AU2023393689A1

AU2023393689A1 - Improved lipase (gcl1) variants

Info

Publication number: AU2023393689A1
Application number: AU2023393689A
Authority: AU
Inventors: Lars Lehmann Hylling Christensen; Iben DAMAGER; Marco Malten; Anders Gunnar SANDSTRÖM; Jesper Vind
Original assignee: Novozymes AS
Current assignee: Novozymes AS
Priority date: 2022-12-14
Filing date: 2023-12-12
Publication date: 2025-05-01
Also published as: CN120265743A; WO2024126483A1; EP4634355A1

Abstract

The present invention relates to lipase variants of a GCL1 lipase. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

Description

IMPROVED LIPASE (GCL1) VARIANTS

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to lipase variants with improved stability in detergents and low malodor generation during lipid stain removal, polynucleotides encoding the variants, methods of producing the variants, and methods of using the variants.

DESCRIPTION OF THE RELATED ART

The ability of a detergent to remove stains from the surface of textiles is an obvious care-about for the customer and various surfactant ingredients play a role in that process. However, there is a desire to reduce the amount of detergent used in household care for several reasons. One reason is that some of the ingredients in detergents are derived from petrochemical resources and face scrutiny due to environmental concerns, most of all for not being sustainable because they are from a non-renewable source and are poorly biodegradable or even persistent in the environment. Another reason is that lowering the detergent concentration in the wash liquor may reduce production cost and will ultimately lead to less transportation of detergents and consequently less burden on the environment. This trend toward compaction of detergents and reduced in-wash concentration of surfactants requires the development of solutions to ensure continued performance of the detergents, including new enzymes and new use of enzymes. Lipases are included in some detergents to improve fat removal. When lipases degrade fat, short-chain fatty acids (e.g., butyric acid and hexanoic acid) can be released, leading to malodor perception. The dosage of lipase is therefore often limited in laundry detergents by the highest acceptable level of malodors, though the malodors can partly be masked by including an ester- free perfume system to the detergent formulation. Lipase has highest activity under semidry conditions, which are present during drying. The challenge with lipase odor generation is largest under wash conditions with low detergent level, since more lipases will be left on the stain after wash.

Bertolini et al (Eur. J. Biochem. 228, 863-869 (1995)) discloses Geotrichum candidum lipase I (GCL I). For unsaturated substrates having long fatty acyl chains (linoleic acid and alpha-linoleic acid) GCL I show higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains.

SEQ ID NO: 1 is disclosed in Shimada et al: cDNA Molecular Cloning of Geotrichum candidum Liase, The Journal of Biochemistry, Volume 106, Issue 3, September 1989, Pages 383-388, (world wide web: doi.org/10.1093/oxfordjournals.jbchem.a122862) and Swisss-Prot: P17573. WO 2022/162043 discloses detergent composition comprisingn GCL1 lipases having SEQ ID NO: 1 wherein the level of detergent in wash is reduced.

Nevertheless, there is a need for low malodor generating lipases that are improved over the known lipases, such as the GCL1 lipases, in particular by having improved thermostability, improved stability in detergent, lower odor generation and/or improved wash performance.

SUMMARY OF THE INVENTION

The invention relates to lipase variants comprising one or more substitutions at one or more positions corresponding to positions 396, 397, 398, 408, 409, 166 and 325 of SEQ ID NO: 1 , wherein the variant has lipase activity and wherein the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 4

The inventors of the present invention have supringly found that lipase variants comprising said one or more substitutions at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408 and 409 of the lipase of SEQ ID NO: 1 have improved properties compared to the parent lipase of SEQ ID NO: 1. The improvements include improved thermostability, improved stability in detergent, lower odor generation and/or improved wash performance as will become evident from the application.

The present invention also relates to isolated polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of producing the variants.

The present invention also relates to detergent compositions comprising a lipase of the present invention, methods of removal of lipid on a textile during a wash cycle, preferably at a reduced surfactant level, comprising contacting a surface, e.g. a textile, with a lipase of the present invention, as well as use of the lipases of the invention removal of lipid stains.

SEQUENCES

SEQ ID NO: 1 is a Geotrichum candidum lipase I (GCL I)

SEQ ID NO: 2 is a variant of SEQ ID NO: 1

SEQ ID NO: 3 is a variant of SEQ ID NO: 1

SEQ ID NO: 4 is a variant of SEQ ID NO: 1

SEQ ID NO: 5 is a commercially available lipase from Novozymes A/S

SEQ ID NO: 6 is a variant of SEQ ID NO: 1

SEQ ID NO: 7-23 are DNase/Nuclease from various organisms

SEQ ID NO: 24-47 are variants of SEQ ID NO: 1

DEFINITIONS

In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

AEP (active enzyme protein):

Enzyme protein which has a catalytic activity. There is various way to determine AEP. For example, AEP can be calculated by dividing total activities by the enzyme’s specific activity, or by active site titration. cDNA:

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

Coding sequence:

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

Control sequences:

The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the variant, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.

Detergent adjunct ingredient:

The detergent adjunct ingredient is different to the lipase of this invention. The precise nature of these additional adjunct components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the operation for which it is to be used. Suitable adjunct materials include, but are not limited to the components described below such as surfactants, builders, flocculating aid, chelating agents, dye transfer inhibitors, enzymes, enzyme stabilizers, enzyme inhibitors, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, s, s, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, builders and co-builders, fabric hueing agents, anti-foaming agents, dispersants, processing aids, solvents, and/or pigments.

Detergent composition:

The term “detergent composition” refers to compositions that find use in the removal of undesired compounds from items to be cleaned, such as textiles. The detergent composition may be used to e.g. clean textiles for both household cleaning and industrial cleaning. The terms encompass any materials/compounds selected for the particular type of cleaning composition desired and the form of the product (e.g., liquid, gel, powder, granulate, paste, bar, or spray compositions) and includes, but is not limited to, detergent compositions (e.g., liquid and/or solid laundry detergents and fine fabric detergents; fabric fresheners; fabric softeners; laundry boosters; and textile and laundry pre-spotters/pre-treatment). In addition to containing the enzyme of the invention, the detergent formulation may contain one or more additional enzymes (such as proteases, amylases, lipases, cutinases, cellulases, DNases, endoglucanases, xyloglucanases, pectinases, pectin lyases, xanthanases, peroxidases, haloperoxygenases, catalases and mannanases, or any mixture thereof), and/or detergent adjunct ingredients such as surfactants, builders, chelators or chelating agents, bleach system or bleach components, polymers (as set forth herein), fabric conditioners, foam boosters, suds suppressors, dyes, perfume, tannish inhibitors, optical brighteners, bactericides, fungicides, soil suspending agents, anti-corrosion agents, enzyme inhibitors or stabilizers, enzyme activators, bluing agents and fluorescent dyes, antioxidants, and solubilizers. The term “detergent composition” may be used interchangeably with the term “detergent”.

Detergent load

Detergent load is the amount of detergent used in a wash cycle.

Enzyme detergency benefit:

The term “enzyme detergency benefit” is defined herein as the advantageous effect an enzyme may add to a detergent compared to the same detergent without the enzyme. Important detergency benefits which can be provided by enzymes are stain removal, such as lipid stains, with no or very little visible soils after washing and/or cleaning. Expression:

The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Expression vector:

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

Extension:

The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a variant, wherein the “extended” variant has lipase activity.

Fatty acid:

A fatty acid is a carboxylic acid with an aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides or phospholipids. When they are not attached to other molecules, they are known as "free" fatty acids. Examples of fatty acids include, but are not limited to, butanoic acid (butyric acid), pentanoic acid (valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthic acid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), decanoic acid (capric acid), dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid), eicosanoic acid (arachidic acid) oleic acid, palmitoleic acid linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. It is to be understood that in the context of this invention, a fatty acid and an acyl group of a lipid are equivalents. When the fatty acid is an acyl group of a lipid, the lipid can be a monoglyceride, diglyceride, triglyceride, phospholipid, sphingolipid, galactolipid, sterolester or wax ester. The acyl group may be saturated or unsaturated, and optionally functional groups (substituents) may be attached. Examples of acyl groups include, but are not limited to, the acyl forms of butanoic acid (butyric acid), pentanoic acid (valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthic acid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), decanoic acid (capric acid), dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid), eicosanoic acid (arachidic acid), linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, oleic acid, palmitoleic acid, and docosahexaenoic acid. Fragment:

The term “fragment” means a variant having one or more amino acids absent from the amino and/or carboxyl terminus of the variant; wherein the fragment has lipase activity.

Fungal:

In the context of the present invention the term “fungal” in relation to polypeptide (such as an enzyme, e.g. a lipase) refers to a polypeptide encoded by and thus directly derivable from the genome of a fungus, where such fungus has not been genetically modified to encode said polypeptide, e.g. by introducing the encoding sequence in the genome by recombinant DNA technology. In the context of the present invention, the term “fungal lipase” or “polypeptide having lipase activity obtained from a fungal source” thus refers to a lipase encoded by and thus directly derivable from the genome of a fungal species, where the fungal species has not been subjected to a genetic modification introducing recombinant DNA encoding said lipase. Thus, the nucleotide sequence encoding the fungal polypeptide having lipase activity is a sequence naturally in the genetic background of a fungal species. The fungal polypeptide having lipase activity encoding by such sequence may also be referred to a wildtype lipase (or parent lipase). In a further aspect, the invention provides polypeptides having lipase activity, wherein said polypeptides are substantially homologous to a fungal lipase. In the context of the present invention, the term “substantially homologous” denotes a polypeptide having lipase activity which is at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, and most preferably at least 99% identical to the amino acid sequence of a selected fungal lipase. The polypeptides being substantially homologous to a fungal lipase may be included in the detergent of the present invention and/or be used in the methods of the present invention.

Half-life

The term “half-life” is the time it takes for an enzyme to lose half of its enzymatic activity under a given set of conditions. It is denoted as T% or T¹Z> and is measured at a suitable time scale. Halflife can be calculated at a given detergent concentration and-storage-temperature (in this case at roomtemperature) for a Wild-type control and/or variants, as the degradation follows an exponential decay and the incubation time (hours) is known.

Half-life improvement factor

The term "Half-life improvement factor" or "HIF" is the improvement of half-life of a variant compared to the parent polypeptide, such as a parent lipase. A half-life improvement factor (HIF) under a given set of storage conditions (detergent concentration and temperature) can be calculated as: where the reference (e.g., the wild-type (wt)) is incubated under the same storage condition as the variant. In the cases where the wild-type is not stable in the give detergent concentration a more stable variant of the wild-type may be used as reference.

A preferred way of calculating HIF is described in the examples herein.

Heterologous:

The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

Host Strain or Host Cell:

A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a variant has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

Improved property:

The term “improved property” means a characteristic associated with a variant that is improved compared to the reference enzyme/parent enzyme. Such improved properties include, but are not limited to reduced odor generation i.e. odor reduction, improved thermostabilty, improved halflife in detergent (’’detergent stability”) and improved wash performance (WP). Some aspects of the invention relate to variants having an improvement factor above 1 when the variant is tested for a property of interest in a relevant assay, wherein the property of the reference enzyme/parent enzyme is given a value of 1. Improved properties may be determined as described in the experimental section.

Introduced:

The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

Isolated:

The term “isolated” means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell. Laundering:

The term “laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition and optionally one or more enzymes. The laundering process can for example be carried out using e.g. a household or an industrial washing machine or can be carried out by hand.

Lipase:

The terms “lipase”, “lipase enzyme”, “lipolytic enzyme”, “lipid esterase”, “lipolytic polypeptide”, and “lipolytic protein” refers to an enzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may have lipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-ester hydrolase activity (EC3.1.1.50). In this context a “lipase substrate” is any substrate which can be hydrolyzed by the lipase of the invention. For purposes of the present invention lipase activity (i.e. the hydrolytic activity of the lipase) may be determined with a pNP assay using substrates with various chain length as described in Example 1.

Lipase variant:

The terms “lipase variant” refers to a lipase where one or more mutations have been introduced when the lipase variant is aligned with a parent lipase.

Malodor:

The term ’’malodor” means an odor which is not desired on clean items. Malodor can be quantified by SPME-GC as released butyric acid or assessed by sensory panel scoring. Unless otherwise specified the term malodour may be used interchangeably with the term odor.

Mature polypeptide:

The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing and/or C-terminal processing (e.g., removal of signal peptide) as well as glycosylation and phosphorylation.

Mature polypeptide coding sequence:

The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having lipase activity.

Mutant:

The term “mutant” means a polynucleotide encoding a variant.

Mutation

The term “mutation” refers to a deletion (including a truncation), insertion (including an extension) or substitution in a parent polypeptide. Native:

The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

Nucleic acid:

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

Nucleic acid construct:

The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.

Operably linked:

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

Parent or parent lipase:

The term “parent”, “parent enzyme” or “parent lipase” means a lipase to which an alteration is made to produce the enzyme variants of the present invention. In the context of the present invention lipase having SEQ ID NO: 1 as well as the lipase having SEQ ID NO: 4 tcan be considered a parent lipase.

Purified:

The term “purified” means a nucleic acid, variant or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

In one aspect, the term "purified" as used herein refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

Accordingly, the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The variant may be "substantially pure", /.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

It is, therefore, preferred that the substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The variant of the present invention is preferably in a substantially pure form (/.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.

Recombinant:

The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

Recover:

The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and/or purification of the polypeptide.

Rhamnolipid:

Rhamnolipid (RL) is a glycolipid that may be used as a biodegradable surfactant. RL may be in the form of mono-rhamnolipid or di-rhamnolipid, which consist of one or two rhamnose groups respectively, wherein the length of the chain may vary: m,n being 4 to 8.

(Appl Microbiol Biotechnol (2005) 68: 718-725).

In the context of the present invention the term “rhamnolipid” includes mono-rhamnolipid or dirhamnolipid, mixtures thereof and varying chain length as well as salts of rhamnolipid.

Sequence difference:

The term "sequence difference" means the percent of amino acid differences between a polypeptide and the polypeptide of SEQ ID NO: 1 , and is calculated as follows:

(Different Residues x 100)/(Length of SEQ ID NO: 1) wherein the different residues comprise any substitution, deletion, or insertion (e.g., an extension at the N-terminus and/or C-terminus) in the sequence.

Sequence identity:

The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)

Signal Peptide:

A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.

Sophorolipid:

The term “sophorolipid” include sophorolipid in the lactone form and the corresponding acidic form as well as mixtures thereof. Further “sophorolipid” also includes salts of sophorolipid.

Subsequence:

The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity.

Substantially the same:

The term “substantially same” in the present invention is within the reasonable understanding of those skilled in the art, and may mean that the level of lipid removal of different detergent compositions is similar or no obvious difference, for example, the difference in the level of lipid removal is within e.g. 1%, 2% or 3% depending on the experimental errors.

Sustainability:

Sustainability and sustainable means use of renewable resources that cause little or no damage to the environment and are biodegradable. Sustainability profile:

In the context of the present invention the term sustainability profile is used for comparing the sustainability of ingredients (e.g. in a detergent composition) where one or more ingredients can replace other less sustainable ingredients while maintaining the performance of the system (e.g. the performance of a detergent composition during wash of an item).

Textile:

The term “textile” means any textile material including yarns, yarn intermediates, fibers, nonwoven materials, natural materials, synthetic materials, and any other textile material, fabrics made of these materials and products made from fabrics (e.g., garments and other articles). The textile or fabric may be in the form of knits, wovens, denims, non-wovens, felts, yarns, and toweling. The textile may be cellulose based such as natural cellulosics, includ-ing cotton, flax/linen, jute, ramie, sisal or coir or manmade cellulosics (e.g. originating from wood pulp) including viscose/rayon, cellulose acetate fibers (tricell), lyocell or blends thereof. The textile or fabric may also be non-cellulose based such as natural polyamides including wool, camel, cashmere, mohair, rabbit and silk or synthetic polymers such as nylon, aramid, polyester, acrylic, polypropylene and spandex/elastane, or blends thereof as well as blends of cellulose based and non-cellulose based fibers. Examples of blends are blends of cotton and/or rayon/viscose with one or more companion material such as wool, synthetic fiber (e.g. polyamide fiber, acrylic fiber, polyester fiber, polyvinyl chloride fiber, polyurethane fiber, polyurea fiber, aramid fiber), and/or cellulose-containing fiber (e.g. rayon/viscose, ramie, flax/linen, jute, cellulose acetate fiber, lyocell). Fabric may be conventional washable laundry, for example stained household laundry. When the term fabric or garment is used it is intended to include the broader term textiles as well. In the context of the present invention, the term “textile” also covers fabrics. In the context of the present invention, the term “textile” is used interchangeably with fabric and cloth.

Variant:

The term “variant” means a polypeptide having enzyme, e.g. lipase, activity comprising a substitution, an insertion (including extension), and/or a deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position. The lipase variants of the present invention have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity, but less than 100% identity, to the polypeptide of SEQ ID NO: 1. In the context of the present invention the terms “variant” and “lipase variant” may be used interchangeably unless it is clear from the context that the variant refers to another enzyme class. Wash cycle:

The term “wash cycle” is defined herein as a washing operation wherein textiles are immersed in the wash liquor, mechanical action of some kind is applied to the textile in order to release stains and to facilitate flow of wash liquor in and out of the textile and finally the superfluous wash liquor is removed. After one or more wash cycles, the textile is generally rinsed and dried.

Wash liquor:

The term “wash liquor” refers to an aqueous solution containing a detergent composition in dilute form, such as as the wash liquor in a laundry process.

Wash performance:

The term “wash performance” is used as detergent composition’s, enzyme’s or polymer’s capability to remove stains present on the object to be cleaned or maintain color and whiteness of textile during wash. The improvement in the wash performance may be quantified by lipid removal or odor generation as described in the Experimental section.

Weight percentage:

Weight percentage is abbreviated w/w%, wt% or w%. The abbreviations are used interchangeably.

Wild-type:

The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild- type sequence).

CONVENTIONS FOR DESIGNATION OF VARIANTS

For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid positions in another lipase. The amino acid sequence of another lipase is aligned with the polypeptide disclosed in SEQ ID NO: 1 , and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IIIPAC single letter or three letter amino acid abbreviation is employed.

Substitutions

For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as “Thr226Ala” or “T226A”. Multiple mutations are separated by addition marks (“+”) or comma “G205R.S411 F” representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively.

Deletions

For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of glycine at position 195 is designated as “Gly195*” or “G195*”. Multiple deletions are separated by addition marks (“+”) or commas e.g., “Gly195* + Ser411*” or “G195* + S411*”.

Insertions

For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after glycine at position 195 is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is indicated as “Gly195GlyLysAla” or “G195GKA”.

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

Multiple alterations

Variants comprising multiple alterations are separated by addition marks (“+”) or by commas e.g., “Arg170Tyr+Gly195Glu”, “R170Y+G195E”, “Arg170Tyr,Gly195Glu” or “R170Y,G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively. Different alterations.

Where different alterations can be introduced at a position, the different alterations are separated by a comma or slash e.g., “Arg170Tyr,Glu” or “Arg170Tyr/Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala + Arg170Gly,Ala” or “Tyr167Gly/Ala + Arg170Gly/Ala” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

Unspecified amino acid

Unless otherwise limited further, the amino acid X (or Xaa) is used herein to represent any of the 20 natural amino acids.

An “X” preceding a position means that any original amino acid at that position may be substituted. For example, X93Q means that any amino acid residue at position 93 other than Q is substituted with Q. This allows for designation of substitution to a particular amino acid in different parent mannanases, where the original amino acid may vary among different parent polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to lipase variants, comprising a mutation, such as a substitution, at one or more positions corresponding to positions 396, 397, 398, 408, 409, 166 and 325 of the polypeptide of SEQ ID NO: 1 , wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the polypeptide of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, the variant has at most 4%, such as at most 3% or 2% sequence differences compared to the parent, e.g., the polypeptide of SEQ ID NO: 1 , wherein the variant has lipase activity.

The variants may further comprise an extension of one or more amino acids at the N-terminal and/or C-terminal ends.

Alternatively, the variants may further comprise a truncation of one or more amino acids at the N- terminal and/or C-terminal ends.

The inventors of the present invention have surprisingly found that the above-mentioned variants have a variety of improved properties, in particular improved stability in detergent (expressed as Half-life Improvement) and improved thermostability over the parent lipase of SEQ ID NO: 1 , while maintaining good wash performance and low odor generation even when the detergent load is reduced compared to current standard detergent load: the current standard load of detergent in wash liquour leads to a surfactant concentration of about 1 g/L whereas the variants of the present invention are uselful in a surfactant concentration of about 0.25 g/L. Variants

In one aspect, the amino acid sequence of the variant of the present invention differs from a parent enzyme such as SEQ ID NO: 1 by 1-20, e.g., 1-10, 1-7 or 1-5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations.

In another aspect, a variant comprises a substitution at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at two positions corresponding to any of positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at three positions corresponding to any of positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at four positions corresponding to any of positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at five positions corresponding to any of positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at six positions corresponding to any of positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, a variant comprises a substitution at each position corresponding to positions 166, 325, 396, 397, 398, 408 and 409 of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4, wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 166 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 166 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Leu, Trp or Gly, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of T166L, T166W and T166G in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 325 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 325 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of the substitution G325D or G325E in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4, and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 396 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 396 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Ala, Arg, Asn, Asp, Gin, Glu, His, lie, Met, Pro, Ser, Trp or Vai, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of G396E, G396Q, G396R, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396D, G396K and G396H in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 397 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 397 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asn, Asp, Glu or Thr, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of S397D, S397N, S397T and S397E in the corresponding positions of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 398 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 398 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Vai, preferably with Ala, Arg, Asp, lie, Leu, Lys, Met, Pro, Ser, Thr, Tyr, or Vai, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of W398P, W398L, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, W398M, W398D in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4, and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 408 of SEQ ID NO: 1 , wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 408 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Ala, Arg, Asp, Gin, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of I408R, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408W, I408V, I408H, I408Y and I408F in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4, and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of a substitution at a position corresponding to position 409, wherein the variant has lipase activity. In another aspect, the amino acid at a position corresponding to position 409 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Ser, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Arg, Asn, Asp, Gin, Glu, His, Lys, Ser or Tyr, wherein the variant has lipase activity. In another aspect, the variant comprises or consists of a substitution selected from the group consisting of L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y and L409S in the corresponding position of the polypeptide of SEQ ID NO: 1 , wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4, and wherein the variant has lipase activity.

In another aspect, the variant comprises or consists of two, three, four, five, six or seven substitutions in the corresponding position of the polypeptide of SEQ ID NO: 1 selected from the list below, wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity: 166+325 166+396 166+397 166+398 166+408 166+409 325+396 325+397 325+398 325+408 325+409 396+397 396+398 396+408 396+409 397+398 397+408

397+409

398+408

398+409

408+409

166+325+396

166+325+397

166+325+398

166+325+408

166+325+409

166+396+397

166+396+398

166+396+408

166+396+409

166+397+398

166+397+408

166+397+409

166+398+408

166+398+409

166+408+409

325+396+397

325+396+398

325+396+408

325+396+409

325+397+398

325+397+408

325+397+409

325+398+408

325+398+409

325+408+409

396+397+398

396+397+408

396+397+409

396+398+408

396+398+409

396+408+409

397+398+408

397+398+409 397+408+409

398+408+409

166+325+396+397

166+325+396+398

166+325+396+408

166+325+396+409

166+325+397+398

166+325+397+408

166+325+397+409

166+325+398+408

166+325+398+409

166+325+408+409

166+396+397+398

166+396+397+408

166+396+397+409

166+396+398+408

166+396+398+409

166+396+408+409

166+397+398+408

166+397+398+409

166+397+408+409

166+398+408+409

325+396+397+398

325+396+397+408

325+396+397+409

325+396+398+408

325+396+398+409

325+396+408+409

325+397+398+408

325+397+398+409

325+397+408+409

325+398+408+409

396+397+398+408

396+397+398+409

396+397+408+409

396+398+408+409

397+398+408+409

166+325+396+397+398 166+325+396+397+408

166+325+396+397+409

166+325+396+398+408

166+325+396+398+409

166+325+396+408+409

166+325+397+398+408

166+325+397+398+409

166+325+397+408+409

166+325+398+408+409

166+396+397+398+408

166+396+397+398+409

166+396+397+408+409

166+396+398+408+409

166+397+398+408+409

325+396+397+398+408

325+396+397+398+409

325+396+397+408+409

325+396+398+408+409

325+397+398+408+409

396+397+398+408+409

166+325+396+397+398+408

166+325+396+397+398+409

166+325+396+397+408+409

166+325+396+398+408+409

166+325+397+398+408+409

166+396+397+398+408+409

325+396+397+398+408+409; and 166+325+396+397+398+408+409 .

In a particular embodiment the variant of the invention comprises a substitution in the corresponding position of the polypeptide of SEQ ID NO: 1 selected from the list below, wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity:

T166L/W/G+G325D/E

T166L/W/G+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K

T166L/W/G+S397D/N/T/E T166L/W/G+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+I409R/K/H/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K

G325D/E+S397D/N/T/E

G325D/E+W398P/L/YA//A/I/R/T/K/S/M/D

G325D/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

G325D/E+I409R/K/H/N/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I409R/K/H/N/E/Q/D/Y/S

S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K

T166L/W/G+G325D+S397D/N/T/E

T166L/W/G+G325D+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+G325D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+G325D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

G325D+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E

G325D+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D

G325D+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

G325D+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I409R/K/H/N/E/Q/D/Y/S

G325D+S397D/N/T/E+W398P/L/YA//A/I/R/T/K/S/M/D G325D+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

G325D+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

G325D+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/WA//H/Y/F

G325D+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

G325D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/ L/W/V/H/Y/F

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/ D/Y/S

S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E

T166L/W/G+G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H

/Y/F

T166L/W/G+G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+G325D/E+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+G325D/E+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+G325D/E+W398P/L/Y //A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/

V/H/Y/F

T166L/W/G+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/

M/S/G/A/K/P/L/WA//H/Y/F

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D+I409R/K/H/N/

E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409

R/K/H/N/E/Q/D/Y/S T166L/W/G+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/

Y/F

T166L/W/G+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W //H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H

/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D

G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W // H/Y/F

G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+S397D/N/T/E+I409R/K/H/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/

S/G/A/K/P/L/W/V/H/Y/F

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D+I409R/K/H/N/E/

Q/D/Y/S

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/

K/H/N/E/Q/D/Y/S

G325D/E+S397D/N/T/E+W398P/L/YA//A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/

F

G325D/E+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

G325D/E+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

G325D/E+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N

/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E

/M/S/G/A/K/P/L/WA//H/Y/F

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I409R/K/H/N

/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I40

9R/K/H/N/E/Q/D/Y/S

G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/

L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/

H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/

T/K/S/M/D

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/

A/K/P/L/W/V/H/Y/F

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I409R/K/H/N/E/Q/D/

Y/S T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/YA//A/I/R/T/K/S/M/D+I40

8R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/Y/V/A/I/R/T/K/S/M/D+I40

9R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H

/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+S397D/N/T/E+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P

/L/W/V/H/Y/F

T166L/W/G+G325D/E+S397D/N/T/E+W398P/L/Y //A/I/R/T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q

/D/Y/S

T166L/W/G+G325D/E+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+

I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+

I408R/D/Q/E/M/S/G/A/K/P/L/WA//H/Y/F

I409R/K/H/N/E/Q/D/Y/S

V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/

M/S/G/A/K/P/L/WA//H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/

Y/F+I409R/K/H/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I4

08R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F

09R/K/H/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/A //M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/A/K/P/L/W //

H/Y/F+I409R/K/H/N/E/Q/D/Y/S

S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

F+I409R/K/H/N/E/Q/D/Y/S

/M/S/G/A/K/P/L/WA//H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/ T/K/S/M/D+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+I408R/D/Q/E/M/S/G/ A/K/P/L/WA//H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+W398P/L/Y //A/I/R/T/K/S/M/D+I40 8R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+S397D/N/T/E+W398P/L/Y //A/I/R/T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P /L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+ I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

G325D/E+G396E/Q/R/W/S/AA//M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/T/K/S/M/D+I4 08R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

T166L/W/G+G325D/E+G396E/Q/R/W/S/A/V/M/P/N/I/D/H/K+S397D/N/T/E+W398P/L/Y/V/A/I/R/ T/K/S/M/D+I408R/D/Q/E/M/S/G/A/K/P/L/W/V/H/Y/F+I409R/K/H/N/E/Q/D/Y/S

In a particular embodiment the variant of the invention comprises a substitution in the corresponding position of the polypeptide of SEQ ID NO: 1 or of the polypeptide of SEQ ID NO: 4 wherein the numbering is according to alignment SEQ ID NO: 1 selected from the list below, wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.:

T166G

T166L

T166L+G396E

T166L+G396R

T166L+G325D/E+G396E+W398Y

T166L+G325D/E+G396E+W398Y+I408D

T166L+G325D/E+G396E+W398Y+I408F

T166L+G325D/E+G396E+W398Y+I408H

T166L+G325D/E+G396E+W398Y+I408Y

T166L+G325D/E+G396E+W398Y+L409D

T166L+G325D/E+G396E+W398Y+L409E

T166L+G325D/E+G396E+W398Y+L409Q

T166L+G325D/E+G396E+W398Y+L409R

T166L+G325D/E+G396E+W398Y+L409S

T166L+G325D/E+G396E+W398Y+L409Y

T166L+G396E+S397D+W398P+I408E T166L+G325D/E+G396E+S397D+W398P+I408E

T166L+G325D/E+G396E+S397D+W398P+I408E

T166L+G325D/E+G396E+W398Y+L409D

T166L+G396E+S397D+W398P+I408E

T166L+W398L+I408E

T166L+W398P+I408E

T166W

T166G+D330N+A332S+P369E+L475P+R483N

T166G+F256V+D330N+A332S+P369E+V473R

T166L+A332S+P369E+S392D+M523R+F543Y

T166L+A332S+P369E+S392D+R482Q+M523R+F543Y

T166L+D189G+P254A+D330N+A332S+P369E+S392D+F543H

T166L+D199R+F256V+A331 V+R408D

T166L+D199R+F256V+R409L+M505I

T166L+D257L+T259D+D330N+A332S+P369E+S392D+F543H

T166L+D257N+A332S

T 166L+ D257N + A332S+ P369 E+ F416W+ F543Y

T166L+D257N+A332S+P369E+S392D+F543Y

T166L+D257N+A332S+P369E+S392D+M523R+F543Y

T166L+D257N+A332S+R483H

T166L+D257N+A332S+S392D+R483H

T166L+D257N+A332S+S399E+R482Q+G510D

T166L+D257N+A332S+S399E+R483H+G510D

T166L+D257N+D330N+A332S+P369E

T166L+D257N+D330N+A332S+P369E+A385D

T166L+D257N+D330N+A332S+P369E+D493N

T166L+D257N+D330N+A332S+P369E+F543Y

T166L+D257N+D330N+A332S+P369E+H517R

T166L+D257N+D330N+A332S+P369E+S392D+M523R+F543Y

T166L+D257N+G325D+A332S+R482Q+F543Y

T166L+D257N+G325D+A332S+T525E+F543Y

T166L+D257N+K307R+G325D+A332S+R482Q

T166L+D330N+A332S+P369E

T166L+D330N+A332S+P369E+A385D

T166L+D330N+A332S+P369E+A385E

T166L+D330N+A332S+P369E+A385E+F543Y

T166L+D330N+A332S+P369E+A385E+S399E

T166L+D330N+A332S+P369E+F543Y T166L+D330N+A332S+P369E+H517R

T166L+D330N+A332S+P369E+L468I

T166L+D330N+A332S+P369E+S392D

T166L+D330N+A332S+P369E+S392D+D527R+F543Y

T166L+D330N+A332S+P369E+S392D+F543H

T166L+D330N+A332S+P369E+S392D+G497D+F543Y

T166L+D330N+A332S+P369E+S392D+N437Q+F543Y

T166L+D330N+A332S+P369E+S392D+R408K+F543Y

T166L+D330N+A332S+P369E+S392D+R482Q+F543Y

T166L+D330N+A332S+P369E+S392D+R482Q+M523R+F543Y

T166L+D330N+A332S+P369E+S392D+S399E

T166L+D330N+A332S+P369E+S399E+F543Y

T166L+D330N+A332S+P369E+S479D

T166L+D330N+A332S+P369E+S479D+F543Y

T166L+F256K+D330N+A332S+P369E+E396D+R482Q+H517R+F543Y

T166L+F256K+D330N+A332S+P369E+S392D+F543Y

T166L+F256K+L306R+D330N+A332S+P369E+E396D+R482Q+F543Y

T166L+F256V+A278V+P369E+R408S+L468I+H517R

T166L+F256V+D257N+D330N+A332S+P369E+L468I

T166L+F256V+D257N+R408E+L468I+G510D

T166L+F256V+D257N+V296E+R408E+L468I

T166L+ F256V+ D257N+ V296E+S392 E+ R408S+ L468I

T 166L+ F256V+ E264Q+ A278V+ P369 E+ R408S+ L4681

T166L+F256V+R408S+L468I

T 166 L+ F256V+S303 P+ K307Y+ R408S+ K440 V+ L4681

T166L+G231C+D257N+A332S+R340C+P369E+R482Q

T166L+H517R

T 166 L+ K210 R+ D330N +A332S+ P369E+S392 D+ F543Y

T166L+L194M+K210R+D330N+A332S+P369E+S392D+F543Y

T166L+L306R+D330N+A332S+P369E+S392D+F543Y

T166L+N200R+D330N+A332S+P369E+S392D+F543Y

T166L+N283Q+A332S+P369E+S392D+N437Q+F543Y

T166L+N283Q+D330N+A332S+P369E+S392D+F543Y

T166L+P254A+Y255C+D257L+T259S+D330N+A332S+P369E+S392D+F543H

T166L+P254A+Y255C+D257M+T259G+D330N+A332S+P369E+S392D+F543H

T166L+P254A+Y255F+D257L+T259G+D330N+A332S+P369E+S392D+F543H

T166L+P254A+Y255F+D257M+T259D+D330N+A332S+P369E+S392D+F543H

T166L+P254A+Y255S+D257T+T259D+D330N+A332S+P369E+S392D+F543H T166L+P254S+D257M+T259G+D330N+A332S+P369E+S392D

T166L+P254S+D257M+T259G+D330N+A332S+P369E+S392D+F543H

T166L+P254S+Y255C+D257L+T259G+D330N+A332S+P369E+S392D+F543H

T166L+P254S+Y255C+D257L+T259N+D330N+A332S+P369E+S392D+F543H

T166L+P254S+Y255C+D257S+T259S+D330N+A332S+P369E+S392D+F543H

T166L+P254S+Y255F+D257L+T259D+D330N+A332S+P369E+S392D+F543H

T166L+P254S+Y255F+D257L+T259D+D330N+A332S+P369E+S392D+F543Y

T166L+P254S+Y255F+D257T+T259S+D330N+A332S+P369E+S392D

T166L+Q226L+D330N+A332S+P369E

T166L+Q226L+D330N+A332S+P369E+F543Y

T166L+Q226L+D330N+A332S+P369E+S392D

T166L+R408E+R409L+Q515E

T166L+S479D

T166L+T235N+N237S+D257N+A332S

T166L+V228I+D257N+A332S+F416W+R483H

T166L+V228I+D257N+A332S+P369E+R482Q

T166L+V228I+D257N+K307R+A332S+T525E

T166L+V228I+F256E

T166L+Y255F+D257L+D330N+A332S+P369E+S392D+F543H

T166L+Y255F+D257L+D330N+A332S+P369E+S392D+F543Y

T166L+Y255L+F256L+D257L+D330N+A332S+P369E+S392D+F543Y

T166V+D330N+A332S+P369E

T166L+A220S+D330N+A332S+P369E+S392D+F543Y

T166L+D175E+D330N+A332S+P369E+S392D+F543Y

T166L+D209E+D330N+A332S+P369E+S392D+F543Y

T166L+D209G+D330N+A332S+P369E+S392D+F543Y

T166L+D209N+D330N+A332S+P369E+S392D+F543Y

T166L+D209T+D330N+A332S+P369E+S392D+F543Y

T166L+D330N+A332S+I357L+P369E+S392D+F543Y

T 166 L+ D330N +A332S+ 1363F+ P369E+S392 D+ F543Y

T166L+D330N+A332S+I363V+P369E+S392D+F543Y

T166L+D330N+A332S+P369E+A385E+S392D+F543Y

T166L+D330N+A332S+P369E+C379F+S380P+A385E+S392D+F543Y

T166L+D330N+A332S+P369E+I378F+C379L+S380P+A385E+S392D+F543Y

T 166L+ D330N + A332S+ P369 E+ P398E+ F543Y

T166L+D330N+A332S+P369E+S392D+A509S+F543Y T166L+D330N+A332S+P369E+S392D+H517R+F543Y

T166L+D330N+A332S+P369E+S392D+L468V+F543Y

T166L+D330N+A332S+P369E+S392D+R409K+F543Y

T166L+D330N+A332S+P369E+S392D+S465N+H517R+F543Y

T166L+D330N+A332S+P369E+S392D+V473I+F543Y

T166L+D330N+A332S+P369E+S392D+V473L+F543Y

T166L+D330N+A332S+P369E+S392D+V496P+H517R+F543Y

T166L+E216Q+D330N+A332S+P369E+S392D+F543Y

T166L+F256E+R408S+L468I

T166L+F256V+A278V+A385E+R408S+L468I+H517R

T166L+F256V+A278V+V296E+R408S+L468I+G510V+H517R

T 166 L+ F256V+ D257I + R408S+ L4681

T166L+ F256V+ D257N+ R408S+ L468I +F543Y

T166L+F256V+D257N+V296E+R408K+L468I

T166L+F256V+D397E+R408S+L468I

T166L+F256V+E264Q+P369E+R408S+L468I

T166L+ F256V+ 1363G+ R408S+ L468I

T 166L+ F256V+ N283Q+ P369 E+ R408S+ N437Q+ L4681

T166L+F256V+P345L+R408S+L468I

T166L+F256V+P369E+R408S+F416W+L468I+H517R

T 166L+ F256V+ P369 E+ R408S+ L4681

T 166 L+ F256V+ P369 E+S392 L+ R408S+ L4681

T166L+F256V+R408S+L468I+F543Y

T166L+F256Y+D330N+A332S+P369E+S392D+F543Y

T166L+I213L+D330N+A332S+P369E+S392D+F543Y

T166L+I213V+D330N+A332S+P369E+S392D+F543Y

T166L+K210S+D330N+A332S+P369E+S392D+F543Y

T166L+K307R+D330N+A332S+P369E+S392D+S465N+F543Y

T166L+L227I+D330N+A332S+P369E+S392D+F543Y

T166L+L227V+D330N+A332S+P369E+S392D+F543Y

T166L+L306I+D330N+A332S+P369E+S392D+F543Y

T166L+L306R+D330N+A332S+P369E+E396D+R408K+R409K+F543Y

T166L+P208K+D330N+A332S+P369E+S392D+F543Y T166L+V228I+D330N+A332S+P369E+S392D+F543Y

T166L+Y230F+D330N+A332S+P369E+S392D+F543Y

T166L+Y255F+F256L+D257S+D330N+A332S+P369E+C379F+S380P+E381 R+A385E+S392D

+V540P+T541 E+F543R+G544A

T166L+Y255L+F256Y+D330N+A332S+P369E+S392D+F543Y

E18D+D119R+A278V+A332S+P369E+S392E+V496P+R511 K+F543Y

F135L+T166L+D330N+A332S+P369E+S392D+F543Y

T23A+D119R+A278V+A332S+P369E+R511 K+F543Y

G325D/E+G396E+S397E+I408K

C379L/F/N

S380P/Y/N

C379L+S380P

C379L+S380Y

C379L+S380N

C379F+S380P

C379F+S380Y

C379F+S380N

C379V+S380P

C379V+S380Y

C379V+S380N

G396A

G396E

G396E+I408D

G396E+I408E

G396E+I408K

G396E+I408P

G396E+I408R

G396E+S397D

G396E+S397D+I408E

G396E+S397E

G396E+S397E+I408E

G396E+S397N

G396E+S397T

G396E+S397T+W398Y

G396E+W398D+I408D

G396E+W398D+I408E

G396E+W398K+I408K G396E+W398K+I408R

G396E+W398L

G396E+W398P

G396E+W398R+I408R

G396E+W398V

G396E+I408D

G396E+S397D

G396E+S397D+I408E

G396E+S397D+W398P+I408E

G396E+S397D+W398P+I408R+L409R

G396E+S397E+I408E

G396E+S397E+W398P+I408E

G396E+W398D+I408D

G396E+W398P+I408E

G396N+S397E+W398Y

G396N+S397T+W398Y

G396E+S397D+W398P+I408S

G396E+S397D+W398L+I408E

G396E+S397D+W398Y+I408E

G396E+S397D+W398P+I408M

G396E+S397D+W398P+I408R

G396E+S397N+W398P+I408E

G396E+W398P+I408E

G396H

G396I

G396M

G396N

G396N+S397D+W398Y

G396N+S397E

G396N+S397E+W398Y

G396N+S397T

G396N+S397T+W398Y

G396N+W398Y

G396P+I408E

G396P+W398L+I408E

G396P+W398P+I408E

G396Q

G396Q+S397D+W398P+I408E G396Q+S397T+W398Y

G396Q+S397D+W398Y

G396Q+S397E+W398Y

G396Q+S397T

G396Q+S397T+W398Y

G396R

G396R+I408D

G396R+I408E

G396R+I408K

G396R+I408R

G396R+S397D

G396R+S397T

G396R+W398K+I408K

G396R+W398R

G396R+W398R+I408K

G396S

G396V

G396W

S397D

S397D+W398Y

S397D+W398P+I408E

S397E+W398P+I408E

S397E+W398Y

S397T

S397T+W398Y

W398A

W398K

W398L

W398P

W398P+I408E

W398Y+I408E

W398R

W398T

W398V

W398Y

I408A

I408E

I408G I408K

I408L

I408M,

I408Q,

I408R, and

I408S.

In a further particular embodiment the variant of the invention comprises a substitutions in SEQ ID NO:4 selected from the list below, wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or at least 98% to the amino acid sequence of the lipase of SEQ ID NO: 4 and wherein the variant has lipase activity.:

A114E+D330N+A332S+P369E

A114E+T166L+D330N+A332S+P369E+S392D+F543Y

A114E+T166L+L306R+D330N+A332S+K343R+P369E+S399K+F543Y

A114K+F256E+P369H+N454L

A114R+D119R+T166L+P369E+G510D

A114R+P369E+R408E+G497D+F543Y

A114R+P369E+R408E+R409L+G497D+F543Y

A114R+P369E+R408E+R409L+G497D+H517R+F543Y

A114R+T166L+Q226L+D257N+A332S+S399E+G510D

A114R+Y129H+T166L+A385E+D397S+R408E+D493N

A114R+Y146L+T166L+D257N+A332S

A114R+Y146L+T166L+D257N+A332S+S399E+G510D

A114Y+H517R

A 132G+ P369A+ R408 E+ R409 L

A224T+F310S+D330N+A332S+P369E

A2E+L72T+V126F+Y129H+P369E+R408E

A30T+L72T+V126F+Y129H+V228I+P369E+R408E

V126F+Y129H+P369E+S392L+R408E

A332S

A332S+P369R+R408E

A332S+P369R+R408K

A509C

A5V+G19D+K53R+V76L+T166L+D257N+G325D+A332S

A69G+A332S

A69G+G115S+F256E+P369H+W374Y A69G+I83P+A202V+F256E+W374V

A69G+I83Y+T183N+F256E

D119R+A278V+A332S+P369E+F543Y

D119R+A278V+S294D+P369E+G510D+H517R+F543Y

D119R+A332S+P369E

D119R+A332S+P369E+G510D

D119R+A385D+H517R

D119R+F256E+A332S+A385D

D119R+F256E+A385D+R482Q

D119R+F256E+A385D+S392E

D119R+F256E+A385D+S392E+R482Q

D119R+F256E+G325D+A385D

D119R+F256E+P369E+G510D

D119R+F256E+P369E+Q515E

D119R+F256E+Q515E

D119R+F256E+S399E+F543Y

D119R+F256K+P369E+G510D

D119R+F256S+P369E+G510D

D119R+F256V379+P369E+G510D

D119R+G325D+K343R+P369E+G510D

D119R+K343R+P369E+D441 H+G510D

D119R+K343R+P369E+G510D+T525E+F543Y

D119R+K343R+P369E+R483H+G510D

D119R+P252H+F256V+T368E+S392E+G510D

D119R+P369E+G510D

D119R+P369E+R408E

D119R+P369E+R408E+G497D+F543Y

D119R+P369E+R408E+G510D

D119R+P369E+S392D+G510D

D119R+P369E+S392E+G510D

D119R+P369E+S392E+S399E+G510D+H517R

D119R+Q248E+K343R+P369E+D441 H+G510D

D119R+S294D+V296E

D119R+S479D+H517R

D119R+T166L+D257N+K307R+A332S+F416W

D119R+T166L+D257N+K307R+A332S+T525E

D119R+T166L+D330N+A332S+P369E+A385E+S392E+R482Q+F543Y

D119R+T166L+F256V+A278V+V296E+A385E+R408S+L468I D119R+T166L+K343R+P369E+G510D+T525E

D119R+T166L+L306R+G325D+D330N+A332S+P369E+A385E+S392E+F543Y

D119R+T368E+P369E+S392E+G510D

D119R+T368E+S392E

D119R+T525E

D119R+V296D+P369E+S392E+G510D

D119R+V296D+S392E

D119R+Y129H+E264Q+P369R+R408E

D119R+Y129H+T166L+A385E+D397S+R408E+D493N

D119R+Y129H+T166L+A385E+D397S+R408E+D493N+H517R

D119R+A202T+A278V+A332S+P369E+Y472K+F543Y

D119R+A278V+A331S+A332S+P369E+R408E+H517R+F543Y

D119R+A278V+A331S+A332S+P369E+S392E+S399E+R408E+F543Y

D119R+A278V+A332S+P369E+A509S+R511K+F543Y

D119R+A278V+A332S+P369E+G510D+R511 K+Q515E+F543Y

D 119R+A278V+A332S+ P369E+ R408K+ F543Y

D119R+A278V+A332S+P369E+R511 K+F543Y

D 119R+A278V+A332S+ P369E+S392 D+ R482Q+ F543Y

D119R+A278V+A332S+P369E+S392D+S399E+R408E+A509V+F543Y

D119R+A278V+A332S+P369E+S392D+S479D+A509S+F543Y

D119R+A278V+A332S+P369E+S392E+F543Y

D119R+A278V+A332S+P369E+S392E+G510D+R511K+F543Y

D119R+A278V+A332S+P369E+S392E+V496P+R511K+F543Y

D119R+A278V+A332S+P369E+T525E+F543Y

D119R+A278V+A332S+P369E+V496P+F543Y

D119R+A278V+G325D+A332S+P369E+A509V+H517R+F543Y

D119R+A278V+L306C+A332S+P369E+A385E+V496P+F543Y

D 119R+A278V+ L306R+A332S+ P369E+A385E+A489V+ V496P+ F543Y

D119R+A278V+S294D+A332S+P369E+S399R+G510D+F543Y

D119R+A278V+S294D+A332S+T368E+P369E+G510D+S522K+F543Y

D119R+A331S+A332S+P369E+R408E

D119R+A331S+A332S+P369E+R408E+H517R

D119R+A331S+P369E+R408E+L475S+H517R

D119R+A331S+P369E+S392E+R408E+F543Y D 119R+D 175P+A278V+A332S+ P369E+ F543Y

D119R+D199R+A278V+L309E+A332S+P369E+S392D+F543Y

D 119R+ D 199R+ N200Q+ V228I + P369E+ R408E

D119R+D257N+A278V+N283Q+A332S+P369E+N437Q+F543Y

D119R+E264M+A278V+A332S+P369E+S392E+G510D+F543Y

D119R+E264Q+A278V+A332S+P369E+S392E+R482Q+F543Y

D119R+F256E+A278V+A332S+P369E+G510D+F543Y

D119R+F256E+A278V+A332S+P369E+S392E+F543Y

D119R+F256E+A278V+A332S+P369E+S479D+V496P+F543Y

D119R+F256K+P369E+S392E+L468I+G510D

D119R+G181 E+P369E+G510D

D119R+I213V+V223I+F256K+P369E+S392E+L468I+G510D

D119R+K210R+V228I+A278V+A332S+K343R+P369E+F543Y

D119R+L227M+F256K+P369E+S392E+L468I+G510D

D119R+N283Q+P369E+N437Q+G510D

D119R+P369E+C379V+G510D

D119R+P369E+R408E+F416W+R482Q+F543Y

D119R+P369E+R408E+R482Q+A509V+F543Y

D119R+P369E+R408E+R482Q+F543Y

D119R+P369E+S392D+R408E+A509V+F543Y

D 119 R+ P369E+S392 E+ R408 E+ A509V+ F543Y

D119R+P369E+S392E+R408E+R482Q+F543Y

D119R+P369E+S392G+R408E+R482Q+F543Y

D119R+Q226H+F256K+P369E+S392E+L468I+G510D

D119R+T166L+F256K+P369E+R408S+L468I

D 119 R+T 166L+ F256S+ V296D+ R408S+ L4681

D 119 R+T 166 L+ K210R+ A278V+ A332S+ P369E+S392 D+ F543Y

D119R+T166L+P369E+S392D+R408E+F543Y

D119R+T166L+Q226L+A278V+V296D+A332S+P369E+F543Y

D119R+V223I+F256K+P369E+S392E+L468I+G510D

D119R+V228I+A278V+A332S+P369E+S392D+G510D+F543Y

D119R+V228I+L306H+K343R+P369E+R408E

D119R+V296K+P369E+R408E+R482Q+F543Y D119R+Y129V+P369M+G510D

D119R+Y146L+A278V+A332S+P369E+F543Y

D119R+Y146L+A278V+A332S+P369E+V496P+G510D+F543Y

D119R+Y146L+E264Q+A278V+A332S+P369E+T525E+F543Y

D119R+Y146L+F256S+A278V+A332S+P369E+F543Y

D175P+P369E+R408E+R409L+G497D+F543Y

D175R+F256N+R408E

D175R+P369E+R408E+R409L

D199R+F256V+T368E+R408D

D199R+H298R+P369E+R408E+G497D+F543Y

D199R+P369E+R408E+G497D+F543Y

D199R+P369E+R408E+R409L+G497D+F543Y

D207N+D330N+A332S+P369E+A385D+F423L

D257N+D330N+S392E

D257N+P369E+R408E+R409L+F543Y

D257N+R408E+R409L+F543Y

D330N+A332S+E334G+P369E

D330N+A332S+H517R

D330N+A332S+K343R+P369E

D330N+A332S+P369E

D330N+A332S+P369E+A385D

D330N+A332S+P369E+A385D+S522K

D330N+A332S+P369E+D397E

D330N+A332S+P369E+D397K

D330N+A332S+P369E+E396D

D330N+A332S+P369E+G544A

D330N+A332S+P369E+H517R

D330N+A332S+P369E+K373N+H517N

D330N+A332S+P369E+K376N

D330N+A332S+P369E+K376Q

D330N+A332S+P369E+K440R

D330N+A332S+P369E+K440S

D330N+A332S+P369E+K440V

D330N+A332S+P369E+K440Y

D330N+A332S+P369E+K501 E

D330N+A332S+P369E+N499G

D330N+A332S+P369E+N499L D330N+A332S+P369E+N499Y

D330N+A332S+P369E+P398D

D330N+A332S+P369E+R408E

D330N+A332S+P369E+R408E+R409L+G497D+F543Y

D330N+A332S+P369E+R408K

D330N+A332S+P369E+R409G

D330N+A332S+P369E+R409K

D330N+A332S+P369E+R444D

D330N+A332S+P369E+R444G

D330N+A332S+P369E+R444K

D330N+A332S+P369E+R444N

D330N+A332S+P369E+R444P

D330N+A332S+P369E+R444Q

D330N+A332S+P369E+R482Q

D330N+A332S+P369E+S392R

D330N+A332S+P369E+S399D

D330N+A332S+P369E+S399E

D330N+A332S+P369E+S399E+L475S+S522R

D330N+A332S+P369E+S399R

D330N+A332S+P369E+S522K

D330N+A332S+P369E+V473L

D330N+A332S+P369E+W374R

D330N+A332S+R408E

D330N+A332S+R408E+H517R

D330N+A332S+R408E+R409L

D330N+P369E+S392E

D441 H+H517R

D493N+H517R

D56E+D175Y+P208Y+F256E

D56E+ F 159V+F256E+Y377G+ L4121

D56E+F256E+A278V+S294Q

D56E+F256E+Y377G+L412I

D56K+D119R+P369E+S392E+R408E+F543Y

E264M+P369E+R408E+R409L+G497D+F543Y

E264S+P369E+R408E+R409L+G497D+F543Y

F159V

F159V+A202V+P369E+A385E+S392E+E396Q+D397T+P398Y+R408I+R409L

F159V+E264Q+A332S F159V+E264Q+S479D

F159V+L468I

F159V+R408E+R409L

F159V+R408E+R409L+H517R

F159V+R408K

F159V+R408K+L468I

F 159V+T166L+ F256E+P369H +W374G+ F458Y

F159V+T166L+V228I+D257N+A332S+P369E

F256E

F256E+A332S+S392E+F543Y

F256E+A332S+S392 E+ R482Q

F256E+D257N+G325D+S392E

F256E+D330N+A332S

F256E+F543Y

F256E+G325D

F256E+G325D+S392E+F543Y

F256E+G497D

F256E+G497D+F543Y

F256E+K307T+K343E+Y472K

F256E+K307T+P369N+W374G

F256E+P369E

F256E+P369E+F543Y

F256E+P369H+W374G+F458Y

F256E+P369N+S392H+Y472K

F256E+Q515E

F256E+Q515E+F543Y

F256E+S294P+A331V+Q515Y

F256E+S294P+P369H+Y377G

F256E+V296D+G325D+S392E

F256G

F256K

F256K+D330N+A332S+P369E+A385Q+S479P

F256L

F256N

F256N+R408E+R409L

F256Q

F256S

F256V F256V+E264Q+A332S+S392E

F256V+ E264Q+ L468I +G510D

F256V+ E264Q+ P369E+ L468I +G510D

F256V+E264Q+T368E+H517R

F256V+E264Q+V296D+S392E+H517R

F256Y

F310S+ D330N +A332S+ P369 E

G115D+T166L+D257N+G325D+A332S+T525E+F543Y

G115E+D119R+A385E

G115E+D119R+T525E

G115E+T166L+D330N+A332S+P369E

G115E+T166L+D330N+A332S+P369E+F543Y

G115Q+D175Y+F256E+S392H

G115S+F256E+P369H+W374Y

G115Y+F256E+K343S+P369H

G115Y+F256E+W374Y+S479G

G115E+D119R+A278V+A332S+K343R+P369E+S479D+F543Y

G145D+T166L+D257N+A332S+S392D+R483H

G19D+D175Y+F256E+K343P

G19D+F256E+N350D+R511 K

G19D+I83R+F256E+V296Q

G19Q+D330N+A332S+P369E

G231C+R340C+R408E+R409L

G262C

G325D+P369E

G520R

G67D+D119R+D189G+F256E+P369E

G78D+D119R+K343R+P369E+D441 H+D504H+G51 OD

G78D+D119R+K343R+P369E+G510D+F543Y

G78D+D119R+P369E+G510D+F543Y

G78D+D119R+S392E

G78D+D119R+T166L+P369E+G510D+F543Y

G78D+D119R+V228I+K343R+P369E+G51 OD

G78D+T166L+D330N+A332S+P369E

G78D+T166L+D330N+A332S+P369E+F543Y

G78D+D119R+P369E+R408E+V473K+F543Y

G78D+G115E+D119R+A278V+A332S+P369E+S392R+F543Y G78D+T166L+V228I+F256V+R408S+L468I

G80K+R408E+R409L

G80H+K81E+D119R+A278V+A332S+P369E+T525E+F543Y

G80N+D119R+A331 S+P369E+R408E+F543Y

G80P+A95V+D119R+A278V+A332S+P369E+F543Y

G80P+D119R+A218T+A278V+A332S+P369E+A385E+R482Q+F543Y

G80P+D119R+A278V+A332S+K343R+T368E+P369E+F543Y

G80P+D119R+A278V+A332S+P369E+F543Y

G80P+D119R+A278V+A332S+T368E+P369E+S392D+F543Y

G80P+D119R+D175P+A278V+A332S+T368E+P369E+G510D+F543Y

G80P+D119R+D175P+A278V+A332S+T368E+P369E+S392D+F543Y

G80P+D119R+F256E+A278V+A332S+P369E+A385E+F543Y

G80P+ D 119R+F256E+A278V+ L309E+A332S+ P369E+ F543Y

G80P+D119R+Y146L+A278V+A332S+P369E+V496P+F543Y

I70S+D330N+A332S+P369E+V442A

I70S+V76L+D330N+A332S+P369E+H517R

I82A+P369E+R408E+R409E+T541 E

I82A+R88G+P369E+R408E+R409L+G544M

182G+ D 119 R+ A278 V+A332S+ P369 E+ F543Y

182G+ D 119 R+ A278 V+ L309 E+A332S+ P369 E+ V496P+ F543Y

182G+ D 119 R+ A278 V+ L309 E+ A332S+T368 E+ P369E+ F543Y

182G+ D 119R+ D 175P+A278V+A332S+T368E+ P369E+F543Y

I82L+T166L+D330N+A332S+P369E+S392D+G510D+F543Y

I82V+T166L+D330N+A332S+P369E+S392D+F543Y

I83P+F256E+A331V+K343Q

I83P+T183A+F256E+T541G

I83R

I83Y+A114K+F256E+W374Y

K121E+P369E+R408E+R409L+G497D+F543Y

K148R+D330N+A332S+P369E

K20M+T166L+D330N+A332S+P369E+S392D+R408K+F543Y

K210R+D330N+A332S+P369E

K210R+P369E+R408E+R409L+G544M

K210R+P369E+R408E+R409L+V496P+G544M

K25R+L72R+R408E+R409L K343P

K40M+D119R+P369E+A385E+G51 OD

K40M+P369E+R408E+G497D+F543Y

K40R+F159V+R408E+R409L

K40R+F256E

K40R+F256E+F543Y

K40R+F256E+G497D

K40R+F256E+Q515E

K40R+T166L+F256V+S279C+R408S+L468I+F543Y

K40R+A114R+T166L+F256V+P398L+R408S+L468I

K40R+G78D+T166L+F256V+D397E+R408S+L468I

K40R+K53R+T166L+D209N+F256V+P369E+R408S+L468I

K40R+K53R+T166L+F256V+P369E+R408S+L468I

K40R+Q50N+T166L+F256V+P369E+R408S+L468I

K40R+T166L+F256E+R408S+L468I

K40R+T166L+F256V+D330N+A332S+S392D+R408S+L468I

K40R+T166L+F256V+R408S+L468I+S479D+F543Y

K40R+T166L+K210R+F256V+R408S+L468I

K53R+D119R+P369K+R483H+G510D

K53R+D119R+T166L+P369E+G510D+T525E

K53R+D119R+T166L+V228I+D257N+A332S

K53R+ D 199R+ F256V+ R408S

K53R+F256V+A331 V+R408S+F416W

K53R+G78D+A114R+D119R+P369E+G510D

K53R+G78D+D119R+P369E+D441 H+G510D

K53R+G78D+D119R+P369E+G51 OD

K53R+G78D+D119R+Q248E+P369E+G510D

K53R+G78D+T166L+ F256V+R408S+ L468I + F543Y

K53R+I70S+G80Q+D330N+A332S+P369E

K53R+N68G+V76L+T166L+D257N+A332S

K53R+T166L+G231C+D257N+K307R+A332S+R340C

K53R+Y146L+T166L+D257N+A332S+T525E

K53R+T166L+F256V+R408S+L468I

K75E+*11OaP+*11ObP+*110cR+D119R+F256E+D257N

K75E+D119R+F256E+A332S

K75E+D119R+F256E+A385D

K75E+D119R+F256E+D257N K75E+D119R+F256E+S392E

K75E+D119R+Y146L+F256E

K75E+F256E+R482Q

K75E+G80H+I82R+I83Q+T166L+D257N+A332S

K75E+I82G+P369E+R408E+R409L+V496P

K75E+L79I+G80H+K81 E+I82R+I83G+T166L+D257N+A332S

K75E+L79I+G80N+K81E+I82G+I83Q+T166L+D257N+A332S

K75E+L79I+G80R+K81 E+I82R+I83G+T166L+D257N+A332S

K75E+L79V+G80E+I82R+I83R+T166L+D257N+A332S

K75E+L79V+G80R+K81 E+I82R+I83R+T166L+D257N+A332S

K75E+L79V+I82G+I83Q+T166L+D257N+A332S

K75E+N86S+D119R+T166L+F256E

K75E+P369E+R408E+R409L+G544M

K75E+P84R+D119R+F256E+D257N

K75E+R408E+R409L

K75E+V147I+L309E+P369E+R408E+R409E

K75E+V76G+K81 E+I82R+I83Q+T166L+D257N+A332S

K75E+V76G+L79V+G80S+I82R+I83Q+T166L+D257N+A332S

K75G+F256K+D330N+A332S+P369E+P398D

K75P+D330N+A332S+P369E

K75Q+L79I+K81 E+I82R+I83G+T166L+D257N+A332S+D493N

K75Q+R408E+R409L

K75Q+V261 E+P369E+R408E+R409L

K75R+T166L+P254A+Y255F+D257L+T259G+D330N+A332S+P369E+S392D+F543H

K75S+D119R+F256E+F543Y

K75S+D119R+F256E+P369E

K75S+D330N+A331 S+A332S+P369E

K75S+F256E+S392E+F543Y

K75S+R408E+R409L

K75S+T166L+F256E+G325D

K75Y+D330N+A332S+P369E

K75E+D119R+A278V+A332S+P369E+F543Y

K75E+D119R+E264M+A278V+A332S+P369E+A385E+F543Y

K75E+D119R+F256E+E264S+A278V+A332S+P369E+F543Y

K75G+ D 119 R+A278V+ A332S+ P369E+ F543Y

K75G+G80P+A114E+D119R+A278V+A332S+P369E+F543Y

K75Q+ D 119R+A278V+A332S+ K343R+ P369E+G510D+ F543Y K75Q+K81 E+D119R+A278V+A332S+K343R+P369E+G510D+F543Y

K81 D+P84E+F256E+K307T

K81Q+A114K+F256E+W374G

K81 Q+N86S+F256E+S294Q

K81 S+L309E+P369E+R408E+R409L+T541 E

L17K+N234R+D330N+A332S+P369E+R408D

L17N + V76 P+T 166 L+ F256V+ R408S+ K440S+ L4681

L17R+K53R+D330N+A331S+A332S+P369E

L306 R+ D330N +A332S+ P369 E

L309E

L309E+P369E+R408E+R409L

L309E+P369E+R408E+R409L+R483H+V496P

L309G+D330N+A332S+P369E

L37E+P369E+R408E+R409L+G497D+F543Y

L468I

L52R+K53R+N55R+N68G+D119R+K343R+P369E+G510D+T525E

L52R+L72E+M523R

L52R+N326D+H517R

L52R+S392E+M523R

L52R+S399E+H517R

L52S

L52S+G78D+T166L+D330N+A332S+P369E

L52S+S392E

L52S+S392E+D397E

L52S+S392E+E396Q

L52S+S392E+R408K

L52S+T166L+D330N+A332S+P369E+F543Y

L72D+F256E+A331V+K343P

L72 D+ F256E+Y377G+S392 H

L72 D+ P84R+ F256E+T541 G

L72E+D119R+A385D

L72E+G78D+T166L+D330N+A332S+P369E

L72E+G80S+K81E+I82R+I83R+T166L+D257N+A332S

L72E+K75E+L79I+G80R+K81E+I82G+I83R+T166L+D257N+A332S

L72E+L79V+K81E+I82R+I83R+T166L+D257N+A332S

L72E+S98D+D119R

L72E+T166L+D330N+A332S+P369E

L72E+V76A+L79I+G80D+I82R+I83R+T166L+D257N+A332S L72E+Y129H+T166L+S392D+D397S+R408E+R409L

L72Q+K75E+L79I+G80R+K81E+I82R+I83R+T166L+D257N+A332S

L72Q+K75Q+V76G+L79I+K81 E+I82R+I83R+T166L+D257N+A332S

L72Q+ V76G+ L79V+ 182G+ 183G+T166L+ D257N+A332S

L72S+D119R+Y129H+P369E+R408E

L72S+Y129H+P369E+R408E

L72T+A114D+V126F+Y129H+P369E+R408E+A480S

L72T+D199R+R408E+R409L

L72T+P369E+R408E

L72T+V126F+ P369E+S392 L+ R408E

L72T+V126F+V228I+P369E+R408E

L72T+V126F+Y129H+N237C+P369E+R408E

L72T+V126F+Y129H+P369E+R408E

L72T+V126F+Y129H+P369E+R408E+A480S

L72T+V126F+Y129H+P369E+R408E+A480S+G497D

L72T+V126F+Y129H+P369E+R408E+F416W

L72T+V126F+Y129H+P369E+R408E+L468I

L72T+V126F+Y129H+P369E+R408E+R482L

L72T+V126F+Y129H+P369E+R408E+S538T

L72T+V126F+Y129H+P369E+S392L+R408E

L72T+V126F+Y129H+V228I+P369E+R408E

L72V+K75E+G80H+K81 E+I82R+I83R+T166L+D257N+A332S

L72V+K75E+L79I+G80E+K81E+I82R+I83R+T166L+D257N+A332S

L72V+K75E+L79I+G80H+I82R+I83R+T166L+D257N+A332S

L72V+K75E+L79V+G80N+K81 E+I82R+I83R+T166L+D257N+A332S

L72V+K75E+V76A+L79I+G80H+K81 E+I82R+I83R+T166L+D257N+A332S

L72V+K75E+V76A+L79V+G80D+I82G+I83R+T166L+D257N+A332S

L72V+K75E+V76A+L79V+G80R+K81E+I82G+I83G+T166L+D257N+A332S

L72V+K75E+V76G+L79I+G80S+I82R+I83G+T166L+D257N+A332S

L72V+K75Q+V76G+G80D+I82R+I83G+T166L+D257N+A332S

L72V+N200H+P369E+R408E+R409L+T541 E

L72V+P369E+R408E+R409L+T541 E

L72 Y+ A331 V+ P369 E+ R408 E+ R409L+T541 E

L72 Y+ R88G+ P369 E+ R408 E+ R409 L+ R483 H

L72E+D119R+A278V+A332S+P369E+S392D+R511 K+F543Y

L72E+G80P+D119R+F256E+A278V+A332S+P369E+F543Y

L72E+K75G+V76L+D119R+A278V+A332S+P369E+F543Y L72I+T166L+D330N+A332S+P369E+S392D+F543Y

L72K+D119R+D257N+P369E+R408E

L72Q+V76G+D119R+A278V+A332S+P369E+L468I+F543Y

L72Q+ V76G+ D 119R+A278V+A332S+ P369E+T525E+F543Y

L72Q+V76G+D119R+A278V+A332S+T368E+P369E+T525E+F543Y

L72Q+ V76G+ D 119R+K210R+A278V+A332S+ P369E+ F543Y

L72S+D119R+G325D+P369E+P398Y+R408E

L72S+D119R+P369E+R408E

L72S+D119R+R340C+P369E+R408E

L72T+D119R+A278V+A332S+T368E+P369E+A385E+F543Y

L72T+D119R+T166L+K343R+P369A+S392D+R408E

L72T+K75E+G80P+D119R+A278V+L306R+A332S+P369E+F543Y

L72T+ K75G+ D 119 R+ D257N + A278V+ A332S+ P369E+ F543Y

L72T+V76L+D119R+Y146L+A278V+A332S+P369E+F543Y

L73G+D330N+A332S+P369E

L73K+ D330N +A332S+ P369E

L73R+D330N+A332S+P369E

L79I+I82R+I83G+T166L+D257N+A332S+F543Y

L79I+Y146L+L309E+P369E+R408E+R409L

L79V+R408E+R409L

L79F+T166L+D330N+A332S+P369E+S392D+F543Y

L87I+D119R+T166L+L306R+G325D+D330N+A332S+P369E+A385E+S392E+F543Y

L87P+A114K+F256E+V296Q

L87V+F256E+N350D+Y472K

L87F+T166L+D330N+A332S+P369E+S392D+F543Y

M94Y+D119R+G181 E+P369E+G51 OD

A95V+D119R+V228I+A278V+A332S+P369E+S392D+G510D+F543Y

N200R+P369E+R408E+R409L+G497D+F543Y

N200R+P369E+R408E+R409L+G497D+H517R+F543Y

N283Q+D330N+A332S+P369E

N283Q+D330N+A332S+P369E+N437Q

N55D+K210R+D330N+A332S+P369E

N55D+K210R+P369E+R408E+R409L+V496P+G544M

N55R+D119R+A385E

N55R+G78D+D119R+F256S+P369E+A385E+G510D N55R+G78D+S392D

N55R+G78D+S399E

N55R+G78D+S399E+H491 Q

N55D+G78D+D119R+A278V+A332S+P369E+A509S+F543Y

N55D+G78D+P90H+D119R+A278V+A332S+P369E+A509S+F543Y

N55D+I82G+D119R+ F256 E+A278V+ A332S+ P369E+ F543Y

N55D+L72T+D119R+A278V+A332S+P369E+A509S+F543Y

N55D+T166L+D330N+A332S+P369E+S392D+F543Y

N55D+V76G+D119R+A278V+A332S+P369E+G510D+F543Y

N55D+V76G+D119R+V228I+A278V+A332S+P369E+F543Y

N68G+A114R+D119R+P369E+G510D

N68G+D119R+K343R+P369E+G510D+T525E

N68G+D119R+P369E+G510D+T525E+F543Y

N68G+D119R+Q248E+K343R+P369E+G510D+T525E

N68G+D119R+T166L+P369E+G510D+T525E

N68G+D119R+T166L+P369E+T525E

N68G+D119R+T166L+V228I+P369E+G510D

N68G+D119R+V228I+G325D+P369E+G510D

N68G+D119R+V228I+G325D+P369E+S465N+G510D

N68G+F256E

N68G+F256E+G325D

N68G+L72T+P369E+R408E+R409L+G497D+F543Y

N68G+P369E+R408E+R409L+G497D+F543Y

N68G+R408E+R409L+Q515E

N68G+T166L+D257N+A332S+R482Q+T525E

N68G+T166L+D257N+G325D+A332S+T525E

N68G+T166L+D257N+K307R+A332S+P369E

N68G+T166L+F256E+D330N+A332S

N68G+T166L+V228I+F256E

N68G+T166L+V228I+F256E+D330N+A332S

N68G+V228I+F256E+D330N+A332S

N68G+V228I+F256E+G325D

N68G+V76L+T166L+D257N+A332S+F416W

N68G+Y146L+T166L+D257N+A332S+P369E

N68G+Y146L+T166L+V228I+D257N+A332S

N68L+K75S+N86L+D330N+A332S+P369E+L475R

N68Q+V76L+N86V+D207N+D330N+A332S+P369E N68G+D119R+A278V+A332S+K343E+P369E+Y472K+G510D+F543Y

N68G+G80P+A114E+D119R+A278V+A332S+P369E+F543Y

N68G+L72T+D119R+A278V+A332S+P369E+F543Y

N68L+D119R+P369E+R408E+F543Y

N68L+D119R+P369E+S392D+R408E+F543Y

N86G+D330N+A332S+P369E+V473L+S522K

N86L+D330N+A332S+P369E+A385Q+S522R

N86L+G115D+D330N+A332S+P369E+A385Q+S522R

N86P+D330N+A332S+P369E+A385E+S522R

N86P+F256K+D330N+A332S+P369E+V473K

N86R+F256E+A331V+K343S

N86R+F256E+K307T+N454L

N86S+D175Y+F256E+S294P

N86V+D330N+A332S+P369E+A385E+L500V

P208V+P369E+R408E+R409L+G497D+G544A

P118Y+T166L+F256V+R408S+L468I

P369A+R408E+R409L

P369E

P369E+G497D+F543Y

P369E+H517R

P369E+R408E

P369E+R408E+G497D+F543Y

P369E+R408E+G497D+M523R+F543Y

P369E+R408E+R409L

P369E+R408E+R409L+F543Y

P369E+R408E+R409L+G497D+A509S+R511 K+F543Y

P369E+R408E+R409L+G497D+F543Y

P369E+R408E+R409L+G497D+H517E+F543Y

P369E+R408E+R409L+G497D+Q515E+H517E+F543Y

P369E+R408E+R409L+G497D+Q515E+I519E+F543Y

P369E+R408E+R409L+G497D+R511 K+F543Y

P369E+R408I+G497D+F543Y

P3S+Y146L+T166L+A332S+P369E+S392D+F543Y

P44T+L72T+V126F+Y129H+E149V+P369E+S392L+R408E

P66H+D119R+Y129H+D330N+A332S+P369E+R408E

P84E+D175Y+F256E+N350D

P84E+S98*+F256E+P369V Q1R+T166L+D257N+A332S+P369E+T525E+F543Y

Q226L

Q226L+A229T+A278V

Q226L+D257N+R408K

Q50N

Q50N+D257N+P369E

Q50 N + F 159V+ F256V+ E264Q

Q50N+F256V+E264Q+G325D+S392E

Q50N+G325D+P369E

Q50N+L52S+P369E+R408E+R409L+G497D+F543Y

Q50N+L52S+T166L+D330N+A332S+P369E+S392D+F543Y

Q50N+Q226L+S392E

Q50N+T166L+F256V+D257N+P369E+R408S+L468I

Q50N+T166L+F256V+P369E+R408S+L468I+H517R

Q50N+T166L+S392E

Q50N+T166L+F256V+P369E+R444S+L468I

Q50N+T166L+L306R+D330N+A332S+P369E+S392D+G407C+R409K+F543Y

Q50N+T166L+L306R+D330N+A332S+P369E+S392D+R409K+F543Y

R408E+R409L+A509C+H517R

R482Q

R88Q+D330N+A331S+A332S+P369E+E396P

R88V+D330N+A332S+P369E

R88Y+D330N+A332S+P369E

S14R+D119R+F159V+P369E+G510D

S14R+D119R+P369E+G510D+T525E

S14R+D119R+T166L+V228I+G510D

S14R+D119R+T166L+V228I+P369E+G510D

S14R+D119R+T166L+V228I+P369E+L427Q+G510D

S14R+K53R+D119R+P369E+G510D+T525E

S14R+N68G+D119R+V228I+P369E+G510D

S14H+T166L+F256V+R408S+L468I

S48N+Q50S+D119R+A278V+A332S+P369E+F543Y

S48N+Q50S+D119R+D257N+A278V+A332S+P369E+F543Y

S294D+S479E+H517R

S294E

S303P+D330N+A332S+P369E

S392D+H517R S392E

S392E+D397E

S392E+H517R

S392E+L468I

S392E+R408H

S392E+S399E+H517R

S479D

S58E+F256E+W374V+T541G

S58E+G115Q+F256E+S479G

S58E+I83P+F256E+Y472K

S58K+A114L+F256E+N454L

S58K+T183A+F256E+F458Y

S58Q+A69G+F256E+Q515Y

S58Q+K81 D+F256E+V442I

S59 R+ L72Q+ P84D+ F256 E+ L4121

S59R+P369E+R408E+R409L+G497D+F543Y

S71 L+L72T+H517R

S98F+F256E+S294Q+A331 V

T178D+D330N+A332S+P369E

T178I+E264Q+P369E

T183A+F256E+A278V+Y472K

T183A+F256E+K343Q+L412I

T46N+D330N+A332S+P369E

V12T+P369E+R408E+R409L+G497D+F543Y

V12T+Q50N+L52S+T166L+D330N+A332S+P369E+S392D+F543Y

V12T+T166L+D330N+A332S+P369E+S392D+F543Y

V228I+F256E

V228I+F256E+D330N+A332S

V228I+R408E+R409L

V261 E

V261E+P369E+R408E+R409L+R483H

V296D+P369D+M523R

V296E+A385E+H517R

V6T+D56K+V76L+D330N+A332S+P369E

V76A+G115D+V261 E+K307R+P369E+R408E+R409L

V76A+G80E+I82R+I83G+T166L+D257N+A332S

V76A+ 182G+ V261 E+ P369 E+ R408 E+ R409 L

V76A+V261 E+K307R+P369E+R408E+R409L V76G+G80K+Y146L+P369E+R408E+R409L

V76G+ 182G+ P369 E+ R408 E+ R409 L+ R483H

V76G+R408E+R409L

V76L+F256K+V296K+D330N+A332S+P369E

V76L+T166L+D257N+K307R+A332S+P369E

V76L+T166L+D257N+K307R+A332S+R482Q

V76L+Y146L+T166L+D257N+A332S+F543Y

V76Q+T166L+F256V+K373N+R408S+K440R+L468I

V76S+Q96K+D119R+P369E+G510D

V76G+ D 119 R+A278V+ A332S+ P369E+ F543Y

V76G+D119R+P369E+G510D

V76I+T166L+D330N+A332S+P369E+S392D+F543Y

V76L+D119R+A278V+A332S+K343R+P369E+A385E+F543Y

V76L+D119R+A278V+A332S+P369E+F543Y

V76L+D119R+Y146L+A278V+A332S+P369E+A385E+F543Y

V76L+G78D+G115E+D119R+D209N+A278V+A332S+P369E+F543Y

V99E+D330N+A332S+P369E

V99F+P369E+R408E+D474R+G497D+F543Y

V99K+D330N+A332S+P369E

V99N+D330N+A332S+P369E

V99R+D330N+A332S+P369E

W374V

Y129H+F159V+T166L+V228I+D397S+R408E+R409L

Y129H+T166L+A385E+D397S+R408E+D474R+D493N

Y129H+T166L+A385E+D397S+R408E+D474R+D493N+H517R

Y129H+T166L+A385E+D397S+R408E+D493N

Y129H+T166L+A385E+D397S+R408E+D493N+H517R

Y129H+T166L+A385E+D397S+R408E+R409L+D493N

Y129H+T166L+D175R+D199R+A385E+D397S+R408E+R409L+D493N

Y129H+T166L+D199R+A331V+A385E+D397S+R408E+D493N

Y129H+T166L+D199R+A385E+D397S+R408E+D493N

Y129H+T166L+D199R+A385E+D397S+R408E+D493N+H517R

Y129H+T166L+D330N+A332S+P369E

Y129H+T166L+E396D+D397S+R408E+R409L+T525E

Y129H+T166L+N200R+A385E+D397S+R408E+D493N+H517R

Y129H+T166L+Q226L+E396D+D397S+R408E+R409L+G520D

Y129H+T166L+Q226L+E396D+D397S+R408E+R409L+R483H+G520D Y129R+T166L+D175R+D330N+A332S+P369E+P398D+R482Q+F543Y

Y146L

Y146L+F256V+E264Q+V296D

Y146L+F256V+E264Q+V296D+T368E

Y146L+F256V+V296D+G325D+P369E

Y146L+K210R+P369E+R408E+R409Q

Y146L+K307R+P369E+R408E+R409L+G544M

Y146L+T166L+A332S+P369E+S392D+F543Y

Y146L+T166L+A332S+P369E+S392D+M523R+F543Y

Y146L+T166L+D257N+A332S+F416W+T525E

Y146L+T166L+D257N+A332S+P369E+S392D+F543Y

Y146L+T166L+D257N+D330N+A332S+P369E+S392D+M523R+F543Y Y146L+T166L+D257N+S291 N+D330N+A332S+P369E+S392D+F543Y Y146L+T166L+F256K+D330N+A332S+K343R+P369E+E396D+F543Y Y146L+T166L+K210R+F256V+R408S+L468I+H517R

Y146L+T166L+L306R+D330N+A332S+P369E+E396D+R409K+F543Y

Y472R

Additional amino acid changes of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein, may be introduced; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a 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 generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like. Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for lipase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et a/., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and/or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

The variants may consist of 520 to 570 amino acids, e.g., 530 to 560, 535 to 555, and 540 to 550 amino acids.

In an embodiment, the variant has improved Half-life compared to the lipase of SEQ ID NO: 1 ,

1.e., the Half-life Improvement Factor (HIF) determined as described in Example 1 is above 1 , preferably above 1.1 , such as above 1.2, above 1.3, above 1.4 or even above 1.5. In a preferred embodiment the HIF is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8. at least 9, at least 10.

In another embodiment, the variant has improved Half-life compared to the lipase of SEQ ID NO:

2, i.e., the Half-life Improvement Factor (HIF) determined as described in Example 2 or Example 3 is above 1 , preferably above 1.1 , such as above 1.2, above 1.3, above 1.4 or even above 1.5. In a preferred embodiment the HIF is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8. at least 9, at least 10.

In an embodiment, the variant has improved thermostability compared to the lipase of SEQ ID NO: 1 , i.e., the Tm when determined as in Example 4 is at least 55°C, such as at least 56°C, at least 57°C, such as at least 58°C at least 59°C, or such as at least 60°C.

In an embodiment, the variant has improved wash performance compared to the lipase of SEQ ID NO: 1 , wherein wash performance is determined as in described in Example 5. In particular the wash performance measured as lard removal is improved for the lipase variants compared to the lipase of SEQ ID NO: 1. In an embodiment, the variant has lower odor generation compared to the commercially available lipase of SEQ ID NO: 5, wherein odor generation is determined as in described in Example 5. In particular the odor generation of the variants is improved (i.e. reduced compared to the lipase of SEQ ID NO: 5.

In an embodiment, the variant has improved Half-life compared to the lipase of SEQ ID NO: 4, i.e., the Half-life Improvement Factor (HIF) determined as described in Example 6 is above 1 , preferably above 1.1 , such as above 1.2, above 1.3, above 1.4 or even above 1.5. In a preferred embodiment the HIF is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8. at least 9, at least 10.

In an embodiment, the variant has improved residual activity compared to the lipase of SEQ ID NO: 4 determined as described in Example 7, i.e., the residual activity relative of the lipase variant to the residual activity of SEQ ID NO: 4 is above 1 , preferably above 1.1 , such as above 1.2, above 1.3, above 1.4 or even above 1.5. In a preferred embodiment the HIF is at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8. at least 9, at least 10.

In an aspect, the variant is isolated.

In another aspect, the variant is purified.

Parent lipase

The parent lipase may be a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 1.

In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have lipase activity. In one aspect, the amino acid sequence of the parent differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 1. In another aspect, the parent comprises or consists of the amino acid sequence of SEQ I D NO: 1.

The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.

In another aspect, the parent is a Geotrichum candidum lipase, e.g., the lipase of SEQ ID NO: 1.

Preparation of Variants

The present invention also relates to methods for obtaining a variant having lipase activity, comprising: (a) introducing into a parent lipase a substitution at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408, 409 of the polypeptide of SEQ ID NO: 1 , wherein the variant has lipase activity; and (b) recovering the variant.

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

Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 7Q: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

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

Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable 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 shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92/06204) and region- directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variant of the present invention. The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.

In an aspect, the polynucleotide is isolated.

In another aspect, the polynucleotide is purified.

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 the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

Promoters

The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology. Terminators

The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.. mRNA Stabilizers

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

Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177: 3465- 3471).

Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.

Leader Sequences

The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

Polyadenylation Sequences

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsinlike protease. Signal Peptides

The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu et al., 2018, Biotechnology Letters 40: 949-955.

Propeptides

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

Regulatory Sequences

It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in filamentous fungi include, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification.

Transcription Factors

The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and/or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, /.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, /.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017/144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7- 23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.

Expression Vectors

The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend 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, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome. For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non-homologous recombination, such as non-homologous end-joining (NHEJ).

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

Host Cells

The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.

A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.

The host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.

The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave- mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470- 1474, Christensen etal., 1988, Bio/TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.

The fungal host cell may be a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth etal., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Fili basidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.

For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium 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 torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

In an aspect, the host cell is isolated.

In another aspect, the host cell is purified.

Methods of Production

The present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.

The present invention also relates to a method for obtaining a lipase variant having at least one improved property compared to the lipase shown in SEQ ID NO: 1.

One aspect of the invention relates to method for obtaining a lipase variant, comprising;

1. introducing into a parent lipase one or more substitutions selected from the group consisting of T166L, T166W, T166G, G396E, G396Q, G396R, G396K, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396H, S397D, S397N, S397T, S397E, W398P, W398L, W398M, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408R, I408W, I408V, W398D, I408H, I408Y, I408F, L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y, L409S, G325E and G325D; and

2. recovering the variant, wherein the variant has lipase activity.

The method preferably comprises introduction of 1-20, e.g. 1-10, 1-7 or 2-5, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions selected from the group consisting of T166L, T166W, T166G, G396E, G396Q, G396R, G396K, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396H, S397D, S397N, S397T, S397E, W398P, W398L, W398M, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408R, I408W, I408V, W398D, I408H, I408Y, I408F, L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y, L409S, G325E and G325D compared to the polypeptide shown in SEQ ID NO: 1. Some aspects of the invention relate to a method for obtaining a lipase variant, comprising introducing into a parent lipase an substitution at one or more positions selected from the group consisting of T166L, T166W, T166G, G396E, G396Q, G396R, G396K, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396H, S397D, S397N, S397T, S397E, W398P, W398L, W398M, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408R, I408W, I408V, W398D, I408H, I408Y, I408F, L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y, L409S, and G325E, G325D, wherein the parent lipase is selected from the group of polypeptides:

1 . a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polypeptide shown in SEQ ID NO: 2,

2. a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polypeptide shown in SEQ ID NO: 3,

3. a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polypeptide shown in SEQ ID NO: 4,

4. a polypeptide having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the polypeptide shown in SEQ ID NO: 6.

The host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small- scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant. The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, a cell- free fermentation broth comprising the polypeptide is recovered.

The variant may be purified by a variety of procedures known in the art to obtain substantially pure variants and/or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

In an alternative aspect, the variant is not recovered.

Detergent Compositions

In one embodiment, the invention is directed to detergent compositions comprising a variant of the present invention as disclosed above in combination with one or more additional cleaning composition components. In one embodiment, the detergent composition comprises a a lipase variant comprising one or more substitutions at one or more positions corresponding to positions 396, 397, 398, 408, 409, 166 and 325 of SEQ ID NO: 1 , wherein the variant has lipase activity and wherein the variant has at least 60% identity, such as 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% identity to the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 6. In one embodiment the detergent composition is in solid form. In another embodiment, the detergent composition is in a liquid or gel form. In another embodiment a bar form. In one embodiment the detergent may be wrapped in water soluble PVOH film. The choice of additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

Detergent Ingredients

The choice of detergent components may include, for textile care, the consideration of the type of textile to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product. Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the skilled artisan.

Any detergent components known in the art for use in detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in detergents may be utilized. The choice of such ingredients is well within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.

Surfactants

The cleaning composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a surfactant system (comprising more than one surfactant) e.g. a mixture of one or more nonionic surfactants and one or more anionic surfactants. In one embodiment the detergent comprises at least one anionic surfactant and at least one nonionic surfactant, the weight ratio of anionic to nonionic surfactant may be from 20:1 to 1 :20. In one embodiment the amount of anionic surfactant is higher than the amount of non-ionic surfactant e.g. the weight ratio of anionic to non-ionic surfactant may be from 10: 1 to 1.1 : 1 or from 5: 1 to 1.5: 1. The amount of anionic to non-ionic surfactant may also be equal and the weight ratios 1 :1. In one embodiment the amount of non-ionic surfactant is higher than the amount of anionic surfactant and the weight ratio may be 1 : 10 to 1 : 1.1. Preferably the weight ratio of anionic to non-ionic surfactant is from 10:1 to 1 :10, such as from 5:1 to 1 :5, or from 5:1 to 1:1.2. Preferably, the weight fraction of non- ionic surfactant to anionic surfactant is from 0 to 0.5 or 0 to 0.2 thus non-ionic surfactant can be present or absent if the weight fraction is 0, but if non-ionic surfactant is present, then the weight fraction of the nonionic surfactant is preferably at most 50% or at most 20% of the total weight of anionic surfactant and non-ionic surfactant. Light duty detergent usually comprises more nonionic than anionic surfactant and there the fraction of non-ionic surfactant to anionic surfactant is preferably from 0.5 to 0.9. The total weight of surfactant(s) is typically present at a level of from about 0.1% to about 60% by weight, such as about 1% to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and may include any conventional surfactant(s) known in the art. When included therein the detergent will usually contain from about 1 % to about 40% by weight of an anionic surfactant, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 15% to about 20%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, typically available as sodium or potassium salts or salts of monoethanolamine (MEA, 2-aminoethan-1-ol) or triethanolamine (TEA, 2,2',2"-nitrilotriethan-1-ol); in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS such as branched alkylbenzenesulfonates (BABS) and phenylalkanesulfonates; olefin sulfonates, in particular alphaolefinsulfonates (AOS); alkyl sulfates (AS), in particular fatty alcohol sulfates (FAS), /.e., primary alcohol sulfates (PAS) such as dodecyl sulfate (SLS); alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates); paraffin sulfonates (PS) including alkane-1 -sulfonates and secondary alkanesulfonates (SAS); ester sulfonates, including sulfonated fatty acid glycerol esters and alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES or MES); alkyl- or alkenylsuccinic acids such as dodecenyl/tetradecenyl succinic acid (DTSA); diesters and monoesters of sulfosuccinic acid; fatty acid derivatives of amino acids. Anionic surfactants may be added as acids, as salts or as ethanolamine derivatives.

When included therein the detergent will usually contain from about 0, 1 % to about 40% by weight of a cationic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12% or from about 10% to about 12%. Non-limiting examples of cationic surfactants include alkyldimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, ester quats, and combinations thereof.

When included therein the detergent will usually contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, from about 8% to about 12%, or from about 10% to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO) e.g. the AEO-series such as AEO-7, alcohol propoxylates, in particular propoxylated fatty alcohols (PFA), ethoxylated and propoxylated alcohols, alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters (in particular methyl ester ethoxylates, MEE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamides, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

When included therein the detergent will usually contain from about 0.01 to about 10 % by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamine oxides, in particular N-(coco alkyl)-N,N-dimethylamine oxide and N- (tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, and combinations thereof.

When included therein the detergent will usually contain from about 0.01 % to about 10 % by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaines such as alkyldimethylbetaines, sulfobetaines, and combinations thereof.

Additional bio-based surfactants may be used e.g. wherein the surfactant is a sugar-based non-ionic surfactant which may be a hexyl-p-D-maltopyranoside, thiomaltopyranoside or a cyclic- maltopyranoside, such as described in EP2516606 B1. Other biosurfactants may include rhamnolipids and sophorolipids.

Hydrotropes

A hydrotrope is a compound that solubilises hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and a hydrophobic character (so-called amphiphilic properties as known from surfactants); however, the molecular structure of hydrotropes generally do not favor spontaneous self-aggregation, see e.g. review by Hodgdon and Kaier (2007), Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming miceller, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care, food, to technical applications. Use of hydrotropes in detergent compositions allow for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.

The detergent may contain 0-10% by weight, for example 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzenesulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycolethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.

Builders and Co-Builders

The detergent composition may contain about 0-65% by weight, such as about 5% to about 50% of a detergent builder or co-builder, or a mixture thereof. The builder and/or co-builder may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any builder and/or co- builder known in the art for use in cleaning detergents may be utilized.

Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP orSTPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Clariant), ethanolamines such as 2- aminoethan-1-ol (MEA), diethanolamine (DEA, also known as 2,2'-iminodiethan-1-ol), triethanolamine (TEA, also known as 2,2',2"-nitrilotriethan-1-ol), and (carboxymethyl)inulin (CMI), and combinations thereof.

The detergent composition may also contain from about 0-50% by weight, such as about 5% to about 30%, of a detergent co-builder. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA/PMA). According to the present invention, these components can be included in lower levels than in currently available detergent compositions. Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic 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), methylglycinediacetic acid (MGDA), glutamic acid-N,N -di acetic acid (GLDA), 1-hydroxyethane-1 ,1-diylbis(phosphonic acid (HEDP),ethylenediaminetetramethylenetetrakis(phosphonic acid)

(EDTMPA),diethylenetriaminepentamethylenepentakis(phosphonic acid) (DTMPA or DTPMPA), N- (2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid- N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2- sulfomethyl)glutamicacid (SMGL), N-(2-sulfoethyl)glutamicacid (SEGL), N-methyliminodiaceticacid (MIDA), a-alanine-N,N-diacetic acid (a-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N- diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PH DA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilicacid-N,N-diaceticacid (SLDA) , taurine-N,N-diaceticacid (TLIDA) and sulfomethyl- N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)ethylenediamine-N,N’,N”-triacetic acid (HEDTA), diethanolglycine (DEG), aminotrimethylenetris(phosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 09/102854 and US 5977053.

Polymers and Dispersants

Generally, detergent compositions may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1 % of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide anti-redeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyleneglycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of polyethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-/V-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include polyethylene oxide and polypropylene oxide (PEO-PPO), diquaternium ethoxy sulfate, styrene/acrylic copolymer and perfume capsules Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.

The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.

Fabric Hueing Agents

The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when said fabric is contacted with a wash liguor comprising said detergent compositions and thus altering the tint of said fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.l.) classifications of 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 W02005/03274, W02005/03275, W02005/03276 and EP1876226 (hereby incorporated by reference). The detergent composition preferably comprises 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 hueing agent. The composition may comprise from 0.0001 wt% to 0.2 wt% fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g. WO 2007/087257 and W02007/087243.

Dye Transfer Inhibiting Agents

The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine /V-oxide polymers, copolymers of /V-vinylpyrrolidone and /V-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels 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 compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01% to about 0.5%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbenesulfonic acid derivative type of fluorescent whitening agents include the 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-(/V-methyl-/\/-2-hydroxy- ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulfonate, 4,4'-bis-(4-phenyl-1 ,2,3-triazol-2- yl)stilbene-2,2'-disulfonate and sodium 5-(2/7-naphtho[1 ,2-d][1 ,2,3]triazol-2-yl)-2-[(E)-2- phenylvinyl]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-(phenyl-styryl)-disulfonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Tinopal CBS-X is a 4.4'-bis-(sulfostyryl)-biphenyl disodium salt also known as Disodium Distyrylbiphenyl Disulfonate. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins.

Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt%.

Soil Release Polymers

The detergent compositions of the present invention may also include one or more soil release polymers which aid 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 release polymers may for example be nonionic or anionic terephthalte based 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. Another type of soil release polymers are amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore, random graft co-polymers are suitable soil release polymers. Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference).

Anti-redeposition Agents

The detergent compositions of the present invention may also include one or more antiredeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.

Rheology Modifiers

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

Other suitable adjunct materials include, but are not limited to, anti-shrink agents, anti-wrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.

Additional Enzymes

The detergent additive as well as the detergent composition may comprise one or more [additional] enzymes such as a protease, a lipase, a cutinase, a cellulase, an amylase, carbohydrase, DNase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

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

Cellulases

Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691 ,178, US 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulases having colour care benefits. Examples of such cellulases are cellulases described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307 and WO 99/001544.

Other cellulases are endo-beta-1 ,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.

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

DNases

The term “DNase” means a polypeptide having DNase (deoxyribonuclease) activity that catalyzes the hydrolytic cleavage of phosphodiester linkages in DNA, thus degrading DNA. The polypeptide having DNase activity is typically of microbial origin, from a bacterium or a fungus. DNases belong to the esterases (EC number 3.1), a subgroup of the hydrolases. The DNases may be classified in several subclasses, in particular EC 3.1.21 and its subclasses. The term “DNase” as used herein is also intended to include nucleases that cleave both RNA and DNA, for example the endoribonucleases in EC 3.1.30 that are active on both RNA and DNA. The terms “DNase” and “a polypeptide with DNase activity” may be used interchangeably throughout the application.

Mannanases

Suitable mannanases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included. The mannanase may be an alkaline mannanase of Family 5 or 26. It may be a wild-type or a variant from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, Paenibacillus illinoisensis or H. insolens. Suitable mannanases are described in WO 1999/064619 and PCT/EP2023/066246. A commercially available mannanase is Mannaway (Novozymes A/S).

Proteases

Suitable proteases may be of any origin, but are preferably of bacterial or fungal origin, optionally in the form of protein engineered or chemically modified mutants. The protease may be an alkaline protease, such as a serine protease or a metalloprotease. A serine protease may for example be of the S1 family, such as trypsin, or the S8 family such as a subtilisin. A metalloprotease may for example be a thermolysin, e.g. from the M4 family, or another metalloprotease such as those from the M5, M7 or M8 families.

The term "subtilases" refers to a sub-group of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate. The subtilases may be divided into six subdivisions, the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.

Although proteases suitable for detergent use may be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases have generally been obtained from bacteria and in particular from Bacillus. Examples of Bacillus species from which subtilases have been derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus gibsonii. Particular subtilisins include subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN’, subtilisin 309, subtilisin 147 and subtilisin 168 and e.g. protease PD138 (described in WO 93/18140). Other useful proteases are e.g. those described in WO 01/16285 and WO 02/16547.

Examples of trypsin-like proteases include the Fusarium protease described in WO 94/25583 and WO 2005/040372, and the chymotrypsin proteases derived from Cellumonas described in WO 2005/052161 and WO 2005/052146.

Examples of metalloproteases include the neutral metalloproteases described in WO 2007/044993 such as those derived from Bacillus amyloliquefaciens, as well as e.g. the metalloproteases described in WO 2015/158723 and WO 2016/075078.

Examples of useful proteases are the protease variants described in WO 89/06279 WO 92/19729, WO 96/34946, WO 98/20115, WO 98/20116, WO 99/11768, WO 01/44452, WO 03/006602, WO 2004/003186, WO 2004/041979, WO 2007/006305, WO 2011/036263, WO 2014/207227, WO 2016/087617 and WO 2016/174234.

Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase™, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Blaze®, Blaze Evity® 100T, Blaze Evity® 125T, Blaze Evity® 150T, Blaze Evity® 200T, Neutrase®, Everlase®, Esperase®, Progress® Uno, Progress® In and Progress® Excel (Novozymes A/S), those sold under the tradename Maxatase™, Maxacai™, Maxapem®, Purafect® Ox, Purafect® OxP, Puramax®, FN2™, FN3™, FN4^ex™, Excellase®, Excellenz™ P1000, Excellenz™ P1250, Eraser™, Preferenz® P100, Purafect Prime, Preferenz P110™, Effectenz P1000™, Purafect®, Effectenz P1050™, Purafect® Ox, Effectenz™ P2000, Purafast™, Properase®, Opticlean™ and Optimase® (Danisco/DuPont), BLAP (sequence shown in Figure 29 of US 5352604) and variants hereof (Henkel AG), and KAP (Bacillus alkalophilus subtilisin) from Kao.

Lipases and Cutinases

Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216, cutinase from Humicola, e.g. H. insolens (WO96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g. P. alcaligenes or P. pseudoalcaligenes (EP218272), P. cepacia (EP331376), P. sp. strain SD705 (W095/06720 & W096/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (W010/065455), cutinase from Magnaporthe grisea (WO10/107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W011/084412), Geobacillus stearothermophilus lipase (W011/084417), lipase from Bacillus subtilis (W011/084599), and lipase from Streptomyces griseus (WO11/150157) and S. pristinaespiralis (W012/137147).

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,

W096/00292, W097/04079, W097/07202, WO00/34450, WO00/60063, W001/92502,

W007/87508 and WO09/109500.

Preferred commercial lipase products include include Lipolase 100T/L, Lipex 100T/L, Lipex 105T, Lipex Evity 100L, Lipex Evity 200L (all Novozymes A/S), Preferenz® L 100 (DuPont).

Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/111143), acyltransferase from Mycobacterium smegmatis (WO05/56782), perhydrolases from the CE 7 family (WO09/67279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO10/100028).

Amylases

Suitable amylases include an alpha-amylase or a glucoamylase and may be of bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Amylases include, for example, alpha-amylases obtained from Bacillus, e.g., a special strain of Bacillus licheniformis, described in more detail in GB 1 ,296,839.

Suitable amylases include amylases having SEQ ID NO: 2 in WO 95/10603 or variants having 90% sequence identity to SEQ ID NO: 3 thereof. Preferred variants are described in WO 94/02597, WO 94/18314, WO 97/43424 and SEQ ID NO: 4 of WO 99/019467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.

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

Other amylases which are suitable are hybrid alpha-amylase comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO: 6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO: 4 of WO 2006/066594 or variants having 90% sequence identity thereof.

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

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme ^TM, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ Amplify; Amplify Prime; (from Novozymes A/S), and Rapidase™ , Purastar™/Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).

Peroxidases/Oxidases

Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

A suitable peroxidase is preferably a peroxidase enzyme comprised by the enzyme classification EC 1.11.1.7, as set out by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), or any fragment derived therefrom, exhibiting peroxidase activity. Suitable peroxidases also include a haloperoxidase enzyme, such as chloroperoxidase, bromoperoxidase and compounds exhibiting chloroperoxidase or bromoperoxidase activity. Haloperoxidases are classified according to their specificity for halide ions. Chloroperoxidases (E.C. 1.11.1.10) catalyze formation of hypochlorite from chloride ions. The haloperoxidase may be a chloroperoxidase. Preferably, the haloperoxidase is a vanadium haloperoxidase, /.e., a vanadate-containing haloperoxidase. In a preferred method the vanadate-containing haloperoxidase is combined with a source of chloride ion.

Haloperoxidases have been isolated from many different fungi, in particular from the fungus group dematiaceous hyphomycetes, such as Caldariomyces, e.g., C. fumago, Alternaria, Curvularia, e.g., C. verruculosa and C. inaequalis, Drechslera, Ulocladium and Botrytis.

Haloperoxidases have also been isolated from bacteria such as Pseudomonas, e.g., P. pyrrocinia and Streptomyces, e.g., S. aureofaciens.

The haloperoxidase may be derivable from Curvularia sp., in particular Curvularia verruculosa or Curvularia inaequalis, such as C. inaequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444.70 as described in WO 97/04102; or from Drechslera hartlebii as described in WO 01/79459, Dendryphiella salina as described in WO 01/79458, Phaeotrichoconis crotalarie as described in WO 01/79461 , or Geniculosporium sp. as described in WO 01/79460.

Suitable oxidases include, in particular, any laccase enzyme comprised by the enzyme classification EC 1.10.3.2, or any fragment derived therefrom exhibiting laccase activity, or a compound exhibiting a similar activity, such as a catechol oxidase (EC 1.10.3.1), an o- aminophenol oxidase (EC 1.10.3.4), or a bilirubin oxidase (EC 1.3.3.5).

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

Suitable examples from fungi include a laccase derivable from a strain of Aspergillus, Neurospora, e.g., N. crassa, Podospora, Botrytis, Collybia, Pomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cinerea, C. comatus, C. friesii, and C. plicatilis, Psathyrella, e.g., P. condelleana, Panaeolus, e.g., P. papilionaceus, Myceliophthora, e.g., M. thermophila, Schytalidium, e.g., S. thermophilum, Polyporus, e.g., P. pinsitus, Phlebia, e.g., P. radiata (WO 92/01046), or Coriolus, e.g., C. hirsutus (JP 2238885). Suitable examples from bacteria include a laccase derivable from a strain of Bacillus.

A laccase derived from Coprinopsis or Myceliophthora is preferred; in particular a laccase derived from Coprinopsis cinerea, as disclosed in WO 97/08325; or from Myceliophthora thermophila, as disclosed in WO 95/33836.

Licheninases

Licheninases (or lichenases) (e.g. EC 3.2.1.73) hydrolyse (1 ,4)-beta-D-glucosidic linkages in beta-D-glucans containing (1 ,3)- and (1 ,4)-bonds and can act on lichenin and cereal beta-D- glucans, but not on beta-D-glucans containing only 1,3- or 1 ,4-bonds.

Pectate Lyases

Pectate lyases catalyze the cleavage of a-1,4-D-galacturonan (i.e., homogalacturonan or polygalacturonic acid) by an eliminative pathway leaving a double bond between C4 and C5 at the +1 subsite and a reducing sugar at the -1 subsite. Pectate lyases may also have pectin lyase activity.

Xanthanases

Xanthan gum is a natural polysaccharide consisting of different sugars which are connected by several different bonds, such as b-D-mannosyl-b-D-1 ,4-glucuronosyl bonds and b-D-glucosyl-b- D-1 ,4-glucosyl bonds. Xanthan gum is at least partly soluble in water and forms highly viscous solutions or gels. Complete enzymatic degradation of xanthan gum requires several enzymatic activities including xanthan lyase activity and endo-beta-1, 4-glucanase activity, preferably a GH9 endoglucanase. Xanthan lyases are enzymes that cleave the b-D-mannosyl-b-D-1 ,4- glucuronosyl bond of xanthan, whereas the GH9 endoglucanase catalyses the hydrolysis of the glycosyl bond to release smaller sugars.

Complete enzymatic degradation of xanthan gum requires enzymatic activities including xanthan lyase activity and xanthan endoglucanase activity as described above. Xanthan lyases and endoglucanases for the degradation of xanthan gum and the use of such enzymes for cleaning purposes, such as the removal of xanthan gum containing stains, and in the drilling and oil industries are known in the art, e.g. from WO 2013/167581 A1.

In the context of the present invention the term Xanthanase (or xanthanase) means the combination of the enzymatic activities including xanthan lyase activity and xanthan endoglucanase activity.

A commercially available Xanthanase is the product Caledonia 100L from Novozymes A/S.

Formulation of Detergent Products

The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. Pouches can be configured as single or multicompartments. It can be of any form, shape and material which is suitable for hold the composition, e.g. without allowing the release of the composition to release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, malto dextrin, poly methacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blended compositions comprising hydrolytically degradable and water soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by MonoSol LLC, Indiana, USA) plus plasticisers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water- soluble film. The compartment for liquid components can be different in composition than compartments containing solids: US2009/0011970 A1.

Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

A liquid or gel detergent, which is not unit dosed, may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.

Granules

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

The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core diameter, measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020).

In an embodiment, the core comprises a variant of the present invention.

The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.

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

The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.

The core may include an inert particle with the variant absorbed into it, or applied onto the surface, e.g., by fluid bed coating.

The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250- 1200 pm.

The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).

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

The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.

The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness. The coating can further contain other materials as known in the art, e.g. , fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.

To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.

The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.

The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water. The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

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

Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO₃ (CH2o°c=92%), NaNO₃ (CH₂O C=73%), Na₂HPO₄ (CH₂o°c=95%), Na₃PO₄ (CH₂5°c=92%), NH₄CI (CH₂Q C = 79.5%), (NH₄)₂HPO₄ (CH₂O C = 93,0%), NH₄H₂PO₄ (CH₂Q C = 93.1%), (NH₄)₂SO₄ (CH₂o°c=81 .1%), KOI (CH₂O C=85%), K₂HPO₄ (CH₂O C=92%), KH₂PO₄ (CH₂O°C=96.5%), KNO₃ (CH₂O°C=93.5%), Na₂SO₄ (CH₂O C=93%), K₂SO₄ (CH₂O C=98%), KHSO₄ (CH₂O C=86%), MgSO₄ (CH₂o°c=9O%), ZnSO₄ (CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH2PO₄, (NH₄)H2PO₄, CuSO₄, Mg(NO₃)2 and magnesium acetate.

The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSC ), magnesium sulfate heptahydrate (MgSC 7H2O), zinc sulfate heptahydrate (ZnSC 7H2O), sodium phosphate dibasic heptahydrate (IX^HPC FW), magnesium nitrate hexahydrate (Mg(NC>3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.

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

The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.

Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,

(a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker).

(b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606.

(c) Absorbed core particles, wherein rather than coating the variant as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116. (d) Extrusion or pelletized products, wherein a variant-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme (Michael S. Showell (editor); Powdered detergents, Surfactant Science Series; 1998; Vol. 71 ; pages 140- 142; Marcel Dekker).

(e) Prilled products, wherein a variant-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). The product obtained is one wherein the variant is uniformly distributed throughout an inert material instead of being concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.

(f) Mixer granulation products, wherein a variant-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. Granulates consisting of variant, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so- called T-granulate. Reinforced particles, are more robust, and release less enzymatic dust.

(g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

(h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.

(i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the variant contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w/w water.

Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.

The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D. Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.

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

In an embodiment, the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, betaglucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.

Liquid Formulations

The present invention also relates to liquid compositions comprising a variant of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and peptide aldehydes).

The liquid detergent composition has a physical form, which is not solid (or gas). It may be a pourable liquid, a paste, a pourable gel or a non-pourable gel. It may be either isotropic or structured, preferably isotropic. It may be a formulation useful for washing in automatic washing machines or for hand washing. It may also be a personal care product, such as a shampoo, toothpaste, or a hand soap.

The liquid detergent composition may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to 70% water, up to 50% water, up to 40% water, up to 30% water, or up to 20% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid detergent. An aqueous liquid detergent may contain from 0-30% organic solvent. A liquid detergent may even be almost non-aqueous, wherein the water content is below 10%, preferably below 5%.

Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.

The detergent composition may take the form of a unit dose product. A unit dose product is the packaging of a single dose in a non-reusable container. It is increasingly used in detergents for laundry. A detergent unit dose product is the packaging (e.g., in a pouch made from a water-soluble film) of the amount of detergent used for a single wash.

Pouches can be of any form, shape and material which is suitable for holding the composition, e.g., without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. Said inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and, hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. Preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be a blend composition comprising hydrolytically degradable and water-soluble polymer blends such as polyactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers like glycerol, ethylene glycerol, Propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry cleaning composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids (see e.g., US 2009/0011970).

In some embodiments, filler(s) or carrier material(s) are included to increase the volume of liquid detergent compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

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

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.001-25% w/w of a variant of the present invention;

(B) 20-80% w/w of polyol; (C) optionally 0.001-2% w/w preservative; and

(D) water.

In another embodiment, the invention relates to liquid formulations comprising:

(A) 0.001-25% w/w of a variant of the present invention;

(B) 0.001-2% w/w preservative;

(C) optionally 20-80% w/w of polyol; and

(D) water.

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

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

In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1 % w/w preservative or 0.1 -0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02-1.5% w/w preservative, 0.05-1% w/w preservative, or 0.1 -0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In another embodiment, the liquid formulation further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha- mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta- xylosidase or any combination thereof.

The liquid detergent composition may comprise a microcapsule, and thus form part of any detergent composition in any form, such as liquid and powder detergents, and soap and detergent bars.

In one embodiment, the invention is directed to liquid detergent compositions comprising a microcapsule, as described above, in combination with one or more additional cleaning composition components.

The microcapsule, as described above, may be added to the liquid detergent composition in an amount corresponding to from 0.0001% to 5% (w/w) active enzyme protein (AEP); preferably from 0.001% to 5%, more preferably from 0.005% to 5%, more preferably from 0.005% to 4%, more preferably from 0.005% to 3%, more preferably from 0.005% to 2%, even more preferably from 0.01% to 2%, and most preferably from 0.01% to 1% (w/w) active enzyme protein.

Laundry Soap Bars

The lipase variants of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and not a liquid, gel or a powder at room temperature. The term solid is defined as a physical form which does not significantly change over time, i.e. if a solid object (e.g. laundry soap bar) is placed inside a container, the solid object does not change to fill the container it is placed in. The bar is a solid typically in bar form but can be in other solid shapes such as round or oval.

The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerine, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na⁺, K⁺ or NH4⁺ and the organic anion may be for example formate, acetate, citrate or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate. The laundry soap bar may also contain complexing agents like EDTA and HEDP, perfumes and/or different type of fillers, surfactants e.g. anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.

The laundry soap bar may be processed in conventional laundry soap bar making equipment such as, but not limited to, mixers, plodders, e.g. a two-stage vacuum plodder, extruders, cutters, logostampers, cooling tunnels and wrappers. The invention is not limited to preparing the laundry soap bars by any single method. The premix of the invention may be added to the soap at different stages of the process. For example, the premix containing a soap, GCL I, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared, and the mixture is then plodded. The GCL I and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.

Use of lipase variants

The lipase variants of the invention are suitable for use in cleaning such as laundry. Thus, some aspect of the invention relates a method for laundering an item, wherein the method comprises the steps of: a. Exposing an item to a wash liquor comprising a lipase variant of the invention; b. Completing at least one wash cycle; and c. Optionally rinsing the item, wherein the item is a textile or a hard surface.

The pH of the liquid solution is in the range of 1 to 11 , such as in the range 5.5 to 11 , such as in the range of 7 to 9, in the range of 7 to 8 or in the range of 7 to 8.5.

The wash liquor may have a temperature in the range of 5°C to 95°C, or in the range of 10°C to 80°C, in the range of 10°C to 70°C, in the range of 10°C to 60°C, in the range of 10°C to 50°C, in the range of 15°C to 40°C or in the range of 20°C to 30°C. In some aspects the temperature of the wash liquor is 30°C.

The lipase variants of the invention may be added to a wash liquor.

The concentration of the lipase variant enzyme in the wash liquor is typically in the range of 0.00001-1000 ppm enzyme protein, such as in the range of 0.00004-100 ppm enzyme protein, such as in the range of 0.00008-100 enzyme protein, in the range of 0.0001-100 enzyme protein, in the range of 0.0002-100 enzyme protein, in the range of 0.0004-100 enzyme protein, in the range of 0.0008-100 enzyme protein, in the range of 0.001-100 ppm enzyme protein, in the range of 0.01-100 ppm enzyme protein, in the range of 1-1000 ppm enzyme protein, preferably in the range of 0.05-50 ppm enzyme protein, more preferably in the range of 0.1-50 ppm enzyme protein, more preferably in the range of 0.1-30 ppm enzyme protein, more preferably in the range of 0.5-20 ppm enzyme protein, and most preferably in the range of 0.5-10 ppm enzyme protein.

In some aspects the lipase variants of the present invention are effective in preventing and/or reducing the malodor. The presence of biofilm, body soil e.g., cell debris, sebum or EPS implies that the laundry items become sticky and therefore soil adheres to the sticky areas. This soil sticked to the laundry has shown difficult to reduce by commercially available detergent compositions. Further, when dirty laundry items are washed together with less dirty laundry items the dirt present in the wash liquor tend to adhere to the laundry (e.g. by re-deposition) in particular if the laundry is sticky as described above. As a result, hereof, the laundry item is more “soiled” after wash than before wash. In some aspects, the lipase variants of the invention have improved deep cleaning properties compared to the parent lipase and in some aspects, the lipase variants of the invention reduce stickiness and/or re-deposition. In particular, it is foreseen that the combined used of the lipases of the present invention and DNase will have a beneficial effect on the removal of biofilm, body soil e.g., cell debris, and sebum. The use of DNase for sebum removal is disclosed in WO 2023/056892 (Novozymes A/S) and at a presentation at the Sepawa conference, October 13, 2021.

In some aspects, the invention relates to the use of a lipase variant according to the invention for deep cleaning of an item, wherein the item is a fabric or a hard surface.

Further, the invention relates to the use of a lipase variant according to the invention for preventing and/or reducing the adherence of soil to an item. In some aspect, the item is textile. When the soil does not adhere to the item, the item appears cleaner. Thus, the invention further relates to the use of a lipase variant according to the invention for maintaining or improving the whiteness of the item.

The present invention further relates to detergent compositions comprising a lipase variant according to the invention preferably with a detergent adjunct ingredient. The detergent composition comprising a lipase variant according to the invention may be used for deep cleaning of an item, for preventing and/or reducing the stickiness of an item, for pretreating stains on the item, for preventing and/or reducing redeposition of soil during a wash cycle, for preventing and/or reducing adherence of soil to an item, for maintaining or improving the whiteness of an item and/or for preventing and/or reducing malodor from an item.

EMBODIMENTS

The invention is further defined by the following numbered paragraphs:

1 . A lipase variant, comprising a mutation, such as a substitution, at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408 and 409 of the polypeptide of SEQ ID NO: 1 , wherein the variant has lipase activity and wherein the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or even 100% sequence identity to the polypeptide of SEQ ID NO: 4, wherein the variant optionally comprises an extension of one or more amino acids at the N-terminal and/or C-terminal ends or a truncation of one or more amino acids at the N-terminal and/or C- terminal ends and wherein the variant has lipase activity. The lipase variant of paragraph 1 comprising a substitution at position 396 and one or more further substitutions at positions selected from the group consisting of 397, 398, 408, 409, 166 and 325, wherein the variant has lipase activity and wherein the variant has at least 60% sequence identity to the polypeptide of SEQ ID NO: 4. The lipase variant of paragraph 1 or 2, wherein the substitution in position 396 is selected from the group consisting of G396E, G396Q, G396R, G396K, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396D and G396H. The lipase variant of paragraphs 1 to 3 comprising a substitution at position 408 selected from the group consisting of I408R, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408W, I408V, W398D, I408H, I408Y and I408F. The lipase variant of any of paragraphs 1 to 4 further comprising one or more substitutions selected from the group consisting of S397D, S397N, S397T, S397E, W398P, W398L, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, W398M, L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y, L409S, G325D, G325E, T166L, T166W and T166G. The lipase variant of any of paragraphs 1 to 5 wherein the variant is selected from the group of variants a) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions G396E, S397D, W398P, I408R and L409R b) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, G325D, G396E, W398Y and L409R c) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions W398P and I408E d) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, G325D, G396E and W398Y e) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitution F543Y f) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D330N, A332S, P369E, G396E, S397D, W398P, I408R, L409R. g) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, P369E, G396E, S397D, W398P, I408E, L409R. h) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, D330N, A332S, P369E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y. i) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, F256V, G396E, S397D, W398P, I408S, L409R, L468I. j) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, A278V, A332S, P369E, G396E, S397D, W398P, I408R, L409R, F543Y. k) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions N68G, D119R, P369E, G396E, S397D, W398P, I408R, L409R, G510D, T525E, F543Y. l) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, P369E, S392E, G396E, S397D, W398P, I408E, L409R, A509V, F543Y. m) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions P369E, S392D, G396E, S397D, W398P, I408R, L409R, R482Q, M523R, F543Y. n) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions Q50N, L52S, T166L, D330N, A332S, P369E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y o) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, K210R, D330N, A332S, P369E, S392D ,G396E, S397D, W398P, I408R, L409R, F543Y. p) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions S48N, Q50S, D119R, D257N, A278V, A332S, P369E, G396E, S397D, W398P, I408R, L409R, F543Y q) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, A278V, A332S, P369E, G396E, S397D, W398P, I408R, L409R, R511K, F543Y. r) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, A278V, A332S, P369E, G396E, S397D, W398P, I408R, L409R, A509S, R511K, F543Y. s) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, A331S, A332S, P369E, G396E, S397D, W398P, I408E, L409R, H517R. t) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, D330N, A332S, P369E, C379L, S380N, A385E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y. u) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, F256V, D257N, E264Q, S392E, G396E, S397D, W398P, I408S, L409R, L468I. v) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions K53E, T166L, E264H, D330N, A332S, P369E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y. w) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, D330N, A332S, P369E, S392D, G396E, S397D, W398P, I408R, L409R, R511 D, F543Y. x) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, D330N, A332S, P369E, A385E, S392D, G396E, S397D, W398P, I408R, L409R, H517R, F543Y. y) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, T166L, L306R, G325D, D330N, A332S, P369E, A385E, S392E, G396E, S397D, W398P, I408R, L409R, F543Y. z) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, D330N, A332S, P369E, C379F, S380Y, A385E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y. aa) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions A114E, T166L, D330N, A332S, P369E, S392D, G396E, S397D, W398P, I408R, L409R, F543Y. bb) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions D119R, A278V, A332S, P369E, S392E, G396E, S397D, W398P, I408R, L409R, V496P, R511 K, F543Y. cc) Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions V76L, D119R, A278V, A332S, K343R, P369E, A385E, G396E, S397D, W398P, I408R, L409R, F543Y wherein the variants have at least at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or even 100% sequence identity to the polypeptide of SEQ ID NO: 4.

7. The lipase variant of paragraph 1 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a polypeptide selected from the group of polypeptides consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

8. The lipase variant of paragraph 1 , comprising a substitution at one or more positions corresponding to positions , 166, 325, 396, 397, 398, 408 and 409 of the polypeptide of SEQ ID NO: 1 , wherein the variant has 1-7 amino acid differences, such as 2-7 amino acid differences, 3-7 amino acid differences, 4-7 amino acid differences, 5-7 amino acid differences, 6-7 amino acid differences or even 7 amino acid differences compared to the parent and wherein the variant has lipase activity. The lipase variant of paragraph 1 , comprising a substitution at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408 and 409 of the polypeptide of SEQ ID NO: 1 , having an amino acid sequence which has at most 2%, such as at most 1 % sequence differences compared to the parent, wherein the variant has lipase activity. The variant of any one of the preceding paragraphs, wherein the variant consists of 500 to 550, e.g., 510 to 550, 520 to 550, 530 to 550, or 540 to 550 amino acids. The lipase variant of any of the preceding paragraphs which has an improved property relative to the lipase of SEQ ID NO: 1 , wherein the improved property is selected from the group consisting of Half-life Improvement Factor, wash performance, odor reduction, thermal stability and detergent stability. A granule, which comprises: a) a core comprising the variant lipase of any one of paragraphs 1-11 , and b) optionally one or more additional enzymes, and, c) optionally a coating consisting of one or more layer(s) surrounding the core. A liquid composition comprising the variant lipase of any one of paragraphs 1-11 and optionally one or more additional enzymes, and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid. The liquid composition of paragraph 13, further comprising a filler or carrier material. The liquid composition of paragraph 13 or 14, further comprising a preservative. A composition comprising the lipase variant of any one of paragraphs 1-11 , the granule of paragraph 12, or the liquid compositions of any one of paragraphs 13-15. A polynucleotide encoding the lipase variant of any one of paragraphs 1-11. The polynucleotide of paragraph 17, which is isolated. The polynucleotide of paragraph 17 or 18, which is purified. A nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 17-19. 21. A recombinant host cell transformed with the polynucleotide of any one of paragraphs 17 to 19.

22. The recombinant host cell of paragraph 21, which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of any one of paragraphs 17-19.

23. The recombinant host cell of paragraph 21 or 22, which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium 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 torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

24. The recombinant host cell of any one of paragraphs 21 to 23, which is isolated.

25. The recombinant host cell of any one of paragraphs 21 to 24, which is purified.

26. A method of producing a lipase variant, comprising a) cultivating the host cell of any one of paragraphs 21-25 under conditions suitable for expression of the variant; and b) recovering recovering the variant 27. A method for obtaining a lipase variant, comprising introducing into a parent lipase a substitution at one or more positions corresponding to positions 166, 325, 396, 397, 398, 408 or 409 of the polypeptide of SEQ ID NO: 1 , and the variant has lipae activity; and recovering the variant.

28. A detergent composition comprising a lipase variant of any of paragraphs 1 to 11 and at least one surfactant and optionally one or more enzymes.

29. The detergent composition according to paragraph 28, wherein the concentration of linear alkylbenzenesulfonates (LAS) is less than 5% w/w, such as less than 4% w/w, such as less than 3% w/w, such as less than 2% w/w, such as less than 1% w/w, such as less than 0.5% w/w such as less than 0.1% w/w.

30. A washing method for textile comprising: a) Exposing a textile to a wash liquor, said wash liquor comprising i. The lipase variant of any of paragraphs 1 to 11 ; ii. At least one surfactant; and iii. Optionally one or more additional enzymes b) completing at least one wash cycle, and optionally rinsing the textile.

31 . The washing method according to paragraph 30, wherein the surfactant level in the wash liquor is below 10 g/L.

32. The detergent composition according to paragraph 28 or 29 or the washing method according to paragraph 30 or 31 , wherein the at least one surfactant is rhamnolipid and/or sophorolipid.

33. The granule according to paragraph 12, the liquid composition according to paragraph 13 to 15, or the composition according to paragraph 16 comprising a DNase having at least 60% sequence identity to any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22 or SEQ ID NO: 23. In particular, the DNase may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22 or SEQ ID NO: 23. 34. The washing method according to paragraph 30 and 31 , comprising a DNase having at least 60% sequence identity to any of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22 or SEQ ID NO: 23. In particular, the DNase may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22 or SEQ ID NO: 23.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

SEQUENCES

SEQ ID NO: 1

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYI CSEASDASLDRVLSLYPGSWSEGAPFRTGILNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 2

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYI

CSEASDASLDRVLSLYPGSPSEGAPFRTGELNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 3

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDDNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYIC

SEASDASLDRVLSLYPESYSEGAPFRTGILNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVNR

WTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMYT

DAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 4

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 6

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDDNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYIC

SEASDASLDRVLSLYPESYSEGAPFRTGIRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVNR

WTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMYT

DAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG SEQ ID NO: 24

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYI

CSEASDASLDRVLSLYPGSPSEGAPFRTGELNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 25

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGERNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 26

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 27

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 28

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYVDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYIC

SEASDASLDRVLSLYPEDPSEGAPFRTGSRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLIFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMY

TDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 29

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGGAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDADREMLQIHMIGNSMREDDFRIEGISNFESDVTLYG

SEQ ID NO: 30

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLELYPEDPSEGAPFRTGERNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDVGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 31

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYQRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSRRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 32

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYNGSKANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 33

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDRVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG SEQ ID NO: 34

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGNYSGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFNSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 35

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGKEMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 36

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDSGKEMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 37

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG GPLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDSSYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLSLYPEDPSEGAPFRTGERNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD

MYTDAGREMLQIRMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 38

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYILN

EASDESLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVNR

WTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMYT

DAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 39

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYVNSTSVGPQSAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPDAAYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTPHVKKWLKYIC

SEASDASLDRVLELYPEDPSEGAPFRTGSRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLIFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMY

TDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLFG

SEQ ID NO: 40

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLEANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPHSAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 41

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKWGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGDEMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 42

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDESLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIRMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 43

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDRKDLFGLLP

QFLGFGPRPDDNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDESLDRVLELYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG SEQ ID NO: 44

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIFY

EASDESLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVNR

WTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDMYT

DAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 45

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPEGTKPDAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRLGPYGFLGGDAITAEGNTNAGLHD

QRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSGG

PLPYFDSTSVGPESAYSRFAQYAGCDASAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLLP

QFLGFGPRPDGNIIPNASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYIC

SEASDASLDRVLDLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDVN

RWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWDM

YTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 46

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKVVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG

GPLPYFDSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL

PQFLGFGPRPDGNIIPDASYELYRSGRYAKVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI

CSEASDASLDRVLELYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV

NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNPGTNLKQWD

MYTDAGKEMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

SEQ ID NO: 47

QAPTAVLNGNEVISGVLEGKVDTFKGIPFADPPVGDLRFKHPQPFTGSYQGLKANDFSSACMQ

LDPGNAISLLDKLVGLGKIIPDNLRGPLYDMAQGSVSMNEDCLYLNVFRPAGTKPRAKLPVMV

WIYGGAFVFGSSASYPGNGYVKESVEMGQPVVFVSINYRTGPYGFLGGDAITAEGNTNAGLH

DQRKGLEWVSDNIANFGGDPDKVMIFGESAGAMSVAHQLVAYGGDNTYNGKQLFHSAILQSG GPLPYFDSTSVGPESAYSRFAQYAGCDVSAGDNETLACLRSKSSDVLHSAQNSYDLKDLFGLL PQFLGFGPRPDGNIIPDASYELYRSGRYARVPYITGNQEDEGTILAPVAINATTTEHVKKWLKYI CSEASDESLDRVLSLYPEDPSEGAPFRTGRRNALTPQFKRIAAIFTDLLFQSPRRVMLNATKDV NRWTYLATQLHNLVPFLGTFHGSDLLFQYYVDLGPSSAYRRYFISFANHHDPNVGTNLKQWD MYTDAGREMLQIHMIGNSMRTDDFRIEGISNFESDVTLYG

EXAMPLES

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

Example A: pNP assay for determination of lipase activity

Enzymes can be assayed for lipase activity using the pNP assay described below.

Principle

The substrate pNP-substrate is hydrolyzed by the lipolytic enzyme under standard conditions. pNP-valerate is used as an example of a saturated short chain fatty acid. Valeric acid as the acyl group may be replaced by a long chain fatty acid such as oleic acid.

Hydrolysis of the pNP-substrate results in a yellow solution, the absorbance of the solution measured at 405 nm is a function of the activity of the lipolytic enzyme.

By varying the pNP substrate the ratio between lipase activity on unsaturated substrates having long fatty acyl chains (e.g., oleic acid) to short acyl chain (e.g., p-nitrophenyl butyrate and/or p- nitrophenyl valerate) can be determined. Variation of substrate may call for adjustment of e.g., buffer system, adjustments that are easily within the purview of the skilled person.

Lipase activity

Enzymes are diluted in Buffer

Substrate: The relevant pNP substrate (e.g. pNp-Valerate Sigma N-4377) 1 mM in Buffer prepared from stock-solution 100 mM in Methanol

Buffer: 50 mM TRIS, 0,4% Triton X-100, is prepared to pH 7,7

Results are calculated as:

[Vmax (enzyme)-Vmax(buffer)] / [slope of standard curve] Microtiter plates (Thermo Scientific 269620 96F without lid microwell plate) for plate reader spectrophotometers (Molecular Devices Spectramax 190) can conveniently be used for determination of lipase activity by standard methods based on use of paranitrophenol-esters.

Example B: Active site titration of lipase variants

Concentrations of micro-purified and conventionally purified lipase variants are determined by burst active site titration. 100 pl lipase diluted in 0.01% Triton X-100 to 0.1 mg/mL (or 50 pl lipase diluted in 0.01% Triton X-100 to 0.1 mg/mL + 0.05 mL 0.01% Triton X-100) is mixed with 100 pl ethyl resorufinyl heptylphosphonate inhibitor dissolved in DSMA (3.8 mg/mL) further diluted with buffer (1 M Tris, 4 mM SDS, pH 7.0) to 0.016 mg/mL in the well of a black microtiter plate. Immediately after mixing kinetics of fluorescence from liberated resorufin is measured every 1.5 minute for 5 hours (until bursts are finalized) (excitation at 515 nm, emission at 590 nm, measured on CLARIOstar (from BMG LABTECH). Measured fluorescences are fitted to the equation:

F = F0 + Burst * (1-exp(-(t + dt) * ln(2) I T¹/₂) + Slope * (t + dt) where F is the measured fluorescence, F0 is the fluorescence background from inhibitor and lipase, t is the time since first fluorescence measurement, dt is the time from mixing of lipase with inhibitor to the first fluorescence measurement. Burst is the fluorescence burst, T1 is the half-time for the exponential burst, and Slope is the slope for the linear change in fluorescence e.g. due to hydrolysis of lipase-ethyl heptylphosphonate complex and/or bleaching of resorufin. From the calculated burst the active lipase concentration is determined using a resorufin standard curve (0-4 pM) included on the microtiter plate.

Model detergents used in the experiments

The following model detergent was used in the experiments conducted to test the stability and performance of the lipase variants of the present invention:

Table 1: Model detergents applied in the experiments

Example 1 : Half-life Improvement Factor (HIF) of variants compared to GCL1 (SEQ ID NO:1) Each variant of SEQ ID NO: 1 , expressed in A oryzae, was grown in four independent biological replicates in separate microtiter plates in defined media (M Dll-2 w. 10 mM NaNOs), for 4 days at 37 °C in 96-well lidded MTPs without agitation. The expression plates also contained GCL1 references expressed in A. oryzae. The filamentous material was removed by pressing a Biopress® plate down into the wells. Expressed enzyme variants in supernatants were diluted in dilution media (0.02% v/v Triton X-100 in H2O), then dispensed into four quadrants per supernatant sample in a 384-well flat black microtiter plate.

Model detergent 2 (Table 1) was diluted to 0.2 % in 200 mM Tris-HCI, 15 °dH, pH 8.0. At four different timepoints ranging from 3 to 41 min, this model detergent solution was dispensed to quadrant wells containing the diluted enzyme sample and shaken briefly. After the incubation period, assay solution (500 pM 4-methylumbelliferyl oleate (4-Mll oleate) in 2.4 % v/v ethanol) was added, and the plate were read immediately using a Tecan Infinite M1000 using kinetic fluorescence measurements settings of A_ex 372 nm; A_em 445 nm for 12 min. The output was saved (V_max, R²) and used for calculating half-life (T%), and initial activity, for data that passed quality filters.

From linear fit of the logarithm of V_max as a function of incubation time it was possible to derive the coefficient s. Half-life were calculated using the equation T%= Half-life improvement factors (HIF) were calculated by the ratio of the half-life variant and the half-life of cultivated references. The initial activity (Relative fluorescence units (RFU) min^-1) was calculated from the mean Vmax of the shortest incubation time in the detergent possible for each variant or reference. This was used as a quality threshold to disregard non-active variants.

Half-life Improvement Factor (HIF) of the variants using SEQ ID NO: 1 as reference was measured as described above, results are listed in Table 2:

Table 2: HIF of variants compared to SEQ ID NO: 1, 0.2% detergent

An increase in HIF reflects that the variants are more stable in detergent, i.e. have improved detergent stabilityt, both during wash and storage. Consequently, increased HIF is important in the search for new variants with increased stability. The results show that the tested variants have increased stability compared to the GCL1 having SEQ ID NO: 1.

Example 2: Half-life Improvement Factor (HIF) of variants compared to improved variant (SEQ ID NO: 2)

The same assay as in Example 1 was used except that the Model detergent 2 was used at dilution to 1%. The GCL1 wildtype (SEQ ID NO: 1) had to low stability at the higher detergent concentration to be used as a reference. Therefore a stabilized variant of SEQ ID NO: 1 with the mutations W398PJ408E (SEQ ID NO: 2) was used as reference for the Half-life Improvement Factor (HIF) calculation. It is clear from Examplel that SEQ ID NO: 2 has a clearly improved stability compared to SEQ ID NO: 1 (HIF is 39.11).

Table 3: HIF of variants compared SEQ ID NO: 2, 1% detergent

An increase in HIF reflects that the variants are more stable in detergent, i.e. , have increased detergent stability, both during wash and storage. Consequently, increased HIF is important in the search for new variants with increased stability. The results show that the tested variants have increased stability compared to the stabilized variant of SEQ ID NO: 1 with the mutations W398PJ408E.

Example 3: Half-life Improvement Factor (HIF) of variants compared to improved variant (SEQ ID NO: 2) The same assay as in example 1 was used except that the Model detergent 2 was used at a dilution to 2%. Furthermore, the already stabilized variant of SEQ ID NO:1 with the mutations W398P 408E (SEQ ID NO: 2) was used as reference for the Half-life Improvement Factor (HIF) calculation, because the wildtype lipase (SEQ ID NO: 1) is too instable at the higher detergent concentration. It is clear from Example 1 that SEQ ID NO: 2 has a clearly improved stability compared to SEQ ID NO: 1 (HIF of variant having SEQ ID NO: 2 is 39.11).

Table 4: HIF of variants compared SEQ ID NO: 2, 2% detergent

An increase in HIF reflects that the variants are more stable in detergent, both during wash and storage. Consequently, increased HIF is important in the search for new variants with increased stability. The results show that the tested variants have increased stability compared to the stabilized variant of SEQ ID NO: 1 with the mutations W398P 408E.

Example 4: Thermal stability of variants

Purified variants of SEQ ID NO: 1 were diluted with 0.01% Triton X-100 to 0.8 mg/ml with concentration calculated from absorbance at 280 nm. 40 pl diluted sample was mixed with 40 pl buffer (0.2 M HEPES 20 mM MgCI2, pH 8.5). Unfolding transition temperatures (Tm) were determined in duplicate on a Prometheus NT.Plex nDSF instrument with heating from 20°C to 95°C at a rate of 3.3°C/min from inflection points for ratios between emission at 330 and 350 nm.

Table 5: Thermal stability of variants and SEQ ID NO: 1

Increased thermal stability (increased Tm) reflects that unfolding or other denaturation of the enzyme appears at a higher temperature and consequently that a better storage stability can be expected. Tm data above shows that Tm of the variants is improved with up to about 25% compared to the GCL having SEQ ID NO: 1 , indicating an improved storage stability of the variants. Example 5: Wash performance and odor measurement

Below the experimental setup for wash performance and odor measurement is described, followed by the experimental results.

Terq-O-tometer (TOM) wash assay

The Tergo-To-Meter (TOM) is a medium scale model wash system that can be applied to test 16 different wash conditions simultaneously. A TOM is basically a large temperature-controlled water bath with up to 16 open metal beakers (1000 mL) submerged into it. Each beaker constitutes one small top loader style washing machine and during an experiment, each of them will contain a solution of a specific detergent/enzyme system and the soiled and unsoiled fabrics its performance is tested on. Mechanical stress is achieved by a rotating stirring arm, which stirs the liquid within each beaker. Because the TOM beakers have no lid, it is possible to withdraw samples during a TOM experiment and assay for information on-line during wash.

The TOM model wash system is mainly used in medium scale testing. In a TOM experiment, factors such as the ballast to soil ratio and the fabric to wash liquor ratio can be varied. Therefore, the TOM provides the link between small scale experiments and the more timeconsuming full-scale experiments in top loader washing machines.

Terq-O-tometer (TOM) wash

Water hardness was adjusted to the strength described below by addition of CaCh, MgCh and NAHCO3. Wash solutions were prepared with desired amount of detergent, temperature and water hardness in a bucket as described below. Detergent was dissolved during magnet stirring for 10 minutes. Wash solution was used within 30 to 60 minutes after preparation.

Temperature and rotation (rpm) in the water bath in the Terg-O-Tometer were set according to the settings below. When temperature was adjusted according to settings (+/- 1°C) wash solution was added to TOM beaker according to the amount described below.

Agitation in the beaker was at 120 rpm. 2 homemade lard stains and 2 CS-10 butter fat stains were added to each of the beakers and wash carried out according to time stated below. 2 replicas of each stain type in each beaker. The swatches were rinsed in cold tap water for 10 minutes and dried in the dark overnight. Lard stains were weighed on an analytical scale (348- AV-50). CS-10 butter fat stains were cut out in 2 cm in diameter and used for odor measurements.

Homemade lard stains: Blue knitted cotton swatches (WFK80A, 5 x 5 cm, from Warwick Equest Ltd, Unit 55, Consett Business Park, Consett, County Durham, DH86BN, United Kingdom) were heated for 100 °C for 20 min, then left at room temperature for min 60 min. Lard (heated in water bath 75°C, 100 pL) was applied on each swatch and heated at 100 °C for 20 min and then left at room temperature for min 60 minutes, weighed on analytical scale (348-AV-50).

CS-10 (butter fat) stains were obtained from Center for Testmaterials BV, P.O. Box 120, 3133 KT Vlaardingen, the Netherlands. Table 6: Experimental conditions in TOM wash

Odor measurements

The butyric acid release (odor) from the lipase washed swatches was measured by Solid Phase Micro Extraction Gas Chromatography (SPME-GC) using the following method:

The cotton textile was washed as specified above and after wash, excess water was removed from the textile using filter paper and the textile was thereafter dried at 25°C for 2 hours. Each

SPME-GC measurement was performed with four pieces of the washed and dried textile (5 mm in diameter), which were transferred to a Gas Chromatograph (GC) vial and the vial was closed. The samples were incubated at 30°C for 24 hours and subsequently heated to 140°C for 30 minutes and stored at 20°C-25°C for at least 4 hours before analysis. The analyses were performed on a Varian 3800 GC equipped with a Stabilwax- DA w/lntegra-Guard column (30m, 0.32mm ID and 0.25um df) and a Carboxen PDMS SPME fiber (85 micro-m). Sampling from each GC vial was done at 50°C for 8 minutes with the SPME fiber in the head-space over the textile pieces and the sampled compounds were subsequently injected onto the column (injector temperature = 250°C). Column flow = 2 ml helium/minute. Column oven temperature gradient: 0 minute = 50°C, 2 minutes = 50°C, 6 minutes 45 seconds = 240°C, 11 minutes 45 seconds = 240°C. Detection was done using a Flame Ionization Detector (FID) and the retention time for butyric acid was identified using an authentic standard.

The odor generation was measured applying the method described above (SPME-GC) after wash with 0.1 ppm lipase of SEQ ID NO: 5 and 0.3 ppm lipase of SEQ ID NO: 1 with the indicated mutations. A Reciprocal Improvement Factor (RIF or rif) above 1 indicates that the odor generation is reduced, e.g., RIF for the lipase of SEQ ID NO: 1 and the mutation G396S is 135.1 , meaning that area under the curve for that lipase is reduced with a factor 135.1 compared to the area under the curved obtained when the lipase of SEQ ID NO: 5 is used.

All lipase variants were dosed at 0.3 ppm, the commercially available lipase of SEQ ID NO: 5 was dosed at a threefold lower level, ie. 0.1 ppm.

Improvement factors (IF) are calculated for removal. If_removal is the percentage removed lard of the lipase variant divided by the percentage removed lard of SEQ ID NO:1. Delta_removal is the percentage removed lard of the lipase variant minus the percentage removed lard of the sample without enzyme addition.

Reciprocal improvement factors are calculated for odor, so a higher value is a reduction in odor. rif_odor is the butyric acid of the commercial lipase of SEQ ID NO: 5 dosed at 0.1 ppm variant divided by the butyric acid of the variant. Value_butyric acid is the butyric acid of the lipase variant minus the butyric acid of the sample without enzyme addition. Delta_butyric acid is the butyric acid of the lipase variant minus the butyric acid of the sample without enzyme addition.

Results

Variants of Seq ID no 1 are generated and their improvements of performance are shown in Table as if_removal. A value above 1 is equal to improvement over the performance of SEQ ID NO: 1 . Further, the odor generation from these enzymes are listed in table 7 as well under rif_odor. The comparison for odor generation is to SEQ ID NO: 5

Table 7: Lard removal and odor generation by variants of SEQ ID NO: 1 and benchmark lipase (N.A.: Not Available)

From the data above it can be seen that all the generated variants have better performance on lard removal than the lipase of SEQ ID NO: 1. Further, the odor generation is compared to the commercially available lipase, SEQ ID NO: 5, at 0.1 ppm. It is clear that the lipase having SEQ ID NO: 1 as well as all the variants have lower odor generation than the commercially available lipase, SEQ ID NO: 5.

Example 6: Half life improvement for purified lipase variants

From Example 3 and Example 4 it is evident that further improvement of half-life as well as thermal stability compared to the variant having SEQ ID NO: 2 can be obtained. The variant of SEQ ID

NO: 1 having the substitutions G396E+S397D+W398P+I408R+L409R (SEQ ID NO: 4) is therefore selected as reference for test of storage stability for new variants.

Purified lipase variants were diluted to 0.1, 0.5 or 1.0 mg/ml with 0.01 % Triton X-100 based on concentrations determined by active site titration (Example B: Active site titration of lipase variants). Each variant was tested in two concentrations. In the wells of a microtiter plate (called detergent plate, Nunc U96 PP 0.5 ml) 30 pl diluted lipase sample was mixed with 270 pl Model detergent 1 or 2 using magnetic bars. The model detergents were diluted with ion-exchanged water to obtain concentration of 80, 160, 320 or 900 g/L. On each microtiter plate the reference SEQ ID NO: 4 was included, the HIF value for SEQ ID NO: 4 was set to 1.

After various incubation times (e.g., 0 min, 10 min, 20 min 30 min, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours) at room temperature, residual lipase activity was measured. 10 pl sample from the detergent plate was withdrawn and diluted to approximately 0.001 % detergent with assay buffer (0.2 M Tris, pH 8.0). 100 pl of this dilution was mixed in a black microtiter plate (Nunc F) with 100 pl substrate solution (125 pM 4-methylumbelliferyl oleate (Sigma 75164) in assay buffer). Fluorescence was read every 10 seconds for 5 minutes (excitation at 372 nm, emission at 445 nm, Biotek Synergy H1 microplate reader). Activity was determined from the initial slope of fluorescence increase by linear regression.

Decrease in activity during incubation with detergent was assumed to be exponential, and halflives (T%) were found from linear regression of logarithm of activity versus incubation time. Halflife improvement factors (HIF) were calculated as ratio between half-life of variant and half-life of the SEQ ID NO: 4 (reference) tested under the same conditions. The following Half-life improvement factors (HIF) were obtained for the variants in Model detergent 1 and Model detergent 2, respectively:

Table 8: HIF of variants in 80g/L Model detergent 1

Table 9: HIF of variants in 160g/L Model detergent 1

Table 10: HIF of variants in 320g/L Model detergent 1

Table 11: HIF of variants in 900g/L Model detergent 1

Table 12: HIF of variants in 80g/L Model detergent 2

Table 13: HIF of variants in 160g/L Model detergent 2

Table 14: HIF of variants in 320g/L Model detergent 2

Table 15: HIF of variants in 900g/L Model detergent 2

Table 16: HIF of variants in 900g/L Model detergent 2

The lipase variants all show an improved stability (half-life) compared to the lipase variant having SEQ ID NO: 4, which in turn showed improved stability compared to the GCL1 lipase having SEQ ID NO: 1.

Example 7: Residual activity for expressed lipase variants

From Example 3 and Example 4 it is evident that further improvement of half-life as well as thermal stability compared to the variant having SEQ ID NO: 2 can be obtained. The variant of SEQ ID NO: 1 having the substitutions G396E+S397D+W398P+I408R+L409R (SEQ ID NO: 4) is therefore selected as reference for test of storage stability for new variants.

Each lipase variant, expressed in A oryzae, was grown in four independent biological replicates in separate microtiter plates in defined media, for 4 days at 37 °C in 96-well lidded MTP’s without agitation. Expressed variants in supernatants were diluted in dilution media (0.02% v/v Triton X- 100 in H2O), then dispensed into four quadrants per supernatant sample in a 384-well flat black microtiter plate. Model detergent 1 or 2 was dispensed to quadrant wells containing the diluted enzyme sample, at different timepoints, to a final concentration of 200 mM Tris-HCI, 15 °dH, pH 8.0, besides the components of the model detergent and the diluted enzyme sample, and briefly shaken. The sample was incubated in the model detergent at the incubation times and detergent concentration designated in Table 17-19 below, and the initial, unstressed reference was obtained by mixing the sample with the detergent just before activity measurement. Detergent incubation and assays were performed at room temperature (about 25 °C).

After the incubation period, assay solution (500 pM 4-methylumbelliferyl oleate in 2.4 % v/v ethanol) was added, and the plate were read immediately using a Tecan Synergy2 using fluorescence measurements settings of A_ex 372 nm; A_em 445 nm for 12 min. The output is saved (Vmax, R²) and used for calculating residual activity, and initial activity, for data that passed quality filters. Residual activities are calculated as a ratio of the activity of the detergent-stressed sample and the initial activity, in model detergent. Grown lipase variant having SEQ ID NO: 4 was used as reference was tested under the same conditions for comparison. The storage stability value for SEQ ID NO: 4 under the specified condition was set to 1.

The following residual activity results were obtained for the variants in Model detergent 1 and Model detergent 2, respectively:

Table 17: Storage stability of variants in 16% Model detergent 1 compared to SEQ ID NO: 4

Table 19: Storage stability of variants in 16% Model detergent 2 compared to SEQ ID NO: 4

The lipase variants all show an improved stability compared to the lipase variant having SEQ ID NO: 4, which in turn showed improved stability compared to the GCL1 lipase variant of SEQ ID NO: 1.

Example 8: Wash performance and odor measurement compared to SEQ ID NO: 4

The variant having SEQ ID NO: 4 showed improved stability compared to the variants having SEQ ID NO: 1 and SEQ ID NO: 2, cf. Examples 3 and 4 above. On top of these improvements, variants of SEQ ID NO: 4 showed an even further improved stability, c.f. Examples 6 and 7. Based on these observations, wash performance and odor measurement of variants of SEQ ID NO: 4 was tested. The experimental set-up and conditions were the same as in Example 5 above with the modification that improvement factor for removal (if_removal) was compared to the variant having SEQ ID NO: 4.

Results The following results were obtained:

Table 20: Lard removal and odor generation by variants of SEQ ID NO: 4 and benchmark lipase From the data above it can be seen that all the generated variants of SEQ ID NO: 4 have better performance on lard removal than the lipase of SEQ ID NO: 4. Further, the odor generation is compared to the commercially available lipase, SEQ ID NO: 5, at 0.1 ppm. It is clear that the variants of the lipase having SEQ ID NO: 4 have lower odor generation than the commercially available lipase, SEQ ID NO: 5.

Claims

1 . A lipase variant comprising one or more mutations, such as one or more substitutions, at one or more positions corresponding to positions 396, 397, 398, 408, 409, 166 and 325 of SEQ ID NO: 1 , wherein the variant has lipase activity and wherein the variant has at least 85% sequence identity to the polypeptide of SEQ ID NO: 4.

2. The lipase variant of claim 1 comprising a substitution at position 396 and one or more further substitutions at positions selected from the group consisting of 397, 398, 408, 409, 166 and 325, wherein the variant has lipase activity and wherein the variant has at least 85% sequence identity to the polypeptide of SEQ ID NO: 4.

3. The lipase variant of claim 1 or 2, wherein the substitution in position 396 is selected from the group consisting of G396E, G396Q, G396R, G396K, G396W, G396S, G396A, G396V, G396M, G396P, G396N, G396I, G396D and G396H.

4. The lipase variant of claims 1 to 3 comprising a substitution at position 408 selected from the group consisting of I408R, I408D, I408E, I408Q, I408M, I408S, I408G, I408A, I408K, I408P, I408L, I408W, I408V, W398D, I408H, I408Y and I408F.

5. The lipase variant of any of claims 1 to 4 further comprising one or more substitutions selected from the group consisting of S397D, S397N, S397T, S397E, W398P, W398L, W398Y, W398V, W398A, W398I, W398R, W398T, W398K, W398S, W398M, L409R, L409K, L409H, L409N, L409E, L409Q, L409D, L409Y, L409S, G325D, G325E, T166L, T166W and T166G.

6. The lipase variant of any of claims 1 to 5 wherein the variant is selected from the group of variants a. Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions G396E, S397D, W398P, I408R and L409R b. Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, G325D, G396E, W398Y and L409R c. Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions W398P and I408E d. Variant of the polypeptide of SEQ ID NO: 1 comprising the substitutions T166L, G325D, G396E and W398Y wherein the variants have at least 80% identity to the polypeptide of SEQ ID NO: 4.

7. The lipase variant of claim 1 having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to a polypeptide selected from the group of polypeptides consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 , SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

8. The lipase variant of any of the preceding claims which has an improved property relative to the lipase of SEQ ID NO: 1 , wherein the improved property is selected from the group consisting of Half-life Improvement Factor, wash performance, odor reduction, thermal stability and detergent stability.

9. A detergent composition comprising a lipase variant of any of claims 1 to 8 and at least one surfactant and optionally one or more enzymes.

10. A washing method for textile comprising: a. Exposing a textile to a wash liquor, said wash liquor comprising i. The lipase variant of any of claims 1 to 8; ii. At least one surfactant; and iii. Optionally one or more additional enzymes b. completing at least one wash cycle, and c. optionally rinsing the textile.

11 . The washing method according to claim 10, wherein the surfactant level in the wash liquor is below 10 g/L.

12. The detergent composition according to claim 9 or the washing method according to claim 10, wherein the at least one surfactant is rhamnolipid and/or sophorolipid.

13. A polynucleotide encoding the variant of any of claims 1 to 8.

14. A nucleic acid construct or expression vector comprising the polynucleotide of claim 13.

15. A recombinant host cell transformed with the polynucleotide of claim 13.

16. A method of producing a lipase variant, comprising: a. cultivating the recombinant host cell of claim 15 under conditions suitable for expression of the variant; and b. recovering the variant.