RU2775700C2 - Detergent compositions containing lipases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and is a water-soluble product with a single dose for washing fabric containing a water-soluble film and a liquid detergent composition for washing, and the liquid detergent composition for washing contains a variant of the parent lipase, which has lipase activity, the sequence of which is at least 70% identical to the sequence with SEQ ID NO: 2, contains substitutions in positions corresponding to F51I,L, T231R and N233R, and optionally one or more substitutions in positions corresponding to G23S, D27N, A40I, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E and P256T; and an auxiliary component of the detergent; while the liquid detergent composition for washing is covered with a water-soluble film; the water-soluble film contains a polymer material selected from polyvinyl alcohols, polyvinylpyrrolidone, polyalkylene oxides, acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetates, polycarboxylic acids and salts, polyaminoacids or peptides, polyamides, polyacrylamide, copolymers of maleic/acrylic acids, polysaccharides, including starch and gelatin, natural gums such as xanthan gum and carrageenan and their mixtures.

EFFECT: invention makes it possible to effectively carry out a method for washing fabrics using a water-soluble product with a single dose according to the invention.

13 cl, 1 dwg, 4 tbl, 13 ex

Description

LINK TO SEQUENCE LISTING

This application contains a sequence listing in machine readable form, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to detergent compositions and single dose water-soluble articles containing detergent compositions containing lipase enzymes, as well as to methods for preparing and using such compositions. The invention also relates to liquid products, detergent compositions and water-soluble single dose articles containing detergent compositions in which the lipase variant is encapsulated in a microcapsule. Such compositions and articles are preferably fabric care and home cleaning products, most preferably laundry products.

BACKGROUND OF THE INVENTION

Detergent manufacturers are still trying to develop cleaning compositions, in particular laundry and dishwashing compositions, which are the most reliable cleaning systems. Lipases are important biocatalysts that have been shown to be very useful in detergent compositions, aiding in the removal of oily soils and stains by hydrolyzing triglycerides to form fatty acids. After hydrolysis, hydrolyzed contaminants are easier to remove from fabric surfaces during washing in the presence of other detergent components. Many known lipases provide good washing performance, however they can also be prone to form odorous short chain fatty acids during washing and/or have poor storage stability.

Stability can be a particular problem, for example, when in contact with incompatible components of the detergent composition or when exposed to temperatures significantly higher or lower than ambient temperature, either during storage or during the washing or processing step. If the stability is not high enough, this leads to a decrease in the activity or effectiveness of the composition as a whole and, in particular, to a loss of enzymatic activity. Thermomyces lanuginosus lipase (another name for Humicola lanuginosa) sold under the tradename LIPOLASE™ and variants thereof are marketed as active ingredients in detergent compositions for removing grease stains by hydrolyzing triglycerides to form fatty acids. However, there is still a need for compositions containing lipases that provide good washing performance, reduce odor generation and/or improve storage stability, provide longer shelf life/increased thermal stability.

SUMMARY OF THE INVENTION

The present invention relates to a detergent composition containing a parental lipase variant having lipase activity, said variant having at least 60% but less than 100% sequence identity to that of SEQ ID NO: 2 and containing (i) one or more substitutions in positions 40, 118, T244E and V60E; and (ii) one or more substitutions at positions corresponding to T231R and N233R. The lipase variant preferably contains substitutions at positions corresponding to both positions 40 and 118, more preferably one or more substitutions at positions corresponding to A40I and R118F.

The present invention also relates to a detergent composition containing a parental lipase variant having lipase activity, said variant having at least 60% but less than 100% sequence identity with SEQ ID NO: 2 and containing one or more substitutions in positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, T231R, N233R, Y220F, T244E and P256T; and an auxiliary detergent component. Preferably, the detergent composition is in the form of a liquid, preferably containing less than 20% by weight of water.

In a further aspect, the invention also relates to a detergent composition comprising a parental lipase variant having lipase activity, said variant having at least 60% but less than 100% sequence identity with SEQ ID NO: 2 and containing (i ) one or more substitutions at positions corresponding to positions 23, 51 and 56, preferably one or more substitutions at positions corresponding to G23S, F51I, L and E56R; and (ii) one or more substitutions at positions corresponding to T231R and N233R; and preferably (iii) one or more substitutions at positions corresponding to 27, 40, 57, 60, 98, 101, 118, 163, 220, 244 and 256, preferably one or more substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E and P256T.

The invention also provides a single dose water soluble product comprising a water soluble film and a cleaning composition containing a lipase variant as described above; and an auxiliary detergent component. The lipase variant may be present in a water soluble single dose product in a liquid composition or in a solid composition. Preferably, the detergent composition containing the lipase variant is a liquid composition.

The present invention also relates to a method for preparing a detergent composition containing a lipase variant, the method comprising the first step of mixing a detergent adjuvant and a lipase variant to form a detergent composition. Preferably, the detergent composition is then formed into a unit dose form, optionally by coating the detergent composition with a film, preferably a water-soluble film. The lipase variant is preferably present in the detergent composition described herein containing less than 20% by weight of water. The water soluble single dose product may comprise one compartment or more than one compartment. The water soluble single dose product contains the lipase variant in one or more compartments, most preferably in a single compartment of a single dose detergent containing multiple compartments.

The present invention also relates to a method for washing fabric, including the following steps:

(i) forming an aqueous cleaning solution containing water and a cleaning composition as described herein;

(ii) (ii) cleaning the surface with an aqueous cleaning solution, preferably at a temperature of 60°C or less, or preferably below 40°C or less, or preferably at a temperature of 30°C or less; and

(iii) (iii) surface rinsing. In a preferred method, a single dose water soluble product according to the invention is combined with sufficient water to dissolve the water soluble film and dilute the laundry detergent composition preferably between 300 and 3000 times to form an aqueous detergent solution.

The sequence of the lipase variant is preferably at least 60%, but less than 100%, identical to the sequence of SEQ ID NO: 2, and contains one or more substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E ,K, K98I, N101D, R118F, G163S, Y220F, T244E and P256T, and one or preferably both substitutions at positions corresponding to T231R and N233R.

The preferred embodiment contains a substitution at the position corresponding to F51I,L. The preferred embodiment contains a replacement in the position corresponding to E56R. The preferred embodiment contains a substitution at the position corresponding to R118F. A preferred embodiment contains one or more substitutions at positions corresponding to F51I,L, E56R and/or R118F. The preferred embodiment contains a replacement in the position corresponding to P256T. The preferred embodiment contains a substitution at the position corresponding to D27N. The preferred embodiment contains a replacement in the position corresponding to G23S. The preferred embodiment contains a replacement at a position corresponding to T244E. The preferred embodiment contains a substitution at the position corresponding to A40I. The preferred embodiment contains a substitution at the position corresponding to K98I. The preferred embodiment contains a substitution at the position corresponding to D57N. The preferred embodiment contains a replacement at the position corresponding to Y220F. The preferred embodiment contains a replacement in a position corresponding to G163S.

In one aspect, the invention relates to a detergent composition containing a lipase enzyme, preferably a lipase variant, encapsulated in a microcapsule. According to one preferred aspect, the microcapsule membrane is prepared by cross-linking a polybranched polyamine with a molecular weight greater than 1 kDa. In a further aspect, the detergent composition comprises a composition of microcapsules with a variant lipase of the invention, preferably enclosed in a compartment formed by a membrane, wherein the membrane is obtained by cross-linking (a) a polybranched polyamine with a molecular weight of more than 800 Da and (b) an aliphatic or aromatic amine with a molecular weight of less than 300 Yes; and the mass ratio (a)/(b) is in the range from 0.1 to 1000.

Lipase. The terms "lipase", "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide" and "lipolytic protein" refer to an enzyme of class EC3.1.1 as defined in Enzyme Nomenclature. It may have lipase activity (triacylglycerolipase, EC 3.1.1.3), cutinase activity (EC 3.1.1.74), sterolesterase activity (EC 3.1.1.13) and/or wax ester hydrolase activity (EC 3.1.1.50). For the purposes of the present invention, lipase activity is determined in accordance with the procedure described in the section on examples. In one aspect, embodiments of the present invention have at least 20%, such as at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least least 95% or 100% lipase activity of the polypeptide of SEQ ID NO: 2.

In one aspect, the invention relates to a single dose water soluble product comprising a water soluble film and a detergent composition containing a lipase variant and a detergent adjuvant comprising a microcapsule formulation, wherein the microcapsule membrane is obtained by cross-linking a polybranched polyamine with a molecular weight greater than 1 kDa, wherein the microcapsule contains a lipase variant of the present invention.

allelic variant. The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variants are formed naturally through mutation and can lead to polymorphism in populations. Gene mutations may be inactive (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

cDNA. The term "cDNA" means a DNA molecule that can be obtained by reverse transcription from a mature spliced mRNA molecule derived from a eukaryotic or prokaryotic cell. cDNA does not have intron sequences that may be present in the corresponding genomic DNA. The primary RNA transcript is an mRNA precursor that undergoes a series of steps, including assembly, to become a mature spliced mRNA molecule.

coding sequence. The term "coding sequence" means a polynucleotide that directly indicates the amino acid sequence of a variant. The boundaries of a coding sequence are typically defined by an open reading frame that starts with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence may be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences. The term "control sequences" means the nucleotide sequences necessary for the expression of a polynucleotide encoding a variant of the present invention. Each control sequence may be native (ie, from the same gene) or foreign (ie, from a different gene) to the polynucleotide encoding the variant, or native or foreign to each other. Such control sequences include, without limitation, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, control sequences include a promoter, as well as transcriptional and translational stop signals. Control sequences can be provided with linkers to introduce specific restriction sites to facilitate ligation of the control sequences to the coding region of the polynucleotide encoding the variant.

Auxiliary component of detergent. The term means any liquid, solid or gaseous material selected for the specific type of detergent composition desired and product form (eg liquid, granule, powder, stick, paste, spray, tablet, gel or foam composition).

Wash composition. The terms "detergent composition" and "detergent composition" are used in relation to mixtures that are intended for use in the washing process to clean contaminated objects. The term is used, in particular, in relation to the washing of fabrics and/or garments (for example, "laundry detergents"). Unless otherwise noted, they may include granular or powder all-purpose or high performance detergents, especially cleansers; liquid, gel or paste all-purpose detergents, especially so-called high-performance liquid detergents (HDL); liquid detergents for delicate fabrics; manual dishwashing detergents or low-efficiency dishwashing detergents, in particular high-foaming detergents; dishwasher detergents, including various types of tablets, granules, liquids and rinses for household and institutional use; liquid cleaning and disinfecting agents; and cleaning aids such as bleach additives and "stain sticks" or pre-treatment agents. In alternative aspects, the term refers to other detergents such as those used for washing dishes, knives, etc. (e.g. "dishwashing detergents"). The term "detergent composition" is not limited to compositions that contain surfactants. In addition to the options necessary for the invention, the term is intended to include detergents, which may contain any auxiliary detergent component, for example, surfactants, modifiers, chelating agents, bleach system or bleach components, polymers, fabric conditioners, foaming agents. , antifoaming agents, dyes, fragrances, tarnish inhibitors, optical brighteners, bactericidal agents, fungicides, soil suspending agents, anticorrosive agents, enzyme inhibitors or stabilizers, enzyme activators, transferases (transferases), hydrolytic enzymes, oxidoreductases, bluing agents and fluorescent dyes, antioxidants, solubilizers and solvents. Detergent compositions can also be used to treat the fabric in either the washing step or the fabric treating step, for example, they can provide a fabric refresh, fabric softening, and/or color restoration step. Detergent compositions described herein, in particular, are included in the composition of a water-soluble product with a single dose.

Expression. The term "expression" includes any step associated with obtaining a variant, including, without limitation, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector. The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a variant and is also operably linked to expression control sequences.

Fragment. The term "fragment" means a polypeptide in which one or more (eg, several) amino acids are missing from the amino acid and/or carboxyl terminus of the polypeptide; wherein the fragment has lipase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of amino acids 1-269 of SEQ ID NO: 2.

conditions of high severity. The term "high stringency conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of hydrodynamically sheared and denatured salmon sperm DNA and formamide 50 % followed by standard Southern blotting procedures within 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 65°C.

Host cell. The term "host cell" means any cell type that is subject to transformation, transfection, transduction, or the like. with a nucleic acid construct or expression vector containing a polynucleotide of the present invention. The term "host cell" includes any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

Improved property. The term "improved property" means a characteristic associated with a variant that has been improved compared to the "parent lipase". Such improved properties include, without limitation, detergent stability, detergent stability in the presence of protease, protease stability, chemical stability, oxidative stability, pH stability, storage stability, and thermal stability.

Dedicated. The term "isolated" means a substance in a form or medium that is not found in nature. Non-limiting examples of isolated substances include: (1) any substance of non-natural origin, (2) any substance, including, without limitation, any enzyme, variant, nucleic acid, protein, peptide or cofactor, which is at least partially removed from one or more or all components of natural origin with which it is associated in nature; (3) any substance modified by man from the same substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components to which it is naturally associated (eg, multiple copies of the gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance) . The isolated substance may be present in the fermentation broth sample.

Low severity conditions. The term "low stringency conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of hydrodynamically sheared and denatured salmon sperm DNA and formamide 25 % followed by standard Southern blotting procedures within 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 50°C.

mature polypeptide. The term "mature polypeptide" means the polypeptide in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like. In one aspect, the mature polypeptide is amino acids 1-269 of SEQ ID NO: 2. It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (i.e., with a different C-terminal and/or N -terminal amino acid) expressed by the same polynucleotide.

The coding sequence of the mature polypeptide. The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having lipase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1-807 of SEQ ID NO: 1.

medium hard conditions. The term "medium stringency conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of hydrodynamically sheared and denatured salmon sperm DNA and formamide 35 % followed by standard Southern blotting procedures within 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 55°C.

Conditions of medium-high hardness. The term "medium-high stringency conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of shear-dehydrated and denatured salmon sperm DNA, and formamide 35% followed by standard Southern blot procedures for 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 60°C.

Mutant. The term "mutant" means a polynucleotide encoding a variant.

Nucleic acid construct. The term "nucleic acid construct" means a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a gene of natural origin or modified to contain nucleic acid segments in such a way that it would not otherwise exist in nature, or that is synthetic , and it contains one or more control sequences.

Functionally related. The term "operably linked" means a configuration in which the control sequence is located at an appropriate position relative to the coding sequence of the polynucleotide such that the control sequence directs the expression of the coding sequence.

Parent or parental lipase. The term "parent" or "parental lipase" means a lipase that has been modified to produce variants of the enzyme of the present invention. The parent lipase may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.

sequence identity. The similarity between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For the purposes of the present invention, sequence identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet 16: 276-277), preferably version 5.0.0 or newer. Parameters include a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 replacement matrix (the BLOSUM62 version of the EMBOSS package). The output of the Needle program labeled "longest identity" (obtained by the nobrief option) is used as the percent identity and is calculated as follows:

(identical residuals x 100)/(length of the align section - total number of gaps in the align section).

For the purposes of the present invention, the identity of two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000 , see above), preferably version 5.0.0 or newer. Parameters include a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL replacement matrix (NCBI NUC4.4 version of the EMBOSS package). The output of the Needle program labeled "longest identity" (obtained by the nobrief option) is used as the percent identity and is calculated as follows:

(identical deoxyribonucleotides x 100)/(length of alignment region - total number of gaps in alignment region).

Sustainability. The stability of a lipase variant of the present invention can be expressed as the residual activity or residual effectiveness of said lipase during or after exposure to various test conditions, such as, for example, storage in a detergent composition, at various temperatures, at various pH values, in the presence of various components, such as as protease, chemicals and/or oxidizing agents (stress conditions), or when used in the washing process. The stability of a lipase variant can be measured against the known activity or potency of the parent lipase, e.g. the lipase shown as SEQ ID NO: 2, or in an alternative embodiment, the known potency or potency of the lipase variant when initially added to a detergent composition, optionally stored cold or frozen, or relative to the lipase variant stored cold or frozen (no load conditions).

Subsequence. The term "subsequence" means a polynucleotide in which one or more (eg, several) nucleotides are missing from the 5' and/or 3' end of the coding sequence of the mature polypeptide; wherein the subsequence encodes a fragment having lipase activity. In one aspect, the sequence contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of nucleotides 1-807 of SEQ ID NO: 1.

Option. The term "variant" means a polypeptide having lipase activity containing a change, i. substitution, insertion and/or deletion at one or more (eg, multiple) positions. Substitution means replacing the amino acid occupying the position with another amino acid; deletion means the removal of the amino acid occupying the position; and insertion means adding an amino acid to a position adjacent to and immediately following the position occupied by the amino acid. Embodiments of the present invention have at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 100% lipase activity of the polypeptide of SEQ ID NO: 2.

Very harsh conditions. The term "very stringent conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of shear-dehydrated and denatured salmon sperm DNA and formamide 50% followed by standard Southern blot procedures within 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 70°C.

Very low severity conditions. The term "very low stringency conditions" means probes of at least 100 nucleotides in length, pre-hybridization and hybridization at 42° C. in 5X SSPE, 0.3% SDS, 200 micrograms/mL of shear-dehydrated and denatured salmon sperm DNA and formamide 25% followed by standard Southern blot procedures within 12-24 hours. The carrier material is then washed three times for 15 minutes using 2X SSC, 0.2% SDS at 45°C.

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

Conventions for naming variants

For the purposes of the present invention, the polypeptide described in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another lipase. The amino acid sequence of another lipase is aligned with SEQ ID NO: 2 and from the results of the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide described in SEQ ID NO: 2 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) implemented in the Needle program from the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0. 0 or newer. Parameters include a gap opening penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 replacement matrix (the BLOSUM62 version of the EMBOSS package).

Identification of the corresponding amino acid residue in another lipase can be accomplished by aligning multiple polypeptide sequences using several computer programs including, without limitation, MUSCLE (Multiple Sequence Comparison by Log-Expectation); version 3.5 or newer; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or newer; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511 -518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900) and EMBOSS EMMA with using ClustalW (version 1.83 or newer; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default settings.

When another enzyme differs from the polypeptide of SEQ ID NO: 2 such that conventional sequence-based comparison does not reveal their similarity (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise comparison algorithms can be used. sequences. Higher sensitivity searches based on sequences can be achieved using search programs that use probabilistic representations of polypeptide families (profiles) to search databases. For example, the PSI-BLAST program generates profiles using an iterative database search process and can detect distant homologues (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even higher sensitivity can be achieved if one or more members of a family or superfamily of polypeptides are contained in the protein structure databases. Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881) use information from multiple sources (PSI-BLAST, secondary structure prediction data , structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural folding of a sequence of interest. Similarly, the method described in Gough et al., 2000, J. Mol. Biol. 313:903-919 can be used to align a sequence of unknown structure with superfamily models present in the SCOP database. These alignments can in turn be used to create homologous models for the polypeptide, and the accuracy of such models can be assessed using various tools designed for this purpose.

For proteins with known structure, several tools and resources are available to identify and create structural alignments. For example, the SCOP protein superfamilies are structurally aligned, and these alignments are available, including for download. Various algorithms can be used to align two or more protein structures, such as distance matrix alignment (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11: 739-747 ) and these algorithms can further be used to search structure databases of interest in structure databases to identify possible structural homologues (eg, Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing embodiments of the present invention, the nomenclature described below is adapted for ease of use. Use the IUPAC accepted one-letter or three-letter abbreviations for amino acids.

Replacements. For amino acid substitutions, the following nomenclature is used: parent amino acid, position, substituting amino acid. Accordingly, for example, a change from threonine to alanine at position 226 is referred to as Thr226Ala or T226A. Multiple mutations are separated from each other by addition signs (+), for example, Gly205Arg + Ser411Phe or G205R + S411F, which means replacements at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively .

Deletions. For deletion of amino acids, the following nomenclature is used: original amino acid, position, *. Accordingly, the glycine deletion at position 195 is referred to as Gly195* or G195*. Multiple deletions are separated from each other by addition signs (+), eg Gly195* + Ser411* or G195* + S411*.

Inserts. With respect to an amino acid insert, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of a lysine after the glycine at position 195 is referred to as Gly195GlyLys or G195GK. Multiple amino acid insertion is referred to as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, insertion of lysine and alanine after glycine at position 195 is referred to as Gly195GlyLysAla or G195GKA.

In such cases, the inserted amino acid(s) residue(s) are numbered by adding lower case letters to the position number of the amino acid residue before the inserted amino acid(s) residue(s). Thus, in the example above, the sequence should be as shown below.

Multiple changes. Variants containing multiple changes are separated from each other by addition signs (+), for example, Arg170Tyr + Gly195Glu or R170Y + G195E, which means the replacement of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively.

Various changes. Where different changes can be included in one position, these different changes are separated by a comma, for example, Arg170Tyr,Glu or R170Y,E means the replacement of arginine with tyrosine or glutamic acid at position 170. Thus, Tyr167Gly,Ala + Arg170Gly,Ala implies the following options:

Tyr167Gly+Arg170Gly, Tyr167Gly+Arg170Ala, Tyr167Ala+Arg170Gly and Tyr167Ala+Arg170Ala.

DETAILED DESCRIPTION OF THE INVENTION

This document describes compositions containing variants of the parent lipase that have lipase activity, the sequences of which are at least 60%, but less than 100% identical to the sequence of SEQ ID NO: 2, for example, obtained from Thermomyces lanuginosus.

In FIG. 1 shows a water soluble single dose product in accordance with the present invention.

In FIG. 1 describes a water soluble single dose product (1) according to the present invention. Water-soluble product (1) with a single dose contains the first water-soluble film (2) and the second water-soluble film (3), which are soldered to each other in the soldering area (4). Detergent composition (5) for washing is contained inside a water-soluble soluble product (1) with a single dose.

Options

The present invention provides a detergent composition and a single dose water soluble product comprising a detergent composition containing a parental lipase variant that has lipase activity that is at least 60% but less than 100% sequence identical to that of SEQ ID NO: 2 and which contains one or more (e.g., multiple) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T .

The lipase variant sequence is preferably at least 60% but less than 100% identical to SEQ ID NO: 2 and contains one or more substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, E and P256T. Preferably, the variant further comprises one or both substitutions at positions corresponding to T231R and/or N233R.

The preferred embodiment contains a replacement in the position corresponding to G23S. The preferred embodiment contains a substitution at the position corresponding to D27N. The preferred embodiment contains a replacement at the position corresponding to A40l. The preferred embodiment contains a substitution at the position corresponding to F51I,L. The preferred embodiment contains a replacement in the position corresponding to E56R. The preferred embodiment contains a substitution at the position corresponding to D57N. The preferred embodiment contains a replacement at the position corresponding to V60E,K. The preferred embodiment contains a substitution at the position corresponding to K98I. The preferred embodiment contains a substitution at the position corresponding to N101D. The preferred embodiment contains a substitution at the position corresponding to R118F. The preferred embodiment contains a replacement in a position corresponding to G163S. The preferred embodiment contains a replacement at the position corresponding to Y220F. The preferred embodiment contains a replacement at a position corresponding to T244E. The preferred embodiment contains a replacement in the position corresponding to P256T.

A preferred embodiment contains one or more substitutions at positions corresponding to F51I,L, E56R and/or R118F.

In a preferred embodiment, an embodiment of the invention contains any of the following set of substitutions:

.

.

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

In a preferred embodiment, the variant contains substitutions corresponding to E56R+T231R+N233R and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, R118F , G163S, Y220F, T244E and P256T.

In a preferred embodiment, the variant contains substitutions at positions corresponding to R118F+T231R+N233R and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I , N101D, G163S, Y220F, T244E and P256T.

In a more preferred embodiment, the variant contains substitutions at positions corresponding to E56R+R118F+T231R+N233R and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E and P256T.

In an even more preferred embodiment, the variant contains substitutions at positions corresponding to E56R+R118F+T231R+N233R+P256T and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, D57N, V60E ,K, K98I, N101D, G163S, Y220F and T244E.

In an even more preferred embodiment, the variant contains substitutions at positions corresponding to F51I,L+E56R+R118F+T231R+N233R and one or more (e.g., multiple) substitutions at positions corresponding to GG23S, D27N, A40I, D57N, V60E,K , K98I, N101D, G163S, Y220F, T244E and P256T.

In an even more preferred embodiment, the variant contains substitutions at positions corresponding to F51I, L+E56R+R118F+T231R+N233R+P256T, and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E ,K, K98I, N101D, G163S, Y220F and T244E.

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

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

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

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

In another even more preferred embodiment, the variant contains substitutions at positions corresponding to D27N+F51I, L+E56R+R118F+T231R+N233R, and one or more (for example, several) substitutions at positions corresponding to G23S, A40I, D57N, V60E, K, K98I, N101D, G163S, Y220F, T244E and P256T.

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

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

In another even more preferred embodiment, the variant contains substitutions at positions corresponding to F51I, L+E56R+D57N+R118F+T231R+N233R+P256T, and one or more (for example, several) substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, K98I, N101D, G163S, Y220F and T244E.

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

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

In a further more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I, L+E56R+D57N+K98I+R118F+G163S+T231R+N233R and one or more (e.g., multiple) substitutions at positions corresponding to G23S, D27N, A40I , V60EK, N101D, Y220F, T244E and P256T.

In a further more preferred embodiment, the variant comprises substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T and one or more (e.g., multiple) substitutions at positions corresponding to G23S, D27N , A40I, V60EK, N101D, Y220F and T244E.

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

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

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

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

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

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

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

In particular, preferred embodiments include variants containing substitutions at positions corresponding to one of the following sets of substitutions:

R118F+T231R+N233R+P256T;

A40I+R118F+T231R+N233R;

F51I+E56R+R118F+T231R+N233R;

F51L+E56R+R118F+T231R+N233R;

E56R+D57N+R118F+T231R+N233R;

E56R+V60K+R118F+T231R+N233R;

G23S+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R;

F51I+E56R+R118F+T231R+N233R;

E56R+R118F+T231R+N233R+P256T;

G23S+D27N+E56R+R118F+T231R+N233R;

G23S+F51I+E56R+R118F+T231R+N233R;

G23S+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R+P256T;

F51I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R;

G23S+D27N+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T;

G23S+F51I+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;

A40I+E56R+R118F+T231R+N233R;

F51L+E56R+R118F+T231R+N233R;

D57N+E56R+R118F+T231R+N233R;

K98I+E56R+R118F+T231R+N233R;

G163S+E56R+R118F+T231R+N233R;

A40I+F51L+E56R+R118F+T231R+N233R;

A40I+D57N+E56R+R118F+T231R+N233R;

A40I+K98I+E56R+R118F+T231R+N233R;

A40I+G163S+E56R+R118F+T231R+N233R;

A40I+E56R+R118F+T231R+N233R+P256T;

F51L+D57N+E56R+R118F+T231R+N233R;

F51L+K98I+E56R+R118F+T231R+N233R;

F51L+G163S+E56R+R118F+T231R+N233R;

F51L+E56R+R118F+T231R+N233R+P256T;

D57N+K98I+E56R+R118F+T231R+N233R;

D57N+G163S+E56R+R118F+T231R+N233R;

D57N+E56R+R118F+T231R+N233R+P256T;

K98I+G163S+E56R+R118F+T231R+N233R;

K98I+E56R+R118F+T231R+N233R+P256T;

G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+D57N+E56R+R118F+T231R+N233R;

A40I+F51L+K98I+E56R+R118F+T231R+N233R;

A40I+F51L+G163S+E56R+R118F+T231R+N233R;

A40I+F51L+E56R+R118F+T231R+N233R+P256T;

A40I+D57N+K98I+E56R+R118F+T231R+N233R;

A40I+D57N+G163S+E56R+R118F+T231R+N233R;

A40I+D57N+E56R+R118F+T231R+N233R+P256T;

A40I+K98I+G163S+E56R+R118F+T231R+N233R;

A40I+K98I+E56R+R118F+T231R+N233R+P256T;

A40I+G163S+E56R+R118F+T231R+N233R+P256T;

F51L+D57N+K98I+E56R+R118F+T231R+N233R;

F51L+D57N+G163S+E56R+R118F+T231R+N233R;

F51L+D57N+E56R+R118F+T231R+N233R+P256T;

F51L+K98I+G163S+E56R+R118F+T231R+N233R;

F51L+K98I+E56R+R118F+T231R+N233R+P256T;

F51L+G163S+E56R+R118F+T231R+N233R+P256T;

D57N+K98I+G163S+E56R+R118F+T231R+N233R;

D57N+K98I+E56R+R118F+T231R+N233R+P256T;

D57N+G163S+E56R+R118F+T231R+N233R+P256T;

K98I+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R;

A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R;

A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R;

A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R;

A40I+D57N+K98I+E56R+R118F+T231R+N233R+P256T;

A40I+D57N+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;

F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;

F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;

F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R;

A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T;

A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T;

A40I+E56R+R118F+T231R+N233R;

E56R+R118F+T231R+N233R+T244E;

G23S+D27N+E56R+R118F+T231R+N233R;

G23S+A40I+E56R+R118F+T231R+N233R;

G23S+F51I+E56R+R118F+T231R+N233R;

G23S+E56R+R118F+T231R+N233R+T244E;

G23S+E56R+R118F+T231R+N233R+P256T;

D27N+A40I+E56R+R118F+T231R+N233R;

D27N+F51I+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R+T244E;

D27N+E56R+R118F+T231R+N233R+P256T;

A40I+F51I+E56R+R118F+T231R+N233R;

A40I+E56R+R118F+T231R+N233R+T244E;

A40I+E56R+R118F+T231R+N233R+P256T;

F51I+E56R+R118F+T231R+N233R+T244E;

F51I+E56R+R118F+T231R+N233R+P256T;

E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+E56R+R118F+T231R+N233R;

G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R;

G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R;

G23S+D27N+F51I+E56R+R118F+T231R+N233R;

G23S+D27N+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+E56R+R118F+T231R+N233R+P256T;

G23S+A40I+F51I+E56R+R118F+T231R+N233R;

G23S+A40I+E56R+R118F+T231R+N233R+T244E;

G23S+A40I+E56R+R118F+T231R+N233R+P256T;

G23S+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+E56R+V60K+R118F+T231R+N233R+T244E+P256T;

G23S+E56R+V60E+R118F+T231R+N233R+T244E+P256T;

D27N+A40I+F51I+E56R+R118F+T231R+N233R;

D27N+A40I+E56R+R118F+T231R+N233R+T244E;

D27N+A40I+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+E56R+R118F+T231R+N233R+P256T;

D27N+E56R+R118F+T231R+N233R+T244E+P256T;

A40I+F51I+E56R+R118F+T231R+N233R+T244E;

A40I+F51I+E56R+R118F+T231R+N233R+P256T;

A40I+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;

F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R;

G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R;

G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R;

G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+A40I+E56R+V60K+R118F+T231R+N233R+T244E:

G23S+D27N+A40I+E56R+V60E+R118F+T231R+N233R+T244E;

G23S+D27N+A40I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+A40I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;

D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;

D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;

A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E;

G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T;

G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T;

G23S+D27N+A40I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;

D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T;

F51I+E56R+R118F+T231R+N233R;

E56R+R118F+T231R+N233R+T244E;

D27N+F51I+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R+T244E;

F51I+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R;

K98I+E56R+R118F+T231R+N233R;

Y220F+E56R+R118F+T231R+N233R;

E56R+R118F+T231R+N233R+T244E;

G23S+D27N+E56R+R118F+T231R+N233R;

G23S+F51I+E56R+R118F+T231R+N233R;

G23S+K98I+E56R+R118F+T231R+N233R;

G23S+Y220F+E56R+R118F+T231R+N233R;

G23S+E56R+R118F+T231R+N233R+T244E;

G23S+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+E56R+R118F+T231R+N233R;

D27N+K98I+E56R+R118F+T231R+N233R;

D27N+Y220F+E56R+R118F+T231R+N233R;

D27N+E56R+R118F+T231R+N233R+T244E;

D27N+E56R+R118F+T231R+N233R+P256T;

F51I+K98I+E56R+R118F+T231R+N233R;

F51I+Y220F+E56R+R118F+T231R+N233R;

F51I+E56R+R118F+T231R+N233R+T244E;

F51I+E56R+R118F+T231R+N233R+P256T;

K98I+Y220F+E56R+R118F+T231R+N233R;

K98I+E56R+R118F+T231R+N233R+T244E;

K98I+E56R+R118F+T231R+N233R+P256T;

Y220F+E56R+R118F+T231R+N233R+T244E;

Y220F+E56R+R118F+T231R+N233R+P256T;

E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R;

G23S+D27N+K98I+E56R+R118F+T231R+N233R;

G23S+D27N+Y220F+E56R+R118F+T231R+N233R;

G23S+D27N+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+E56R+R118F+T231R+N233R+P256T;

G23S+F51I+K98I+E56R+R118F+T231R+N233R;

G23S+F51I+Y220F+E56R+R118F+T231R+N233R;

G23S+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+K98I+Y220F+E56R+R118F+T231R+N233R;

G23S+K98I+E56R+R118F+T231R+N233R+T244E;

G23S+K98I+E56R+R118F+T231R+N233R+P256T;

G23S+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+K98I+E56R+R118F+T231R+N233R;

D27N+F51I+Y220F+E56R+R118F+T231R+N233R;

D27N+F51I+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+E56R+R118F+T231R+N233R+P256T;

D27N+K98I+Y220F+E56R+R118F+T231R+N233R;

D27N+K98I+E56R+R118F+T231R+N233R+T244E;

D27N+K98I+E56R+R118F+T231R+N233R+P256T;

D27N+Y220F+E56R+R118F+T231R+N233R+T244E;

D27N+Y220F+E56R+R118F+T231R+N233R+P256T;

D27N+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+K98I+Y220F+E56R+R118F+T231R+N233R;

F51I+K98I+E56R+R118F+T231R+N233R+T244E;

F51I+K98I+E56R+R118F+T231R+N233R+P256T;

F51I+Y220F+E56R+R118F+T231R+N233R+T244E;

F51I+Y220F+E56R+R118F+T231R+N233R+P256T;

F51I+E56R+R118F+T231R+N233R+T244E+P256T;

K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

K98I+E56R+R118F+T231R+N233R+T244E+P256T;

Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R;

G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R;

G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+K98I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;

G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E;

G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T;

G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;

D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R;

G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E;

G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T;

G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T;

G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T.

The sequence of the lipase variant is preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identical to the parent lipase.

Variant sequence is preferably at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, identical to the sequence of SEQ ID NO: 2.

Variant may have 1-40, 1-30, 1-20, such as 1-12, such as 1-11, such as 1-10, such as 1-9, such as 1-8, such as 1-7, such as 1-6, for example 1-5, for example 1-4, for example 1-3 or for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 subs.

Compared to the parent lipase, the variant may have one or more of the following properties: increased wash performance, reduced odor generation, improved storage stability, longer shelf life, and/or increased thermal stability.

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

Changes in amino acids may be minor, i.e. be conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, usually 1-30 amino acids long; small extensions at the amino or carboxyl end, for example an amino-terminal methionine residue; low molecular weight linker peptide containing up to 20-25 residues; or a slight elongation that facilitates clearance by changing net charge or other function, such as a polyhistidine tract, antigenic epitope, or binding domain.

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

In an alternative embodiment, the changes in amino acids are of a nature that results in changes in the physicochemical properties of the polypeptides. For example, changes in amino acids can improve the thermal stability of a polypeptide, change substrate specificity, change optimal pH, and the like.

Essential amino acids in a polypeptide can be identified using procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latest technology, single alanine mutations are introduced into each residue of the molecule, and the resulting mutant molecules are tested for lipase activity to identify amino acid residues that are critical for the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, ie. techniques such as nuclear magnetic resonance, crystallography, electrographic analysis, or photoaffinity labeling, in association with mutation of the putative amino acid contact site. 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 identification of essential amino acids can also be carried out by alignment with a related polypeptide.

Variants may contain or consist of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, or at least 95% of the amino acids of SEQ ID NO: 2.

Parental lipases

The parent lipase may be selected from the group consisting of the following:

a) a polypeptide whose sequence is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or 100% identical to the sequence of SEQ ID NO: 2;

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

c) a polypeptide encoded by a polynucleotide whose sequence is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence of SEQ ID NO : 1, and

d) a polypeptide fragment of SEQ ID NO: 2.

In one aspect of the invention, the parent lipase has a sequence identity with the polypeptide of SEQ ID NO: 2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 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%, which has lipase activity.

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

In another aspect, the parent consists of or contains the amino acid sequence of SEQ ID NO: 2.

In another aspect, the parent is a fragment of a polypeptide with SEQ ID NO: 2, containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 %, at least 80%, at least 85%, at least 90% or at least 95% of the amino acids of the sequence SEQ ID NO: 2.

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

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

A polynucleotide of SEQ ID NO: 1 or a subsequence, as well as a polypeptide of SEQ ID NO: 2 or a fragment, can be used to design nucleic acid probes for identifying and cloning DNA encoding a parent molecule from strains of various genera or species according to methods well known in the field. In particular, such probes can be used to hybridize to the genomic DNA or cDNA of a cell of interest, using standard Southern blot techniques to identify and isolate the appropriate gene from it. Such probes can be significantly shorter than the full sequence, but should be at least 15, such as at least 25, at least 35, or at least 70 nucleotides in length. The length of the nucleic acid probe is preferably at least 100 nucleotides, such as at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides or at least 900 nucleotides. Both DNA and RNA probes can be used. As a rule, probes for determining the corresponding gene are labeled (for example, with 32P, 3H, 35S, biotin or avidin). Such probes are within the scope of the present invention.

A genomic DNA library or cDNA derived from such other strains may be screened for DNA that hybridizes to the probes described above and encodes a parent. Genomic or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation methods. DNA from libraries or isolated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. A carrier material is used in Southern blotting to identify a clone or DNA that hybridizes to SEQ ID NO: 1 or a subsequence thereof.

For the purposes of the present invention, hybridization means that the polynucleotide hybridizes with a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1, (ii) the coding sequence of the polypeptide of SEQ ID NO: 1, (iii) the full length complementary sequence; or (iv) subsequences; in conditions from very low to very high stiffness. Molecules to which a nucleic acid probe hybridizes under such conditions can be detected, for example, by X-ray film or any other detection method known in the art.

In one aspect, the nucleic acid probe is the coding sequence for the polypeptide of SEQ ID NO: 1. In another aspect, the nucleic acid probe contains at least 50%, at least 55%, at least 60%, at least 65%, of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the nucleotides of the sequence of SEQ ID NO: 1. In another aspect, the nucleic acid probe is a polynucleotide that encodes a polypeptide of SEQ ID NO: 2; its polypeptide; or fragment. In another aspect, the nucleic acid probe is SEQ ID NO: 1.

In another embodiment, the parent is encoded by a polynucleotide whose sequence is at least 60% identical to the polypeptide-encoding sequence of SEQ ID NO: 1, e.g., at least 65%, at least 70%, at least 75%, at least 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%.

The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused to the N-terminal or C-terminal portion of a region of another polypeptide.

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

The fused polypeptide may further comprise a cleavage site located between the two polypeptides. Following secretion of the fusion protein, the site is cleaved, releasing two polypeptides. Examples of cleavage sites include, without limitation, those described in Martin et al., 2003, J. Ind. microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

The parental lipase can be obtained from microorganisms of any kind. For the purposes of the present invention, the term "derived from" as used herein in relation to a given source means that the parental form encoded by a polynucleotide is produced by the source or strain into which the polynucleotide from the source has been inserted. In one aspect, the parent form is secreted in an extracellular way.

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

In one aspect, the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtiluringis, or Bac lipase. .

In another aspect, the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. lipase. zooepidemicus.

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

In another aspect, the parent is a Thermobifida alba or Thermobifida fusca lipase (formerly known as Thermomonaspora fusca).

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

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

В другом аспекте родитель представляет собой липазу Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum Chrysosporium tropicum Chrysosporium zonatum Fusarium bactridioides Fusarium cerealis Fusarium crookwellense Fusarium culmorum Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum , Trichoderma reesei or Trichoderma viride.

In another aspect, the parent is a Thermomyces lanuginosus lipase, for example, in particular, the lipase of SEQ ID NO: 2.

It should be understood that for the aforementioned species, the variants used in the invention cover both perfect and imperfect stages, as well as other taxonomic equivalents, such as anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of the respective equivalents.

Strains of these species can be freely purchased from a number of crop collections such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS) and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

The parental lipase can be identified and obtained from other sources, including microorganisms isolated from natural sources (eg, soil, compost, water, etc.) or DNA samples obtained directly from natural materials (eg, soil, compost, water). etc.), using the above probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding the parent can then be obtained by similarly screening a library of genomic DNA or cDNA obtained from other organisms, or a mixed DNA sample. Once the polynucleotide encoding the parent has been detected by the probe(s), the polynucleotide can be isolated or cloned using techniques well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

Getting Options

A variant suitable for the present invention can be obtained by methods for preparing lipase variants, including: (a) introducing substitutions at positions corresponding to G23S, D27N, A40I, F51I, L, E56R, D57N, K98I, R118F, G163S, T231R, N233R, Y220F , T244E and P256T; (b) selecting a variant that has lipase activity and, compared to the parent lipase, has one of the desired properties listed above; and (c) variant highlighting.

Variants can be generated using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, permutation, and the like.

Site-directed mutagenesis is a technique in which one or more (eg, multiple) mutations are made at one or more specific sites in a polynucleotide encoding a parental lipase.

Site directed mutagenesis can be performed in vitro by PCR using oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis, which involves restriction enzyme cleavage at a site in a plasmid containing a polynucleotide encoding a parental lipase and then ligation of the oligonucleotide containing the mutation to the polynucleotide. Typically, both the plasmid and the oligonucleotide are cleaved with the same restriction enzyme, allowing the plasmid and insert to be ligated to each other. See, for example, Scherer and Davis, 1979, Proc. Natl. Acad. sci. USA 76: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

Site directed mutagenesis can also be performed in vivo by methods known in the art. See, for example, US2004/0171154. 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.

Any site-directed mutagenesis procedure can be used in the present invention. There are many kits available on the market that you can use to get options.

The construction of synthetic genes entails the in vitro synthesis of a given polynucleotide molecule to encode the desired polypeptide. Gene synthesis can be performed using a number of technologies such as the microarray multiplex technology described in Tian et al. (2004, Nature 432: 1050-1054), and similar technologies in which oligonucleotides are synthesized and assembled onto photoprogrammable microfluidic chips.

Single or multiple amino acid substitutions, deletions and/or insertions can be made and tested using known methods of mutagenesis, recombination and/or rearrangement followed by an appropriate screening procedure as described in Reidhaar Olson and Sauer, 1988, Science 241: 53- 57; Bowie and Sauer, 1989, Proc. Natl. Acad. sci. USA 86: 2152-2156; WO95/17413 or WO95/22625 Other possible methods include reduced fidelity PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US Pat. No. 5,223,409; WO 92/06204) and mutagenesis targeting region (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high throughput automated screening methods to detect the activity of cloned, mutated polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenesis DNA molecules that encode active polypeptides can be isolated from host cells and rapidly sequenced using standard techniques known in the art. These methods allow you to quickly determine the value of individual amino acid residues in the polypeptide.

Construction of semi-synthetic genes is accomplished by combining aspects of synthetic gene construction and/or site-directed mutagenesis and/or random mutagenesis and/or shuffling. A typical method of semi-synthetic construction is the use of polynucleotide fragments synthesized in combination with PCR technologies. Thus, certain regions of genes can be synthesized de novo, while other regions can be amplified with primers for site-directed mutagenesis, while other regions can be amplified by reduced precision PCR or other types of PCR. The polynucleotide subsequences can then be shuffled.

The lipase variant can be obtained by a method comprising: (a) culturing the host cell under conditions suitable for expression of the variant; and (b) variant highlighting.

Nucleic acid constructs

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

The polynucleotide can be manipulated in a variety of ways to allow expression of the variant. Manipulation of the polynucleotide prior to insertion into the vector may be desirable or necessary, depending on the expression vector. Methods for modifying polynucleotides using recombinant DNA technology are well known in the art.

The control sequence may be a promoter, a polynucleotide that is recognized by the host cell to express the polynucleotide. The promoter contains transcriptional control sequences that mediate the expression of the variant. A promoter can be any polynucleotide that exhibits transcriptional activity in a host cell, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

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

Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are those derived from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-fast alpha-amylase, Aspergillus niger glycoamylase, or Aspergillus awamori (glaA) genes. , Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO00/56900), Daria Fusarium venenatum (WO00/56900), Quinn Fusarium venenatum506 (WO00/56900), Quinn Fusarium venenatum506 ), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, endoglucanase Trichoderma reesei kanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, and the NA2-tpi promoter a leader sequence from the Aspergillus triosephosphate isomerase gene; Non-limiting examples include modified promoters from the Aspergillus niger neutral alpha-amylase gene in which the untranslated leader sequence is replaced by an untranslated leader sequence from the Aspergillus nidulans or Aspergillus oryzae triosephosphate isomerase gene); and their mutant, truncated and hybrid promoters.

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

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

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

Terminators preferred for filamentous fungal host cells are derived from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

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

The control sequence may also be a stabilizing region of the mRNA, located downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

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

The control sequence may also be a leader, an untranslated region of an mRNA that is required for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the variant. Any leader that is functional in the host cell can be used.

The leaders favored by filamentous fungal host cells are derived from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase genes.

Leaders that are preferred by yeast host cells are derived from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP) genes.

The control sequence may also be a polyadenylation sequence, ie. a sequence operably linked to the 3' end of a variant coding sequence and recognized by the host cell during transcription as a signal to add polyadenosine to the transcribed mRNA. Any polyadenylation sequence that will be functional in the host cell may be used.

Polyadenylation sequences preferred for filamentous fungal host cells are derived from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

Polyadenylation sequences that can be used for yeast host cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

The control sequence may also be the signal peptide coding region, ie. a region that encodes a signal peptide associated with the N-terminal portion of the variant and directs the variant into the secretory pathway of the cell. The 5' end of a coding sequence of a polynucleotide may congenitally contain a signal peptide coding sequence that is naturally linked in a translational reading frame to a coding sequence segment that encodes a variant. In an alternative embodiment, the 5' end of the coding sequence may contain a sequence encoding a signal peptide that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not have its own signal peptide coding sequence. In an alternative embodiment, the foreign signal peptide coding sequence can simply replace the self signal peptide coding sequence to enhance secretion of the variant. However, any signal peptide encoding sequence that directs the expressed variant into the secretory pathway of the host cell can be used.

Effective signal peptide coding sequences for bacterial host cells are signal peptide coding sequences derived from Bacillus NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ( nprT, nprS, nprM) and prsA Bacillus subtilis. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

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

Useful signal peptides for yeast host cells are derived from the Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase genes. Other signal peptide coding sequences that can be used are described by Romanos et al., 1992, supra.

The control sequence may also be a sequence encoding a propeptide that encodes a propeptide located at the N-terminus of the variant. The resulting polypeptide is known as a proenzyme or propolypeptide (or in some cases a zymogen). The propolypeptide is essentially inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence can be derived from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha factor.

When both the signal peptide sequence and the propeptide are present, the propeptide sequence is located near the N-terminus of the variant, and the signal peptide sequence is located near the N-terminus of the propeptide sequence.

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

Expression vectors

The application also describes recombinant expression vectors containing a polynucleotide encoding a variant used in the present invention, a promoter and transcription and translation stop signals. Various nucleotide and control sequences can be combined together to form a recombinant expression vector, which can include one or more suitable restriction sites to allow for insertion or replacement of a variant-encoding polynucleotide at such sites. In an alternative embodiment, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide into an appropriate expression vector. When creating an expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked to the appropriate control sequences for expression.

The recombinant expression vector can be any vector (eg, plasmid or viral) that can be easily subjected to recombinant DNA procedures and that is capable of expressing the polynucleotide. The choice of vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

The vector may be an autonomously replicating vector, ie. a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, examples include, for example, a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for self-replication. In an alternative embodiment, the vector may be a vector which, when introduced into a host cell, integrates into the genome and replicates along with the chromosome(s) into which it is integrated. In addition, one vector or plasmid, or two or more vectors or plasmids, which together contain complete DNA for introduction into the host cell genome, or transposon, can be used.

The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced or the like. cells. A selectable marker is a gene whose product provides resistance to biocides or viruses, resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the Bacillus licheniformis or Bacillus subtilis dal genes, or antibiotic resistance markers such as resistance to ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline. Suitable markers for yeast host cells include, without limitation, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in the mycelial fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine 5'-phosphate decarboxylase ), sC (sulfate adenyl transferase), and trpC (anthranilate synthase), as well as their equivalents. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the gene of Streptomyces hygroscopicus bar.

The vector preferably contains element(s) which(s) allow(s) to integrate the vector into the genome of the host cell or to carry out autonomous replication of the vector in the cell, regardless of the genome.

For integration into the genome of the host cell, the vector may rely on the polynucleotide sequence encoding the variant, or on any other element of the vector for integration into the genome by homologous or non-homologous recombination. In an alternative embodiment, the vector may contain additional polynucleotides to direct integration by homologous recombination into the genome of the host cell at precisely defined location(s) on the chromosome(s). To increase the likelihood of integration at a well-defined site, the integration elements should contain a sufficient number of nucleic acids, for example, 100-10,000 base pairs, 400-10,000 base pairs, and 800-10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to increase the likelihood of homologous recombination. The integration elements can be any sequence homologous to the target sequence in the genome of the host cell. In addition, the integration elements may be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the genome of the host cell through non-homologous recombination.

For autonomous replication, the vector may further comprise an origin of replication allowing the vector to autonomously replicate in the host cell of interest. The origin of replication can be any plasmid replicator that functions in the cell and mediates autonomous replication. The term "origin of replication" or "plasmid replicator" means a polynucleotide that allows for in vivo replication of a plasmid or vector.

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

Examples of origins of replication for use in a yeast host cell are 2 μm origins, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

Examples of origins of replication suitable for a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/ 24883). Isolation of the AMA1 gene and construction of plasmids or vectors containing the gene can be carried out according to the methods described in WO 00/24883.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase variant production. An increase in the copy number of a polynucleotide can be achieved by integrating at least one additional copy of the sequence into the genome of the host cell or by incorporating an amplifiable selectable marker gene with a polynucleotide, where cells containing amplified copies of the selectable marker gene and, accordingly, additional copies of the polynucleotide , can be selected by culturing the cells in the presence of a suitable selective agent.

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

Host cells

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

The host cell may be any cell suitable for recombinant production of the variant, eg prokaryotes or eukaryotes.

The prokaryotic host cell can be any gram positive or gram negative bacterium. Gram-positive bacteria include, without limitation, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, without limitation, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell can be any Bacillus cell, including, without limitation, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis.

The bacterial host cell may also be any Streptococcus cell, including, without limitation, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. zooepidemicus.

The bacterial host cell may also be any Streptomyces cell, including, without limitation, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

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

The host cell may also be a eukaryote, such as a mammalian, insect, plant or fungal cell.

The host cell may be a fungal cell. In the context of this document, the term "fungi" includes the phyla Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota, as well as Oomycota and all mitosporous fungi (as defined in Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995 , CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. In the context of this document, the term "yeast" includes ascosporogenic yeasts (endomycetes), basidiosporogenic yeasts, and yeasts belonging to imperfect fungi (blastomycetes). Because the classification of yeasts may change in the future, for the purposes of the present invention, yeasts are defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

Дрожжевая клетка-хозяин может представлять собой клетку Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces или Yarrowia, например Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis или Yarrowia lipolytica.

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

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

For example, the filamentous fungal host cell may be Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis, pangiea, Ceriporiopsis gilvescens, Ceriporiopsis, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum , Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulos um Fusarium trichothecioides Fusarium venenatum Humicola insolens Humicola lanuginosa Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride.

Fungal cells can be transformed using essentially known methods, including the formation of protoplasts, transformation of protoplasts and regeneration of the cell wall. Suitable methods for transforming Aspergillus and Trichoderma host cells are described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. sci. USA 81: 1470-1474; and Christensen et al., 1988, Bio/Technology 6: 1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78: 147-156, and WO96/00787. Yeast can be transformed using the procedures described in Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153; 163; and Hinnen et al., 1978, Proc. Natl. Acad. sci. USA 75: 1920.

Production methods

The present application also describes methods for obtaining a lipase variant of the present invention, including: (a) culturing the host cell of the present invention under conditions suitable for expression of the variant; and (b) variant highlighting.

The host cells are cultured in a nutrient medium suitable for obtaining the variant by methods known in the art. For example, the cell can be cultured by shake flask culture or by small scale or large scale fermentation (including continuous, semi-continuous, batch or solid phase fermentation) in laboratory or industrial fermenters carried out in a suitable medium under conditions suitable for expression and/or isolation of the variant. Cultivation is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts by methods known in the art. Suitable media can be purchased from commercial suppliers or obtained in accordance with published compositions (for example, in the catalogs of the American Type Culture Collection). When secreting a variant using a nutrient medium, such a variant can be isolated directly from the medium. In addition to secretion, the variant can be isolated from cell lysates.

The variant can be detected by methods known in the art that are specific to particular variants. These detection methods include, without limitation, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, enzyme assays can be used to determine the activity of variants such as those described in the examples.

The variant can be isolated using methods known in the art. For example, the variant can be isolated from the culture medium by conventional means, including, without limitation, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

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

In an alternative embodiment, the variant is not recovered, but a host cell of the present invention expressing the variant is used as the source of the variant.

Composition of microcapsules

In the present invention, a lipase, preferably a lipase variant, is optionally encapsulated in a microcapsule. The microcapsule membrane is preferably prepared by cross-linking a polybranched polyamine with a molecular weight greater than 1 kDa. The membrane formed by cross-linking a polybranched polyamine is able to separate the enzyme from surfactants, in particular anionic surfactants, in the detergent, which are known to adversely affect the stability of enzymes.

Preferably, when using encapsulated enzymes in a detergent, the capsules are capable of releasing the enzyme substantially immediately upon dilution of the detergent in water, such as for laundry or dishwashing. Preferred microcapsules are excellent in this respect and are preferably capable of releasing the entire encapsulated enzyme within one minute.

Preferred microcapsules do not require a polymer in the core to release the enzyme when reconstituted in water. Furthermore, the invention does not require the enzyme(s) to be deposited in the microcapsule core to prevent premature release as described in WO 97/24177.

By encapsulating a lipase variant as defined herein and optionally other enzymes in a microcapsule with a semi-permeable membrane and having water activity within these capsules (prior to addition to liquid detergent) greater than activity in liquid detergent when added to detergent ( water is released to the outside) the capsules will undergo (partial) breakdown, thus leaving a more concentrated and more viscous enzyme contained within the capsules. Destruction of the membrane can also lead to a decrease in permeability. In such a situation, additional stabilizers/polymers can be added, especially those that are unable to permeate the membrane. Breakdown and the resulting increase in viscosity will reduce diffusion/prevent diffusion of undesired components (eg surfactants or sequestrants) into the capsules and thus improve the storage stability of the enzyme in the detergent liquid. Components in liquid detergent that are sensitive to the enzyme (eg, components that act as a substrate for the enzyme) are also protected from degradation by the enzyme. During washing, the liquid detergent is diluted with water, thus increasing the activity of the water. The water will then diffuse into the capsules (osmosis). The capsules will swell and the membrane will either become permeable to the enzyme so that it can exit the capsules, or the capsules will simply burst and the enzyme will be released. This concept is very effective in protecting enzymes from aggressive components in liquid detergent and vice versa, and also protects enzyme sensitive components in liquid detergent from enzymes.

Examples of detergent components that are enzyme sensitive and may be degraded include (corresponding enzyme in brackets): xanthan gum (xanthanase), ester polymers (lipase), hydrogenated castor oil (lipase), fragrance (lipase), methyl ester sulfonate surfactants (lipase), cellulose and cellulose derivatives (eg CMC) (cellulase), dextrin and cyclodextrin (amylase).

In addition, sensitive detergent ingredients can be encapsulated and thus stabilized in the microcapsules described herein. Sensitive detergent ingredients are susceptible to degradation during storage. Such detergent ingredients include bleaching compounds, bleach activators, fragrances, polymers, modifiers, surfactants, and the like.

Generally, the microcapsules described herein can be used to separate incompatible/sensitive components/compounds in a cleaning composition.

The addition of microcapsules to the detergent composition can be used to change the appearance of the detergent composition or unit dose product, for example by providing an opaque effect (using small microcapsules) or a clearly visible particulate effect (using large microcapsules). The microcapsules may optionally be colored.

Microcapsules can be used to reduce the amount of enzyme dust during handling and processing of enzyme products.

Unless otherwise indicated, all percentages in all cases are given in percent by weight (wt.%) in this document.

Obtaining a microcapsule

Preferred microcapsules are generally made by forming water droplets into a continuum that is immiscible with water (i.e., typically by making a water-in-oil emulsion) and then forming a membrane using interfacial polymerization by adding a crosslinker. After final curing, the capsules are collected and further rinsed and formed by methods known in the art. Subsequently, the composition of the capsules is added to the detergent.

The active substance, the main constituents of the membrane and a possible additional component to be encapsulated are in the aqueous phase. In the continuum are the components that stabilize the water droplets against sticking (emulsifiers, emulsion stabilizers, surfactants, etc.) and a crosslinking agent is also added through the continuum.

The emulsion can be prepared by any of the methods known in the art, such as mechanical agitation, drop methods, membrane emulsification, microfluidic methods, sonication, etc. In some cases, simply mixing the phases will automatically result in emulsification, often referred to as self-emulsification. It is preferred to use methods that result in a size distribution within a narrow range.

The crosslinking agent(s) is then typically added to the emulsion, either directly or more commonly by preparing a solution of the crosslinking agent in a solvent which is soluble in the continuous phase. The emulsion and crosslinker or solution can be mixed by standard methods used in this field, for example, by simple mixing or by carefully controlling the flow of the emulsion and crosslinker solution through an inline mixer.

In some cases, curing of the capsules may be required to complete membrane formation. Curing can be carried out by simply stirring the capsules for some time in order to complete the interfacial polymerization reaction. In other cases, membrane formation can be stopped by adding a reaction quencher.

The capsules can be subjected to subsequent modification, for example, with reactive components on the membrane to prevent or reduce the flocculation of particles in the detergent, as described in WO 99/01534.

The resulting capsules can be isolated or concentrated by methods known in the art, for example by filtering, centrifuging, distilling or decanting the capsule dispersion.

The composition of the resulting capsules can be further modified, for example by adding surfactants to give the product the desired properties for storage, transport and post-processing and adding to the detergent. Other agents that may be included in the microcapsules include rheology modifiers, biocides (eg Proxel), acid/base to adjust the pH (which also adjusts the pH within the microcapsules) and water to adjust the water activity.

The method for forming a capsule may include the following steps:

- obtaining the initial water and oil phases;

- formation of a water-in-oil emulsion;

- formation of the membrane through interfacial polymerization;

- optional subsequent modification;

- optional isolation and / or addition of the composition;

- adding to detergent.

The process may be either a batch process, or a continuous or semi-continuous process.

As described herein, a microcapsule is a small water sphere, and a substantially uniform membrane is provided around it. The material inside the microcapsule is referred to as the core, internal phase, or filler, while the membrane is sometimes referred to as the shell, coating, or wall. Preferred microcapsules described herein have diameters from 0.5 microns to 2 millimeters. Preferably, the average microcapsule diameter is in the range of 1 µm to 1000 µm, more preferably in the range of 5 µm to 500 µm, even more preferably in the range of 10 µm to 500 µm, even more preferably in the range of 50 µm to 500 µm, and most preferably in the range of 50 µm to 200 µm. In an alternative embodiment, the microcapsule diameter ranges from 0.5 µm to 30 µm; or in the range from 1 µm to 25 µm. The microcapsule diameter is measured in the oil phase after completion of the polymerization. The diameter of the capsule may vary depending on the activity of water in the surrounding chemical environment.

Microencapsulation of enzymes that can be used in the present invention can be carried out by interfacial polymerization, in which two reactants in the polymerization reaction meet at the interface and quickly react with each other. The basis of this method is the reaction of a polyamine with an acid derivative, usually an acid halide, acting as a crosslinking agent. The polyamine is preferably substantially water soluble (when in free base form). Under suitable conditions, thin, flexible membranes rapidly form at the interface. One way to carry out polymerization is to use an aqueous solution of an enzyme and a polyamine that are emulsified with a non-aqueous solvent (and an emulsifier) and add a solution containing an acid derivative. An alkaline agent may be present in the enzyme solution to neutralize the acid formed during the reaction. Polymer (polyamide) membranes are formed immediately at the interface between the emulsion droplets. The polymeric membrane of the microcapsule is generally cationic in nature and thus binds/complexes with anionic type compounds.

The diameter of the microcapsules is determined by the droplet size of the emulsion, which is controlled, for example, by the stirring speed.

Emulsion

An emulsion is a temporary or permanent dispersion of one liquid phase within a second liquid phase. The second liquid is essentially called the continuous phase. Surfactants are commonly used to facilitate the formation and stabilization of emulsions. Not all surfactants have the same ability to stabilize the emulsion. The type and amount of surfactant must be selected to provide optimum emulsion performance, especially with regard to formulation and physical stability of the emulsion, as well as stability upon dilution and post-treatment. Physical stability refers to maintaining the emulsion in the form of a dispersion. Processes such as sticking, aggregation, adsorption to container walls, settling, and segregation are forms of physical instability and should be avoided. Examples of suitable surfactants are described in WO 97/24177, pages 19-21; and in WO 99/01534.

Emulsions can be further divided into simple emulsions, in which the dispersed liquid phase is a simple homogeneous liquid, or more complex emulsions, in which the dispersed liquid phase is a heterogeneous combination of liquid or solid phases, such as double emulsion or multiple emulsion. For example, a water-in-oil double emulsion or multiple emulsion can be formed in which the aqueous phase itself further comprises an emulsified oil phase; an emulsion of this type can be referred to as an oil-in-water-in-oil (o/w/o) emulsion. In an alternative embodiment, a water-in-oil emulsion can be formed in which the aqueous phase contains a dispersed solid phase and is often referred to as a slurry-emulsion. Other more complex emulsions may be described. Because of the complexity of describing such systems, the term "emulsion" is used to describe both simple and more complex emulsions without necessarily limiting the form of the emulsion or the type and amount of phases present.

Polyamine

The stiffness/flexibility and permeability of the membrane mainly depends on the choice of polyamine. The preferred polyamine used for encapsulation as described herein contains a polybranched polyamine. Each branch, preferably terminating in a basic amino acid, acts as an anchor point in the membrane network, thereby providing the beneficial features of the invention. A polybranched polyamine as described herein preferably contains a polyamine having more than two branch points and more than two reactive amino groups (capable of interacting with the crosslinker, ie primary and secondary amino groups). To obtain the preferred microcapsules, polybranched polyamine is used as a starting material in the preparation of the emulsion, i. it is not formed in situ from other starting materials. In order to provide the attractive properties of the preferred microcapsules, the starting material must be a poly-branched polyamine structure.

There is a close relationship between the number of branch points and the number of primary amines, since primary amines will always be located at the end of the branch: a linear amine can only contain two primary amines. Each branch point that is hypothetically introduced into such a linear diamine will allow the introduction of one or more primary amines at the end of the introduced branch(s). In this context, the main amino group is meant to be part of a branch, i.e. the end point of the branch. For example, it should be understood that tris(2-aminoethyl)amine and 1,2,3-propanetriamine are single branch point molecules. In preferred microcapsules, the polyamine has at least four primary amines. Branch points can be introduced from an aliphatic hydrocarbon chain, as in the previous examples, or from unsaturated carbon bonds, such as, for example, 3,3'-diaminobenzidine, or from tertiary amino groups, such as N,N,N',N'- tetrakis-(2-aminoethyl)ethylenediamine.

Preferably, the multibranched polyamine is not a peptide or protein. Preferably, the reactive amino groups comprise at least 15% of the molecular weight of the polybranched polyamine, for example more than 20% or more than 25%. Preferably, the molecular weight of the polybranched polyamine is at least 1 kDa; more preferably, the molecular weight of the polybranched polyamine is at least 1.3 kDa. Preferably, the polybranched polyamine is polyethyleneimine (PEI) and modifications thereof having more than two branch points and more than two reactive amino groups; moreover, reactive amino groups make up at least 15% of the molecular weight of PEI, for example, more than 20% or more than 25%. Preferably the molecular weight of PEI is at least 1 kDa. To obtain a microcapsule, combinations of various polybranched polyamines can be used.

Advantageous properties (eg, enzyme storage stability, reduced enzyme leakage, reduced penetration of detergent ingredients into the capsule) of the microcapsule can be improved by adding one or more smaller amines with a molecular weight of less than 1 kDa. The low molecular weight amine is preferably substantially water soluble (when in free base form) and may be a material such as ethylenediamine, hexamethylenediamine, hexanediamine, diethylenetetramine, ethylenetetramine, diaminebenzene, piperazine, tetramethylenepentamine, or preferably diethylenetriamine (DETA). Low molecular weight amines can be added in an amount of up to 50%, preferably up to 40%, up to 30%, up to 20%, up to 10% or up to 5 wt.% of the total content of low molecular weight amine and polybranched polyamine when obtaining a microcapsule of the invention.

A preferred crosslinker contains a molecule having at least two groups/sites capable of interacting with amines to form covalent bonds. The crosslinking agent is preferably oil soluble and may be in the form of an acid anhydride or an acid halide, preferably an acid chloride. For example, it may be adipoyl chloride, sebacoyl chloride, dodecanedioic acid chloride, phthaloyl chloride, terephthaloyl chloride, isophthaloyl chloride, or trimesoyl chloride; but preferably the crosslinking agent is terephthaloyl chloride or trimesoyl chloride.

In a specifically contemplated embodiment, the composition of the microcapsules of the invention is the composition described in WO 2014/177709 (incorporated herein by reference), with a lipase variant of the invention. As also mentioned above, the microcapsules may further include an alcohol such as a polyol. The preferred microcapsules (a) use polyethyleneimine. The preferred microcapsule (b) uses ethyleneamine or alkanolamine. In a preferred embodiment, (b) is selected from the group consisting of ethylenediamine, diethylenetriamine, triethylenetetramine, bis(3-aminopropyl)amine, monoethanolamine, diethanolamine, triethanolamine, hexamethylenediamine, diaminobenzene, piperazine and tetraethylenepentamine, more preferably (b) is selected from the group, consisting of diethylenetriamine, triethylenetetramine, bis(3-aminopropyl)amine, monoethanolamine and diethanolamine. The microcapsule preferably contains a source of Mg2+, Ca2+ or Zn2+ ions, such as a slightly soluble salt of Mg2+, Ca2+ or Zn2+. As the crosslinking agent, an acid chloride such as isophthaloyl chloride, terephthaloyl chloride or trimesoyl chloride is preferably used. In a preferred embodiment, the membrane is obtained by interfacial polymerization.

Suitable microcapsules are described in WO 2015/1144784 (incorporated herein by reference) with a lipase variant as described herein.

Detergent composition

Preferred detergent compositions are laundry detergent compositions for cleaning and/or treating fabric. Preferred detergent compositions are in liquid form. The present invention particularly relates to a water soluble single dose product comprising a water soluble film and a detergent composition, preferably a liquid laundry detergent composition.

Single dose water soluble product

The water-soluble film and liquid detergent composition are described in more detail below.

The water soluble single dose product comprises a water soluble film shaped such that the single dose product comprises at least one internal compartment surrounded by a water soluble film. The unit dose article may comprise a first water soluble film and a second water soluble film sealed together to form an internal compartment. The single dose water soluble product is designed so that the cleaning composition does not leak out of the compartment during storage. However, when the single dose water soluble product is added to the water, the water soluble film dissolves and releases the contents of the inner compartment into the cleaning solution.

The compartment is to be understood as the enclosed interior space within the unit dose article that holds the cleaning composition. During production, the first water-soluble film may be in the form of an open compartment into which the detergent composition is added. Then, a second water-soluble film is applied over the first film in such a way as to cover the separation opening. Thereafter, the first and second films are sealed together along the sealing area.

The single dose article may comprise more than one compartment, even at least two compartments, or even at least three compartments. The compartments may be superimposed on each other, i. e. one compartment is located on top of the other. In this orientation, the unit dose article will comprise three films: top, middle, and bottom. In an alternative embodiment, the compartments may be arranged side by side, ie. oriented next to each other. The compartments may even be in a "tire and rim" orientation, ie. the first compartment is located next to the second compartment, however, the first compartment at least partially surrounds the second compartment, but does not completely cover the second compartment. In an alternative embodiment, one compartment may be completely surrounded by another compartment.

If the single dose article contains at least two compartments, one of the compartments may be smaller than the other. If the unit dose article contains at least three compartments, two compartments may be smaller than the third compartment, and preferably the smaller compartments overlap the larger compartments. The superimposed compartments are preferably oriented side by side.

In a multi-compartment configuration, the detergent composition according to the present invention may be contained in at least one of the compartments. It may, for example, be contained in only one compartment, or it may be contained in two compartments or even three compartments.

All compartments may contain the same or different compositions. Different compositions may have the same shape, or they may have different shapes.

The water-soluble single dose article may comprise at least two internal compartments, wherein the liquid laundry detergent composition is contained in at least one of the compartments, preferably wherein the single dose article comprises at least three compartments, the detergent composition being contained in at least at least one of the departments.

In FIG. 1 describes a water soluble single dose product (1) according to the present invention. Water-soluble product (1) with a single dose contains the first water-soluble film (2) and the second water-soluble film (3), which are soldered to each other in the soldering area (4). The liquid detergent composition (5) for washing is contained inside a water-soluble soluble product (1) with a single dose.

Water soluble film

The film used in the present invention is water soluble or dispersible. The water-soluble film preferably has a thickness of 20 to 150 µm, preferably 35 to 125 µm, even more preferably 50 to 110 µm, most preferably about 76 µm.

The film preferably has a water solubility of at least 50%, preferably at least 75%, or even at least 95%, as measured by the method set forth herein, after applying a glass filter with a maximum pore size of 20 µm:

5 grams ± 0.1 gram of film material is added to a pre-weighed 3 L beaker and 2 L ± 5 ml of distilled water is added. It is stirred vigorously in a magnetic stirrer, model Labline #1250 or equivalent, and a 5 cm magnetic stirrer set at 600 rpm for 30 minutes at 30°C. The mixture is then filtered through a folded quality sintered glass filter with a pore size as defined above (max. 20 µm). Dry the water from the collected filtrate by any standard method and determine the mass of the remaining material (which is the dissolved or dispersed fraction). The percentage solubility or dispersibility can then be calculated.

Preferred film materials are polymeric materials. The film material can be obtained, for example, by casting, blow molding, extrusion or blown extrusion of a polymeric material, as is known in this field.

Preferred polymers, copolymers or derivatives thereof suitable for use as a pouch material are selected from polyvinyl alcohols, polyvinylpyrrolidone, polyalkylene oxides, acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetates, polycarboxylic acids and salts, polyamino acids or peptides, polyamides, polyacrylamide, maleic/acrylic acid copolymers, polysaccharides including starch and gelatin, natural gums such as xanthan gum and carrageenan. More preferred polymers are selected from polyacrylates and water-soluble acrylate copolymers, methylcellulose, sodium carboxymethylcellulose, dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, and most preferably selected from polyvinyl alcohols, polyvinyl alcohol-hydroxypropyl methylcellulose (HPMC) copolymers, and combinations thereof. The level of polymer in the pouch material, such as PVA polymer, is preferably at least 60%. The polymer may have any weight average molecular weight, preferably from about 1,000 to 1,000,000, more preferably from about 10,000 to 300,000, even more preferably from about 20,000 to 150,000.

Preferably, the water-soluble film comprises a polyvinyl alcohol polymer or copolymer, preferably a mixture of polyvinyl alcohol polymers and/or polyvinyl alcohol copolymers, preferably selected from sulfonated and carboxylated anionic polyvinyl alcohol copolymers, especially carboxylated anionic polyvinyl alcohol copolymers, most preferably a mixture of polyvinyl alcohol homopolymer and carboxylated anionic copolymer polyvinyl alcohol.

Preferred films have good solubility in cold water, namely unheated distilled water. Preferably such films have good solubility at temperatures of 24°C, even more preferably at 10°C. By good solubility is meant that the film exhibits a water solubility of at least 50%, preferably at least 75% or even at least 95%, measured by the method set forth herein, after applying a glass filter with a maximum pore size of 20 µm, described above.

Films supplied by Monosol under trade names M8630, M8900, M8779, M8310 are preferred.

The film can be opaque, transparent or translucent. The film may contain a printed area.

The printable area may be produced using standard techniques such as flexographic printing or inkjet printing.

The film may contain an aversive agent, such as a bittering agent. Suitable bittering agents include, without limitation, naringin, sucrose octaacetate, quinine hydrochloride, denatonium benzoate, or mixtures thereof. The aversive agent can be used in the film at any suitable concentration. Suitable concentrations include, without limitation, 1 to 5000 ppm, or even 100 to 2500 ppm, or even 250 to 2000 ppm.

Detergent composition

The single dose water soluble article contains a detergent composition, preferably a liquid laundry detergent composition. The term "liquid laundry detergent composition" refers to any laundry detergent composition containing a liquid capable of wetting and treating fabric, and includes, without limitation, liquids, gels, pastes, dispersions, and the like. The liquid composition may include solids or gases in suitably subdivided form, but the liquid composition excludes forms that are not generally liquid, such as tablets or granules.

Without wishing to be bound by theory, it is preferable that the optionally encapsulated lipase variants be provided as a liquid laundry detergent, as this provides faster release into the detergent solution. This is especially preferred in fast and cold wash cycles where the risk of getting stuck in the undissolved powder paste phase is minimized.

The liquid detergent composition may be used in a fabric hand washing operation or may be used in an automatic fabric washing operation.

Auxiliary detergent component

The detergent composition, preferably a liquid laundry detergent composition, contains a detergent adjunct component, for example, selected from surfactants, polymers, modifiers, dye transfer inhibiting agents, dispersants, enzymes, enzyme stabilizers, catalytic materials, bleaches, bleach activators, hydrogen peroxide, hydrogen peroxide sources, preformed peracids, polymeric dispersants, anti-redeposition agents, brighteners, defoamers, cosmetic colors, coloring pigments, opacifying agents, fragrances, fragrance delivery systems, builders, hydrotropes, processing aids, solvents, pigments, flocculating agents, chelating agents and mixtures thereof.

The detergent auxiliary component preferably contains a surfactant, in particular in the case of a cleaning composition, the surfactant preferably contains anionic and/or nonionic surfactants, preferably in a weight ratio of 50:1 to 1:10 or more preferably from 20:1 to 1:2.

The detergent composition preferably contains up to 50%, preferably from 5% to 50%, more preferably from 7.5% to 45%, even more preferably from 10% to 40%, or even more preferably from 12% to 37%, most preferably from 15% to 30% non-foaming anionic surfactant by weight of detergent composition. Preferably, the non-foaming anionic surfactant comprises a linear alkyl benzene sulfonate, an alkoxylated alkyl sulfate, or a mixture thereof. More preferably, the non-foaming anionic surfactant is a mixture of linear alkylbenzene sulfonate and alkoxylated alkyl sulfate, more preferably a mixture of linear alkylbenzene sulfonate and ethoxylated alkyl sulfate.

The weight ratio of linear alkylbenzene sulfonate to alkoxylated alkyl sulfate, more preferably linear alkylbenzene sulfonate to ethoxylated alkyl sulfate, is 1:2 to 20:1, preferably 1.1:1 to 15:1, more preferably 1.2:1 to 10:1 , even more preferably from 1.3:1 to 5:1, most preferably from 1.4:1 to 3:1.

The weight ratio of linear alkylbenzenesulfonate to ethoxylated alkyl sulfate is 1:10 to 20:1, preferably 1:7 to 3:1, more preferably 1:5 to 1.5:1.

Preferably, the weight ratio of total anionic surfactant (ie, total anionic surfactant present in the liquid composition) to nonionic surfactant in the liquid composition is between 5:1 and 23:1.

Preferably, the non-foaming anionic surfactant is neutralized with an amine, preferably selected from monoethanolamine, diethanolamine, triethanolamine or a mixture thereof, more preferably monoethanolamine.

The detergent composition preferably contains from 0% to 40%, preferably from 0.01% to 30%, more preferably from 0.1% to 20%, most preferably from 0.15% to 15% of non-ionic surfactant by weight of liquid detergent composition for washing. Preferably, the nonionic surfactant is selected from alcohol alkoxylates obtained by oxosynthesis of alcohol alkoxylate, Guerbet alcohol alkoxylates, alkylphenol alcohol alkoxylates, or mixtures thereof.

The detergent composition preferably contains from 1.5% to 20%, more preferably from 2% to 15%, even more preferably from 3% to 10%, most preferably from 4% to 8% of soap by weight of the liquid detergent composition, preferably a fatty salt. acid, more preferably an amine-neutralized fatty acid salt, and preferably the amine is an alkanolamine, more preferably selected from monoethanolamine, diethanolamine, triethanolamine or a mixture thereof, more preferably monoethanolamine.

The detergent composition preferably contains a cationic polysaccharide, preferably selected from cationically modified hydroxyethyl cellulose, cationically modified hydroxypropyl cellulose, cationically and hydrophobically modified hydroxyethyl cellulose, cationically and hydrophobically modified hydroxypropyl cellulose, or a mixture thereof, more preferably cationically modified hydroxyethyl cellulose, cationically and hydrophobically -modified hydroxyethylcellulose or mixtures thereof. The cationic polysaccharide is preferably present in an amount of 0.05% to 3%, preferably 0.1% to 2%, more preferably 0.2% to 1%, most preferably 0.25% to 0.75% by weight liquid detergent composition for washing.

The detergent composition preferably contains an alkoxylated polyethyleneimine, preferably an ethoxylated polyethyleneimine. The liquid laundry detergent composition preferably contains 0.1% to 10%, preferably 0.5% to 7%, more preferably 1% to 5% ethoxylated polyethyleneimine, based on the weight of the liquid laundry detergent composition.

The water soluble single dose article preferably contains 20% or less or more preferably 15% or less water by weight of the single dose article, preferably 0.1% to 15%, more preferably 1% to 12.5% water by weight of the article with a single dose.

Preferably the water soluble single dose article contains 10% to 60%, preferably 12% to 50%, most preferably 15% to 40% non-aqueous solvent based on the weight of the liquid laundry detergent composition, with preferably the non-aqueous solvent selected from 1,2- propanediol, glycerin, sorbitol, dipropylene glycol, tripropylene glycol or mixtures thereof.

Enzymes. The composition may contain one or more enzymes, optionally encapsulated in microcapsules as described herein, that improve cleaning performance and/or tissue care. Examples of suitable enzymes include, without limitation, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannases, pectate lyases, keratinases, reductases, oxidases, phenol oxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases. , malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, or mixtures thereof. A typical combination is a mixture of enzymes, which may contain, for example, a protease and a lipase in combination with an amylase. When present in the composition, the above additional enzymes may be present in an amount of 0.00001 to 2% by weight, 0.0001 to 1% by weight, or 0.001 to 0.5% by weight of the enzyme protein based on the weight of the composition.

In general, the properties of the selected enzyme(s) should be compatible with the detergent selected (i.e. optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.) and the enzyme(s) should (should) be present in effective amounts.

The lipase variant may optionally be encapsulated in a microcapsule. In addition, the detergent composition of the invention may further contain one or more additional enzymes in encapsulated form. Examples of suitable enzymes are selected from the group consisting of protease, amylase, additional lipase, cellulase, mannase, pectinase, DNase, laccase, peroxidase, haloperoxidase, perhydrolase, and combinations thereof.

The enzyme(s) in the composition may include one or more enzymes suitable for inclusion in a laundry or dishwashing detergent (detergent enzymes), such as a protease (e.g., subtilisin or metalloprotease), lipase, cutinase , amylase, carbohydrase, cellulase, pectinase, mannase, arabinase, galactanase, xanthase, xylanase, DNase, perhydrolase, oxidoreductase (eg laccase, peroxidase, peroxygenase and/or haloperoxidase). Preferred detergent enzymes are protease (eg subtilisin or metalloprotease), lipase, amylase, lyase, cellulase, pectinase, mannanase, DNase, perhydrolase, and oxidized hydrases (eg laccase, peroxidase, peroxygenase and/or haloperoxidase); or their combinations. More preferred detergent enzymes are protease (eg subtilisin or metalloprotease), lipase, amylase, cellulase, pectinase and mannanase; or their combinations.

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

In one aspect, preferred enzymes will include cellulase. Suitable cellulases include enzymes of bacterial or fungal origin. They also include chemically modified or engineered protein mutants. Suitable cellulases include those of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., for example the fungal cellulases derived from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum described in US4435307, US5648263, US5691178, US5959/07 and 2WO99/07.

Particularly suitable cellulases are alkaline or neutral cellulases, which have advantages relating to the care of colored fabrics. Examples of such cellulases are those described in EP0495257, EP0531372, WO96/11262, WO96/29397, WO98/08940. Other examples are cellulase variants such as those described in WO94/07998, EP0531315, US5457046, US5686593, US5763254, WO95/24471, WO98/12307 and PCT/DK98/00299

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

In one aspect, preferred enzymes will include a protease. Suitable proteases include proteases of bacterial, fungal, plant, viral or animal origin, eg plant or microbial origin. Microbial origin is preferred. They also include chemically modified or engineered protein mutants. It may be an alkaline protease such as a serine protease or a metalloprotease. The serine protease may, for example, belong to the S1 family, such as trypsin, or the S8 family, such as subtilisin. The metalloprotease may be, for example, thermolysin, for example, of the M4 family, or another metalloprotease, for example, from the M5, M7 or M8 families.

The term "subtilase" refers to a subgroup of serine protease in accordance with the publications of 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 the presence of serine at the active site, which forms a covalent adduct with the substrate. Subtilases can be divided into 6 subgroups, i.e. the subtilisin family, the termitase family, the proteinase K family, the lantibiotic peptidase family, the kexin family, and the pyrolysin family.

Examples of subtilases are described bacteria of the genus Bacillus, e.g. Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279, and the protease PD138 described in (WO93/18140). Other suitable proteases may be those described in WO92/175177, WO01/016285, WO02/026024 and WO02/016547. Examples of trypsin-like proteases are trypsin (eg, porcine or bovine origin) and Fusarium protease described in WO89/06270, WO94/25583 and WO05/040372, and Cellumonas derived chymotrypsin proteases described in WO05/052161 and WO05/052146.

An additional preferred protease is the alkaline protease from Bacillus lentus DSM 5483 as described in WO95/23221, for example, and variants thereof as described in WO92/21760, WO95/23221, EP1921147 and EP1921148.

Examples of metalloproteases are the neutral metalloprotease described in WO07/044993 (Genencor Int.), eg derived from Bacillus amyloliquefaciens.

Examples of suitable proteases are those described in: WO92/19729, WO96/034946, WO98/20115, WO98/20116, WO99/011768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO05/0WO0613 /036263, WO11/036264, especially variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100 , 101, 102, 103, 104, 106, 118, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235 , 236, 245, 248, 252 and 274 using BPN' numbering. More preferably, subtylase variants may contain 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, Q235H, Q235H , N252K, T274A (using BPN' numbering).

Suitable commercially available protease enzymes include proteases sold under the trade names Alcalase®, Blaze®; Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase ® and Esperase®, all of which may be sold as Ultra® or Evity® (Novozymes A/S), proteases sold under the tradenames Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, Preferenz™, Purafect MA ®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, Effectenz™, FN2®, FN3®, FN4®, Excellase®, Opticlean® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N.V.) , BLAP® (sequence shown in FIG. 29 in US5352604), as well as variants thereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.

In one aspect, preferred enzymes will include amylase. Suitable amylases may be alpha-amylase or glucoamylase and may be of bacterial or fungal origin. They also include chemically modified or engineered protein mutants. Amylases include, for example, alpha-amylases derived from Bacillus, for example from a special strain of Bacillus licheniformis, described in more detail in GB1296839.

Suitable amylases include the amylases of SEQ ID NO: 3, WO95/10603, or variants whose sequence is 90% identical to SEQ ID NO: 3. Preferred variants are described in WO94/02597, WO94/18314, WO97/43424 and SEQ ID NO: 4 in WO99/019467, for example, variants with 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.

Various suitable amylases include the amylases of SEQ ID NO: 6 in WO02/010355 or variants with 90% sequence identity to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having deletions at positions 181 and 182 and substitutions at position 193.

Other suitable amylases are a hybrid alpha-amylase containing residues 1-33 of B. amyloliquefaciens-derived alpha-amylase shown in SEQ ID NO: 6 WO2006/066594 and residues 36-483 of B. licheniformis alpha-amylase shown in SEQ ID NO: 4 WO2006/066594, or variants with 90% sequence identity. Preferred variants of such hybrid alpha-amylase are those that have a substitution, deletion or insertion at one or more of the following positions: G48, T49, G107, H156, A181, N190, M197, I201, A209 and Q264. The most preferred hybrid alpha-amylase variants containing residues 1-33 of alpha-amylase derived from B. amyloliquefaciens as shown in SEQ ID NO: 6 of WO2006/066594 and residues 36-483 of SEQ ID NO: 4 are variants with substitutions:

M197T;

H156Y+A181T+N190F+A209V+Q264S or

G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S.

Other suitable amylases are the amylases having SEQ ID NO: 6 in WO99/019467, or variants whose sequence is 90% identical to SEQ ID NO: 6. Preferred variants of SEQ ID NO: 6 are those having a substitution, deletion, or insertion in one or more of the following positions: R181, G182, H183, G184, N195, I206, E212, E216 and K269. Particularly preferred amylases are those that have a deletion at positions R181 and G182 or positions H183 and G184.

Additional amylases can be used that have SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 7 of WO96/023873, or variants whose sequence is 90% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7. Preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 7 are those that have a substitution, a deletion or an 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 variants having a deletion at positions 181 and 182, or positions 183 and 184. The most preferred variants of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 7 are variants having a deletion at positions 183 and 184 and a substitution at one or more of positions 140, 195, 206, 243 , 260, 304 and 476.

Other amylases can be used that have SEQ ID NO: 2, WO08/153815, SEQ ID NO: 10, WO01/66712, or variants whose sequence is 90% identical to SEQ ID NO: 2 in WO08/153815, or whose sequence is on 90% identical to that of SEQ ID NO: 10 in WO01/66712. Preferred variants of SEQ ID NO: 10 in WO01/66712 are those that have a substitution, deletion, or insertion at one or more of the following positions: 176, 177, 178, 179, 190, 201, 207, 211, and 264.

Other suitable amylases are the amylases having SEQ ID NO: 2 in WO09/061380, or variants whose sequence is 90% identical to SEQ ID NO: 2. Preferred variants of SEQ ID NO: 2 are those that are truncated at the C-terminus 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 a substitution 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 a deletion at position R180 and/or S181 or T182 and/or G183. The most preferred variants of SEQ ID NO: 2 are those having substitutions:

N128C+K178L+T182G+Y305R+G475K;

N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;

S125A+N128C+K178L+T182G+Y305R+G475K or

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

Other suitable amylases are the alpha-amylase of SEQ ID NO: 12 in WO01/66712, or a variant whose sequence is at least 90% identical to SEQ ID NO: 12. Preferred amylase variants are those having a substitution, deletion, or insertion in one or more of the following positions of SEQ ID NO: 12 in WO01/66712: 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. Specific preferred amylases include variants having a D183 and G184 deletion and substitutions R118K, N195F, R320K and R458K, and a variant additionally having substitutions at one or more positions selected from the group: M9, G149, G182, G186, M202, T257 , Y295, N299, M323, E345 and A339. Most preferred is a variant that additionally has substitutions at all of these positions.

Other examples are amylase variants such as those described in WO2011/098531, WO2013/001078 and WO2013/001087.

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

Suitable additional lipases and cutinases include enzymes of bacterial or fungal origin. They also include chemically modified or engineered protein mutant enzymes. Examples include lipase from Thermomyces, such as from T. lanuginosus (formerly Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, such as H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of which have since been renamed Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (WO95/06720 and WO96/27002), P. wisconsinensis (WO96/12012), Streptomyces GDSL-type lipase (WO10/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (US5,389,536 ), lipase from Thermobifida fusca (WO11/084412, WO13/033318), lipase from Geobacillus stearothermophilus (WO11/084417), lipase from Bacillus subtilis (WO11/084599) and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (WO12 /137147).

Other examples are lipase variants such as those described in EP407225, WO92/05249, WO94/01541, WO94/25578, WO95/14783, WO95/30744, WO95/35381, WO95/22615, WO96/00292, WO97/04079, WO97 /07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.

Preferred commercial formulations of lipase include Lipolase™, Lipex™; Lipolex™; Lipolase Ultra™; Lipex Evity 100L; Lecitase™; Lipoprime™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades) and Bacillus sp., Solvay.

Other examples are lipases, sometimes referred to as acyltransferases or perhydrolases, for example, acyltransferases homologous to Candida antarctica lipase A (WO10/111143), acyltransferase from Mycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and perhydrolase variants M. smegmatis, specifically the S54V variant used in the commercial Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

In one aspect, other preferred enzymes include microbial-derived endoglucanases exhibiting endo-beta-1,4-glucanase activity (EC3.2.1.4), including a bacterial polypeptide endogenous to a member of the genus Bacillus whose sequence is at least 90%, 94% , 97% or 99% identical to the amino acid sequence of SEQ ID NO: 2 in US7141403, and mixtures thereof. Suitable endoglucanases are commercially available under the tradenames Celluclean® and Whitezyme® (Novozymes).

Other preferred enzymes include pectate lyases, available under the trade names Pectawash®, Pectaway®, Xpect®, and mannases, available under the trade names Mannaway® (Novozymes) and Purabrite® (Danisco/DuPont).

The detergent enzyme(s) can be included in the detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive containing all of these enzymes. The detergent additive of the invention, i. e. a single additive or a combined additive, may be in the form of a formed mixture, for example, in the form of a granulate, liquid, suspension, and the like. Preferred detergent additive formulations are granulates, in particular non-dusting granules, liquids, in particular stabilized liquids, or suspensions.

Non-dusting granules can be obtained, for example, as described in US4106991 and US4661452, and they can optionally be coated by methods known in the field. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol, PEG) with average molar masses from 1000 to 20000; ethoxylated nonylphenols having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and in which 15 to 80 ethylene oxide units are present; fatty alcohols; fatty acid; and mono-, di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application using fluidized-bed boiling techniques are given in GB1483591. Liquid enzyme preparations can, for example, be stabilized by adding a polyol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid, according to established methods. Protected enzymes can be obtained in accordance with the method described in EP238216.

In general, the properties of the selected enzyme(s) should be compatible with the selected detergent (i.e., optimum pH, compatibility with other enzymatic and non-enzymatic ingredients, etc.) and the enzyme(s) should (should) be present in effective amounts.

Polymers

Preferably, the auxiliary detergent component is a polymer or a mixture of polymers. The detergent composition may contain 0-10% by weight, for example 0.5-5%, 2-5%, 0.5-2% or 0.2-1% polymer. Any polymer known in the art suitable for use in detergents can be used. The polymer may function as an additional modifier as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or antifoam properties. Some polymers may have more than one of the above properties and/or more than one of the following motifs. Examples of polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI ) and polycarboxylates such as PAA, PAA/PMA, polyaspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly/ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidonevinylimidazole (PVPVI). Additional examples of polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternium ethoxysulfate.

Examples of modifiers include citrate, chelating agents such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl or alkenyl succinic acid. Additional specific examples include 2,2',2''-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine-N,N-diacetic acid (MGDA), glutamate-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid, N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartate- N-monoacetic acid (ASMA), aspartate-N,N-diacetic acid (ASDA), aspartate-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(sulfomethyl)aspartic acid (SMAS), N-( 2-sulfoethyl)aspartic acid (SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methylimidodiacetic acid (MIDA), serine-N,N-diacetic acid ( SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilate-N,N-diacetic acid (ANDA), sulfanilate-N,N-diacetic acid (SLDA) , taurine-N,N-diacetic acid (TUDA) and N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), diethanolglycine (DEG), and combinations and salts thereof. Phosphonates suitable for use in the present invention include 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetrakis(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis(methylenephosphonic acid) (DTMPA or DTPMPA or DTPMP), nitrilotris( methylenephosphonic acid) (ATMP or NTMP), 2-phosphonobutane-1,2,4-tricarboxylic acid (PBTC), hexamethylenediaminetetrakis(methylenephosphonic acid) (HDTMP).

The composition may also contain 0-50 wt.%, for example from about 5% to about 30%, additional detergent modifier. Non-limiting examples of additional modifiers include polyacrylate homopolymers or copolymers thereof such as poly(acrylic acid) (PAA) or (acrylic acid/maleic acid) copolymer (PAA/PMA) or polyaspartic acid.

Dyes for fabric

The detergent composition of the present invention may also include fabric coloring agents, such as dyes or pigments, which, when included in detergent compositions, can be deposited on fabric upon contact of said fabric with a cleaning solution containing said detergent compositions, and thus change the shade of said fabric. by absorbing/reflecting visible light. Fluorescent whitening agents emit at least some visible radiation. In contrast, fabric-staining agents change the hue of the surface because they absorb at least a portion of the visible light spectrum. Suitable fabric coloring agents include dyes, dye-clay conjugates, and may also include pigments. Suitable dyes include low molecular weight dyes and polymer dyes. Suitable low molecular weight dyes include low molecular weight dyes selected from the group consisting of dyes that are classified according to the color index (C.I.) in the groups such as direct blue, direct red, direct violet, acid blue, acid red, acid violet, basic blue, basic violet and basic red, or mixtures thereof, for example as described in WO 2005/03274, WO 2005/03275, WO 2005/03276 and EP 1876226 (incorporated herein by reference). The detergent composition preferably contains from about 0.00003 wt.% to about 0.2 wt.%, from about 0.00008 wt.% to about 0.05 wt.%, or even from about 0.0001 wt.% to about 0 04 wt% fabric coloring agent. The composition may contain from 0.0001 to 0.2% by weight of the fabric staining agent, and this may be particularly preferred if the composition is in the form of a single dose pouch. Suitable coloring agents are also described in, for example, WO 2007/087257 and WO 2007/087243.

Dispersing agents

The detergent compositions of the present invention may also contain dispersants. In particular, powder detergents may contain dispersants. Suitable water-soluble organic materials include homo- or copolymer acids or salts thereof, wherein the polycarboxylic acid contains at least two carboxyl radicals separated from each other by no more than two carbon atoms. Suitable dispersants are described, for example, in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.

Dye transfer inhibitors

The detergent compositions of the present invention may also include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, without limitation, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, N-vinylpyrrolidone-N-vinylimidazole copolymers, polyvinyloxazolidones, and polyvinylimidazoles, or mixtures thereof. When used in the inventive composition, dye transfer inhibitors may be present at concentrations of from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

Fluorescent whitening agent

The detergent composition may preferably also contain additional components that can change the color of the articles to be cleaned, such as a fluorescent bleaching agent or optical brighteners. The clarifier, if present, is preferably present at a concentration of from about 0.01% to about 0.5%. Any fluorescent bleaching agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent bleaching agents are those belonging to the classes of diaminostilbene sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl distyryl derivatives. Examples of fluorescent bleaching agents based on diaminostilbene sulfonic acid derivatives include sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4 4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2.2'-disulfonate, 4,4'-bis-(2-anilino-4-(N-methyl-N-2- hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1,2,3-triazol-2-yl)stilbene-2,2' -disulfonate and 5-(2H-naphtho[1,2-d][1,2,3]triazol-2-yl)-2-[(E)-2-phenylvinyl]sodium benzenesulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino)stilbene-2,2'-disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenylstyryl)-disulfonate. A preferred fluorescent whitening agent is also the commercially available Parawhite KX available from Paramount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is 4,4'-bis-(sulfostyryl)-biphenyl disodium salt, also known as disodium distyryl biphenyl disulfonate. Other fluorescent agents suitable for use in the present invention include 1-3-diarylpyrazolines and 7-alkylaminocoumarins.

Suitable concentrations of fluorescent brightener are lower concentrations of about 0.01, 0.05, about 0.1, or even about 0.2 wt.% to higher concentrations of 0.5 or even 0.75 wt.%.

Dirt-releasing polymers

The detergent compositions may also include one or more soil releasing polymers that aid in the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil releasing polymers can be, for example, non-ionic or anionic terephthalate polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see for example chapter 7 in Powdered Detergents, Surfactant science series volume 71, Marcel Dekker, Inc.

Preferably, the auxiliary detergent component contains polyether terephthalate. Preferably, the polyester terephthalate contains a backbone grafted with one or more anionic groups, more preferably containing an anionic polyether propylene terephthalate.

Suitable anionic polyesters are polyesters derived from terephthalic acid, 5-sulfoisophthalic acid or a salt of 5-sulfoisophthalic acid, from ethylene glycol or polyethylene glycol, propylene glycol or polypropylene glycol and polyalkylene glycol monoalkyl ether, and optionally from other additional monomers.

Another type of soil release polymers are amphiphilic alkoxylated grease removal polymers containing a core structure and a plurality of alkoxylate groups attached to the core structure. The core structure may comprise a polyalkyleneimine structure or a polyalkanolamine structure as detailed in WO 2009/087523 (incorporated herein by reference). In addition, random graft copolymers are suitable soil releasing polymers. Suitable graft copolymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (incorporated herein by reference).

Preferably, the detergent adjunct contains an amphiphilic graft polymer, preferably based on polyalkylene oxides and vinyl esters, preferably based on water-soluble polyalkylene oxides as a graft base and side chains to form a vinyl ester component by polymerization, said polymer having an average of < 1 graft site per 50 alkylene oxide groups, more preferably the molar ratio of grafted to ungrafted alkylene oxide groups is from 0.002 to 0.05, preferably from 0.002 to 0.035, more preferably from 0.003 to 0.025, most preferably from 0.004 to 0.02. Preferred graft polymers have an average molecular weight Mw of 3,000 to 100,000. Preferred polymers contain 20 to 70%, preferably 25 to 60% by weight of a polyalkylene oxide polymer, preferably a water-soluble polyalkylene oxide, as the graft base. Preferably, the polyalkylene oxide graft base is polyethylene glycol.

The polymer preferably contains from 30 to 80 wt.% vinyl ester component, and preferably the vinyl ester component contains vinyl acetate, vinyl propionate or a mixture thereof and optionally C1-C8 alkyl acrylate, more preferably from 70% to 100 wt.% vinyl acetate and from 0 to 30 wt. % C1-C8 alkyl acrylate.

Other soil releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 2003/040279 (incorporated herein by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides, and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose ester, and mixtures thereof. Preferably, the detergent auxiliary contains carboxymethyl cellulose or a derivative thereof, preferably selected from carboxymethyl cellulose, hydrophobically modified carboxymethyl celluloses or a mixture thereof, with hydrophobically modified carboxymethyl cellulose being particularly preferred.

Anti-Redeposition Agents

The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethylene glycol (PEG), acrylic acid homopolymers, acrylic acid copolymers. acid and maleic acid; and ethoxylated polyethyleneimines. The cellulose-based polymers described above in the description of soil releasing polymers can also function as anti-redeposition agents.

Rheology Modifiers

The detergent compositions of the present invention may also include one or more rheology modifiers, builders or thickeners other than thinning agents. The rheology modifiers are selected from the group consisting of non-polymeric crystalline, hydroxy-functional materials, polymeric rheology modifiers that impart shear thinning properties to the aqueous liquid matrix of the liquid detergent composition. The rheology and viscosity of the detergent can be modified and adjusted using methods known in the art, for example as shown in EP 2169040.

Other suitable adjuvants include, but are not limited to, anti-shrinkage agents, anti-crease agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, fragrances, pigments, defoamers, solvents, and builders. means for liquid detergents and/or means providing the elasticity of the structure.

Preparation method

It will be apparent to those skilled in the art how to prepare the water soluble single dose product and liquid laundry detergent composition of the present invention using well known manufacturing methods.

Washing method

A further aspect of the present invention is a method for washing fabrics, comprising the following steps:

a. combining the water soluble single dose article of the present invention with sufficient water to dissolve the water soluble film and dilute the laundry detergent composition, preferably 300 to 3000 times, preferably 300 to 800 times, to form a cleaning solution;

b. combining the cleaning solution with at least one fabric to be washed.

Enzyme stabilizers and/or rheology modifiers

The compositions of the present invention preferably contain enzyme stabilizers, such as polyols, polymers, reversible enzyme inhibitors, divalent cations, enzyme substrates, antioxidants, and the like. Water soluble stabilizers are preferred.

To create a local environment within the capsule that is more "friendly" to the encapsulated enzyme/compound, slow dissolving stabilizers can be added, thereby increasing stability during storage.

Examples of reversible protease inhibitors are boronic acids, peptide aldehydes and their derivatives, as well as high molecular weight protein inhibitors (such as BASI/RASI inhibitors, see WO 2009/095425). An example of metalloprotease inhibitors is described in WO 2008/134343. Protease inhibitors are described in more detail below under the heading "Protease inhibitors".

The stabilizing polymers may be based, for example, on polyvinylpyrrolidone, polyvinyl acetate, polyvinyl alcohol and their copolymers. Stabilizing polyols can be smaller molecules such as glycerol, sorbitol, propylene glycol and the like, but also larger molecules such as polyethylene glycol, polysaccharides and the like.

The stabilizing divalent cations Ca2+, Mg2+ and Zn2+ are well known in the art. Thus, in one embodiment, the composition of the invention contains a source of Ca2+, Mg2+ or Zn2+ ions. Preferably, the Ca2+, Mg2+ or Zn2+ ion source is a slightly soluble (slowly dissolving) salt of Ca2+, Mg2+ or Zn2+. Slightly soluble means that the solubility in pure water at 20°C is less than 5 g/l, 2 g/l, 1 g/l, 0.5 g/l, 0.2 g/l, 0.1 g/l, or 0.05 g/l. Preferred Ca2+, Mg2+ or Zn2+ salts are calcium carbonate, magnesium carbonate, zinc carbonate, calcium sulfate, calcium sulfite, magnesium sulfite, zinc sulfite, calcium phosphate, dicalcium phosphate, magnesium phosphate, zinc phosphate, calcium citrate, magnesium citrate, zinc citrate, calcium oxalate, magnesium oxalate, zinc oxalate, calcium tartrate, magnesium tartrate or zinc tartrate.

In addition, slowly dissolving acids or bases can be used to create a local pH within the microcapsule, which is more "friendly" to the encapsulated enzyme/compound.

In most cases, enzymes are stabilized by the addition of their substrates (eg protein for proteases, starch for amylases, etc.). Antioxidants or reducing agents can be used to reduce the oxidation of enzymes, such as thiosulfate, ascorbate, and the like. The total dose required for these stabilizers per gram of detergent is much lower than when stabilizers are added to the continuous phase of the detergent, since they are concentrated in the internal phase of the capsule and in many cases will either not diffuse outwards during storage or will diffuse slowly depending on the structure and molecular weight of the stabilizer. Especially high molecular weight stabilizers (eg greater than 1 kDa or greater than 2 kDa, more preferably greater than 5 kDa) will provide better overall performance. Thus, high molecular weight inhibitors, polymers, polyols, cations, enzyme substrates and antioxidants are preferred.

The enzyme can be protected by adding a scavenger protein. Thus, components that destabilize the enzyme by interacting with amino acid groups (eg, amines) on the protein may therefore interact with the added scavenger or sacrificial protein. Preferred is an absorbent protein with a sufficiently high molecular weight that remains inside the capsules.

The stability of the enzyme can be further improved by adding a rheology modifying component, thereby increasing the viscosity of the internal phase of the capsule. The increased internal viscosity will slow the diffusion of enzyme destabilizers into the capsule (and/or slow the diffusion of enzyme stabilizers out of the capsule) and thus prolong the life of the enzyme. Examples of such viscosity modifiers are polymers such as polyethylene glycol (PEG), polyethylene oxide (PEO), hydrophilic polyurethane, polyvinylpyrrolidone (PVP) and PVP-vinyl acetate copolymers, starch, hyaluronic acid, water-soluble cellulose derivatives such as carboxymethylcellulose, water-soluble gums such as gum arabic, locust bean gum, guar gum or xanthan gum, and the like; and combinations or copolymers thereof. Most preferred are non-ionic high molecular weight polymers with a molecular weight greater than 1 kDa or greater than 2 kDa, more preferably greater than 5 kDa. Non-ionic polymers are preferred because in most cases they are better compatible with the reactive membrane polymer than ionic polymers.

High viscosity can be obtained by making capsules using a high viscosity aqueous phase or (which is more modern) making capsules in which the increase in viscosity occurs only after the emulsion/capsules have been made. This "activated" viscosity increase is preferred, since the production of high viscosity aqueous phase emulsions can be challenging. Activated viscosity increase can be done in situ when added to a detergent, if the internal phase of the capsule has a higher water activity than the detergent to which it is added, as a result, water (but not the rheology modifier) will diffuse out of the capsules, increasing the viscosity internal phase after adding to the detergent. This can also be done using the diffusion of salt or other low molecular weight components, for example, using a component that will increase viscosity with decreasing salt concentration by adding to the detergent (for example, a polymer that precipitates at an initial high salt content, but is soluble when the salt concentration salt is reduced due to diffusion of the salt when added to the detergent). Another way to activate the increase in viscosity is to use components in which the viscosity depends on pH. For some interfacial polymerization processes (eg, amine-halo acid reaction), the pH of the internal phase will change during encapsulation if the pH of the amine-halo acid decreases during the interfacial polymerization. This can be used to activate the increase in viscosity. Many rheology modifiers, such as polyacrylates, exhibit a maximum viscosity at a particular pH value or range. Lubrizol's Carbopol 934 and Scott Bader's Texipol 63-258 are examples of rheology modifiers in which the viscosity increases significantly as pH decreases from 11 to 8 or as pH increases from 4 to 8. Another type of polymer with different viscosities at low pH and at high pH is a partially hydrolyzed polyacrylamide. Another possibility is to use rheology modifiers that are temperature dependent, resulting in an emulsion/encapsulation at one temperature and subsequently changing the temperature to increase the viscosity. Viscosity can also be induced by light or ultrasound. Another method is to use shear thinning rheology modifiers such that the viscosity is low at high shear when emulsifying and high at shear reduction.

Another stabilization technique for enzyme encapsulation is to precipitate the enzyme in capsules during storage, for example by adding precipitants such as salt or polyethylene glycol (PEG). The same "activated stabilization" described above can be realized, for example, by adding PEG, which, after being added to the detergent, is concentrated by diffusion of water outward to the extent that the enzyme will precipitate. Thus, the enzyme may be in solution during the processing of the capsules, but precipitate when added to the detergent.

Enzymes can also be used in precipitated or crystalline form in the preparation of microcapsules.

The present invention is further described with the help of the following examples, which should not be construed as limiting the scope of the present invention.

EXAMPLES

Example 1 Analysis of p-nitrophenyl (pNP)

The hydrolytic activity of the lipase can be determined by kinetic analysis using p-nitrophenylacyl esters as a substrate.

84, 2605

№ по кат. C3:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) разводят до конечной концентрации 1 мМ 25 мМ в аналитическом буферном растворе (50 мМ Трис; pH 7,7; 0,4% Triton X-100).100 mM stock solution in DMSO of each of the substrates p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12), and p-nitrophenyl palmitate (C16) (all from Sigma-Aldrich) Danmark A/S, Kirkebjerg

84, 2605

cat. no. C3:N-9876, C6: N-0502, C10: N-0252, C12: N-2002, C16: N-2752) are diluted to a final concentration of 1 mM 25 mM in assay buffer (50 mM Tris; pH 7, 7; 0.4% Triton X-100).

Lipase variants, parental lipase and appropriate controls, eg Lipolase™ (SEQ ID NO: 2) in 50 mM Hepes; pH 8.0; 10 ppm Triton X-100; +/-20 mM CaCl2 is added to the substrate solution at the following final protein concentrations: 0.01 mg/ml; 5 x 10-3 mg/ml; 2.5 x 10-4 mg/ml; and 1.25 x 10-4 mg/mL in NUNC 96-well plates (cat. no. 260836, Kamstrupvej 90, DK-4000, Roskilde). Buffer solution is also used as a negative control. The release of p-nitrophenol by hydrolysis of p-nitrophenylacyl can be monitored at 405 nm for 5 minutes at 10 second intervals on a Spectra max 190 spectrometer (Molecular Devices GmbH, Bismarckring 39, 88400 Biberach an der Riss, GERMANY). Hydrolytic activity against one or more substrates of the variant can be compared to the activity of the parent lipase.

Example 2 Construction of Variants by Site-Directed Mutagenesis

Site-directed variants were constructed from Thermomyces lanuginosus lipase (TLL) (SEQ ID NO: 2). Variants were generated by standard PCR cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) along with appropriately designed mutagenic oligonucleotides that introduce the desired mutations into the resulting sequence.

The mutagenic oligonucleotides were designed according to the DNA sequence flanking the desired mutation site(s) separated from each other by base pairs DNA insertions/deletions/substitutions were purchased from an oligonucleotide supplier such as Life Technologies.

To test for TLL variants, the mutated DNA encoding the variant was integrated into a competent strain of A. oryzae by homologous recombination, fermented using standard protocols (yeast extract media, 3-4 days, 30°C) and purified by chromatography. Thus, the options listed in the table below were designed and manufactured.

Options SEQ ID NO: 2

Example 3 Relative Wash Efficiency (RE(wash))

Washing experiments were performed using automatic mechanical force analysis (AMSA) to evaluate the effectiveness of washing in washing. The AMSA plate has a number of slotted holes for test solutions and a lid that tightly presses the laundry sample, which allows washing the textile product through all the slotted holes. During washing, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution into contact with the textile and mechanical force is applied in the form of regular periodic vibrations. For additional description, see WO02/42740, especially in the section on special embodiments of the method on pages 23-24.

Washing experiments were carried out under the experimental conditions described below.

Detergent: 3.3 g/l detergent B or 0.8 g/l detergent J The volume of the test solution: 160 µl Wash time: 20 minutes Temperature: 30°C Lipase dose: 0 ppm or 0.35 ppm Test material: Cream Annatto dyed cotton EMPA221 was prepared as described in WO06/125437, except for the replacement of annatto turmeric (Annatto: A-320-WS, Chr. Hansen A/S, Boege Alle' 10-12, DK-2970, Hersholm, Denmark and EMPA221: EMPA, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland)

Water hardness was adjusted to 15 F or 6 F by adding CaCl2, MgCl2 and NaHCO3 (Ca2+:Mg2+:HCO3-=4:1:7.5 or 2:1:4.5).

After washing, the textiles were washed in tap water, and excess water was removed from the textiles using filter paper, and immediately thereafter, the textiles were dried at 100° C. for 15 minutes.

Washing efficiency was measured by the color change of the soiled textile after washing. The soil was mixed with annatto. Annatto contains the dye norbixin, which acts as a pH indicator with a pH-dependent color change. Lipase activity leads to the release of free fatty acids from the acylglycerols of the cream, which leads to a decrease in pH and, as a result, to a discoloration of the pH indicator normyxin. Thus, the effectiveness of lipase for washing can be expressed as the degree of color change of reflected-emitted light from a soiled textile after washing under white light illumination.

Color measurements were taken on a professional flatbed scanner (EPSON EXPRESSION 11000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark) which was used to image the soiled textile after washing. To obtain a light intensity value based on the scanned images, the 24-bit image pixel values were converted to values for red, green, and blue (RGB) colors.

The color change due to lipase activity was performed as a change in green light reflection-emission (G) compared to a light intensity value (Int) calculated as follows:

The relative wash efficiency (OE(wash)) of the lipase relative to the reference lipase was calculated as follows:

ME(wash) = (G/Int(tested lipase) - G/Int(no enzyme))/(G/Int (this. lipase) - G/Int (no enzyme)).

A lipase is considered to exhibit a higher wash performance if it is better than the reference variant (ME(wash) > 1). In the context of the present invention, the reference enzyme is a lipase shown as SEQ ID NO: 2.

Odor detection by measurement on a solid phase microextraction gas chromatograph

Butyric acid release (odor) from the lipase-washed samples was measured by solid phase microextraction gas chromatography (SPME-GC) using the following method.

The Cream Annatto-dyed EMPA221 textile was washed as above and after washing, excess water was removed from the textile using filter paper, after which the textile was dried at 25° C. for 2 hours. Each SPME-GC measurement was performed with four pieces of washed and dried textile (5 mm diameter) which were transferred to a gas chromatograph (GC) vial and then the vial was capped. Samples were incubated at 30°C for 24 hours and subsequently heated to 140°C for 30 minutes and stored at 20-25°C for at least 4 hours prior to analysis. Analyzes were performed on a Varian 3800 GC column equipped with Stabilwax-DA, lntegra-Guard column (30 m, 0.32 mm id and 0.25 µm df), and Carboxen PDMS SPME fiber (85 µm). Sampling of each vial for GC was performed at 50°C for 8 minutes with SPME fiber in the vapor phase over pieces of textile and the selected compounds were then injected onto the column (injector temperature = 250°C). Column flow = 2 ml helium/minute. Column oven temperature gradient: 0 minutes = 50°C, 2 minutes = 50°C, 6 minutes 45 seconds = 240°C, 11 minutes 45 seconds = 240°C. A flame ionization detector (FID) was used and butyric acid retention time was determined using an authentic standard.

The relative odor release (OE(smell)) of lipase is the ratio between the amount of butyric acid released (peak area) from a lipase-washed sample and the amount of butyric acid released (peak area) from a reference lipase-washed sample, after which both values are corrected for the amount of butyric acid released. acid (peak area) from a non-lipase-washed sample (blank). The relative odor potency (OE(smell)) of a polypeptide is calculated according to the following formula:

OE(smell) = (smell(lipase tested) - odor(no enzyme))/(smell(this lipase) - odor(no enzyme)),

where odor is the measured butyric acid (peak area) released from the surface of the textile.

Benefit/Risk Index (ME(wash)/ME(smell))

The Benefit/Risk Ratio (BRF), which describes the effectiveness of washing (benefit) versus odor release (risk), can be defined as ME(wash)/ME(smell). If the Benefit/Risk score for the lipase is greater than 1, the lipase has a higher wash performance with respect to odor release compared to the reference lipase (SEQ ID NO: 2).

Example 4 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Analysis)

Thermal unfolding of SEQ ID NO: 1 variants was monitored with Sypro Orange dye (Invitrogen, S-6650) using a real-time PCR analyzer (Applied Biosystems; Step-One-Plus).

In a 96-well white PCR plate, 15 µl of sample (purified enzyme diluted in 100 mM EPPS, 0.01% Troton-X-100; pH 8.0) was mixed (1:1) with Sypro Orange (conc = 10X ; stock solution from supplier = 5000X) in water.

The plate was sealed with an optical PCR cap. The PCR analyzer was set to a scan rate of 76°C per hour starting at 25°C and ending at 96°C.

Fluorescence was monitored every 20 seconds using a built-in blue LED for excitation and a ROX filter (610 nm, emission). Tm values were calculated as the maximum value of the first derivative (dF/dK) (Gregory et al., 2009, J. Biomol. Screen. 14: 700).

Example 5 Construction of Variants by Site-Directed Mutagenesis

Site-directed variants were constructed from Thermomyces lanuginosus lipase (TLL) (SEQ ID NO: 2). Variants were generated by standard PCR cloning of DNA fragments (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989) along with appropriately designed mutagenic oligonucleotides that introduce the desired mutations into the resulting sequence.

The mutagenic oligonucleotides were designed according to the DNA sequence flanking the desired mutation site(s) separated from each other by base pairs DNA insertions/deletions/substitutions were purchased from an oligonucleotide supplier such as Life Technologies. To test for TLL variants, the mutated DNA encoding the variant was integrated into a competent strain of A. oryzae by homologous recombination, fermented using standard protocols (yeast extract media, 3-4 days, 30°C) and purified by chromatography. Thus, the options listed in the table below were designed and manufactured.

Example 6 Relative Wash Efficiency (RE(wash))

Washing experiments were performed using automatic mechanical force analysis (AMSA) to evaluate the effectiveness of washing in washing. The AMSA plate has a number of slotted holes for test solutions and a lid that tightly presses the laundry sample, which allows washing the textile product through all the slotted holes. During washing, the plate, test solutions, textile and lid are vigorously shaken to bring the test solution into contact with the textile and mechanical force is applied in the form of regular periodic vibrations. For additional description, see WO 02/42740, especially in the section on special embodiments of the method on pages 23-24.

Washing experiments were carried out under the experimental conditions described below.

Detergent: 3.3 g/l detergent A or 0.8 g/l detergent B The volume of the test solution: 160 µl Wash time: 20 minutes Temperature: 30°C Lipase dose: 0 ppm or 0.35 ppm Test material: Cream Annatto dyed cotton EMPA221 was prepared as described in WO 06/125437, except for the replacement of annatto turmeric (Annatto: A-320-WS, Chr. Hansen A/S, Boege Alle' 10-12, DK-2970 , Hersholm, Denmark, and EMPA221: EMPA, Lerchenfeldstrasse 5, CH-9014, St. Gallen, Switzerland)

Water hardness was adjusted to 15 °F or 6 °F by adding CaCl2, MgCl2 and NaHCO3 (Ca2+:Mg2+:HCO3- = 4:1:7.5 or 2:1:4.5).

After washing, the textiles were washed in tap water, and excess water was removed from the textiles using filter paper, and immediately thereafter, the textiles were dried at 100° C. for 15 minutes.

Washing efficiency was measured by the color change of the soiled textile after washing. The soil was mixed with annatto. Annatto contains the dye norbixin, which acts as a pH indicator with a pH-dependent color change. Lipase activity leads to the release of free fatty acids from the acylglycerols of the cream, which leads to a decrease in pH and, as a result, to a discoloration of the pH indicator normyxin. Thus, the effectiveness of lipase for washing can be expressed as the degree of color change of reflected-emitted light from a soiled textile after washing under white light illumination.

Color measurements were taken on a professional flatbed scanner (EPSON EXPRESSION 11000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark) which was used to image the soiled textile after washing. To obtain a light intensity value based on the scanned images, the 24-bit image pixel values were converted to values for red, green, and blue (RGB) colors.

The color change due to lipase activity was performed as a change in green light reflection-emission (G) compared to a light intensity value (Int) calculated as follows:

The relative wash efficiency (OE(wash)) of the lipase relative to the reference lipase was calculated as follows:

ME(wash) = (G/Int(tested lipase) - G/Int(no enzyme))/(G/Int (this. lipase) - G/Int (no enzyme)).

A lipase is considered to exhibit a higher wash performance if it is better than the reference variant (ME(wash) > 1). In the context of the present invention, the reference enzyme is a lipase shown as SEQ ID NO: 2.

Odor detection by measurement on a solid phase microextraction gas chromatograph

Butyric acid release (odor) from the lipase-washed samples was measured by solid phase microextraction gas chromatography (SPME-GC) using the following method.

The Cream Annatto-dyed EMPA221 textile was washed as above and after washing, excess water was removed from the textile using filter paper, after which the textile was dried at 25° C. for 2 hours. Each SPME-GC measurement was performed with four pieces of washed and dried textile (5 mm diameter) which were transferred to a gas chromatograph (GC) vial and then the vial was capped. Samples were incubated at 30°C for 24 hours and subsequently heated to 140°C for 30 minutes and stored at 20-25°C for at least 4 hours prior to analysis. Analyzes were performed on a Varian 3800 GC column equipped with Stabilwax-DA, lntegra-Guard column (30 m, 0.32 mm id and 0.25 µm df), and Carboxen PDMS SPME fiber (85 µm). Sampling of each vial for GC was performed at 50°C for 8 minutes with SPME fiber in the vapor phase over pieces of textile and the selected compounds were then injected onto the column (injector temperature = 250°C). Column flow = 2 ml helium/minute. Column oven temperature gradient: 0 minutes = 50°C, 2 minutes = 50°C, 6 minutes 45 seconds = 240°C, 11 minutes 45 seconds = 240°C. A flame ionization detector (FID) was used and butyric acid retention time was determined using an authentic standard.

The relative odor release (OE(smell)) of lipase is the ratio between the amount of butyric acid released (peak area) from a lipase-washed sample and the amount of butyric acid released (peak area) from a reference lipase-washed sample, after which both values are corrected for the amount of butyric acid released. acid (peak area) from a non-lipase-washed sample (blank). The relative odor efficiency (OE(smell)) is calculated according to the following formula:

OE(smell) = (smell(lipase tested) - odor(no enzyme))/(smell(this lipase) - odor(no enzyme)),

where odor is the measured butyric acid (peak area) released from the surface of the textile.

Benefit/Risk Index (ME(wash)/ME(smell))

The Benefit/Risk Index (BRF), which describes the effectiveness of washing (benefit) versus odor release (risk), can be defined as ME(wash)/ME(smell). If the benefit/risk score for the lipase is greater than 1, the lipase has a higher wash performance with respect to odor release compared to the reference lipase (SEQ ID NO: 2).

Example 7 Protein Thermal Unfolding Analysis (TSA, Thermal Shift Analysis)

Thermal unfolding of SEQ ID NO: 2 variants was monitored with Sypro Orange dye (Invitrogen, S-6650) using a real-time PCR analyzer (Applied Biosystems; Step-One-Plus).

In a 96-well white PCR plate, 15 µl of sample (purified enzyme diluted in 100 mM EPPS, 0.01% Troton-X-100; pH 8.0) was mixed (1:1) with Sypro Orange (conc = 10X ; stock solution from supplier = 5000X) in water.

The plate was sealed with an optical PCR cap. The PCR analyzer was set to a scan rate of 76°C per hour, starting at 25°C and ending at 96°C.

Fluorescence was monitored every 20 seconds using a built-in blue LED for excitation and a ROX filter (610 nm, emission). Tm values were calculated as the maximum value of the first derivative (dF/dK) (Gregory et al., 2009, J. Biomol. Screen. 14: 700).

EXAMPLES OF DETERGENT

Examples 1-5

Detergent composition for washing with a single dose. Such single dose formulations may comprise one or more compartments.

Example 6 Single Dose Multiple Compartment Composition

Below are single dose laundry detergent formulations of the present invention with multiple compartments. In these examples, one dose has three compartments, but similar compositions may include two, four or five compartments. The film used to encapsulate the compartments is polyvinyl alcohol.

Raw materials and notes for detergent examples 1-7

Linear alkylbenzenesulfonate with an average length of aliphatic carbon chain C11-C18

C12-18 Dimethylhydroxyethylammonium chloride

AE3S is C12-15 alkylethoxy(3)sulfate

AE7 is a C12-15 alcohol ethoxylate with an average degree of ethoxylation of 7

AE9 is a C12-16 alcohol ethoxylate with an average degree of ethoxylation of 9

HSAS is a mid-branched primary alkyl sulfate with a carbon chain length of about 16-17 as described in US Patents 6,020,303 and US 6,060,443

Polyacrylate MW 4500 supplied by BASF

Carboxymethylcellulose is Finnfix® V available from CP Kelco, Arnhem, The Netherlands

CHEC is a cationically modified hydroxyethyl cellulose polymer.

Phosphonate chelating agents are, for example, diethylenetetraaminepentaacetic acid (DTPA) hydroxyethanediphosphonate (HEDP)

S-ACMC is carboxymethyl cellulose conjugated to C.I. Reactive Blue 19, product name AZO-CM-CELLULOSE

Dirt release agent is Repel-o-tex® PF

The copolymer of acrylic acid and maleic acid has a molecular weight of 70,000 and an acrylate:maleate ratio of 70:30

The color pigment is Liquitint® Violet CT polymer color pigment available from Milliken, Spartanburg, South Carolina, USA.

The amphiphilic random graft copolymer is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and a plurality of polyvinyl acetate side chains. The molecular weight of the polyethylene oxide backbone is about 6,000, and the weight ratio of polyethylene oxide to polyvinyl acetate is from about 40 to 60, with no more than 1 grafting point per 50 ethylene oxide units.

2 Polyethyleneimine (MM = 600) with 20 ethoxylate groups per -NH group.

3 The amphiphilic alkoxylated polymer is polyethyleneimine (MM 600) prepared from a polymer derivatized to contain 24 ethoxylate groups per -NH group and 16 propoxylate groups per -NH group.

4lipase and amylase are shown as milligrams of active enzyme per 100 g of detergent.

a Proxel GXL, 20% aqueous dipropylene glycol solution of 1,2-benzisothiazolin-3-one, supplied by Lonza.

b Fatty acid ester of N,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride. The iodine number of this material in the parent fatty acid is between 18 and 22. The material obtained from Evonik contains impurities in the form of a free fatty acid, a monoester form of a fatty acid ester and N,N-bis(hydroxyethyl)-N,N-dimethylammonium chloride and fatty acid esters of N,N-bis(hydroxyethyl)-N-methylamine.

c MP10® supplied by Dow Corning, 8% activity

d as described in US 8,765,659, expressed as 100% encapsulated aromatic oil

e Rheovis® CDE, a cationic polymer thickener available from BASF

f N,N-dimethyloctanamide and N,N-dimethyldecanamide in a weight ratio of about 55:45, trade name Steposol® M-8-10, Stepan Company

The dimensions and values described herein should not be understood as being strictly limited to the exact numerical values listed. On the contrary, unless otherwise indicated, each such dimension shall mean both the indicated value and a functionally equivalent range within which that value falls. For example, a size described as "40 mm" implies "about 40 mm".

--->

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35 40 45

Ala Thr Phe Leu Tyr Ser Phe Glu Asp Ser Gly Val Gly Asp Val Thr

50 55 60

Gly Phe Leu Ala Leu Asp Asn Thr Asn Lys Leu Ile Val Leu Ser Phe

65 70 75 80

Arg Gly Ser Arg Ser Ile Glu Asn Trp Ile Gly Asn Leu Asn Phe Asp

85 90 95

Leu Lys Glu Ile Asn Asp Ile Cys Ser Gly Cys Arg Gly His Asp Gly

100 105 110

Phe Thr Ser Ser Trp Arg Ser Val Ala Asp Thr Leu Arg Gln Lys Val

115 120 125

Glu Asp Ala Val Arg Glu His Pro Asp Tyr Arg Val Val Phe Thr Gly

130 135 140

His Ser Leu Gly Gly Ala Leu Ala Thr Val Ala Gly Ala Asp Leu Arg

145 150 155 160

Gly Asn Gly Tyr Asp Ile Asp Val Phe Ser Tyr Gly Ala Pro Arg Val

165 170 175

Gly Asn Arg Ala Phe Ala Glu Phe Leu Thr Val Gln Thr Gly Gly Thr

180 185 190

Leu Tyr Arg Ile Thr His Thr Asn Asp Ile Val Pro Arg Leu Pro Pro

195 200 205

Arg Glu Phe Gly Tyr Ser His Ser Ser Pro Glu Tyr Trp Ile Lys Ser

210 215 220

Gly Thr Leu Val Pro Val Thr Arg Asn Asp Ile Val Lys Ile Glu Gly

225 230 235 240

Ile Asp Ala Thr Gly Gly Asn Asn Gln Pro Asn Ile Pro Asp Ile Pro

245 250 255

Ala His Leu Trp Tyr Phe Gly Leu Ile Gly Thr Cys Leu

260 265

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Claims (190)

1. A water soluble single dose fabric laundry product comprising a water soluble film and a liquid laundry detergent composition, wherein the liquid laundry detergent composition contains a parent lipase variant that has lipase activity that has at least 70% sequence identity with SEQ ID NO: 2, contains substitutions at positions corresponding to F51I,L, T231R and N233R, and optionally one or more substitutions at positions corresponding to G23S, D27N, A40I, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S , Y220F, T244E and P256T; and an auxiliary component of the detergent; wherein the liquid laundry detergent composition is coated with a water-soluble film; wherein the water-soluble film contains a polymeric material selected from polyvinyl alcohols, polyvinylpyrrolidone, polyalkylene oxides, acrylamide, acrylic acid, cellulose, cellulose ethers, cellulose esters, cellulose amides, polyvinyl acetates, polycarboxylic acids and salts, polyamino acids or peptides, polyamides, polyacrylamide, maleic/acrylic acid copolymers, polysaccharides including starch and gelatin, natural gums such as xanthan gum and carrageenan, and mixtures thereof. 2. The single dose water-soluble product of claim 1, wherein said lipase variant contains one or more substitutions at positions corresponding to G23S, D27N, A40I, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E and P256T. 3. The single dose water soluble product of claim 1 or 2, wherein the variant contains F51I,L and substitutions corresponding to any of the following sets of substitutions:

. 4. A water soluble single dose product according to any preceding claim, wherein the variant contains substitutions corresponding to E56R+T231R+N233R and one or more substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T244E, and P256T; or wherein the variant contains substitutions at positions corresponding to R118F+T231R+N233R and one or more substitutions at positions corresponding to G23S, D27N, A40I, E56R, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T; or wherein the variant contains substitutions at positions corresponding to E56R+R118F+T231R+N233R and one or more substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E, and P256T; or wherein the variant contains substitutions at positions corresponding to E56R+R118F+T231R+N233R+P256T and one or more substitutions at positions corresponding to G23S, D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, and T244E; or in which the variant contains substitutions at positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R, and one or more substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F, T244E and P256T. 5. The single dose water soluble product of any preceding claim, wherein the variant further comprises P256T. 6. A water soluble single dose product according to any one of the preceding claims, in which the variant contains substitutions at positions corresponding to G23S+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to D27N, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to D27N+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, A40I, D57N, V60E,K, K98I, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to A40I+F51I,L+E56R+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, D57N, V60E,K, K98I, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, K98I, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, Y220F and T244E; or wherein the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+T244E+P256T and one or more substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D and Y220F; or in which the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+V60E,K+K98I+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, A40I, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions at positions corresponding to F51I,L+E56R+D57N+N101D+K98I+R118F+T231R+N233R+P256T, and one or more substitutions at positions corresponding to G23S, D27N, A40I, V60E,K, N101D, G163S, Y220F and T244E; or in which the variant contains substitutions in positions corresponding to F51I,L+E56R+D57N+V60E,K+K98I+N101D+R118F+T231R+N233R+P256T, and one or more substitutions in positions corresponding to G23S, D27N, A40I, G163S, Y220F and T244E. 7. The single dose water-soluble product according to any preceding claim, wherein the variant contains substitutions at positions corresponding to one of the following sets of substitutions: F51I+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+D27N+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; F51L+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R; F51L+D57N+E56R+R118F+T231R+N233R; F51L+K98I+E56R+R118F+T231R+N233R; F51L+G163S+E56R+R118F+T231R+N233R; F51L+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R; A40I+F51L+G163S+E56R+R118F+T231R+N233R; A40I+F51L+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+E56R+R118F+T231R+N233R; F51L+D57N+G163S+E56R+R118F+T231R+N233R; F51L+D57N+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R; F51L+K98I+E56R+R118F+T231R+N233R+P256T; F51L+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R; A40I+F51L+D57N+K98I+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+K98I+G163S+E56R+R118F+T231R+N233R+P256T; F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+D57N+K98I+G163S+E56R+R118F+T231R+N233R+P256T; A40I+F51L+E56R+D57N+K98I+R118F+G163S+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; A40I+F51I+E56R+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+A40I+F51I+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E; A40I+F51I+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+K98I+N101D+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60K+R118F+T231R+N233R+T244E+P256T; G23S+D27N+A40I+F51I+E56R+V60E+R118F+T231R+N233R+T244E+P256T; F51I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R; F51I+K98I+E56R+R118F+T231R+N233R; F51I+Y220F+E56R+R118F+T231R+N233R; F51I+E56R+R118F+T231R+N233R+T244E; F51I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R; G23S+F51I+K98I+E56R+R118F+T231R+N233R; G23S+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R; D27N+F51I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+E56R+R118F+T231R+N233R+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R; F51I+K98I+E56R+R118F+T231R+N233R+T244E; F51I+K98I+E56R+R118F+T231R+N233R+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+P256T; G23S+D27N+F51I+K98I+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+K98I+Y220F+E56R+R118F+T231R+N233R+T244E+P256T; G23S+D27N+F51I+E56R+K98I+R118F+Y220F+T231R+N233R+T244E+P256T. 8. A water-soluble single dose product according to any one of the preceding claims, wherein one or more lipase enzymes are encapsulated in a microcapsule, preferably the microcapsule membrane being prepared by cross-linking a polybranched polyamine with a molecular weight greater than 1 kDa. 9. A water-soluble single dose article according to any one of the preceding claims, wherein the cleaning composition is in the form of a liquid, preferably containing less than 20% by weight of water. 10. A water soluble single dose article according to any one of the preceding claims, wherein the cleaning composition comprises a polymer selected from carboxymethyl cellulose, hydrophobically modified carboxymethyl cellulose, or mixtures thereof. 11. The water soluble single dose article of any preceding claim, wherein the non-foaming anionic surfactant comprises a linear alkyl benzene sulfonate, an alkoxylated alkyl sulfate, or a mixture thereof. 12. The single dose water soluble product according to any one of the preceding claims, wherein the liquid laundry detergent composition comprises: a. a cationic polysaccharide selected from cationically modified hydroxyethyl cellulose, cationically modified hydroxypropyl cellulose, cationically and hydrophobically modified hydroxyethyl cellulose, cationically and hydrophobically modified hydroxypropyl cellulose, or mixtures thereof; b. alkoxylated polyethyleneimine; c. their mixture. 13. The method of washing fabrics, including the following steps: a. combining the water soluble single dose product of any preceding claim with sufficient water to dissolve the water soluble film and dilute the laundry detergent composition to form a detergent solution; b. bringing the cleaning solution into contact with at least one fabric to be washed.

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