Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38627—Preparations containing enzymes, e.g. protease or amylase containing lipase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
  • C11D2111/10—Objects to be cleaned
  • C11D2111/12—Soft surfaces, e.g. textile
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi

Definitions

• 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.
• 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.
• GCL I Geotrichum candidum lipase I
• 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.
• 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
• 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.
• 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
• 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 cDNA:
• 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 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 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.
• 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.
• 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.
• adjunct ingredient is different to the lipase of this invention.
• 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 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).
• 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.
• additional enzymes such as proteases, amylases, lipases, cut
• Detergent load is the amount of detergent used in a wash cycle.
• 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 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.
• 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.
• 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 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.
• 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.
• 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.
• butanoic acid butyric acid
• pentanoic acid valeric acid
• a fatty acid and an acyl group of a lipid are equivalents.
• 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.
• 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:
• 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 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.
• 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.
• 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).
• the invention provides polypeptides having lipase activity, wherein said polypeptides are substantially homologous to a fungal lipase.
• 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 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 T 1 Z> 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 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 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.
• heterologous means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell.
• 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.
• 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 means a characteristic associated with a variant that is improved compared to the reference enzyme/parent enzyme.
• 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).
• WP wash performance
• 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.
• 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 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 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).
• a “lipase substrate” is any substrate which can be hydrolyzed by the lipase of the invention.
• lipase activity i.e. the hydrolytic activity of the lipase
• 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 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 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 means a polynucleotide that encodes a mature polypeptide having lipase activity.
• mutant means a polynucleotide encoding a variant.
• mutant refers to a deletion (including a truncation), insertion (including an extension) or substitution in a parent polypeptide.
• nucleic acid or polypeptide naturally occurring in a host cell.
• 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 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 means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
• 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 means a lipase to which an alteration is made to produce the enzyme variants of the present invention.
• 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).
• 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.
• 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.
• the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present.
• 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.
• 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.
• 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.
• 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 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.
• 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.
• recombinant is synonymous with “genetically modified” and “transgenic”.
• 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
• Rhamnolipid 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.
• rhamnolipid includes mono-rhamnolipid or dirhamnolipid, mixtures thereof and varying chain length as well as salts of rhamnolipid.
• 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:
• sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
• 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.
• the Needle program 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:
• 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.
• the nobrief option must be specified in the command line.
• the output of Needle labeled “longest identity” is calculated as follows:
• 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 include sophorolipid in the lactone form and the corresponding acidic form as well as mixtures thereof. Further “sophorolipid” also includes salts of sophorolipid.
• sequence 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 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 and sustainable means use of renewable resources that cause little or no damage to the environment and are biodegradable.
• Sustainability profile :
• 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).
• 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.
• 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.
• 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.
• Fabric may be conventional washable laundry, for example stained household laundry.
• fabric or garment it is intended to include the broader term textiles as well.
• textile also covers fabrics.
• textile is used interchangeably with fabric and cloth.
• 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;
• 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.
• 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:
• 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 refers to an aqueous solution containing a detergent composition in dilute form, such as as the wash liquor in a laundry process.
• 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 is abbreviated w/w%, wt% or w%. The abbreviations are used interchangeably.
• 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.
• naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature.
• 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).
• 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.
• gap open penalty 10
• gap extension penalty 0.5
• EBLOSUM62 EMBOSS version of BLOSUM62
• an amino acid insertion 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”.
• 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).
• the sequence would thus be:
• 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.
• addition marks 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.
• commas e.g., “Arg170Tyr+Gly195Glu”, “R170Y+G195E”, “Arg170Tyr,Gly195Glu” or “R170Y,
• Arg170Tyr,Glu or “Arg170Tyr/Glu” represents a substitution of arginine at position 170 with tyrosine or glutamic acid.
• “Tyr167Gly,Ala + Arg170Gly,Ala” or “Tyr167Gly/Ala + Arg170Gly/Ala” designates the following variants: “Tyr167Gly+Arg170Gly”, “Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.
• amino acid X (or Xaa) is used herein to represent any of the 20 natural amino acids.
• X preceding a position means that any original amino acid at that position may be substituted.
• 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.
• 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.
• a mutation such as a substitution
• 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.
• 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 expressed as Half-life Improvement
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the variant comprises or consists of a substitution at a position corresponding to position 409, wherein the variant has lipase activity.
• 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.
• 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.
• 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+408 325+409 396+397 396+408 396+409 397+398 397+398 397+398 397+398 3
• 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+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+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+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/
• 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/
• 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 //
• 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/
• 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.:
• 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.:

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