit-risk factor (BRF) compared to the parent lipase, in particular SEQ ID NO: 2, 4, 6 or 8, respectively.

6. A particle comprising: (a) comprising a core of a variant as claimed in any one of claims 1 to 5, and optionally (b) a coating consisting of one or more layers surrounding the core, or The pellets contain: (a) core, and (b) a coating consisting of one or more layers surrounding the core, wherein the coating comprises the variant of any one of claims 1-5.

7. A liquid composition comprising a variant as claimed in any one of claims 1 to 5 and an enzyme stabilizer, for example a polyol such as propylene glycol or glycerol, a sugar or a sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, for example an aromatic borate ester or a phenylboronic acid derivative such as 4-formylphenylboronic acid.

8. A composition comprising the variant of any one of claims 1 to 5, the particles of claim 6, or the liquid composition of claim 7.

9. A polynucleotide encoding the variant according to any one of claims 1 to 5. 10 . A nucleic acid construct or an expression vector comprising the polynucleotide according to claim 9 .

11. A recombinant host cell transformed with the polynucleotide according to claim 9.

12. A method for producing a lipase variant, the method comprising: a. Cultivating the recombinant host cell according to claim 11 under conditions suitable for expressing the variant; as well as b. optionally recovering the variant.

13. A method for hydrolyzing a lipase substrate, the method comprising mixing the substrate with the lipase variant of any one of claims 1 to 5 or the composition of claim 8 under conditions that favor hydrolysis of the substrate by the lipase variant.

14. A method for removing lipid stains from a surface, the method comprising: The stain is contacted with the lipase variant according to any one of claims 1 to 5 or the composition according to claim 8, followed by rinsing the surface and optionally drying.

15. A method for washing clothes, the method comprising the following steps: i) by washing the laundry with a lipase according to any one of claims 1 to 5, a granule according to claim 6, a liquid composition according to claim 7, or a composition according to claim 8; ii) rinsing the garment; and iii) optionally drying the garment.

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