WO2016046334A1 - Utilisation d'enzyme pour le nettoyage - Google Patents

Utilisation d'enzyme pour le nettoyage Download PDF

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Publication number
WO2016046334A1
WO2016046334A1 PCT/EP2015/072021 EP2015072021W WO2016046334A1 WO 2016046334 A1 WO2016046334 A1 WO 2016046334A1 EP 2015072021 W EP2015072021 W EP 2015072021W WO 2016046334 A1 WO2016046334 A1 WO 2016046334A1
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Prior art keywords
seq
lipase
protease
variant
cleaning
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PCT/EP2015/072021
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English (en)
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Peter Klindt MOGENSEN
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Novozymes A/S
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Publication of WO2016046334A1 publication Critical patent/WO2016046334A1/fr

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    • 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

Definitions

  • the present invention relates to the use of a lipase variant or detergent compositions comprising a lipase variant for cleaning in place, a detergent composition comprising a lipase variant and a method for cleaning-in-place.
  • Cleaning-in-place is commonly used for cleaning storage tanks, bioreactors, fermenters, mix vessels, pipelines and other equipment used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing.
  • WO97/02753 concerns a solution comprising a protease and a lipase for cleaning-in-place.
  • the solution has been found effective in cleaing process equipment containing residues of milk or burnt milk.
  • WO2002/081755 relates to a method of cleaning dairy pipelines using an enzymatic prewash and an acidic secondary wash.
  • the present invention concerns the use of a variant of a parent lipase for cleaning-in- place, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO: 2.
  • the invention further concerns a detergent composition
  • a detergent composition comprising a variant of a lipase, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2, and which composition is free of borate.
  • the invention concerns a method for cleaning-in-place, wherein a variant of a parent lipase is circulated, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO: 2.
  • 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.
  • lipase activity triacylglycerol lipase, EC3.1 .1 .3), phospholipase activity (Phospholipase A(2) EC3.1 .1 .4 (Lecithinase A, Phosphatidase, Phosphatidolipase, Phosphatidylcholine 2-acylhydrolase, Phospholipase A2) or (Phospholipase A(1 ), EC3.1.1 .32 Phospholipase A1 )), cutinase activity (EC3.1.1 .74), sterol esterase activity (EC3.1.1.13) and/or wax-ester hydrolase activity (EC3.1.1 .50).
  • Phospholipase A(2) EC3.1 .1 .4 (Lecithinase A, Phosphatidase, Phosphatidolipase, Phosphatidylcholine 2-acylhydrolase, Phospholipa
  • lipase activity is determined according to the procedure described in the Examples.
  • the variants of the present invention have at least 20%, e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the lipase activity of the polypeptide of SEQ ID NO: 2.
  • allelic variant means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences.
  • An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
  • cDNA 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.
  • CIP Cleaning-in-place
  • Process equipment can be processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, centrifuges, evaporators, extruders, coolers, storage tanks, sieves, hydroclones, filter units and filter membranes.
  • CIP can also be used in road tankers transporting liquid food such as milk or beer, or in equipment used in slaughter houses.
  • Cleaning-Out-of-Place "Cleaning-Out-of-Place” or "COP" is a method for cleaning equipment which is dismantled.
  • COP is dismantling of equipment and cleaning it separate from the rest of the equipment for example by soaking in wash liquor and optionally cleaning the equipment manually by brush or the like.
  • One example could be dismantling of filters and soaking the filter membrane in a wash liquor optionally comprising the variant lipase of the invention.
  • Open-Plant-Cleaning Open-Plant-Cleaning
  • OPC Open-Plant-Cleaning
  • Cleaning performance is a measure for the removal of lipids and protein from the equipment.
  • lipase degrades lipids present in the equipment free fatty acids are released from the glycerol backbone.
  • 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,
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a variant of the present invention.
  • Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the variant or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • 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.
  • 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 means a linear or circular DNA molecule that comprises a polynucleotide encoding a variant and is operably linked to control sequences that provide for its expression.
  • fragment means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a polypeptide; wherein the fragment has lipase activity.
  • a fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of amino acids 1 to 369 of SEQ ID NO: 2.
  • High stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Improved property means a characteristic associated with a variant that is improved compared to the parent lipase.
  • improved properties include, but are not limited to, detergent stability, stability in detergent with protease present, protease stability, chemical stability, oxidation stability, pH stability, stability under storage conditions, and thermostability.
  • Isolated means a substance in a form or environment which does not occur in nature.
  • isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • An isolated substance may be present in a fermentation broth sample.
  • Low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • the mature polypeptide is amino acids 1 to 269 of SEQ ID NO: 2. It is known in the art that a host cell may produce a mixture of two or more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having lipase activity.
  • the mature polypeptide coding sequence is nucleotides 1 to 807 of SEQ ID NO: 1 .
  • Medium stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
  • Medium-high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • Mutant means a polynucleotide encoding a variant.
  • 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, which comprises one or more control sequences.
  • Operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Organic particles By the term “organic particles is meant particles of organic origin that can be present in equipment used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing. Examples of organic particles are lipids, protein, carbohydrates. One example of organic particles that can be removed is lipids or fragments thereof. The lipase variant degrades the lipids, which will then be re moved by the circulation of wash liquor or by rinsing with water. Another example of organic particles that can be removed is protein. The lipase variant can therefore be used together with a protease which degrades and removes the protein present in the equipment.
  • parent or parent lipase means a lipase to which an alteration is made to produce the enzyme variants of the present invention.
  • the parent lipase may be a naturally occurring (wild-type) polypeptide or a variant or fragment thereof.
  • 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 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.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide sequences is determined 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 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
  • the stability of a lipase may be expressed as the residual activity or the residual performance of said lipase during or after exposure to various test conditions such as e.g. storage in a detergent composition, at various temperatures, at various pH, in the presence of different components such as protease, chemicals, and/or oxidative substances (stress conditions).
  • the stability of a variant lipase can be measured relative to a known activity or performance of a parent lipase, or alternatively to a known activity or performance of the variant lipase when initially added to the detergent composition optionally stored cold or frozen or relative to the variant lipase stored cold or frozen (unstressed conditions).
  • Subsequence means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having lipase activity.
  • a subsequence contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% but less than 100% of the number of nucleotides 1 to 807 of SEQ ID NO: 1.
  • variant means a polypeptide having lipase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) 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 an amino acid adjacent to and immediately following the amino acid occupying a position.
  • the variants of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lipase activity of the polypeptide of SEQ ID NO: 2.
  • Very high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
  • Very low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
  • wash liquor is defined herein as a solution or mixture of water and a parent lipase, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2 the cleaning performance is improved.
  • the preferred lipase variant comprises substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the wash liquor can further comprise detergent ingredients such as calcium chloride, sodium formate, sorbitol, glycerol and mono propylene glycol and optionally surfactants.
  • Wild-type lipase means a lipase expressed by a naturally occurring microorganism, such as a bacterium, yeast, or filamentous fungus found in nature.
  • the polypeptide disclosed in SEQ ID NO: 2 is used to determine the corresponding amino acid residue in another lipase.
  • the amino acid sequence of another lipase is aligned with SEQ ID NO: 2, and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 2 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.
  • Identification of the corresponding amino acid residue in another lipase can be determined by an alignment of multiple polypeptide sequences using several computer programs including, but not limited to, MUSCLE (multiple sequence comparison by log- expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 51 1 -518; Katoh and Toh, 2007, Bioinformatics 23: 372- 374; Katoh et al., 2009, Methods in Molecular Biology 537:_39-64; Katoh and Toh, 2010, Bioinformatics 26:_1899-1900), and EMBOSS EMMA employing ClustalW (1 .83 or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680), using their respective default parameters.
  • MUSCLE multiple sequence comparison
  • GenTHREADER Programs such as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19: 874-881 ) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence.
  • sources PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials
  • Gough et a/., 2000, J. Mol. Biol. 313: 903-919 can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the polypeptide, and such models can be assessed for accuracy using a variety of tools developed for that purpose.
  • proteins of known structure For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable.
  • Two or more protein structures can be aligned using a variety of algorithms such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 1 1 : 739-747), and implementation of these algorithms can additionally be utilized to query structure databases with a structure of interest in order to discover possible structural homologs (e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).
  • 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 “Gly195Glyl_ys” 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 "Gly195Glyl_ysAla" 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: G - K - A
  • variants comprising multiple alterations are separated by addition marks ("+"), e.g., "Arg170Tyr+Gly195Glu” or “R170Y+G195E” representing a substitution of arginine and glycine at positions 170 and 195 with tyrosine and glutamic acid, respectively.
  • the present invention concerns the use of enzymes in cleaning-in-place (CIP) applications.
  • CIP cleaning-in-place
  • the inventor has surprisingly found that the use of a new lipase variant improves the cleaning performance in cleaning-in-place applications.
  • the inventor has found that by using a variant of a parent lipase, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2 the cleaning performance is improved.
  • the cleaning performance may be improved so steady state of pH is reached after 30 minutes when measured with Assay (I).
  • the cleaning performance may be even further improved so steady state of pH is reached after 25 minutes, after 20 minutes, after 15 minutes after 10 minutes, after 9 minutes, after 8 minutes, after 7 minutes, after 6 minutes or after 5 minutes when measured with Assay (I).
  • Further advantages by the process is that the use of the lipase variant in cleaning-in-place allows use of mild alkaline detergents, use of lower temperatures and provides a faster method than in traditional CIP.
  • the present CIP process is low foaming and allows organic particles to be removed easier and therefore results in a more efficient cleaning. As the CIP process is performed faster and at a lower temperature, the energy consumption used for CIP is low.
  • the preferred lipase variant comprises substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the variant is selected from the group consisting of:
  • polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to
  • SEQ ID NO: 2 a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i);
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • the cleaning-in-place can be further improved by using one or more enzyme in combination with the lipase variant.
  • the one ore more further enzymes may be selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, and/or mixtures thereof.
  • the further enzyme is a protease.
  • the protease can be protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the lipase variant is used for cleaning-in- place together with a protease.
  • the protease can have at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3.
  • the protease is the protease of SEQ ID NO: 3.
  • the further enzyme is a lipase.
  • the lipase can be a polypeptide having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 8 or a variant thereof wherein the polypeptide comprises the following substitutions T231 R and N233R.
  • the further enzyme is a phosphorlipase, such as a Phospholipase A1 or a Phospholipase A2.
  • the phosphorlipase can be the phosphorlipase in SEQ ID NO: 9 or the phosphorlipase in SEQ ID NO: 10 or phosphorlipases having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • the lipase variant may be used together with other cleaning compounds such as at least one surfactant, at least one surfactant system, at least one soap or any mixtures thereof. By combining the use of the lipase variant with a further cleaning compound the cleaning performance may be further enhanced.
  • the surfactant can be one or more non-ionic surfactants or one ore more amphoteric surfactant or a mixture thereof.
  • the non-ionic surfactant can be selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end-blocked ethoxylates, polypropylene glycols and polyethylene glycols.
  • amphoteric surfactant is selected from alkylimidazoline, alkylbetaines, alkylamidobetaines and protein derivatives.
  • the lipase variant can be used in cleaning-in-place for removing organic particles in equipment used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing.
  • organic particles that can be removed is lipids.
  • the lipase variant degrades the lipids, which will then be re moved by the circulation of wash liquor or by rinsing with water.
  • organic particles that can be removed is protein.
  • the lipase variant can therefore be used together with a protease which degrades and removes the protein present in the equipment.
  • the lipase variant is therefore very efficient for cleaning-in-place of equipment use for food and beverage manufacturing.
  • the lipase variant may be used together with a protease for example in dairy equipment or brewing equipment, such as road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes.
  • a protease for example in dairy equipment or brewing equipment, such as road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes.
  • filter membranes such as semipermeable membranes used for e.g. ultrafiltration, have shown very difficult and time-consuming to clean.
  • the lipase variant in CIP optionally together with a protease, the cleaning of equipment like membranes are much faster and more efficient and time is saved for cleaning. As such membranes tend to clog during use daily cleaning is needed.
  • the inventor has found that the use of a lipase variant together with a protease ensures a gentle, effective and fast cleaning of filter membranes.
  • One advantage is that the life-time of the filter units is significantly increase by use of the lipase variant of the invention, for example when used together with a protease.
  • the cleaning performance of the inventive lipase optionally used together with a protease when used on lipid products containing at least 18% fat is at least 50% better than the cleaning performance of SEQ ID NO: 4 and SEQ ID NO: 7 used together on lipid products containing at least 18% fat.
  • the cleaning performance is at least 60% better than the cleaning performance of SEQ ID NO: 4 and SEQ ID NO: 7 used together, such as at least 70% better, at least 80% better, at least 90% better or at least 100% better than the cleaning performance of SEQ ID NO: 4 and SEQ ID NO: 7 used together.
  • the invention further concerns a detergent composition
  • a detergent composition comprising variant of a lipase, which variant has lipase activity and has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2, and which composition is free of boron and/or borates.
  • the variant of lipase can further comprise substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the variant of the detergent composition is selected from the group consisting of:
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • the composition comprises a variant of lipase further comprising substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the composition further comprises at least one of the following ingredients: a sugar alcohol, a calcium salt, a formate salt and/or a propylene glycol.
  • the sugar alcohol may be selected from the group consisting of sorbitol, xylitol, mannitol, galactitol fucitol, iditol, maltitol, lactitol and inositol.
  • the calcium salt is selected from the group of calcium chloride, calcium formate, calcium sulphate.
  • the formate salt is selected from the group consisting of sodium formate, calcium formate, potassium formate.
  • the propylene glycol is selected from the group consisting of mono propylene glycol, glycerol, sorbitol, ethylene glycol or poly ethylene glycol (PEG).
  • the detergent composition may further comprise glycerol and water.
  • the detergent composition further comprises one or more further enzymes, such as a protease. Thereby the cleaning-in-place can be further improved.
  • the one ore more further enzymes may be selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, and/or mixtures thereof.
  • the composition further comprises a protease.
  • the protease can be protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the detergent composition comprises a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3.
  • the protease is the protease of SEQ ID NO: 3.
  • the composition further comprises a lipase.
  • the lipase can be a polypeptide having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 8 or a variant thereof wherein the polypeptide comprises the following substitutions T231 R and N233R.
  • the further enzyme is a phosphorlipase, such as a Phospholipase A1 or a Phospholipase A2.
  • the phosphorlipase can be the phosphorlipase in SEQ ID NO: 9 or the phosphorlipase in SEQ ID NO: 10 or phosphorlipases having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • the ratio between the lipase variant and the protease enzyme is in the range of 1 to 9, in the range of 2 to 8 or in the range of 3 to 7.
  • the composition comprises:
  • the composition comprises at least one surfactant, at least one surfactant system, at least one soap or any mixtures thereof.
  • the surfactant can be one or more non-ionic surfactants or one ore more amphoteric surfactant or a mixture thereof.
  • the non-ionic surfactant can be selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end-blocked ethoxylates, polypropylene glycols and polyethylene glycols.
  • amphoteric surfactant is selected from alkylimidazoline, alkylbetaines, alkylamidobetaines and protein derivatives.
  • the surfactant may be present in an amount from about 1 % to about 40% by weight of a 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 a surfactant.
  • the composition is a liquid composition.
  • the composition can further comprise a protease inhibitor.
  • the protease inhibitor inhibits the action of the protease and thus leads to a more shelf-stable detergent composition.
  • the protease inhibitor is 4-formyl-phenyl-boronic acid.
  • the protease inhibitor is a peptide aldehyde of the formula P-(A) y -L-(B) x -B°-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
  • i. H is hydrogen
  • iii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the (B) x amino acid
  • v. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the (A) y amino acid, with the proviso that if L is absent then A is absent;
  • P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of Ci -6 alkyl, C 6- io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, substituent's R';
  • R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -IM H2, -NHR", -NR" 2 , -C0 2 H, -CONH 2 , -CONHR", -CONR” 2 , -
  • R" is a Ci -6 alkyl group.
  • the hydrosulfite adduct of a peptide aldehyde is of the formula P- (A) y -L-(B) x -N(H)-CHR-CH(OH)-S0 3 M, wherein
  • i. M is hydrogen or an alkali metal
  • ii. x is 1 , 2 or 3 for (B) x , and B is independently a single amino acid connected to B° via the C-terminal of the (B) x amino acid
  • iv. y is 0, 1 or 2 for (A) y , and A is independently a single amino acid residue connected to L via the /V-terminal of the (A) y amino acid, with the proviso that if L is absent then A is absent;
  • v. P is selected from the group consisting of hydrogen and an /V-terminal protection group, with the proviso that if L is absent then P is an /V-terminal protection group;
  • R is independently selected from the group consisting of Ci -6 alkyl, C 6- io aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, substituent's R';
  • R' is independently selected from the group consisting of halogen, -OH, -OR", - SH, -SR", -NH 2 , -NHR", -NR" 2 , -C0 2 H, -CONH 2 , -CONHR", -CONR” 2 , -
  • R" is a Ci -6 alkyl group.
  • M is Na or K and R is a C7 arylalkyl substituted with -OH.
  • B0 is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr) and valine (Val).
  • B1 can be selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val).
  • B2 can be selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val).
  • Al alanine
  • Arg arginine
  • Cpd capreomycidine
  • cysteine Cys
  • Gly isoleucine
  • lie leucine
  • NIe norleucine
  • Nva norvaline
  • Phe proline
  • Pro serine
  • Ser serine
  • Thr threonine
  • valine Val
  • B3 can be selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • x can be 1 , 2 or 3.
  • A1 can be selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • A2 can be selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • L can be absent and A is absent.
  • P is selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl (MEO-CO), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t- butyloxycarbonyl (Boc), adamantyloxycarbonyl, p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl (MeS02), ethylsulfonyl (EtS02), benzylsulfonyl (PhCH2S02), methylphosphoric acid
  • the peptide aldehyde adduct is L-Alaninamide, N- [(phenylmethoxy)carbonyl]glycyl-N-[2-hydroxy-1 -[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
  • the detergent composition comprises a protease inhibitor, which protease inhibitor is one of the following peptide aldehydes or a adduct thereof: Cbz-Arg-Ala- Tyr-H, Ac-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Tyr-H, Cbz-Gly-Ala-Leu-H, Cbz-Val-Ala-Leu-H, Cbz-Gly- Ala-Phe-H, Cbz-Gly-Ala-Val-H, Cbz-Gly-Gly-Tyr-H, Cbz-Gly-Gly-Phe-H, Cbz-Arg-Val-Tyr-H, Cbz-Leu-Val-Tyr-H, Ac-Leu-Gly-Ala-Tyr-H, Ac-Phe-Gly-Ala-Tyr-H, Ac-Tyr-Gly-Ala-Tyr-H, Ac
  • the invention further relates to a method for cleaing-in-place, wherein a variant of a parent lipase is circulated in a production or a process equipment, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO: 2.
  • the method provides an good cleaning performance which it at the same time allows use of mild alkaline detergents, use of lower temperatures and provides a faster method than in traditional CIP, e.g. cleaning with the commercially available detergent CIPzyme.
  • the method comprises cleaning-in-place of production or process equipment, which method comprises the following steps:
  • step b Optionally stop circulation under step b and allow the production equipment to soak in the wash liquor;
  • steps (a) to (e) are carried out one or two times.
  • the present invention concerns the use of enzymes in cleaning-in-place (CIP) applications.
  • CIP cleaning-in-place
  • the inventor has surprisingly found that the use of a new lipase variant improves the cleaning performance in cleaning-in-place applications.
  • the temperature of the wash liquor can be in the range of 10-60°C, such as in the range of 30-60°C, in the range of 40-60°C or in the range of 50-60°C.
  • the initial pH of the wash liquor can be in the range of 7-1 1 , such as the pH of the wash liquor being 7, 8, 9, 10 or 1 1.
  • the method further comprises dismantling of the production or process equipment and soaking the equipment in a wash liquor.
  • the time period for circulating the wash liquor in the equipment is in the range of 5 minutes to 90 minutes, such as in the range of 5 minutes to 80 minutes, in the range of 5 minutes to 70 minutes, in the range of 5 minutes to 60 minutes, in the range of 5 minutes to 50 minutes, in the range of 5 minutes to 40 minutes, in the range of 5 minutes to 30 minutes, in the range of 5 minutes to 25 minutes, in the range of 5 minutes to 20 minutes, in the range of 5 minutes to 15 minutes, in the range of 5 minutes to 10 minutes.
  • the lipase variant may comprise substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the variant is selected from the group consisting of:
  • polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 2;
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • the cleaning-in-place can be further improved by using one or more enzyme in combination with the lipase variant.
  • one or more enzyme in combination with the lipase variant.
  • a protease can be used in addition to the lipase variant under step b defined above.
  • enzymes that can be used in the method can be selected from 38. hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, or mixtures thereof.
  • the method further comprises using a protease.
  • the protease can be protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the method further comprises using a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3.
  • the protease is the protease of SEQ ID NO: 3.
  • the method further comprises using a lipase.
  • the lipase can be a polypeptide having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 8 or a variant thereof wherein the polypeptide comprises the following substitutions T231 R and N233R.
  • the further enzyme is a phosphorlipase, such as a Phospholipase A1 or a Phospholipase A2.
  • the phosphorlipase can be the phosphorlipase in SEQ ID NO: 9 or the phosphorlipase in SEQ ID NO: 10 or phosphorlipases having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.
  • the ratio between the lipase variant and the protease enzyme is in the range of 1 to 9, in the range of 2 to 8 or in the range of 3 to 7.
  • a traditional CIP process uses harch chemicals like strong acids and strong bases and takes about 5-15 hours, e.g. 10 -15 hours or 5-10 hours.
  • a final cleaning comprises the use of sterilants and/or sanitizers.
  • the cleaning performance may be improved so steady state of pH is reached after 30 minutes when measured with Assay I.
  • the cleaning performance may be improved so steady state of pH is reached after circulating the wash liquor for 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes or 5 minutes when measured with Assay I.
  • the cleaning performance can be further increased by using at least one surfactant, at least one surfactant system, at least one soap, or any mixtures thereof.
  • the production or process equipment is used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing, such as for cleaning dairy equipment or brewing equipment.
  • the method can be used for cleaning road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes, such as for cleaning of semipermeable membranes.
  • the method may further comprise in-activating enzymes remaining in the production equipment after steps (a) to (e) have been carried out.
  • the enzymes can be in-activated by circulating an acidic solution in the equipment, rinsing the equipment by circulating water in the equipment and subsequently drain water.
  • the acidic solution used for in-acitivating the enzymes have a pH in the range of 1 to 3, such as in the range of 1 .5 to 2.5, such as 1.5-2.25 or 1 .5 -2.0.
  • the acidic solution may comprise nitric acid and citric acid. This combination of acids are better for the environment that using strong acids.
  • the present method for cleaning-in-place can be used for removing organic particles in equipment used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing. However, the method is particular useful for equipment used for food and beverage manufacturing.
  • the lipase variant may be used together with a protease for cleaning-in-place of for example dairy equipment or brewing equipment, such as road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes.
  • dairy equipment or brewing equipment such as road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes.
  • the equipment may be dismantled and soaked in the wash liquor.
  • filter membranes such as semipermeable membranes used for e.g. ultrafiltration, are difficult.
  • the membranes can soaked in the wash liquor during the cleaning-in-place or if dismantled for further cleaning.
  • the lipase variant in CIP optionally together with a protease, the cleaning of equipment like membranes are much faster and more efficient som time is saved for cleaning.
  • the lipase variant further comprises substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the variant is selected from the group consisting of:
  • polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 2;
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • the concentration of the concentration of the lipase variant in the wash liquor is in the range of 0.005-10 mg enzyme protein/liter, such as in the range of 0.005-9.0 mg enzyme protein/liter, in the range of 0.005-8.0 mg enzyme protein/liter, in the range of 0.005-7.0 mg enzyme protein/liter, in the range of 0.005-6.0 mg enzyme protein/liter, in the range of 0.005-5 mg enzyme protein/liter, in the range of 0.005-4 mg enzyme protein/liter, in the range of 0.005-3 mg enzyme protein/liter, in the range of 0.005-2.5 mg enzyme protein/liter, in the range of 0.005-2.0 mg enzyme protein/liter, in the range of 0.005-1 .5 mg enzyme protein/liter, in the range of 0.005-1 .0 mg enzyme protein/liter, in the range of 0.005-0.9 mg enzyme protein/liter, in the range of 0.005-0.8 mg enzyme protein/liter, in the range of 0.005-0.7 mg enzyme protein/liter, in the range of 0.005-0.6
  • the concentration of the lipase variant in the wash liquor is in the range of 0.01 -10 to mg enzyme protein/liter, such as in the range of 0.02-9.0 mg enzyme protein/liter, in the range of 0.03-8.0 mg enzyme protein/liter, in the range of 0.04-7.0 mg enzyme protein/liter, in the range of 0.05-6.0 mg enzyme protein/liter, in the range of 0.06-5.0 mg enzyme protein/liter, in the range of 0.07-4.0 mg enzyme protein/liter, in the range of 0.08-3.0 mg enzyme protein/liter, in the range of 0.09-2.9 mg enzyme protein/liter, in the range of 0.1 -2.85 mg enzyme protein/liter,
  • the concentration of the concentration of the protease in the wash liquor is in the range of 0.02-40 mg enzyme protein/liter, 0.5-35 mg protein/liter, 1 -30 mg enzyme protein/liter, such as in the range of 1 -25 mg enzyme protein/liter, in the range of 1 -20 mg enzyme protein/liter, in the range of 1 -17 mg enzyme protein/liter, in the range of 1 -16 mg enzyme protein/liter, in the range of 1 -15 mg enzyme protein/liter, in the range of 1 -14 mg enzyme protein/liter or in the range of 1 -13.5 mg enzyme protein/liter.
  • the concentration of the concentration of the protease in the wash liquor is in the range of 5-30 mg enzyme protein/liter, such as in the range of 6-25 mg enzyme protein/liter, in the range of 7-20 mg enzyme protein/liter, in the range of 8-17 mg enzyme protein/liter, in the range of 9-16 mg enzyme protein/liter, in the range of 10-15 mg enzyme protein/liter or in the range of 1 1 -14 mg enzyme protein/liter.
  • the present invention provides lipase variants, comprising substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2, wherein the variant has lipase activity.
  • the variants further comprise substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • the variant has 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%, but less than 100%, to the amino acid sequence of the parent lipase.
  • the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 2.
  • the number of substitutions in the variants of the present invention is 1 - 40, e.g., 1 -30, 1 -20, 1 -10 and 1 -5, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 substitutions.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ I D NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at two positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91 T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at three positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91 T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at four positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at five positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at six positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at seven positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at eight positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91 T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • a variant comprises substitutions at positions corresponding to T231 R+N233R and at nine positions corresponding to any of positions D96E, D1 1 1A, D254S, G163K, P256T, G91T, G38A, D27R, and N33Q of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 96.
  • the amino acid at a position corresponding to position 96 is substituted with Glu, Gly, Ser, or Val, preferably with Glu.
  • the variant comprises or consists of the substitution D96E of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 1 1 1 .
  • the amino acid at a position corresponding to position 1 1 1 is substituted with Ala, Gly, lie, Leu, Met, or Val, preferably with Ala.
  • the variant comprises or consists of the substitution D1 1 1A of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 254.
  • the amino acid at a position corresponding to position 254 is substituted with Ser, or Thr, preferably with Ser.
  • the variant comprises or consists of the substitution D254S of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 163.
  • the amino acid at a position corresponding to position 163 is substituted with Asp, Glu, His, or Lys.
  • the variant comprises or consists of the substitution G163K of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 256.
  • the amino acid at a position corresponding to position 256 is substituted with Lys, Ser, or Thr, preferably with Thr.
  • the variant comprises or consists of the substitution P256T of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 91 .
  • the amino acid at a position corresponding to position 91 is substituted with Ala, Asn, Gin, Glu, lie, Leu, Ser, Thr, Trp, or Val, preferably with Thr.
  • the variant comprises or consists of the substitution G91 T of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 38.
  • the amino acid at a position corresponding to position 38 is substituted with Ala, Arg, Asn, Asp, Gin, Glu, lie, Leu, Met, or Val, preferably with Ala.
  • the variant comprises or consists of the substitution G38A of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 27.
  • the amino acid at a position corresponding to position 27 is substituted with Arg, His, or Lys, preferably with Arg.
  • the variant comprises or consists of the substitution D27R of SEQ ID NO: 2.
  • the variant comprises or consists of substitutions at positions corresponding to T231 R+N233R and position 33.
  • the amino acid at a position corresponding to position 33 is substituted with Gin, Lys, Ser, or Thr, preferably with Gin.
  • the variant comprises or consists of the substitution N33Q of SEQ ID NO: 2.
  • the variant comprises or consists of the substitutions at positions corresponding to T231 R+N233R and one of 27+33; 27+38; 27+91 ; 27+96; 27+1 1 1 ; 27+163; 27+254; 27+256; 33+38; 33+91 ; 33+96; 33+1 1 1 ; 33+163; 33+254; 33+256; 38+91 ; 38+96; 38+1 1 1 ; 38+163; 38+254; 38+256; 91 +96; 91 +1 1 1 ; 91 +163; 91 +254; 91 +256; 96+1 1 1 ; 96+163; 96+254; 96+256; 111 +163; 111 +254; 111 +256; 163+254; 163+256; or 254+256, such as those described above.
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38; 27+33+91; 27+33+96; 27+33+111; 27+33+163; 27+33+254; 27+33+256; 27+38+91; 27+38+96; 27+38+111; 27+38+163;
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38+91; 27+33+38+96; 27+33+38+111;
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38+91+96; 27+33+38+91+111 27+33+38+91+163; 27+33+38+91+254; 27+33+38+91+256; 27+33+38+96+111
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38+91+96+111; 27+33+38+91+96+163
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38+91+96+111+163
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of 27+33+38+91+96+111+163+254; 27+33+38+91+96+111 +163+256; 27+33+38+91+96+111 +254+256; 27+33+38+91+96+163+254+256; 27+33+38+91+111+163+254+256; 27+33+38+96+111 +163+254+256; 27+33+91 +96+111 +163+254+256; 27+33+91 +96+111 +163+254+256;
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and 27+33+38+91+96+111+163+254+256 such as those described above.
  • the variant comprises or consists of T231R+N233R and one or more
  • substitutions selected from the group consisting of D27R, N33Q, G38A, G91T,
  • D96E D111A, G163K, D254S, and P256T.
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of D27R+N33Q; D27R+G38A; D27R+G91T;
  • D27R+D96E D27R+D111A; D27R+G163K; D27R+D254S; D27R+P256T; N33Q+G38A;
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of D27R+N33Q+G38A; D27R+N33Q+G91T; D27R+N33Q+D96E; D27R+N33Q+D111 A; D27R+N33Q+G163K; D27R+N33Q+D254S;
  • D27R+N33Q+P256T D27R+G38A+G91T; D27R+G38A+D96E; D27R+G38A+D111 A;
  • the variant comprises or consists of the substitutions at positions corresponding to T231 R+N233R and one of D27R+N33Q+G38A+G91T D27R+N33Q+G38A+D96E; D27R+N33Q+G38A+D1 1 1 A; D27R+N33Q+G38A+G163K
  • the variant comprises or consists of the substitutions at positions corresponding to T231 R+N233R and one of D27R+N33Q+G38A+G91T+D96E
  • the variant comprises or consists of the substitutions at positions corresponding to T231 R+N233R and one of D27R+N33Q+G38A+G91 T+D96E+D1 1 1A
  • the variant comprises or consists of the substitutions at positions corresponding to T231 R+N233R and one of D27R+N33Q+G38A+G91 T+D96E+D1 1 1A+G163K; D27R+N33Q+G38A+G91 T+D96E+D1 1 1 A+D254S;
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and one of
  • the variant comprises or consists of the substitutions at positions corresponding to T231R+N233R and
  • the variants comprise or consist of the substitutions at positions corresponding to the following of SEQ ID NO: 2:
  • the variants may further comprise one or more additional substitutions at one or more
  • amino acid changes may be 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; 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.
  • amino acids amino acids that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill,
  • amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered.
  • 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 mutant 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 al., 1992, FEBS Lett. 309: 59-64.
  • the identity of essential amino acids can also be inferred from an alignment with a related polypeptide.
  • the variants may consist or contain at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids of SEQ ID NO: 2.
  • the variant has improved stability in detergent with protease present compared to the parent lipase.
  • the variant has improved detergent stability compared to the parent lipase.
  • the variant has improved protease stability compared to the parent lipase.
  • the variant has improved chemical stability compared to the parent lipase.
  • the variant has improved oxidation stability compared to the parent lipase.
  • the variant has improved pH stability compared to the parent lipase. In an embodiment, the variant has improved stability under storage conditions compared to the parent lipase.
  • the variant has improved thermostability compared to the parent lipase.
  • the parent lipase may be (a) a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 , (ii) the full-length complement of (i); or (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the polypeptide coding sequence of SEQ ID NO: 1.
  • the parent has a sequence identity to the polypeptide of SEQ I D NO: 2 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.
  • the amino acid sequence of the parent differs by up to 40 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 from the polypeptide of SEQ ID NO: 2.
  • the parent comprises or consists of the amino acid sequence of SEQ ID NO: 2.
  • the parent is a fragment of the polypeptide of SEQ ID NO: 2 containing at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of amino acids of SEQ ID NO: 2.
  • the parent is an allelic variant of the polypeptide of SEQ ID NO:
  • the parent is encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium- high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 , (ii) the full-length complement of (i) (Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).
  • the polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof may be used to design nucleic acid probes to identify and clone DNA encoding a parent from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length.
  • the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a parent.
  • Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1 ; (ii) the polypeptide coding sequence of SEQ ID NO: 1 ; (iii) the full-length complement thereof; or (iv) a subsequence thereof; under very low to very high stringency conditions.
  • Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
  • the nucleic acid probe is the polypeptide coding sequence of SEQ ID NO: 1 . In another aspect, the nucleic acid probe is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the number of nucleotides of SEQ ID NO: 1 . In another aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2; the polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1.
  • the parent is encoded by a polynucleotide having a sequence identity to the polypeptide coding sequence 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%.
  • the polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.
  • the parent may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention.
  • Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator.
  • Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et ai, 1994, Science 266: 776-779).
  • a fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides.
  • cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et ai, 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.
  • the parent may be obtained from microorganisms of any genus.
  • 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.
  • the parent is secreted extracellularly.
  • the parent may be a bacterial lipase.
  • the parent may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, Streptomyces or Thermobifida lipase, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma lipase.
  • the parent is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis lipase.
  • the parent is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus lipase.
  • the parent is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans lipase.
  • the parent is a Thermobifida alba or Thermobifida fusca (formerly known as Thermomonaspora fusca) lipase.
  • the parent may be a fungal lipase.
  • the parent may be a yeast lipase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia lipase; or a filamentous fungal lipase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora
  • the parent is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis lipase.
  • the parent is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium neg
  • the parent is a Thermomyces lanuginosus lipase, e.g., the lipase of SEQ ID NO: 2.
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • Acute Culture Collection ATCC
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the parent may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art.
  • a polynucleotide encoding a parent may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a parent has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
  • the present invention also relates to methods for obtaining lipase variants comprising: (a) introducing substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T, G38A, D27R, and N33Q of SEQ ID NO: 2; (b) selecting the variant which has lipase activity and in comparison with the parent lipase has improved stability; and (c) 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 (e.g., several) mutations are introduced at one or more defined sites in a polynucleotide encoding the parent lipase.
  • 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 lipase 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.
  • Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US2004/0171 154; 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.
  • 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; W095/17413; or W095/22625.
  • Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner ei 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.
  • the present invention also relates to isolated polynucleotides encoding a variant of the present invention.
  • the present invention relates to a nucleic acid construct comprising the polynucleotide of the invention.
  • the present invention relates to an expression vector comprising the polynucleotide of the invention.
  • the present invention relates to a host cell comprising the polynucleotide of the invention.
  • the present invention relates to a method of producing a lipase variant, comprising: (a) cultivating the host cell of the invention under conditions suitable for expression of the variant; and (b) recovering the variant.
  • 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.
  • the control sequence may be a promoter, a polynucleotide which is recognized by a host cell for expression of the polynucleotide.
  • 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.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
  • E. coli lac operon E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene ⁇ dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25).
  • promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase ⁇ glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO96/00787), Fusarium venenatum amyloglucosidase (WO00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO00
  • useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae galactokinase (GAL1 ), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1 ), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GAL1 Saccharomyces cerevisiae galactokinase
  • ADH1 alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • TPI Saccharomyces cerevisia
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator sequence 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.
  • Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ⁇ aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • Preferred terminators 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 trypsin-like protease.
  • Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1 ), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
  • Other useful terminators for yeast host cells are described by Romanos et al. , 1992, supra.
  • 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.
  • 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 sequence 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 are obtained from the genes for
  • Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1 ), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
  • ENO-1 Saccharomyces cerevisiae enolase
  • Saccharomyces cerevisiae 3-phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
  • Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase ADH2/GAP
  • the control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the variant-encoding sequence 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 trypsin-like protease.
  • 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.
  • 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.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant.
  • 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 bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ⁇ nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • 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.
  • Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
  • 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 polypeptide 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 (W095/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of the variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • regulatory sequences that regulate expression of the variant relative to the growth of the host cell.
  • regulatory systems 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 systems in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • filamentous fungi the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used.
  • Other examples of regulatory sequences are those that allow for gene amplification.
  • these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals.
  • the polynucleotide encoding the variant would be operably linked with the regulatory sequence.
  • 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.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • 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.
  • the vector may be one that, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • 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.
  • bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin or tetracycline resistance.
  • Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.
  • Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • Preferred for use in an Aspergillus cell are Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.
  • the vector preferably contains an element(s) 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.
  • the vector may rely on the polynucleotide's sequence encoding the variant or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • 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.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ⁇ permitting replication in Bacillus.
  • origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1 , ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
  • AMA1 and ANSI examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991 , Gene 98: 61 -67; Cullen et ai, 1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a variant.
  • 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.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.
  • 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 term "host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.
  • the host cell may be any cell useful in the recombinant production of a variant, e.g., a prokaryote or a eukaryote.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • Gram- positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces.
  • Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.
  • the bacterial host cell may be any Bacillus cell including, but not limited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.
  • the bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.
  • the bacterial host cell may also be any Streptomyces cell, including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
  • the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 -1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol.
  • the introduction of DNA into an E. coli cell may be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al, 1988, Nucleic Acids Res. 16: 6127-6145).
  • the introduction of DNA into a Streptomyces cell may be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol.
  • DNA into a Pseudomonas cell may be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391 -397), or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71 : 51 -57).
  • the introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981 , Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991 , Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see, e.g., Clewell, 1981 , Microbiol. Rev. 45: 409-436).
  • any method known in the art for introducing DNA into a host cell can be used.
  • the host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
  • the host cell may be a fungal cell.
  • "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).
  • the fungal host cell may be a yeast cell.
  • yeast as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
  • the yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.
  • 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 et al., 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, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
  • 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 zona
  • Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se.
  • Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP238023, Yelton et ai, 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, and Christensen et a/., 1988, Bio/Technology 6: 1419-1422.
  • Suitable methods for transforming Fusarium species are described by Malardier et ai, 1989, Gene 78: 147-156, and WO96/00787.
  • Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J.N. and Simon, M.I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et ai, 1983, J. Bacteriol. 153: 163; and Hinnen et ai, 1978, Proc. Natl. Acad. Sci. USA 75: 1920.
  • the present invention also relates to methods of producing a variant, comprising: (a) cultivating a host cell of the present invention under conditions suitable for expression of the variant; and (b) recovering the variant.
  • the host cells are cultivated in a nutrient medium suitable for production of the variant using methods known in the art.
  • the cell 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 performed in a suitable medium and under conditions allowing the variant to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. 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 variants. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the variant such as those described in the examples.
  • the variant may be recovered using methods known in the art.
  • the variant may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the variant may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure variants.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure
  • the variant is not recovered, but rather a host cell of the present invention expressing the variant is used as a source of the variant.
  • the invention is directed to detergent compositions comprising an enzyme of the present invention in combination with one or more additional detergent composition components.
  • additional components is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the detergent composition may comprise one or more non-ionic surfactants or one ore more amphoteric surfactant.
  • the surfactant should be selected so that the surfactant is a low- foaming surfactant, the surfactant does not precipitate with calcium and it does not dissolve the surface or coatings in the equipment, e.g. it does not dissolve the membranes used in ultra filtration processes.
  • Such membranes can be made of polyethersulphone.
  • the nonionic surfactant can be selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end-blocked ethoxylates, polypropylene glycols, amineoxide and polyethylene glycols.
  • amphoteric surfactants can be selected from alkylimidazoline, alkylbetaines, alkylamidobetaines and protein derivatives.
  • the surfactant may be use in an amount from about 5% to about 40% by weight of a 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 a surfactant.
  • 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 level of builder is typically 40-65%, particularly 50-65%.
  • 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 CIP detergents may be utilized.
  • Non-limiting examples of builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., SKS-6 from Hoechst), 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.
  • zeolites 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)inul
  • the detergent composition may also contain 0-50% by weight, such as about 5% to about 30%, of a detergent co-builder.
  • the detergent composition may include include a co-builder alone, or in combination with a builder, for example a zeolite builder.
  • co-builders include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copoly(acrylic acid/maleic acid) (PAA PMA).
  • Further non-limiting examples include citrate, chelators such as aminocarboxylates, aminopolycarboxylates and phosphonates, and alkyl- or alkenylsuccinic acid.
  • NTA 2,2',2"-nitrilotriacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • IDS iminodisuccinic acid
  • EDDS ethylenediamine-/V,/V-disuccinic acid
  • MGDA methylglycinediacetic acid
  • GLDA glutamic acid-/V,/V-diacetic acid
  • HEDP ethylenediaminetetra(methylenephosphonic acid)
  • DTMPA or DTPMPA N-(2- hydroxyethyl)iminodiacetic acid
  • ASMA aspartic acid-/V-monoacetic acid
  • ASDA aspartic acid- ⁇ /,/V-diacetic acid
  • ASDA aspartic acid-/V
  • the cleaning composition may also include an oxidising agents sanitizing.
  • suitable bleaching agents include bleaching compounds capable of liberating an active halogen species, such as CI2, Br2,— OCI- and/or— OBr-, under conditions typically encountered during the cleansing process.
  • Suitable oxidising agents for use in the present cleaning compositions include, for example, chlorine-containing compounds such as a chlorine, a hypochlorite, and chloramine.
  • Exemplary halogen-releasing compounds include the alkali metal dichloroisocyanurates, chlorinated trisodium phosphate, the alkali metal hypochlorites, monochloramine and dichloramine, and the like. Encapsulated chlorine sources may also be used to enhance the stability of the chlorine source in the composition).
  • An oxidising agent may also be a peroxygen or active oxygen source such as hydrogen peroxide, perborates, sodium carbonate peroxyhydrate, phosphate peroxyhydrates, potassium permonosulfate, and sodium perborate mono and tetrahydrate, with and without activators such as tetraacetylethylene diamine, and the like.
  • the composition can include an effective amount of an oxidising agent.
  • the concentrate includes a bleaching agent, it can be included in an amount of about 0.1 wt. % to about 60 wt. %, about 1 wt. % to about 20 wt. %, about 3 wt. %) to about 8 wt. %, and about 3 wt. % to about 6 wt. %.
  • Stabilizing agents that can be used in the cleaning composition include, but are not limited to: primary aliphatic amines, betaines, borate, calcium ions, sodium citrate, citric acid, sodium formate, glycerine, malonic acid, organic diacids, polyols, propylene glycol, benzalkoliumchloride, BIT, MIT and mixtures thereof.
  • the concentrate need not include a stabilizing agent, but when the concentrate includes a stabilizing agent, it can be included in an amount that provides the desired level of stability of the concentrate. Exemplary ranges of the stabilizing agent include up to about 20 wt. %, between about 0.5 wt. % to about 15 wt. % and between about 2 wt. % to about 10 wt. %.
  • Dispersants include, but are not limited to: primary aliphatic amines, betaines, borate, calcium ions, sodium citrate, citric acid, sodium formate, glycerine, malonic acid
  • Dispersants that can be used in the cleaning composition include maleic acid/olefin copolymers, polyacrylic acid, and its copolymers, and mixtures thereof.
  • the concentrate need not include a dispersant, but when a dispersant is included it can be included in an amount that provides the desired dispersant properties.
  • Exemplary ranges of the dispersant in the concentrate can be up to about 20 wt. %, between about 0.5 w.% and about 15 wt. %, and between about 2 wt. % and about 9 wt. %.
  • compositions of the invention may optionally include a hydrotrope that aides in compositional stability and aqueous formulation.
  • a hydrotrope that aides in compositional stability and aqueous formulation.
  • the suitable hydrotrope couplers which can be employed are non-toxic and retain the active ingredients in aqueous solution throughout the temperature range and concentration to which a concentrate or any use solution is exposed.
  • hydrotrope coupler may be used provided it does not react with the other components of the composition or negatively affect the performance properties of the composition.
  • hydrotropic coupling agents or solubilizers which can be employed include anionic surfactants such as alkyl sulfates and alkane sulfonates, linear alkyl benzene or naphthalene sulfonates, secondary alkane sulfonates, alkyl ether sulfates or sulfonates, alkyl phosphates or phosphonates, dialkyl sulfosuccinic acid esters, sugar esters (e.g., sorbitan esters), amine oxides (mono-, di-, or tri-alkyl) and C8-C10 alkyl glucosides.
  • Preferred coupling agents for use in the present invention include n-octanesulfonate, available as NAS 8D from Ecolab Inc., n-octyl dimethylamine oxide, and the commonly available aromatic sulfonates such as the alkyl benzene sulfonates (e.g. xylene sulfonates) or naphthalene sulfonates, aryl or alkaryl phosphate esters or their alkoxylated analogues having 1 to about 40 ethylene, propylene or butylene oxide units or mixtures thereof.
  • n-octanesulfonate available as NAS 8D from Ecolab Inc.
  • n-octyl dimethylamine oxide and the commonly available aromatic sulfonates such as the alkyl benzene sulfonates (e.g. xylene sulfonates) or naphthalene sulfonates, aryl or alka
  • C6-C24 alcohol alkoxylates alkoxylate means ethoxylates, propoxylates, butoxylates, and co-or-terpolymer mixtures thereof
  • C6-C14 alcohol alkoxylates having 1 to about 15 alkylene oxide groups (preferably about 4 to about 10 alkylene oxide groups)
  • C6-C24 alkylphenol alkoxylates preferably C8-C10 alkylphenol alkoxylates
  • C6-C24 alkylpolyglycosides preferably C6-C20 alkylpolyglycosides having 1 to about 15 glycoside groups (preferably about 4 to about 10 glycoside groups)
  • C6-C24 fatty acid ester ethoxylates, propoxylates or glycerides and C4-C12 mono or dialkanolamides.
  • a preferred hydrotope is sodium cum
  • composition of an optional hydrotrope can be present in the range of from about 0 to about 25 percent by weight.
  • Water conditioning agents function to inactivate water hardness and prevent calcium and magnesium ions from interacting with soils, surfactants, carbonate and hydroxide. Water conditioning agents therefore improve detergency and prevent long term effects such as insoluble soil redepositions, mineral scales and mixtures thereof. Water conditioning can be achieved by different mechanisms including sequestration, precipitation, ion-exchange and dispersion (threshold effect).
  • the water conditioning agents which can be used include inorganic water soluble water conditioning agents, inorganic water insoluble water conditioning agents, organic water soluble conditioning agents, and organic water insoluble water conditioning agents.
  • Exemplary inorganic water soluble water conditioning agents include all physical forms of alkali metal, ammonium and substituted ammonium salts of carbonate, bicarbonate and sesquicarbonate; pyrophosphates, and condensed polyphosphates such as tripolyphosphate, trimetaphosphate and ring open derivatives; and, glassy polymeric metaphosphates of general structure Mn+2Pn03n+1 having a degree of polymerization n of from about 6 to about 21 in anhydrous or hydrated forms; and, mixtures thereof.
  • Exemplary inorganic water insoluble water conditioning agents include aluminosilicate builders.
  • Exemplary water soluble water conditioning agents include aminpolyacetates, polyphosphonates, aminopolyphosphonates, short chain carboxylates and polycarboxylates.
  • Organic water soluble water conditioning agents useful in the compositions of the present invention include aminpolyacetates, polyphosphonates, aminopolyphosphonates, short chain carboxylates and a wide variety of polycarboxylate compounds.
  • Aminopolyacetate water conditioning salts suitable for use herein include the sodium, potassium lithium, ammonium, and substituted ammonium salts of the following acids: ethylenediaminetetraacetic acid, N-(2-hydroxyethyl)-ethylenediamine triacetic acid, N-(2- hydroxyethyl)-nitrilodiacetic acid, diethylenetriaminepentaacetic acid, 1 ,2- diaminocyclohexanetetracetic acid and nitrilotriacetic acid; and, mixtures thereof.
  • Polyphosphonates useful herein specifically include the sodium, lithium and potassium salts of ethylene diphosphonic acid; sodium, lithium and potassium salts of ethane-1 -hydroxy-1 ,1 - diphosphonic acid and sodium lithium, potassium, ammonium and substituted ammonium salts of ethane-2-carboxy-1 ,1 -diphosphonic acid, hydroxymethanediphosphonic acid, carbonyldiphosphonic acid, ethane-1 -hydroxy-1 ,1 ,2-triphosphonic acid, ethane-2-hydroxy-1 ,1 ,2- triphosphonic acid, propane-1 ,1 ,3,3-tetraphosphonic acid propane-1 ,1 ,2,3-tetraphophonic acid and propane 1 ,2,2,3-tetraphosphonic acid; and mixtures thereof.
  • Examples of these polyphosphonic compounds are disclosed in British Pat. No. 1 ,026,366. For more examples see U.S. Pat. No. 3,213,030 to Diehl issued Oct. 19, 1965 and U.S. Pat. No. 2,599,807 to Bersworth issued Jun. 10, 1952.
  • Aminopolyphosphonate compounds are excellent water conditioning agents and may be advantageously used in the present invention. Suitable examples include soluble salts, e.g. sodium, lithium or potassium salts, of diethylene thiamine pentamethylene phosphonic acid, ethylene diamine tetramethylene phosphonic acid, hexamethylenediamine tetramethylene phosphonic acid, and nitrilotrimethylene phosphonic acid; and, mixtures thereof.
  • Water soluble short chain carboxylic acid salts constitute another class of water conditioner for use herein. Examples include citric acid, gluconic acid and phytic acid. Preferred salts are prepared from alkali metal ions such as sodium, potassium, lithium and from ammonium and substituted ammonium.
  • Suitable water soluble polycarboxylate water conditioners for this invention include the various ether polycarboxylates, polyacetal, polycarboxylates, epoxy polycarboxylates, and aliphatic-, cycloalkane- and aromatic polycarboxylates.
  • the detergent additive as well as the detergent composition may comprise one or more enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
  • enzymes such as a protease, lipase, cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.
  • the properties of the selected enzyme(s) should be compatible with the selected detergent, (i.e., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) should be present in effective amounts.
  • 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.
  • 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 96/1 1262, 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 WO99/001544.
  • cellulases are endo-beta-1 ,4-glucanase enzyme having a sequence of at least
  • cellulases include CelluzymeTM, and CarezymeTM (Novozymes A/S) Carezyme PremiumTM (Novozymes A/S), Celluclean TM (Novozymes A/S), Celluclean ClassicTM (Novozymes A/S), CellusoftTM (Novozymes A/S), WhitezymeTM (Novozymes A/S), ClazinaseTM, and Puradax HATM (Genencor International Inc.), and KAC-500(B)TM (Kao Corporation).
  • 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 from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens.
  • Suitable mannanases are described in WO 1999/064619. A commercially available mannanase is Mannaway (Novozymes A/S).
  • Suitable proteases include those of bacterial, fungal, plant, viral or animal origin e.g. vegetable or microbial origin. Microbial origin is preferred. Chemically modified or protein engineered mutants are included. It 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 subtilisin. A metalloproteases protease may for example be a thermolysin from e.g. family M4 or other metalloprotease such as those from M5, M7 or M8 families.
  • subtilases refers to a sub-group of serine protease according to Siezen et al., Protein Engng. 4 (1991 ) 719-737 and Siezen et al. Protein Science 6 (1997) 501 -523.
  • Serine proteases are a subgroup of proteases characterized by having a serine in the active site, which forms a covalent adduct with the substrate.
  • the subtilases may be divided into 6 sub-divisions, i.e. the Subtilisin family, the Thermitase family, the Proteinase K family, the Lantibiotic peptidase family, the Kexin family and the Pyrolysin family.
  • subtilases are those derived from Bacillus such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in; US7262042 and WO09/021867, and subtilisin lentus, subtilisin Novo, subtilisin Carlsberg, Bacillus licheniformis, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 described in WO89/06279 and protease PD138 described in (WO93/18140).
  • trypsin-like proteases are trypsin (e.g. of porcine or bovine origin) and the Fusarium protease described in WO89/06270, W094/25583 and WO05/040372, and the chymotrypsin proteases derived from Cellumonas described in WO05/052161 and WO05/052146.
  • a further preferred protease is the alkaline protease from Bacillus lentus DSM 5483, as described for example in W095/23221 , and variants thereof which are described in WO92/21760, W095/23221 , EP1921 147 and EP1921 148.
  • metalloproteases are the neutral metalloprotease as described in WO07/044993 (Genencor Int.) such as those derived from Bacillus amyloliquefaciens.
  • Examples of useful proteases are the variants described in: W092/19729, WO96/034946, WO98/201 15, WO98/201 16, WO99/01 1768, WO01/44452, WO03/006602, WO04/03186, WO04/041979, WO07/006305, W01 1/036263, W01 1/036264, especially the variants with substitutions in one or more of the following positions: 3, 4, 9, 15, 27, 36, 57, 68, 76, 87, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 106, 1 18, 120, 123, 128, 129, 130, 160, 167, 170, 194, 195, 199, 205, 206, 217, 218, 222, 224, 232, 235, 236, 245, 248, 252 and 274 using the BPN' numbering.
  • subtilase variants may comprise the mutations: S3T, V4I, S9R, A15T, K27R, * 36D, V68A, N76D, N87S,R, * 97E, A98S, S99G,D,A, S99AD, S101 G,M,R S103A, V104I,Y,N, S106A, G1 18V,R, H120D,N, N123S, S128L, P129Q, S130A, G160D, Y167A, R170S, A194P, G195E, V199M, V205I, L217D, N218D, M222S, A232V, K235L, Q236H, Q245R, N252K, T274A (using BPN' numbering).
  • Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase Tm , Durazym Tm , Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect Prime®, , Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, , FN2®, FN3® , FN4®, Excellase®, Eraser®, Opticlean® and Optimase® (Danisco/DuPont), AxapemTM (
  • 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.
  • Thermomyces e.g. from T. lanuginosus (previously named Humicola lanuginosa) as described in EP258068 and EP305216
  • cutinase from Humicola e.g. H
  • strain SD705 (WO95/06720 & WO96/27002), P. wisconsinensis (WO96/12012), GDSL-type Streptomyces lipases (W010/065455), cutinase from Magnaporthe grisea (W010/107560), cutinase from Pseudomonas mendocina (US5,389,536), lipase from Thermobifida fusca (W01 1/084412), Geobacillus stearothermophilus lipase (W01 1/084417), lipase from Bacillus subtilis (W01 1/084599), and lipase from Streptomyces griseus (W01 1/150157) and S. pristinaespiralis (W012/137147).
  • lipase variants such as those described in EP407225, WO92/05249, WO94/01541 , W094/25578, W095/14783, WO95/30744, W095/35381 , W095/22615, WO96/00292, WO97/04079, WO97/07202, WO00/34450, WO00/60063, WO01/92502, WO07/87508 and WO09/109500.
  • Preferred commercial lipase products include include LipolaseTM, LipexTM; LipolexTM and LipocleanTM (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).
  • lipases sometimes referred to as acyltransferases or perhydrolases, e.g. acyltransferases with homology to Candida antarctica lipase A (WO10/1 1 1 143), 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 (W010/100028).
  • Suitable amylases which can be used together with the detergent composition of the invention may be 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, 21 1 , 243, 264, 304, 305, 391 , 408, and 444.
  • 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.
  • 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 I D NO: 4 of WO 2006/066594 or variants having 90% sequence identity thereof.
  • Preferred variants of this hybrid alpha-amylase are those having a substitution, a deletion or an insertion in one of more of the following positions: G48, T49, G107, H156, A181 , N190, M197, 1201 , A209 and Q264.
  • 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 SEQ ID NO: 4 are those having the substitutions:
  • amylases which are suitable are amylases having SEQ ID NO: 6 in WO
  • SEQ ID NO: 6 are those having a substitution, a deletion or an insertion in one or more of the following positions: R181 , G182, H183, G184, N195, I206, E212, E216 and K269.
  • Particularly preferred amylases are those having deletion in positions R181 and G182, or positions H183 and G184.
  • Additional amylases which can be used are those having SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 2 or SEQ ID NO: 7 of WO 96/023873 or variants thereof having 90% sequence identity to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7.
  • Preferred variants of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 7 are those having a substitution, a deletion or an insertion in one or more of the following positions: 140, 181 , 182, 183, 184, 195,
  • SEQ ID NO: 1 SEQ ID NO: 1 or SEQ ID NO: 7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
  • amylases which can be used are amylases having SEQ ID NO: 2 of WO 08/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO: 2 of WO 08/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712.
  • Preferred variants of SEQ ID NO: 10 in WO 01/66712 are those having a substitution, a deletion or an insertion in one of more of the following positions: 176, 177, 178, 179, 190, 201 ,
  • amylases having SEQ ID NO: 2 of WO 09/061380 or variants having 90% sequence identity to SEQ ID NO: 2 thereof.
  • Preferred variants of SEQ ID NO: 2 are those having a truncation of the C-terminus and/or a substitution, a deletion or an insertion in one of more of the following positions: Q87, Q98, S125, N128, T131 , T165, K178, R180, S181 , T182, G183, M201 , F202, N225, S243, N272, N282, Y305, R309, D319, Q320, Q359, K444 and G475.
  • More preferred variants of SEQ ID NO: 2 are those having the substitution in one of more of the following positions: Q87E,R, Q98R, S125A, N128C, T131 I, T165I, K178L, T182G, M201 L, F202Y, N225E,R, N272E,R, S243Q,A,E,D, Y305R, R309A, Q320R, Q359E, K444E and G475K and/or deletion in position R180 and/or S181 or of T182 and/or G183.
  • Most preferred amylase variants of SEQ ID NO: 2 are those having the substitutions:
  • C-terminally truncated and optionally further comprises a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • amylases having SEQ ID NO: 1 of W013184577 or variants having 90% sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 of W013184577 are amylases having SEQ ID NO: 1 of W013184577 or variants having 90% sequence identity to SEQ ID NO: 1 thereof.
  • SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: K176, R178, G179, T180, G181 , E187, N192, M199, I203, S241 , R458, T459, D460, G476 and G477. More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: K176L, E187P, N 192FYH, M199L, I203YF, S241 QADN,
  • amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprises a substitution at position 241 and/or a deletion at position 178 and/or position 179.
  • amylases having SEQ ID NO: 1 of W010104675 or variants having 90% sequence identity to SEQ ID NO: 1 thereof.
  • Preferred variants of SEQ ID NO: 1 are those having a substitution, a deletion or an insertion in one of more of the following positions: N21 , D97, V128 K177, R179, S180, 1181 , G182, M200, L204, E242, G477 and G478.
  • SEQ ID NO: 1 More preferred variants of SEQ ID NO: 1 are those having the substitution in one of more of the following positions: N21 D, D97N, V128I K177L, M200L, L204YF, E242QA, G477K and G478K and/or deletion in position R179 and/or S180 or of 1181 and/or G182. Most preferred amylase variants of SEQ ID NO: 1 are those having the substitutions:
  • variants optionally further comprises a substitution at position 200 and/or a deletion at position 180 and/or position 181.
  • amylases are the alpha-amylase having SEQ ID NO: 12 in WO01/66712 or a variant having at least 90% sequence identity to SEQ ID NO: 12.
  • Preferred amylase variants are those having a substitution, a deletion or an insertion in one of more of the following positions of SEQ ID NO: 12 in WO01/66712: R28, R1 18, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484.
  • Particular preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R1 18K, N195F, R320K and R458K, and a variant additionally having substitutions in one or more position selected from the group: M9, G149, G182, G186, M202, T257, Y295, N299, M323, E345 and A339, most preferred a variant that additionally has substitutions in all these positions.
  • amylase variants such as those described in WO201 1/098531 ,
  • amylases are DuramylTM, TermamylTM, FungamylTM, Stainzyme
  • RapidaseTM PurastarTM/EffectenzTM, Powerase
  • Preferenz S1000 Preferenz S100 and
  • Preferenz S1 10 (from Genencor International Inc./DuPont).
  • a peroxidase according to the invention is a peroxidase enzyme comprised by the enzyme classification EC 1.1 1.1.7, 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.
  • IUBMB Nomenclature Committee of the International Union of Biochemistry and Molecular Biology
  • Suitable peroxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinopsis, e.g., from C. cmerea (EP 179,486), and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • a peroxidase according to the invention also includes 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 .1 1.1 .10) catalyze formation of hypochlorite from chloride ions.
  • the haloperoxidase of the invention is a chloroperoxidase.
  • the haloperoxidase is a vanadium haloperoxidase, i.e., a vanadate-containing haloperoxidase.
  • 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. maequalis, Drechslera, Ulocladium and Botrytis.
  • Caldariomyces e.g., C. fumago
  • Alternaria Curvularia
  • Curvularia e.g., C. verruculosa and C. maequalis
  • 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 is derivable from Curvularia sp., in particular Curvularia verruculosa or Curvularia maequalis, such as C. maequalis 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.
  • Curvularia verruculosa or Curvularia maequalis such as C. maequalis CBS 102.42 as described in WO 95/27046; or C. verruculosa CBS 147.63 or C. verruculosa CBS 444
  • An oxidase according to the invention 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).
  • a catechol oxidase EC 1 .10.3.1
  • an o-aminophenol oxidase EC 1 .10.3.4
  • 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, Fomes, Lentinus, Pleurotus, Trametes, e.g., T. villosa and T. versicolor, Rhizoctonia, e.g., R. solani, Coprinopsis, e.g., C. cmerea, 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
  • peroxidases examples include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO
  • peroxidases include GuardzymeTM (Novozymes A/S).
  • the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
  • a detergent additive of the invention i.e., a separate additive or a combined additive, can be formulated, for example, as a granulate, liquid, slurry, etc.
  • Non-dusting granulates may be produced, e.g. as disclosed in US 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • waxy coating materials are 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.
  • PEG polyethyleneglycol
  • film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
  • Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • a variant of a parent lipase for cleaning-in-place which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO: 2.
  • variant further comprises substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • hemicellulases peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, or mixtures thereof.
  • one or more enzymes selected from: hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate
  • the protease is a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • protease is the protease of SEQ ID NO: 3.
  • phosphorlipase is the phosphorlipase in SEQ ID NO: 9 or the phosphorlipase in SEQ ID NO: 10.
  • polypeptides having at least 90%, such as at least 95%, sequence identity to SEQ ID NO: 8 or a variant thereof wherein the polypeptide comprises the following substitutions T231 R and N233R. 13. Use according to any of the preceding paragraphs, further comprising use of at least one surfactant, at least one surfactant system, at least one soap, or any mixtures thereof.
  • the surfactant is one or more non-ionic surfactants or one ore more amphoteric surfactant or a mixture thereof.
  • non-ionic surfactant is selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end- blocked ethoxylates, polypropylene glycols and polyethylene glycols.
  • amphoteric surfactant is selected from alkylimidazoline, alkylbetaines, alkylamidobetaines and protein derivatives. 17. Use according to any of the preceding paragraphs, wherein the cleaning-in-place removes organic particles in equipment used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing.
  • cleaning-in-place comprises cleaning equipment for food and beverage manufacturing.
  • the cleaning-in-place comprises cleaning dairy equipment or brewing equipment.
  • the cleaning-in-place comprises cleaning of road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes.
  • 21 Use according to paragraph 20, wherein the cleaning-in-place comprises cleaning of semipermeable membranes. 22. Use according to any of paragraphs 17-21 , wherein the organic particles comprises lipids and proteins.
  • the cleaning performance is at least 60% better than the cleaning performance of SEQ ID NO: 4 and SEQ ID NO: 7 used together, such as at least 70% better, at least 80% better, at least 90% better or at least 100% better than the cleaning performance of SEQ ID NO: 4 and SEQ ID NO: 7 used together.
  • Detergent composition comprising a variant of a lipase, which variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91 T and G38A of SEQ ID NO: 2, and which composition is free of borate.
  • composition according to paragraph 25 wherein the composition further comprises at least one of the following ingredients: a sugar alcohol, a calcium salt, a formate salt and/or a polyole.
  • composition according to any of the preceding composition paragraphs wherein the variant is selected from the group consisting of: a. polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO: 2; b.
  • polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i);
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • composition according to any of the preceding composition paragraphs wherein the sugar alcohol is selected from the group consisting of: sorbitol, xylitol, mannitol, galactitol fucitol, iditol, maltitol, lactitol and inositol.
  • the calcium salt is selected from the group of calcium chloride, calcium formate, calcium sulphate.
  • composition according to any of the preceding composition paragraphs wherein the propylene glycol is selected from the group consisting of mono propylene glycol, glycerol, sorbitol, ethylene glycol or poly ethylene glycol (PEG).
  • the propylene glycol is selected from the group consisting of mono propylene glycol, glycerol, sorbitol, ethylene glycol or poly ethylene glycol (PEG).
  • composition according to any of the preceding composition paragraphs wherein the composition further comprises glycerol and water.
  • composition according to any of the preceding composition paragraphs further comprising a protease.
  • composition according to paragraph 34 wherein the protease is a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • composition according to any of the preceding composition paragraphs further comprising a phosphorlipase.
  • composition according to paragraph 38 wherein the phosphorlipase is a
  • Phospholipase A1 or a Phospholipase A2.
  • composition according to any of the preceding composition paragraphs further comprising at least one surfactant, at least one surfactant system, at least one soap, or any mixtures thereof.
  • the surfactant is one or more non-ionic surfactants or one ore more amphoteric surfactant or a mixture thereof. 45.
  • non-ionic surfactant is selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end-blocked ethoxylates, polypropylene glycols and polyethylene glycols.
  • the non-ionic surfactant is selected from the group consisting of glycerol derivatives, sorbitan, glucose, sucrose derivatives, fatty acid ethoxylates, fatty acid ethoxylates propoxylates, fatty alcohol ethoxylates, alkyl phenol ethoxylates, fatty alcohol ethoxylates propoxylates, fatty esters of polyalcohol ethoxylates, end-blocked
  • composition according to paragraph 44 wherein the amphoteric surfactant is selected from alkylimidazoline, alkylbetaines, alkylamidobetaines and protein derivatives.
  • the surfactant is present in an amount from about 1 % to about 40% by weight of a 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 a surfactant.
  • the composition is a liquid composition.
  • composition according to paragraph 49 wherein the protease inhibitor is 4- formyl-phenyl-boronic acid or a peptide aldehyde of the formula P-(A)y-L-(B)x-B0-H or a hydrosulfite adduct or hemiacetal adduct thereof, wherein:
  • i. H is hydrogen
  • ii. B0 is a single amino acid residue with L- or D-configuration of the formula -NH-
  • iii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to B0 via the C-terminal of the (B)x amino acid
  • v. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the N-terminal of the (A)y amino acid, with the proviso that if L is absent then A is absent;
  • P is selected from the group consisting of hydrogen and an N-terminal protection group, with the proviso that if L is absent then P is an N-terminal protection group;
  • hydrosulfite adduct of a peptide aldehyde is of the formula P-(A)y-L-(B)x-N(H)-CHR-CH(OH)-S03M, wherein
  • i. M is hydrogen or an alkali metal
  • ii. x is 1 , 2 or 3 for (B)x, and B is independently a single amino acid connected to BO via the C-terminal of the (B)x amino acid
  • iv. y is 0, 1 or 2 for (A)y, and A is independently a single amino acid residue connected to L via the N-terminal of the (A)y amino acid, with the proviso that if L is absent then A is absent;
  • v. P is selected from the group consisting of hydrogen and a N-terminal protection group, with the proviso that if L is absent then P is an N-terminal protection group;
  • R is independently selected from the group consisting of C1 -6 alkyl, C6-10 aryl or C7-10 arylalkyl optionally substituted with one or more, identical or different, substituent's R';
  • R" is a C1 -6 alkyl group.
  • composition according to any of paragraphs 50-51 wherein M is Na or K and R is a C7 arylalkyl substituted with -OH.
  • B0 is selected from the group consisting of D- or L-form of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (lie), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m- tyrosine, p-tyrosine (Tyr) and valine (Val).
  • i. B1 can be selected from the group consisting of alanine (Ala), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr) and valine (Val),
  • ii. B2 can be selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), cysteine (Cys), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val), or
  • iii. B3 can be selected from the group consisting of isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • A1 can be selected from the group consisting of alanine (Ala), arginine (Arg), capreomycidine (Cpd), glycine (Gly), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), threonine (Thr), tyrosine (Tyr), tryptophan (Trp) and valine (Val) or, ii.
  • A2 can be selected from the group consisting of arginine (Arg), isoleucine (lie), leucine (Leu), norleucine (NIe), norvaline (Nva), phenylalanine (Phe), phenylglycine, Tyrosine (Tyr), tryptophan (Trp) and valine (Val).
  • P is selected from the group consisting of formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, fluorenylmethyloxycarbonyl (Fmoc), methoxycarbonyl (MEO-CO), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl (Boc), adamantyloxycarbonyl, p-methoxybenzyl carbonyl (Moz), benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylamino carbonyl (MeNCO), methylsulfonyl (Me
  • a method for cleaning-in-place wherein a variant of a parent lipase is circulated in a production or a process equipment, wherein the variant has lipase activity, has at least 60% but less than 100% sequence identity with SEQ ID NO: 2, and comprises substitutions at positions corresponding to T231 R+N233R and at least one or more (e.g., several) of D96E, D1 1 1A, D254S, G163K, P256T, G91T and G38A of SEQ ID NO: 2.
  • Method according to paragraph 60 wherein the method comprises cleaning-in- place of production or process equipment, which method comprises the following steps: a. Optionally pre-rinse by circulating water in the equipment and subsequently drain water;
  • step b Optionally stop circulation under step b and allow the production equipment to soak in the wash liquor;
  • steps (a) to (e) are carried out one or two times.
  • Method according to any of the preceding method paragraphs wherein the method further comprises dismantling of the production or process equipment and soaking the equipment in a wash liquor.
  • the time period for circulating the wash liquor in the equipment is in the range of 5 minutes to 90 minutes, such as in the range of 5 minutes to 80 minutes, in the range of 5 minutes to 70 minutes, in the range of 5 minutes to 60 minutes, in the range of 5 minutes to 50 minutes, in the range of 5 minutes to 40 minutes, in the range of 5 minutes to 30 minutes, in the range of 5 minutes to 25 minutes, in the range of 5 minutes to 20 minutes, in the range of 5 minutes to 15 minutes, in the range of 5 minutes to 10 minutes.
  • variant further comprises substitutions at positions corresponding to D27R and/or N33Q of SEQ ID NO: 2.
  • variant is selected from the group consisting of: a. a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least
  • a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% sequence identity to SEQ ID NO: 1 ;
  • Method according to any of the preceding method paragraphs further comprising the use of one or more enzymes selected from: hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ⁇ -glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, chlorophyllases, amylases, or mixtures thereof.
  • one or more enzymes selected from: hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases
  • Methods according to any of the preceding method paragraphs further comprising the use of a protease.
  • the protease is a protease having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • protease is the protease of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • Phospholipase A1 or a Phospholipase A2.
  • Method according to any of the preceding method paragraphs further comprising use of at least one surfactant, at least one surfactant system, at least one soap, or any mixtures thereof.
  • Method according to any of the preceding method paragraphs wherein the production or process equipment is used in biotech manufacturing, pharmaceutical manufacturing and food and beverage manufacturing.
  • Method according to any of the preceding method paragraphs wherein the production or process equipment is used for cleaning dairy equipment or brewing equipment. 82. Method according to any of the preceding method paragraphs, wherein the production or process equipment is used for cleaning of road tankers, processing tanks, storage tanks, pipelines, heat-exchangers, homogenizers, filter units and filter membranes. 83. Method according to any of the preceding method paragraphs, wherein the production or process equipment is used for cleaning of semipermeable membranes. 84.
  • the concentration of the lipase variant in the wash liquor is in the range of 0.005-10 mg enzyme protein/liter, such as in the range of 0.005-9.0 mg enzyme protein/liter, in the range of 0.005-8.0 mg enzyme protein/liter, in the range of 0.005-7.0 mg enzyme protein/liter, in the range of 0.005-6.0 mg enzyme protein/liter, in the range of 0.005-5 mg enzyme protein/liter, in the range of 0.005-4 mg enzyme protein/liter, in the range of 0.005-3 mg enzyme protein/liter, in the range of 0.005-2.5 mg enzyme protein/liter, in the range of 0.005-2.0 mg enzyme protein/liter, in the range of 0.005-1 .5 mg enzyme protein/liter, in the range of 0.005-1 .0 mg enzyme protein/liter, in the range of 0.005-0.9 mg enzyme protein/liter, in the range of 0.005-0.8 mg enzyme protein/liter, in the range of 0.005-0.7 mg enzyme protein/liter
  • the concentration of the protease in the wash liquor is in the range of 0.02-40 mg enzyme protein/liter, 0.5-35 mg protein/liter, 1 -30 mg enzyme protein/liter, such as in the range of 1 -25 mg enzyme protein/liter, in the range of 1 -20 mg enzyme protein/liter, in the range of 1 -17 mg enzyme protein/liter, in the range of 1 -16 mg enzyme protein/liter, in the range of 1 -15 mg enzyme protein/liter, in the range of 1 -14 mg enzyme protein/liter or in the range of 1 -13.5 mg enzyme protein/liter, or the concentration of the concentration of the protease in the wash liquor is in the range of 5-30 mg enzyme protein/liter, such as in the range of 6-25 mg enzyme protein/liter, in the range of 7-20 mg enzyme protein/liter, in the range of 8-17 mg enzyme protein/liter, in the range of 9-16 mg enzyme protein/liter, in the range of 10-15 mg enzyme protein/liter or in the range of 1 1 -14 mg enzyme protein/
  • Method according to paragraph 86 wherein the cleaning performance is improved so steady state of pH is reached after circulating the wash liquor for 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes or 5 minutes when measured with Assay I. 88. Method according to any of the preceding method paragraphs, wherein the method further comprises in-activating enzymes in the production or process equipment by circulating an acidic solution in the equipment, rinsing by circulating water in the equipment and subsequently drain water.
  • the measurement is non-destructive and may be measured directly on-line, without taking samples out of the plant, either by inserting a fixed pH electrode in the processing equipment, or by introducing the electrode wherever open access to the wash liquor is possible, e.g. in the balance tank.
  • Timing Measurements are taken so frequently that a trend curve can be plotted showing the dynamics of the measured values, and hence the dynamics of the lipid and protein hydrolysis, (e.g. every 1 -5 minutes) in the beginning of the process (e.g. in the first 30 minutes), and less frequently (e.g. every 5-10 minutes) later in the process.
  • Test sample Samples of circulation water are drawn from the plant after enzymatic CIP, inactivation and rinse/neutralization by water.
  • Phenolphtalein solution 1 % Phenolphtalein in 1.0 M Sodium hydroxide.
  • the measurement is performed in a clear test tube. Reagent is added by a pipette or dropper bottle. The time for color change from pink to white is measured by a watch.
  • Procedure Pre-heat the reagent and the test sample separately until they both are at 40 °C. Add 20 mL of pre-heated test sample to 5 mL of pre-heated reagent. Mix until it gets a homogeneous pink color. Start timing and incubation at 40 °C. Measure the time when the solution becomes white (compare against a 1 :5 diluted milk).
  • the incubation time may be extended for better sesisitivity.
  • the enzymatic hydrolysis of fats and proteins release free fatty- and amino acids.
  • the velocity of release of acids, and hence the enzymatic activity can be measured by measuring the velocity of a given alkaline solution that need to be added in order to keep pH constant. This can be used to compare different enzyme solutions under different working conditions.
  • pH set-point 7.00; 8.00, 9.00; or 10.00 (or whatever pH is relevant)
  • Titrant NaOH, 0.100 M (20 mL auto-burette)
  • Lipase hydrolyses p-Nitrophenyl-valerate.
  • the hydrolysis cleaves the ester bond producing Valerate and p-Nitrophenol (pNP).
  • pNP is measured photometric at 405 nm.
  • Equipment Thermo Scientific Gallery.
  • alpha-Olephinsulphonate e.g. Stepan Bio-Terge AS-90 beads
  • Substrate pNP valerate, 132 mg/L in TRIS buffer, pH 7.70 Standard is Lipase 2: 16.5 mg enzyme protein /g standard preparation.
  • the lipase to measure must be diluted by TRIS buffer, pH 7.70, to an activity inside the linear dynamic range of the method before the activity measurement.
  • Pre-heat substrate and the diluted lipase sample to 37 °C.
  • the protease inhibitor usee is L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[2- hydroxy-1 -[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
  • Example 1 L-Alaninamide, N-[(phenylmethoxy)carbonyl]glycyl-N-[2- hydroxy-1 -[(4-hydroxyphenyl)methyl]-2-sulfoethyl]-, sodium salt (1 :1 ).
  • the cleaning solution comprised 7920 mg of Protease 1 and 1716 mg of Lipase 1 (i.e. an enzyme dosage of 0.075%) and 6 liters of a commercial alkaline liquid detergent Fluxclean No. 10 (Holchem, UK, potassium polyphosphate 1 -5%, potassium hydroxide 1 -5%, triethanolamine 1 -5%

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Abstract

La présente invention concerne l'utilisation d'une variante de lipase pour le nettoyage en place.
PCT/EP2015/072021 2014-09-25 2015-09-24 Utilisation d'enzyme pour le nettoyage WO2016046334A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11370999B2 (en) 2017-01-19 2022-06-28 Diversey, Inc. Formulations and method for low temperature cleaning of dairy equipment
US20220290070A1 (en) * 2016-02-05 2022-09-15 Ecolab Usa Inc. Detergent compositions for cleaning in the cosmetic and pharmaceutical industry
US11529588B2 (en) 2017-06-30 2022-12-20 Diversey, Inc. Membrane cleaning solution and method of accelerated membrane cleaning using the same

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002753A1 (fr) * 1995-07-12 1997-01-30 Novo Nordisk A/S Nettoyage en place a l'aide d'une solution contenant une protease et une lipase
WO2001044452A1 (fr) * 1999-12-15 2001-06-21 Novozymes A/S Variants de subtilase a performance de nettoyage amelioree sur des taches d'oeuf
US20050020466A1 (en) * 2000-06-29 2005-01-27 Man Victor F. Stable liquid enzyme compositions
WO2009107091A2 (fr) * 2008-02-29 2009-09-03 The Procter & Gamble Company Composition de détergent contenant une lipase
WO2013098205A2 (fr) * 2011-12-29 2013-07-04 Novozymes A/S Compositions détergentes
WO2014184164A1 (fr) * 2013-05-14 2014-11-20 Novozymes A/S Compositions détergentes

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997002753A1 (fr) * 1995-07-12 1997-01-30 Novo Nordisk A/S Nettoyage en place a l'aide d'une solution contenant une protease et une lipase
WO2001044452A1 (fr) * 1999-12-15 2001-06-21 Novozymes A/S Variants de subtilase a performance de nettoyage amelioree sur des taches d'oeuf
US20050020466A1 (en) * 2000-06-29 2005-01-27 Man Victor F. Stable liquid enzyme compositions
WO2009107091A2 (fr) * 2008-02-29 2009-09-03 The Procter & Gamble Company Composition de détergent contenant une lipase
WO2013098205A2 (fr) * 2011-12-29 2013-07-04 Novozymes A/S Compositions détergentes
WO2014184164A1 (fr) * 2013-05-14 2014-11-20 Novozymes A/S Compositions détergentes

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220290070A1 (en) * 2016-02-05 2022-09-15 Ecolab Usa Inc. Detergent compositions for cleaning in the cosmetic and pharmaceutical industry
US11685878B2 (en) 2016-02-05 2023-06-27 Ecolab Usa Inc. Detergent compositions for cleaning in the cosmetic and pharmaceutical industry
US11746305B2 (en) * 2016-02-05 2023-09-05 Ecolab Usa Inc. Detergent compositions for cleaning in the cosmetic and pharmaceutical industry
US11370999B2 (en) 2017-01-19 2022-06-28 Diversey, Inc. Formulations and method for low temperature cleaning of dairy equipment
US11529588B2 (en) 2017-06-30 2022-12-20 Diversey, Inc. Membrane cleaning solution and method of accelerated membrane cleaning using the same

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