EP4630528A1 - Protease variants and polynucleotides encoding same - Google Patents

Protease variants and polynucleotides encoding same

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Publication number
EP4630528A1
EP4630528A1 EP23817746.3A EP23817746A EP4630528A1 EP 4630528 A1 EP4630528 A1 EP 4630528A1 EP 23817746 A EP23817746 A EP 23817746A EP 4630528 A1 EP4630528 A1 EP 4630528A1
Authority
EP
European Patent Office
Prior art keywords
seq
variant
protease
substitutions
variants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23817746.3A
Other languages
German (de)
French (fr)
Inventor
Bena-Marie LUE
Rolf Thomas LENHARD
Esben Peter Friis
Julie Bille RANNES
Christian Lundager GYLSTORFF
Vibeke Skovgaard Nielsen
Katrine THYGESEN
Carl Mikael BAUER
Lars Lehmann Hylling Christensen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP4630528A1 publication Critical patent/EP4630528A1/en
Pending legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned
    • C11D2111/12—Soft surfaces, e.g. textile
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned
    • C11D2111/14—Hard surfaces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)

Definitions

  • the present invention relates to protease variants.
  • the present invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, detergent compositions comprising the variants, and use of the variants in a cleaning process.
  • enzymes have been implemented in detergent compositions for many decades. Enzymes used in such compositions include proteases, lipases, amylases, cellulases, mannanases as well as other enzymes or mixtures thereof. Commercially, the most important enzymes are proteases.
  • proteases used for, e.g., laundry and dishwashing detergents are engineered variants of naturally occurring wild type proteases.
  • proteases have been described in the art with alterations relative to a parent protease resulting in improvements such as better wash performance, thermal stability, storage stability, and catalytic activity.
  • proteases make further improvement of proteases advantageous. For example, washing conditions such as temperature and pH tend to change over time and are also different in different countries or regions of the world. In addition, many stains are still difficult to completely remove under conventional washing conditions. Thus, new protease variants having improved wash performance under various conditions remain commercially relevant. In addition, new protease variants with improved storage stability are warranted to maintain sufficient proteolytic activity and thus wash performance of the protease variant after a period of storage in a detergent composition, either during manufacturing and distribution of the detergent composition and/or following storage of the detergent composition by the end consumer before use.
  • new protease variants displaying improved mildness towards companion enzymes are warranted to avoid unnecessary proteolytic degradation and preserve enzymatic activity of such enzymes.
  • new protease variants with improved stain removal rate are desirable as they are faster acting and thus particularly suitable for cleaning methods, such as, e.g., laundering and dishwashing methods, involving shorter wash cycles.
  • the present invention relates to protease variants with improved properties.
  • the variants of the invention exhibit improved wash performance as well as improved storage stability under several different temperature and pH conditions.
  • the variants of the invention also provide improved mildness towards companion enzymes, which expands the compatibility of the variants of the invention with other enzymes and reduces the need for co-formulation with protease inhibitors.
  • the variants of the invention also display improved stain removal rate, making them particularly suitable for cleaning methods with short wash cycles.
  • the variants of the invention are particularly suitable for high pH liquid detergents having pH 10 or above and for laundry soap bars.
  • high pH liquid detergents the variants of the invention display improved wash performance and improved storage stability.
  • the variants of the invention provide improved proteinaceous stain removal and reduce the need for stabilizing agents, which further decreases production costs associated with manufacturing of laundry soap bars.
  • the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants has a TM-score of at least 0.80 but less than 1.0 compared to the three-dimensional structure of the parent protease, wherein the three- dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.
  • the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at at least three, e.g., at least four, at least five
  • the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60% but less than 100% sequence identity to the parent protease; and wherein the variant has protease activity.
  • the present invention relates to a polynucleotide encoding a variant of the first aspect or the second aspect.
  • the present invention relates to a nucleic acid construct or expression vector comprising a polynucleotide of the third aspect.
  • the present invention relates to a recombinant host cell comprising in its genome a nucleic acid construct or expression vector according to the fourth aspect.
  • the present invention relates to a method for obtaining a variant according to the first aspect or the second aspect, comprising (a) introducing into a parent protease substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further introducing substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has protease activity; and (b) recovering the variant.
  • the present invention relates to a method of producing a variant of the first aspect or the second aspect, comprising (a) cultivating a recombinant host cell of the fifth aspect under conditions suitable for expression of the variant; and (b) recovering the variant.
  • the present invention relates to a detergent composition comprising a variant of the first aspect or the second aspect.
  • the present invention relates to a method of cleaning an object, comprising contacting the object with a detergent composition of the eighth aspect under conditions suitable for cleaning the object.
  • the present invention relates to use of a variant of the first aspect or the second aspect or a detergent composition of the eighth aspect in a cleaning process.
  • Figure 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:2.
  • Figure 2 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:3.
  • Figure 3 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:4.
  • Figure 4 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:5.
  • Figure 5 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:6.
  • Figure 6 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:7.
  • Figure 7 is an overview of corresponding substitutions in SEQ ID NOs:1-7.
  • SEQ ID NO:1 is Subtilisin Savinase (Savinase®) from Bacillus lentus.
  • SEQ ID NO:2 is Subtilisin BPN’ from Bacillus amyloliquefaciens.
  • SEQ ID N0:3 is Subtilisin Carlsberg (Alcalase®) from Bacillus licheniformis.
  • SEQ ID NO:4 is a protease from Bacillus gibsonii.
  • SEQ ID NO:5 is a protease from Bacillus gibsonii.
  • SEQ ID NO:6 is a protease from Bacillus sp. TY-145.
  • SEQ ID NO:7 is a protease from Actinomadura keratinilytica.
  • SEQ ID NO:8 is a stabilized variant of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E.
  • SEQ ID NO:9 is SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.
  • SEQ ID NQ:10 is SEQ ID NO:3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P.
  • SEQ ID NO:11 is SEQ ID NO:3 with the substitutions P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E.
  • SEQ ID NO:12 is SEQ ID NO:3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
  • SEQ ID NO:13 is SEQ ID NO:5 with the substitutions T9E, N42R, N74D, V199I, Q200L, Y203W, N253D, S255W, and Q256E.
  • SEQ ID NO:14 is SEQ ID NO:5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, and S255W.
  • protease activity means an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds.
  • the EC 3.4 group includes several sub-groups, including EC 3.4.21 (serine endopeptidase), which further contains several sub-groups, including EC. 3.4.21.62 (subtilisin).
  • protease activity may be determined according to Protease Activity Assay I or Protease Activity Assay II described in the Examples herein.
  • AlphaFold is a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 model (Varadi et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, 2021). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.
  • AlphaFold produces a per-residue estimate of its confidence on a scale from 0 to 100.
  • This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction.
  • cDNA means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA.
  • the initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a variant.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the variant, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
  • 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 refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
  • control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
  • extension means an addition of one or more amino acids to the amino and/or carboxyl terminus of a variant, wherein the “extended” variant has protease activity.
  • fragment means a variant having one or more amino acids absent from the amino and/or carboxyl terminus of the variant; wherein the fragment has protease activity.
  • Fusion polypeptide is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a variant of the present invention.
  • a fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together.
  • Techniques for producing fusion polypeptides are known in the art and include 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 al., 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. Examples of 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 al., 2000, J.
  • heterologous means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell.
  • heterologous means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.
  • Host Strain or Host Cell is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a variant has been introduced.
  • Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides.
  • the term "host cell” includes protoplasts created from cells.
  • Improved property means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, mildness, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, thermostability, and wash performance.
  • Isolated means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc.
  • An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature.
  • An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell.
  • Mature polypeptide means a polypeptide in its mature form following N-terminal processing and/or C-terminal processing (e.g., removal of signal peptide).
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide having protease activity.
  • Mutant means a polynucleotide encoding a variant.
  • Native means a nucleic acid or polypeptide naturally occurring in a host cell.
  • Nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a variant. Nucleic acids may be single stranded or double stranded and may be chemically modified. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
  • operably linked means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner.
  • a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
  • Parent or parent protease means a protease to which an alteration is made to produce the protease variants of the present invention.
  • purified means a nucleic acid, variant or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
  • a purified nucleic acid or variant is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis).
  • a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique.
  • the term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
  • the term “purified” as used herein refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term “purified” refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.
  • the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present.
  • purified as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide.
  • the variant may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant.
  • the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation.
  • a "substantially pure polypeptide” may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.
  • the substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation.
  • the variant of the present invention is preferably in a substantially pure form i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.
  • Recombinant is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature.
  • the term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
  • the term “recombinant” is synonymous with “genetically modified” and “transgenic”.
  • Recover means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheet or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hydro cyclones or similar), or by precipitating the polypeptide and using relevant solidliquid separation methods to harvest the polypeptide from the broth media by use of
  • Sequence Identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
  • the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the Needle program In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line.
  • the output of Needle labeled “longest identity” is calculated as follows:
  • the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later.
  • the parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix.
  • the nobrief option must be specified in the command line.
  • the output of Needle labeled “longest identity” is calculated as follows:
  • Signal Peptide A "signal peptide” is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
  • the mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.
  • Structural Similarity The relatedness between two amino acid sequences has conventionally been described by the parameter “sequence identity”. However, since the biological function of a polypeptide is defined by its three-dimensional structure rather than its amino acid sequence, a better way of assessing a functional relationship between polypeptides is by comparing their three-dimensional structures. Thus, for the purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.
  • a three-dimensional structure of any polypeptide may be obtained experimentally via, e.g.,
  • TM-score is calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):
  • TM-score where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, d, is the distance between the /th pair of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.
  • LN is always the length of the reference protein, indicating the use of a fixed reference length L to prevent artificially large TM-scores from alignment of substructures:
  • TM-score A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, Protein Eng., 11 , 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).
  • TM-align is applied.
  • TM-score is integrated in the TM-align software, which is available from the author’s website.
  • the version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:
  • ⁇ query.pdb> is the name of the PDB file containing coordinates of the query polypeptide
  • ⁇ reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide.
  • the TM-score is calculated and reported in the output, along with several other parameters from the alignment.
  • the maximal TM-score is 1 , e.g., 1.0, corresponding to identical three-dimensional structures.
  • Subsequence means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.
  • variant means a polypeptide having protease activity comprising a substitution, an insertion (including extension), and/or a deletion (e.g., truncation), at one or more positions.
  • a substitution means replacement of the amino acid occupying a position with a different amino acid;
  • a deletion means removal of the amino acid occupying a position; and
  • an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.
  • Wild-type in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature.
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).
  • the polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid positions in another protease.
  • the amino acid sequence of another protease is aligned with the polypeptide disclosed in SEQ ID NO:1 , and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • substitutions For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of Thr at position 226 with Ala is designated as “T226A”. Multiple mutations are separated by addition marks (“+”) or by commas, e.g., “G205R+S411 F” or “G205R,S411 F”, representing substitutions at positions 205 and 411 of Gly (G) with Arg (R) and Ser (S) with Phe (F), respectively. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted may be indicated with X, e.g., “X226A”.
  • Deletions For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of the amino acid Gly at position 195 is designated as “Gly195*”. Multiple deletions are separated by addition marks (“+”) or by commas, e.g., “G195*+S411*” or “G195*,S411*”. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be deleted may be indicated with X, e.g., “X195*”.
  • 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 Lys after the amino acid Gly at position 195 is designated “G195GK”. Because the amino acid residue at a given position varies from parent to parent, the insertion of lysine after the amino acid at position 195 may be indicated with “X195*”.
  • An insertion of multiple amino acids is designated [original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.].
  • the insertion of Lys and Ala after the amino acid Gly at position 195 is indicated as “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). In the above example, the sequence would thus be:
  • an insertion of an amino acid residue such as lysine after the amino acid at position 195 may be indicated by “195aK”, and the insertion of two or more additional amino acid residues such as Lys and Ala after the amino acid at position 195 may be indicated by “195aK,195bA”.
  • Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “R170Y+G195E” representing a substitution of Arg and Gly at positions 170 and 195 with Tyr and Glu, respectively.
  • alterations where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “R170Y.E” represents a substitution of Arg at position 170 with Tyr or Glu.
  • R170Y.E represents a substitution of Arg at position 170 with Tyr or Glu.
  • Y167G,A+R170G,A designates the following variants: “Y167G+R170G”, Y167G+R170A”, “Y167A+R170G”, and “Y167A+R170A”.
  • the present invention relates to protease variants with improved properties.
  • the variants of the invention exhibit improved wash performance as well as improved storage stability under several different temperature and pH conditions.
  • the variants of the invention also provide improved mildness towards companion enzymes, which expands the compatibility of the variants of the invention with other enzymes and reduces the need for co-formulation with protease inhibitors.
  • the variants of the invention also display improved stain removal rate, making them particularly suitable for cleaning methods with short wash cycles.
  • the variants of the invention are particularly suitable for high pH liquid detergents having pH 10 or above and for laundry soap bars.
  • high pH liquid detergents the variants of the invention display improved wash performance and improved storage stability.
  • the variants of the invention provide improved proteinaceous stain removal and reduce the need for stabilizing agents, which further decreases production costs associated with manufacturing of laundry soap bars.
  • the present invention also relates to polynucleotides encoding variants of the invention, nucleic acid constructs and expression vectors comprising such polynucleotides, recombinant host cells expression variants of the invention, methods for obtaining variants of the invention, methods of producing variants of the invention, detergent compositions comprising variants of the invention, and uses of variants of the invention.
  • the present invention relates to variants of a parent protease, wherein the variants comprise substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants have a TM-score of at least 0.80, e.g., at least
  • the variant has a TM-score of at least 0.90, e.g., at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
  • the variant has a TM-score of at least 0.95, e.g., at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three- dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
  • the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
  • the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.
  • TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.
  • the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold.
  • a three-dimensional structure is publicly available.
  • a three- dimensional structure of SEQ ID NO:1 (Savinase®) is available under UniProt accession number P29600 or, alternatively, PDB accession number 1SVN.
  • a three-dimensional structure of SEQ ID NO:2 (BPN’) is available under UniProt accession number P00782.
  • a three-dimensional structure of SEQ ID NO:3 (Alcalase®) is available under UniProt accession number P00780.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.
  • the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.
  • the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.
  • the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.
  • the present invention relates to variants of a parent protease, wherein the variants comprise substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variants have protease activity.
  • the variants comprise substitutions at positions
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
  • the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1 .
  • the parent is SEQ ID NO:1 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1.
  • the parent is SEQ ID NO:2 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:2.
  • the parent is SEQ ID NO:3 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3.
  • the parent is SEQ ID NO:4 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:4.
  • the parent is SEQ ID NO:5 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:5.
  • the parent is SEQ ID NO:6 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:6.
  • the parent is SEQ ID NO:7 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:7.
  • the number of substitutions in the variants of the present invention is 5-30, e.g., 5-25, 5-20, 5-15 and 5-10, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, substitutions.
  • the number of substitutions in the variants of the present invention is 5-11 , such as 5, 6, 7, 8, 9, 10, or 11 substitutions.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises nine substitutions at positions corresponding to each of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • the variants of the invention comprise a substitution at a position corresponding to position 95 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 95 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp.
  • the variants of the invention comprise a substitution at a position corresponding to position 209 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 209 of SEQ ID NO:1 is substituted with Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.
  • the variants of the invention may comprise a substitution at a position corresponding to position 9 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 9 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Trp, Tyr, or Vai, preferably with Glu.
  • the variants of the invention may comprise a substitution at a position corresponding to position 42 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 42 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, His, lie, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Arg.
  • the variants of the invention may comprise a substitution at a position corresponding to position 74 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 74 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.
  • the variants of the invention may comprise a substitution at a position corresponding to position 199 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position of SEQ ID NO:199 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with lie.
  • the variants of the invention may comprise a substitution at a position corresponding to position 200 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 200 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Vai, preferably with Leu.
  • the variants of the invention may comprise a substitution at a position corresponding to position 203 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 203 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Trp.
  • the variants of the invention may comprise a substitution at a position corresponding to position 253 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 253 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asp.
  • the variants of the invention may comprise a substitution at a position corresponding to position 255 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 255 of SEQ ID NO:1 may comprise a substitution at a position corresponding to position 255 of SEQ ID NO:1.
  • SEQ ID NO:1 255 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Vai, preferably with Trp.
  • the variants of the invention may comprise a substitution at a position corresponding to position 256 of SEQ ID NO:1.
  • the amino acid at a position corresponding to position 256 of SEQ ID NO:1 may comprise a substitution at a position corresponding to position 256 of SEQ ID NO:1.
  • SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Glu.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least four substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least five substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ I D NO: 1 and further comprise at least six substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to substitutions G95D and A209K of SEQ ID NO:1 and further comprise at least seven substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least eight substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprising nine substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • the present invention relates to variants of SEQ ID NO:1 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., S9E), X42R (e.g., N42R), X74D (e.g., N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., S253D), X255W (e.g., N255W), and X256E (e.g., L256E); wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the substitutions
  • the variant comprises or consists of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.
  • the present invention relates to variants of SEQ ID NO:2 comprising the substitutions X97D (e.g., G97D) and X215K (e.g., G215K) and further comprising at least three, e.g., at least four, at least five, at least six, or seven, substitutions selected from the group consisting of X9E (e.g., S9E), X43R (e.g., K43R), X76D (e.g., N76D), X206L (e.g., Q206L), X209W (e.g., L209W), X261W (e.g., F261W), and X262E (e.g., Y262E); wherein position numbering is based on the numbering of SEQ ID NO:2; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at
  • the variant comprises or consists of SEQ ID NO:2 with the substitutions S9E, K43R, N76D, G97D, Q206L, L209W, G215K, F261W, and Y262E.
  • the present invention relates to variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) and X214K (e.g., A214K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g.,Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the variants have a sequence identity of at least 60%, e.g.,
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
  • the present invention relates to variants of SEQ ID NO:4 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., T42R), X74D (e.g., N74D), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein position numbering is based on the numbering of SEQ ID NO:4; wherein the variants have a sequence identity of at least 60%, e.g., at
  • the variant comprises or consists of SEQ ID NO:4 with the substitutions T9E, T42R, N74D, G95D, Q200L, Y203W, A209K, N253D, S255W, and Q256E.
  • the present invention relates to variants of SEQ ID NO:5 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., V209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., N42R), X74D (e.g.
  • X199I e.g., V199I
  • X200L e.g., Q200L
  • X203W e.g., Y203W
  • X253D e.g., N253D
  • X255W e.g., S255W
  • X256E e.g., Q256E
  • position numbering is based on the numbering of SEQ ID NO:5; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:5; and wherein the variant have protease activity.
  • the variant comprises or consists of SEQ ID NO:5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, S255W, and Q256E.
  • the present invention relates to variants of SEQ ID NO:6 comprising the substitutions X107D (e.g., G107D) and X245K (e.g., N245K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X12E (e.g., K12E), X44R (e.g., D44R), X86D (e.g., S86D), X235I (e.g., V235I), X236L (e.g., E236L), X297D (e.g., T297D), X299W (e.g., D299W), and X300E (e.g., D300E); wherein position numbering is based on the numbering of SEQ ID NO:6; wherein the variants have a sequence identity of at least 60%, e.g.,
  • the variant comprises or consists of SEQ ID NO:6 with the substitutions K12E, D44R, S86D, G107D, V235I, E236L, N245K, T297D, D299W, and D300E.
  • the present invention relates to variants of SEQ ID NO:7 comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K) and further comprising at least three, e.g., at least four, at least five, or six, substitutions selected from the group consisting of X12E (e.g., D12E), X51 R (e.g., G51 R), X206L (e.g., T206L), X266D (e.g., T266D), X268W (e.g., N268W), and X269E (e.g., L269E); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
  • the present invention relates to variants of SEQ ID NO:7 comprising the substitutions X99D (e.g. , N99D) and X215K (e.g. , N215K) and further comprising the substitutions X12E (e.g., D12E), X51 R (e.g., G51 R), and X206L (e.g., T206L); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:7; and wherein the variants have protease activity.
  • the variant comprises or consists of SEQ ID NO:7 with the substitutions D12
  • the present invention relates to variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) or X214K (e.g., A214K), preferably X96D (e.g., N96D) and X214K (e.g., A214K), and further comprising at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting of X68S (e.g., A68S), X77N (e.g., T77N), X78I (e.g., T78I), X127S (e.g., G127S), X128P (e.g., A128P), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A68S (e
  • the variant comprises the substitutions N96D and A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting ofA68S, T77N, T78I, G127S, A128P, G165Q, N184Q, A202V, N217S, and S258P.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P.
  • the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E).
  • X9E e.g., P9E
  • X43R e.g., N43R
  • X204I e.g., V204I
  • X205L e.g., Y205L
  • X208W e.g., Y208W
  • X258D
  • the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258P, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258D, F260W, and Y261 E.
  • the present invention relates to variants of SEQ ID NO:3 comprising the substitution X214K (e.g., A214K) and further comprising at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of X9T, (e.g., P9T), X17H, (e.g., Q17H), X77N (e.g., T77N), X78I (e.g., T78I), X96D (e.g., N96D), X103F (e.g., Y103F), X127T (e.g., G127T), X128K (e.g., A128K), X129Q, (e.g., S129Q), X165Q (e.g., X
  • the variant comprises the substitution A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P.
  • substitutions selected from the group consisting of P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, A214K, and S258P.
  • the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E).
  • X9E e.g., P9E
  • X43R e.g., N43R
  • X204I e.g., V204I
  • X205L e.g., Y205L
  • X208W e.g., Y208W
  • X258D
  • the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261 E.
  • the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
  • the variants of the invention may comprise further substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97E), 99 (e.g., S99E), 116 (e.g., G116N) , and 246 (e.g., N246L) of SEQ ID NO:1.
  • the variants comprise one or more further substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L) of SEQ ID NO:1.
  • the variants of the invention may further comprise an extension of one or more amino acids at the N-terminal and/or C-terminal ends.
  • the variants of the invention may further comprise a truncation of one or more amino acids at the N-terminal and/or C-terminal ends.
  • amino acid changes introduced into parent proteases to provide variants according to the present invention 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 aminoterminal 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.
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine).
  • Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York.
  • 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 molecules are tested for protease 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 a!., 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, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity.
  • protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and/or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.
  • the variants of the invention have improved stability under storage conditions (/.e., improved storage stability) compared to a reference protease.
  • storage stability is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
  • the reference protease is a parent protease.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the variant has improved storage stability compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:7.
  • the variant has improved storage stability compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1.
  • storage stability is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%.
  • the variant has improved storage stability compared to SEQ ID NO:8.
  • the variant has improved storage stability compared to SEQ ID NO:11.
  • the variant has improved storage stability compared to SEQ ID NO: 13.
  • the variant has improved storage stability in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
  • the variant has improved storage stability in a liquid detergent having pH 7-11 , preferably pH 7-10, most preferably pH 8-10.
  • the variant has improved storage stability at a temperature of IQ- 40 °C, preferably 10-30 °C, most preferably 15-25 °C.
  • the variant has improved improved storage stability at a temperature of 10-60 °C, more preferably 30-55 °C, most preferably 45-55 °C.
  • the variant has improved storage stability in liquid detergent as determined according to Example 7 herein.
  • the variant has improved storage stability in liquid detergent as determined according to Example 11 herein, preferably in Model O detergent having pH 8-10 or preferably in Model B detergent having pH 7-8.
  • the variants of the invention have improved wash performance compared to a reference parent protease.
  • wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
  • the reference protease is a parent protease.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the variant has improved wash performance compared to SEQ ID NO:1.
  • the variant has improved wash performance compared to SEQ ID NO:2.
  • the variant has improved wash performance compared to SEQ ID NO:3.
  • the variant has improved wash performance compared to SEQ ID NO:4.
  • the variant has improved wash performance compared to SEQ ID NO:5.
  • the variant has improved wash performance compared to SEQ ID NO:6.
  • the variant has improved wash performance compared to SEQ ID NO:7.
  • the variant has improved wash performance compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1.
  • wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%.
  • the variant has improved wash performance compared to SEQ ID NO:8.
  • the variant has improved wash performance compared to SEQ ID NO:11.
  • the variant has improved wash performance compared to SEQ ID NO: 13.
  • the variant has improved wash performance in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
  • the variant has improved wash performance in a liquid detergent having pH 7-11 , preferably pH 7-10, most preferably pH 8-10.
  • the variant has improved wash performance at a washing temperature of 10-60 °C, preferably 10-40 °C, more preferably 10-30 °C, most preferably 15- 25 °C. In one embodiment, the variant has improved wash performance as determined in Example 1 , Example 2, Example 3, Example 4, Example 5, Example 6, or Example 10 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 1 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 2 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 3 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 4 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 5 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 6 herein.
  • the variant has improved wash performance in liquid detergent as determined according to Example 10 herein, preferably in Model O detergent having pH 8-10 or preferably in Model B detergent having pH 7-8.
  • the variants of the invention have improved mildness compared to a reference protease.
  • improved mildness means that a protease variant is less aggressive towards other enzymes in the detergent matrix (also referred to as companion enzymes or secondary enzymes), thereby providing improved residual activity of a companion enzyme after storage with a protease variant of the invention in a detergent composition.
  • mildness is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
  • the reference protease is a parent protease.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2 , SEQ ID NO:3
  • the variant has improved mildness compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:7.
  • the variant has improved mildness compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1.
  • mildness is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%.
  • the variant has improved mildness compared to SEQ ID NO:8.
  • the variant has improved mildness compared to SEQ ID NO:11.
  • the variant has improved mildness compared to SEQ ID NO: 13.
  • the variant provides improved residual activity of a companion enzyme.
  • the residual activity of the companion enzyme is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
  • the companion enzyme is selected from the group consisting of amylase (e.g., alpha-amylase), arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, DNase, galactanase, haloperoxygenase, lipase, mannanase, oxidase (e.g., laccase or peroxidase), pectinase, pectin lyase, protease, xylanase, xanthanase or xyloglucanase.
  • the companion enzyme is an alpha-amylase.
  • the residual activity is determined according to Example 8 herein.
  • the variant provides improved residual activity of a companion enzyme, preferably an alpha-amylase, after storage in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
  • a companion enzyme preferably an alpha-amylase
  • the variant provides improved residual activity of a companion enzyme, preferably an alpha-amylase, after storage with a protease variant of the invention when stored in a liquid detergent at a temperature of 10-60 °C, preferably 15-50 °C, most preferably 20-40 °C.
  • a companion enzyme preferably an alpha-amylase
  • the variant has improved mildness in liquid detergent as determined according to Example 8 herein.
  • the variant provides improved residual activity of an alphaamylase in liquid detergent as determined according to Example 8 herein.
  • the variant has improved mildness in Model O liquid detergent.
  • the variant provides improved residual activity of an alphaamylase in Model O liquid detergent.
  • the variants of the invention have improved strain removal rate compared to a reference protease.
  • improved stain removal rate means that the variant is relatively faster at removing proteinaceous soil compared to the parent protease within a given timeframe.
  • stain removal rate is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
  • the reference protease is a parent protease.
  • the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the variant has improved stain removal rate compared to SEQ ID NO:1.
  • the variant has improved stain removal rate compared to SEQ ID NO:2.
  • the variant has improved stain removal rate compared to SEQ ID NO:3.
  • the variant has improved stain removal rate compared to SEQ ID NO:4.
  • the variant has improved stain removal rate compared to SEQ ID NO:5.
  • the variant has improved stain removal rate compared to SEQ ID NO:6.
  • the variant has stain removal rate mildness compared to SEQ ID NO:7.
  • the variant has improved stain removal rate compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 .
  • stain removal rate is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%.
  • the variant has improved stain removal rate compared to SEQ ID NO:8.
  • the variant has improved stain removal rate compared to SEQ ID NO:11.
  • the variant has improved stain removal rate compared to SEQ ID NO: 13.
  • the variant has improved improved stain removal rate at a washing temperature of 10-60 °C, more preferably 15-50 °C, most preferably 20-40 °C.
  • the variant has improved stain removal rate in liquid detergent as determined according to Example 9 herein.
  • the variant of the invention may be a fusion polypeptide comprising a variant of the invention.
  • the variant of the invention is isolated.
  • the variant of the invention is purified.
  • the parent protease may be a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
  • the parent protease is a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
  • the parent protease is a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:1.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:1.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:2.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:2.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:3 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:3.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:3.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:4 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:4.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:4.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:5 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:5.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:5 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:5.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:6 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:6.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:6.
  • the parent has a sequence identity to the polypeptide of SEQ ID NO:7 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 protease activity.
  • the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:7.
  • the parent comprises or consists of the polypeptide of SEQ ID NO:7.
  • the parent may be a fusion polypeptide or cleavable fusion polypeptide.
  • 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 al., 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 al., 2000, J. Biotechnol. 7Q: 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 is a Bacillus lentus protease, e.g., the protease of SEQ ID NO:1 .
  • the parent is a Bacillus amyloliquefaciens protease, e.g., the protease of SEQ ID NO:2.
  • the parent is a Bacillus licheniformis protease, e.g., the protease of SEQ ID NO:3.
  • the parent is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:4.
  • the parent is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:5. In one aspect, the parent is a Bacillus sp. TY145 protease, e.g., the protease of SEQ ID NO:6. In one aspect, the parent is a Actinomadura keratinilytica protease, e.g., the protease of SEQ ID NO:7.
  • the present invention also relates to methods for obtaining a variant having protease activity, comprising: (a) introducing into a parent protease substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further introducing substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 , wherein the variant has protease activity; and (b) recovering the variant.
  • the variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
  • Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.
  • Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually, the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 7Q: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.
  • Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004/0171154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.
  • Any site-directed mutagenesis procedure can be used in the present invention.
  • Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing several techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.
  • Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625.
  • 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 polynucleotides encoding a variant of the present invention.
  • the polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.
  • the polynucleotide is isolated.
  • the polynucleotide is purified.
  • 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 recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the variant.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier, and Song et al., 2016, PLOS One 11(7): e0158447.
  • promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells may be obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • aprH Bacillus clausii alkaline protease
  • AmyL Bacillus licheniformis alpha-amylase
  • rrnB Escherichia coli ribosomal RNA
  • Preferred terminators for filamentous fungal host cells may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
  • Preferred terminators for yeast host cells may be 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, Yeast 8: 423-488.
  • 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.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue etal., 1995, J. Bacterid. 177: 3465-3471).
  • mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.
  • the control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell.
  • the leader is operably linked to the 5’-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.
  • Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.
  • Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells may be 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 polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
  • Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum 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 filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu etal., 2018, Biotechnology Letters 40: 949-955
  • 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 variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • regulatory sequences that regulate expression of the variant relative to the growth of the host cell.
  • regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems.
  • yeast the ADH2 system or GAL1 system may be used.
  • the Aspergillus niger glucoamylase promoter In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used.
  • Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, 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 control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence.
  • the transcription factor may function alone and/or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase.
  • Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor.
  • the transcription factor may regulate the expression of a protein of interest either directly, /.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, /.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor.
  • Suitable transcription factors for fungal host cells are described in WO 2017/144177.
  • Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7- 23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.
  • 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, /.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, 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.
  • the vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
  • homologous recombination such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ non-homologous end-joining
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier.
  • the choice of a host cell will to a large extent depend upon the gene encoding the variant and its source.
  • the recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.
  • the host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.
  • the host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.
  • the prokaryotic host cell may be any Gram-positive or Gram-negative bacterium.
  • Grampositive 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 Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.
  • Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.
  • 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.
  • Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.
  • the host cell may be a fungal cell.
  • “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
  • Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen et al., 1988, Bio/TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75.
  • any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.
  • the fungal host cell may be a 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 yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).
  • 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, Fili basidium, 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 is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.
  • 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
  • the host cell is isolated.
  • the host cell is purified.
  • the present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.
  • the host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art.
  • the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and/or isolated.
  • Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.
  • the variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant.
  • the variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • the whole fermentation broth is recovered.
  • a cell- free fermentation broth comprising the polypeptide is recovered.
  • the variant may be purified by a variety of procedures known in the art to obtain substantially pure variants and/or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).
  • the variant is not recovered.
  • the present invention also relates to enzyme granules/particles comprising a variant of the invention.
  • the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core diameter, measured as equivalent spherical diameter can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020).
  • the core comprises a variant of the present invention.
  • the core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
  • a binder such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core may include an inert particle with the variant absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • the coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%.
  • the amount may be at most 100%, 70%, 50%, 40% or 30%.
  • the coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
  • the coating should encapsulate the core unit by forming a substantially continuous layer.
  • a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas.
  • the layer or coating should, in particular, be homogeneous in thickness.
  • the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • fillers e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • a salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
  • the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm.
  • the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.
  • the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
  • the salt coating may comprise a single salt or a mixture of two or more salts.
  • the salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
  • the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
  • simple organic acids e.g., 6 or less carbon atoms
  • Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum.
  • anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
  • alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
  • the salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
  • the salt coating may be as described in WO 00/01793 or WO 2006/034710.
  • the salt may be in anhydrous form, or it may be a hydrated salt, /.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595.
  • Specific examples include anhydrous sodium sulfate (Na2SO 4 ), anhydrous magnesium sulfate (MgSO 4 ), magnesium sulfate heptahydrate (MgSO 4 7H2O), zinc sulfate heptahydrate (ZnSO 4 7H2O), sodium phosphate dibasic heptahydrate (Na2HPO 4 7H2O), magnesium nitrate hexahydrate (Mg(NO 3 )2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
  • Na2SO 4 anhydrous sodium sulfate
  • MgSO 4 magnesium sulfate heptahydrate
  • ZnSO 4 7H2O zinc sulfate heptahydrate
  • the salt is applied as a solution of the salt, e.g., using a fluid bed.
  • the coating materials can be waxy coating materials and film-forming coating materials.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol, PEG
  • 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
  • mono- and di- and triglycerides of fatty acids are given in GB 1483591
  • the granule may optionally have one or more additional coatings.
  • suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • PEG polyethylene glycol
  • MHPC methyl hydroxy-propyl cellulose
  • PVA polyvinyl alcohol
  • enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.
  • the cores may be subjected to drying, such as in a fluid bed drier.
  • drying preferably takes place at a product temperature of from 25 to 90°C.
  • the cores comprising the variant contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme negatively.
  • the cores preferably contain 0.1-10% w/w water.
  • Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.
  • the granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
  • the present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.
  • the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
  • the one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta- mannosidase (mannanase), phytase, phospholipase A1 , phospholipase
  • the present invention also relates to liquid compositions comprising a variant of the invention.
  • the composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • an enzyme stabilizer include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • filler(s) or carrier material(s) are included to increase the volume of such compositions.
  • suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like.
  • Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.
  • the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative.
  • the invention relates to liquid formulations comprising:
  • the invention relates to liquid formulations comprising:
  • the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulphate, potassium sulphate, magnesium sulphate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulphate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate.
  • a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulphate, potassium sulphate, magnesium sulphate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate
  • the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.
  • MPG propylene glycol
  • the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol.
  • the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600.
  • MPG propylene glycol
  • the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).
  • polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).
  • the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
  • the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative.
  • the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02- 1.5% w/w preservative, 0.05-1% w/w preservative, or 0.1-0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
  • the liquid formulation further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
  • the one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha- mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospho
  • the present invention also relates to a fermentation broth formulation or a cell composition comprising a variant of the present invention.
  • the fermentation broth formulation or the cell composition further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the variant of the present invention which are used to produce the variant of interest), cell debris, biomass, fermentation media and/or fermentation products.
  • the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
  • fermentation broth refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification.
  • fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium.
  • the fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation.
  • the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation.
  • the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
  • the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof.
  • the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
  • the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris.
  • the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
  • the fermentation broth formulation or cell composition may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • a preservative and/or anti-microbial agent including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
  • the cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation.
  • the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis.
  • the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells.
  • the microbial cells present in the cell-killed whole broth or cell composition can be permeabilized and/or lysed using methods known in the art.
  • a whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
  • the whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
  • the invention also relates to a composition comprising a variant of the invention, e.g., a detergent or cleaning composition.
  • the invention also relates to a composition comprising a variant of the invention and further comprising: one or more detergent components; and/or one or more additional enzymes.
  • the composition is a detergent composition comprising one or more detergent components, in particular one or more non-naturally occurring detergent components.
  • the present invention also relates to a composition
  • a composition comprising a variant of the present invention and further comprising one or more additional enzymes selected from the group consisting of amylases (e.g., alpha-amylases), catalases, cellulases (e.g., endoglucanases), cutinases, DNases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, lichenases and xyloglucanases, or any mixture thereof.
  • amylases e.g., alpha-amylases
  • catalases e.g., cellulases (e.g., endoglucanases), cutinases, DNases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxid
  • a detergent composition may, e.g., be in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.
  • the detergent composition is a liquid composition.
  • the detergent composition is a powder composition.
  • the detergent composition is a laundry soap bar.
  • the invention also relates to use of a composition of the present in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.
  • a detergent composition is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below.
  • the choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.
  • a detergent composition comprises a variant of the invention and one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers.
  • non-naturally occurring detergent components such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers
  • the variant of the invention may be added to a detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.
  • An automatic dish wash (ADW) composition may for example include 0.001 %-30%, such as 0.01 %-20%, such as 0.1-15%, such as 0.5-10% of enzyme protein by weight of the composition.
  • a granulated composition for laundry may for example include 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.
  • a liquid composition for laundry may for example include 0.0001 %-10%, such as 0.001- 7%, such as 0.1 %-5% of enzyme protein by weight of the composition.
  • the enzymes such as the variant of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708 or the variants according to the invention may be stabilized using peptide aldehydes or ketones such as described in WO 2005/105826 and WO 2009/118375.
  • a polyol such as propylene glycol or glycerol
  • a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphen
  • the variants of the invention may be formulated in liquid laundry compositions such as a liquid laundry compositions composition comprising: a) at least 0.01 mg of active variant per liter detergent, b) 2 wt% to 60 wt% of at least one surfactant c) 5 wt% to 50 wt% of at least one builder
  • the detergent composition may be formulated into a granular detergent for laundry.
  • Such detergent may comprise; a) at least 0.01 mg of active protease variant per gram of composition b) anionic surfactant, preferably 5 wt % to 50 wt % c) nonionic surfactant, preferably 1 wt % to 8 wt % d) builder, preferably 5 wt % to 40 wt %, such as carbonates, zeolites, phosphate builder, calcium sequestering builders or complexing agents.
  • the detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof.
  • the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants.
  • the surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%.
  • the surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized. Surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away.
  • the detergent When included therein, the detergent will usually contain from about 1% to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant.
  • anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS
  • the detergent When included therein, the detergent will usually contain from about 0% to about 10% by weight of a cationic surfactant.
  • cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.
  • the detergent When included therein, the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%.
  • a non-ionic surfactant for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%.
  • Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations
  • the detergent When included therein, the detergent will usually contain from about 0% to about 10% by weight of a semipolar surfactant.
  • semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N- (tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.
  • AO amine oxides
  • the detergent When included therein, the detergent will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant.
  • zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.
  • the detergent composition may contain about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof.
  • the level of builder is typically 40-65%, particularly 50-65%.
  • Builders and chelators soften, e.g., the wash water by removing the metal ions form the liquid.
  • 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 laundry 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 iminodiethanol), triethanolamine (TEA, also known as 2,2’,2”-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.
  • 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,
  • the detergent composition may also contain 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof.
  • the detergent composition may 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).
  • PAA/PMA poly(acrylic acid)
  • 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-N,N’-disuccinic acid
  • MGDA methylglycinediacetic acid
  • GLDA glutamic acid-N,N-diacetic acid
  • HEDP 1-hydroxyethane- 1 ,1-diphosphonic acid
  • EDTMPA ethylenediaminetetra-(methylenephosphonic acid)
  • DTPMPA or DTMPA diethylenetriaminepentakis (methylenephosphonic acid)
  • EDG N-(2- hydroxyethyl)iminodiacetic acid
  • ASMA aspartic acid-N-monoacetic acid
  • ASDA aspartic acid-N, N-diacetic acid
  • ASDA aspartic acid-N-mon
  • the variants of the invention may also be formulated into a dish wash composition, preferably an automatic dish wash composition (ADW), comprising: a) at least 0.01 mg of active protease variant according to the invention, and b) 10-50 wt % builder preferably selected from citric acid, methylglycine-N, N-diacetic acid (MGDA) and/or glutamic acid-N, N-diacetic acid (GLDA) and mixtures thereof, and c) at least one bleach component.
  • ADW automatic dish wash composition
  • the detergent may contain 0-50% by weight, such as about 0.1 % to about 25%, of a bleaching system.
  • Bleach systems remove discolor often by oxidation, and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof.
  • Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof.
  • bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra- hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator.
  • bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid.
  • the peracid thus formed constitutes the activated bleach.
  • Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides.
  • Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4- (dodecanoyloxy) benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4- (decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO 98/17767.
  • TAED tetracetylethylene diamine
  • ISONOBS sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate
  • DOBS 4-(decanoyloxy)benzenesulfonate
  • NOBS 4-(nonanoyloxy)-benzenesulfonate
  • ATC acetyl triethyl citrate
  • ATC or a short chain triglyceride like triacetin has the advantage that it is environmentally friendly as it eventually degrades into citric acid and alcohol.
  • acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are efficient bleach activators.
  • ATC provides a good building capacity to the laundry additive.
  • the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type.
  • the bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP).
  • PAP 6-(phthalimido)peroxyhexanoic acid
  • the bleaching system may also include a bleach catalyst or a booster.
  • bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1 ,4,7-trimethyl-1 ,4,7-triazacyclononane (Me3-TACN) or 1 ,2,4,7-tetramethyl-1 ,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2, 2', 2"- nitrilotris(ethane-1 ,2-diylazanylylidene-KN-methanylylidene)triphenolato-K3O]manganese(
  • the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula:
  • each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propyl heptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and isopentadecyl.
  • Suitable bleaching systems are described, e.g., in WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242.
  • Suitable photobleaches may for example be sulfonated zinc phthalocyanine. Hydrotropes
  • a hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment).
  • hydrotropes have both hydrophilic and hydrophobic characters (so-called amphiphilic properties as known from surfactants); however, the molecular structures of hydrotropes generally do not favour spontaneous self-aggregation, see, e.g., review by Hodgdon and Kaier, 2007, Current Opinion in Colloid & Interface Science 12: 121-128.
  • Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micellar, lamellar or other well defined meso-phases.
  • hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases.
  • many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers.
  • Hydrotropes are classically used across industries from pharma, personal care and food to technical applications.
  • Use of hydrotropes in detergent compositions allows for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.
  • the detergent may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope.
  • a hydrotrope Any hydrotrope known in the art for use in detergents may be utilized.
  • Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
  • the detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized.
  • the polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs.
  • Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (P
  • exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate.
  • PEO-PPO polypropylene oxide
  • diquaternium ethoxy sulfate diquaternium ethoxy sulfate.
  • Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
  • the detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when the fabric is contacted with a wash liquor comprising the detergent compositions and thus altering the tint of the fabric through absorption/reflection of visible light.
  • fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum.
  • Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments.
  • Suitable dyes include small molecule dyes and polymeric dyes.
  • Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Color Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO 2005/003274, WO 2005/003275, WO 2005/003276 and EP 1876226 (hereby incorporated by reference).
  • the detergent composition preferably comprises from about 0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05 wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueing agent.
  • the composition may comprise from 0.0001 wt % to 0.2 wt. % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch.
  • Suitable hueing agents are also disclosed in, e.g., WO 2007/087257 and WO 2007/087243.
  • a detergent additive or detergent composition comprising the variant of the invention may comprise one or more enzymes such as an amylase (e.g., alpha-amylase), arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, DNase, galactanase, haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and/or peroxidase, pectinase, pectin lyase, protease, xylanase, xanthanase or xyloglucanase.
  • an amylase e.g., alpha-amylase
  • arabinase e.g., carbohydrase
  • cellulase e.g., endoglucanase
  • cutinase e.g., DNase,
  • the properties of the selected enzyme(s) should be compatible with the selected detergent (e.g., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.).
  • cellulase means one or more (e.g., several) enzymes that hydrolyze a cellulosic material.
  • the terms “cellulase” and the expression “polypeptide having cellulase activity” are used interchangeably.
  • Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172.
  • Such enzymes include endoglucanase(s) (e.g., EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
  • Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated.
  • the cellulase may for example be a mono-component or a mixture of mono-component endo-1 ,4-beta- glucanase also referred to as endoglucanase.
  • Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, My- celiophthora, Fusarium, Thielavia, Trichoderma, and Acremonium.
  • Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., T. reesei or T. viride.
  • Suitable cellulases are from Thielavia, e.g., Thielavia terrestris as described inWO 96/29397, or the fungal cellulases produced from Myceliophthora thermophila and Fusarium ox- ysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89/09259 and WO 91/17244. Also relevant are cellulases from Bacillus as described in WO 02/099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits.
  • cellulases examples include EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940.
  • Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307.
  • cellulases are endo-beta-1 , 4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
  • Suitable cellulases comprise a stabilized linker between the core and the CBM. Particularly useful are such cellulase having at least 80% identity to SEQ ID NO: 397, SEQ ID NO: 398 or SEQ ID NO: 399 of WO 2023/061928.
  • cellulases include Carezyme®, Carezyme® Premium, Cel- luzyme®, Carezyme Elite®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme® Celluclean® Classic, and Cellusoft® (Novozymes A/S); Puradax®, Puradax HA, Puradax EG, Revitalenz 1000, Revitalenz 200, and Revitalenz 2000 (Dupont Industrial Biosciences); KAC- 500(B)TM (Kao Corporation); and Biotouch DCL and Biotouch FLX1 (AB Enzymes).
  • the two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481.
  • Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.
  • the most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appt. Chem. 59: 257-68).
  • the composition may comprise one or more additional proteases including 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 metalloprotease may for example be a thermolysin from, e.g., family M4 or other metalloprotease such as those from M5, M7 or M8 families.
  • metalloproteases are the neutral metalloproteases as described in WO 2007/044993 (Genencor I nt.) such as those derived from Bacillus amyloliquefaciens.
  • Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, DuralaseTM, DurazymTM, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress® Excel, Progress® Key, and Progress® Uno (Novozymes A/S), those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, Preferenz® P200, Pre
  • 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 EP 258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO 96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P.
  • Thermomyces e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216
  • cutinase from Humicola e.
  • sp. strain SD705 (WO 95/06720 & WO 96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyces lipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO 2010/107560), cutinase from Pseudomonas mendocina (US 5,389,536), lipase from Thermobifida fusca (WO 2011/084412), Geobacillus stearothermophilus lipase (WO 2011/084417), lipase from Bacillus subtilis (WO 2011/084599), and lipase from Streptomyces griseus (WO 2011/150157) and S. pristinaespiralis (WO 2012/137147).
  • lipase variants such as those described in EP 407225, WO 92/05249, WO 94/01541 , WO 94/25578, WO 95/14783, WO 95/30744, WO 95/35381 , WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.
  • Preferred commercial lipase products 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 (WO 2010/111143), acyltransferase from Mycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE 7 family (WO 2009/067279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).
  • Suitable amylases which can be used together with the variants 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 99/19467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.
  • amylases having SEQ ID NO:6 in WO 02/10355 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-amylases comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006/066594 or variants having 90% sequence identity thereof.
  • 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 having the sequence of SEQ ID NO:6 in WO 99/19467 or variants thereof having 90% sequence identity to SEQ ID NO:6.
  • Preferred variants of 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 96/23873 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, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/23873 for numbering. More preferred variants are those having a deletion in two positions selected from 181 , 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184.
  • Most preferred amylase variants of SEQ ID NO:1 , SEQ ID NO:2 or SEQ ID NO:7 are those having 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 2008/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO:2 of WO 2008/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712.
  • Preferred variants of SEQ ID NQ: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 , 207, 211 and 264.
  • amylases having SEQ ID NO:2 of WO 2009/061380 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:
  • variants are C-terminally truncated and optionally further comprise a substitution at position 243 and/or a deletion at position 180 and/or position 181.
  • amylases having SEQ ID NO:1 of WO 2013/184577 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: K176, R178, G179, T180, G181 , E187, N192, M199, I203, S241 , R458, T459, D460, G476 and G477.
  • 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: K176L, E187P, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or a deletion in position R178 and/or S179 or of T180 and/or G181.
  • Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions: E187P+I203Y+G476K E187P+I203Y+R458N+T459S+D460T+G476K and optionally further comprise a substitution at position 241 and/or a deletion at position 178 and/or position 179.
  • amylases having SEQ ID NO:1 of WO 2010/104675 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.
  • 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 a deletion in position R179 and/or S180 or of 1181 and/or G182.
  • Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions N21 D+D97N+V128I, and optionally further comprise 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 WO 01/66712 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 WO 01/66712: R28, R118, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484.
  • amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, 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 WO 2011/098531 , WO 2013/001078 and WO 2013/001087.
  • Commercially available amylases are DuramylTM, TermamylTM, FungamylTM, StainzymeTM, Stainzyme PlusTM, NatalaseTM, Liquozyme X and BANTM (from Novozymes A/S), and RapidaseTM, PurastarTM/EffectenzTM, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).
  • Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
  • peroxidases include GuardzymeTM (Novozymes A/S).
  • any detergent components known in the art for use in laundry detergents may also be utilized.
  • Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination.
  • Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
  • the detergent compositions of the present invention can also contain dispersants.
  • powdered detergents may comprise dispersants.
  • Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
  • Suitable dispersants are for example described in Powdered Detergents, Surfactant Science Series, volume 71 , Marcel Dekker, Inc., 1997.
  • the detergent compositions of the present invention may also include one or more dye transfer inhibiting agents.
  • Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01% to about 5% or even from about 0.1 % to about 3% by weight of the composition.
  • Fluorescent whitening agent The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 05%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives.
  • diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6- ylamino) stilbene-2,2'-disulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'- disulphonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2, 1 ,3-triazol-2-yl)stilbene-2,2'-disulphonate; 4,4'- bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-d
  • Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland.
  • Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate.
  • Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate.
  • fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India.
  • Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins.
  • Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.
  • Soil release polymers may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics.
  • the soil release polymers may for example be nonionic or anionic terephthalate based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc.
  • Another type of soil release polymers is amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure.
  • the core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference).
  • random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference).
  • Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 03/040279 (both are hereby incorporated by reference).
  • Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
  • the detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines.
  • CMC carboxymethylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • PEG polyethyleneglycol
  • homopolymers of acrylic acid copolymers of acrylic acid and maleic acid
  • the cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
  • adjunct materials include, but are not limited to, anti-shrink agents, antiwrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
  • the detergent enzyme(s), /.e., a variant of the invention and optionally one or more additional enzymes, 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 comprising one or more enzymes can be formulated, for example, as a granulate, liquid, slurry, etc.
  • Preferred detergent additive formulations include granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
  • the detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • a bar e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • a regular or compact powder e.g., a granule, a paste, a gel, or a regular, compact or concentrated liquid.
  • There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit.
  • Pouches can be configured as single or multiple compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g., without allowing the release of the composition from the pouch prior to water contact.
  • the pouch is made from water soluble film which encloses an inner volume. The inner volume can be divided into compartments of the pouch.
  • Preferred films are polymeric materials, preferably polymers which are formed into a film or sheet.
  • Preferred polymers, copolymers or derivates thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC).
  • the level of polymer in the film for example PVA is at least about 60%.
  • the preferred average molecular weight will typically be about 20,000 to about 150,000.
  • Films can also be of blend compositions comprising hydrolytically degradable and water-soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod, of Gary, Indiana, US) plus plasticizers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof.
  • the pouches can comprise a solid laundry detergent composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film.
  • the compartment for liquid components can be different in composition than compartments containing solids. See, e.g., US 2009/0011970.
  • Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
  • a liquid or gel detergent which is not unit dosed may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water.
  • Other types of liquids including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel.
  • An aqueous liquid or gel detergent may contain from 0-30% organic solvent.
  • a liquid or gel detergent may be non-aqueous.
  • the enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles.
  • laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars.
  • the types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps.
  • the laundry soap bar has a physical form which is solid and thus not a liquid, gel or powder at room temperature.
  • the laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerin, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na + , K + , or NH4 + and the organic anion may be for example formate, acetate, citrate, or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
  • protease inhibitors such as peptide aldehydes (or hydrosulfite adduct
  • the laundry soap bar may also contain complexing agents such as EDTA and HEDP, perfumes and/or different type of fillers, surfactants, e.g., anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
  • the laundry soap bar may be processed in conventional laundry soap bar making equipment such as, but not limited to, mixers, plodders, e.g., a two-stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers.
  • a premix containing a soap, the enzyme of the invention, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded.
  • the enzyme and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form.
  • the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
  • Enzymes such as variants of the present invention in the form of granules, comprising an enzyme-containing core and optionally one or more coatings, are commonly used in granular (powder) detergents.
  • granular (powder) detergents are commonly used in granular (powder) detergents.
  • Various methods for preparing the core include, for example, a) spray drying of a liquid enzyme-containing solution, b) production of layered products with an enzyme coated as a layer around a pre-formed inert core particle, e.g.
  • a fluid bed apparatus c) absorbing an enzyme onto and/or into the surface of a pre-formed core, d) extrusion of an enzyme-containing paste, e) suspending an enzyme-containing powder in molten wax and atomization to result in prilled products, f) mixer granulation by adding an enzymecontaining liquid to a dry powder composition of granulation components, g) size reduction of enzyme-containing cores by milling or crushing of larger particles, pellets, etc., and h) fluid bed granulation.
  • the enzyme-containing cores may be dried, e.g., using a fluid bed drier or other known methods for drying granules in the feed or enzyme industry, to result in a water content of typically 0.1 -10% w/w water.
  • the enzyme-containing cores are optionally provided with a coating to improve storage stability and/or to reduce dust formation.
  • a coating typically an inorganic salt coating, which may, e.g., be applied as a solution of the salt using a fluid bed.
  • Other coating materials that may be used are, for example, polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • PEG polyethylene glycol
  • MHPC methyl hydroxy-propyl cellulose
  • PVA polyvinyl alcohol
  • the granules may contain more than one coating, for example a salt coating followed by an additional coating of a material such as PEG, MHPC or PVA.
  • the present invention thus also relates to enzyme granules/particles comprising the variant of the invention.
  • the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
  • the core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core comprises one or more polypeptides having protease activity of the present invention.
  • the core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
  • the core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
  • a binder such as synthetic polymer, wax, fat, or carbohydrate.
  • the core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
  • the core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
  • the core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
  • the core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule.
  • the optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • the coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1 %, at least 5%, at least 10%, or at least 15%.
  • the amount may be at most 100%, 70%, 50%, 40% or 30%.
  • the coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
  • the coating should encapsulate the core unit by forming a substantially continuous layer.
  • a substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating/enclosing has few or none uncoated areas.
  • the layer or coating should, in particular, be homogeneous in thickness.
  • the coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
  • a salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
  • the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm.
  • the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.
  • the salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
  • the salt coating may comprise a single salt or a mixture of two or more salts.
  • the salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
  • the salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate.
  • simple organic acids e.g., 6 or less carbon atoms
  • Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum.
  • anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate.
  • alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
  • the salt in the coating may have a constant humidity at 20 °C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate).
  • the salt coating may be as described in WO 00/01793 or WO 2006/034710.
  • the salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595.
  • Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSC ), magnesium sulfate heptahydrate (MgSC - tW), zinc sulfate heptahydrate (ZnSC - tW), sodium phosphate dibasic heptahydrate (Na2HPO4'7H2O), magnesium nitrate hexahydrate (Mg(NC>3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
  • Na2SO4 anhydrous magnesium sulfate
  • MgSC magnesium sulfate heptahydrate
  • ZnSC - tW zinc sulfate heptahydrate
  • the salt is applied as a solution of the salt, e.g., using a fluid bed.
  • the coating materials can be waxy coating materials and film-forming coating materials.
  • waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
  • PEG poly(ethylene oxide) products
  • PEG polyethyleneglycol, PEG
  • 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
  • mono- and di- and triglycerides of fatty acids are given in GB 1483591
  • the granule may optionally have one or more additional coatings.
  • suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
  • PEG polyethylene glycol
  • MHPC methyl hydroxy-propyl cellulose
  • PVA polyvinyl alcohol
  • enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
  • the core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
  • Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.
  • the cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the enzyme contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, it will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w/w water.
  • Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
  • the granulate may further one or more additional enzymes.
  • Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes.
  • Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
  • the enzyme may also be a protected enzyme prepared according to the method disclosed in EP 238,216.
  • the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase.
  • the one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta- mannosidase (mannanase), phytase, phospholipase A1 , phospholipase
  • the present invention is also directed to methods for using the variants of the invention or compositions comprising said variants in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing.
  • the invention is also directed to methods for using the variants according to the invention or compositions thereof in cleaning hard surfaces such as floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dishwashing).
  • the variants of the present invention may be added to and thus become a component of a detergent composition.
  • one aspect of the invention relates to the use of a variant of the invention in a cleaning process such as laundering and/or hard surface cleaning.
  • a detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations or be formulated for hand or machine dishwashing operations.
  • the cleaning process or the textile care process may for example be a laundry process, a dishwashing process, or cleaning of hard surfaces such as bathroom tiles, floors, tabletops, drains, sinks and washbasins.
  • Laundry processes can for example be household laundering but may also be industrial laundering.
  • the invention relates to a process for laundering of fabrics and/or garments, where the process comprises treating fabrics with a washing solution containing a detergent composition and at least one protease variant of the invention.
  • the cleaning process or a textile care process can for example be carried out in a machine washing or manually.
  • the washing solution can for example be an aqueous washing solution containing a detergent composition.
  • the variants of the invention are used in a cleaning process, e.g., a laundry process, that comprises a short wash cycle, typically a wash cycle of not more than about 30 minutes, such as not more than about 20 minutes, e.g., not more than about 15 minutes or not more than about 10 minutes. It has surprisingly been found that the subtilase variants of the invention are remarkably effective in short wash cycles lasting, for example, only about 10-20 minutes. This may be useful in, e.g., top-loading washing machines that often have short wash cycles or for hand-washing of laundry.
  • the variants of the invention are used in a cleaning process, e.g., a laundry process, where the wash water is used for more than one portion of laundry.
  • the wash water containing a detergent with a variant of the invention may be used in a first wash cycle for a first portion of laundry, and then reused one or more times for additional wash cycles with new portions of laundry. It has been found that detergents containing a variant of the invention are able to substantially maintain cleaning performance on protease-sensitive stains even after three wash cycles or more. This may for example be useful for laundry washed by hand and/or in regions with water scarcity.
  • the invention further concerns the use of variants of the invention in a proteinaceous stain removing process.
  • the proteinaceous stains may be stains such as food stains, e.g., baby food, cocoa, egg or milk, or other stains such as sebum, blood, ink or grass, or a combination hereof.
  • the present invention provides a method of cleaning a fabric, dishware or a hard surface with a detergent composition comprising a variant of the invention.
  • the method of cleaning comprises contacting an object with a detergent composition comprising a protease variant of the invention under conditions suitable for cleaning the object.
  • a detergent composition comprising a protease variant of the invention under conditions suitable for cleaning the object.
  • the detergent composition is used in a laundry or a dish wash process.
  • Another embodiment relates to a method for removing stains from fabric or dishware which comprises contacting the fabric or dishware with a composition comprising a protease of the invention under conditions suitable for cleaning the object.
  • the object being cleaned may be any suitable object such as a textile or a hard surface such as dishware or a floor, table, wall, etc.
  • compositions and methods of treating fabrics e.g., to desize a textile
  • a protease variant of the invention can be used in any fabrictreating method which is well known in the art (see, e.g., US 6,077,316).
  • the feel and appearance of a fabric is improved by a method comprising contacting the fabric with a protease variant in a solution.
  • the fabric is treated with the solution under pressure.
  • the detergent compositions of the present invention are suited for use in laundry and hard surface applications, including dishwashing. Accordingly, the present invention includes a method for laundering a fabric or washing dishware, comprising contacting the fabric/dishware to be cleaned with a solution comprising the detergent composition according to the invention.
  • the fabric may comprise any fabric capable of being laundered in normal consumer use conditions.
  • the dishware may comprise any dishware such as crockery, cutlery, ceramics, plastics such as melamine, metals, china, glass and acrylics.
  • the solution preferably has a pH from about 5.5 to about 11.5.
  • the compositions may be employed at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution.
  • the water temperatures typically range from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C and about 90°C.
  • the water to fabric ratio is typically from about 1 :1 to about 30:1.
  • the composition may be formulated as described in, e.g., ⁇ NQ) 92/19709, WO 92/19708 and US 6,472,364.
  • the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).
  • barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV) e.g., barium (II), scandium (II), iron (
  • the detergent compositions provided herein are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 12.5, such as from about 5.0 to about 11.5, or from about 6.0 to about 10.5.
  • granular or liquid laundry products are formulated to have a pH from about 6 to about 8.
  • Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
  • the present invention relates to novel protease variants exhibiting increased stability and/or improved wash performance (exemplified by improved removal of proteinaceous stains) in liquid detergent and soap bar compositions compared with the parent protease.
  • Standard textiles or swatches were obtained from Center for Testmaterials BV (P.O. Box 120, 3133 KT Vlaardingen, The Netherlands), Warwick Equest Ltd (Consett, DH8 6BN, United Kingdom) and EMPA (Ueberlandstrasse 129, 8600 Dubendorf, Switzerland).
  • the following detergents used can be composed according to the description herein.
  • the protease(s) to be tested with the swatches can then be added to the model detergent base for testing.
  • Recombinant B. subtilis constructs encoding protease polypeptides were inoculated into and cultivated in a complex medium (TBgly) under antibiotic selection for 24 h at 37 °C.
  • Shake flasks containing a rich media PS-1 : 100 g/L sucrose (Danisco cat.no. 109-0429), 40 g/L crust soy (soybean flour), 10 g/L Na2HPO4 l2H2O (Merck cat.no. 106579), 0.1 ml/L Dowfax63N10 (Dow) were inoculated in a ratio of 1 :100 with the overnight culture.
  • Shake flask cultivation was performed for 4 days at 30 °C shaking at 270 rpm.
  • the culture broth is centrifuged at 26,000 x g for 20 minutes and the supernatant is carefully decanted from the precipitate.
  • the supernatant is filtered through a Nalgene 0.2 pm filtration unit to remove the remains of the host cells.
  • the pH in the 0.2 pm filtrate is adjusted to pH 8 with 3 M Tris base and the pH-adjusted filtrate is applied to a M EP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris/HCI, 1 mM CaCh, pH 8.0.
  • the column After washing the column with the equilibration buffer, the column is step-eluted with 20 mM CHsCOOH/NaOH, 1 mM CaCh, pH 4.5. Fractions from the column are analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 and peak fractions are pooled. The pH of the pool from the MEP Hypercel column is adjusted to pH 6 with 20% (v/v) CH3COOH or 3 M Tris base and the pH-adjusted pool is diluted with deionized water to the same conductivity as 20 mM MES/NaOH, 2 mM CaCh, pH 6.0.
  • the diluted pool is applied to an SP-Sepharose® Fast Flow column (GE Healthcare) equilibrated in 20 mM MES/NaOH, 2 mM CaCh, pH 6.0. After washing the column with the equilibration buffer, the protease variant is eluted with a linear NaCI gradient (0 0.5 M) in the same buffer over five column volumes. Fractions from the column are analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 and active fractions are analyzed by SDS-PAGE. Fractions in which only one band is observed on the Coomassie stained SDS-PAGE gel are pooled as the purified preparation and used for further experiments.
  • the proteolytic activity of a variant of the invention can be determined by a method employing the Suc-AAPF-pNA substrate.
  • Suc-AAPF-pNA is an abbreviation for N-Succinyl-Alanine- Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can be cleaved by endo-proteases. Following proteolytic cleavage, a free pNA molecule having a yellow color is liberated and can be measured by visible spectrophotometry at wavelength 405 nm.
  • the Suc- AAPF-PNA substrate may be purchased from Bachem.
  • a sample containing the variant to be analyzed is diluted in residual activity buffer (100 mM Tris, pH 8.6).
  • the assay is performed by transferring 30 pl of diluted enzyme samples to 96 well microtiter plate and adding 70 pl substrate working solution (0.72 mg/ml in 100 mM Tris, pH 8.6).
  • the solution is mixed at room temperature and absorption at 405 nm is measured over time, e.g., every 20 sec. over 5 minutes.
  • the slope (absorbance per minute) of the time-dependent absorption curve is directly proportional to proteolytic activity.
  • the proteolytic activity of a detergent composition comprising a variant of the invention can be determined by a method employing N,N-dimethyl casein (DMC) as substrate.
  • DMC N,N-dimethyl casein
  • carboxylic acids and primary amines are produced.
  • the produced amines then react under alkaline conditions with 2,4,6-tri-nitrobenzene-sulphonic acid (TNBS, Sigma) to form a colored complex which can be measured at 405 nm.
  • TNBS 2,4,6-tri-nitrobenzene-sulphonic acid
  • a detergent sample containing a variant of the invention is dissolved in 0.08 M sodium sulfite buffer and stirred for 10 minutes, after which the sample is filtered (Whatman filter no. 54 or similar). Sample dilutions are made using buffer (0.05 M boric acid + 0.16 M sodium sulfite + 0.15 M potassium chloride + 0.0225% (w/v) Brij® L23, pH 9.00).
  • Reagents including 1) 3.2 g/L DMC substrate + 0.1 M sodium dihydrogen phosphate monohydrate + 0.07 M Borax + 0.02% (w/v) Brij® L23, pH 8.00, 2) 0.1% TNBS and 3) 0.1 % TNBS + 0.4% DSAA are employed in running the analysis using a Konelab 30 Analyzer (ThermoFisher Scientific) according to the assay parameters outlined in Table 19. Activity values may then be calculated based on a standard curve.
  • AMSA Automatic Mechanical Stress Assay
  • the wash performance is measured as the brightness of the color of the textile washed.
  • the brightness can be expressed as the intensity of the light reflected from the textile sample when illuminated with white light.
  • the intensity of the reflected light is lower than that of a clean textile. Therefore, the intensity of the reflected light can be used to measure wash performance of the proteases of interest.
  • Color measurements are made with a professional flatbed scanner (Epson Expression 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark), which is used to capture an image of the washed textile sample.
  • a specially designed software application is used ⁇ Novozymes Color Vector Analyzer).
  • the program retrieves the values from the image and converts them into values for red, green, and blue (RGB).
  • the intensity value (Int) may be calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
  • the Terg-o-tometer is a medium scale wash assay that can be applied to simultaneously test up to 16 different conditions at the same time. Briefly, it consists of 16 x 2 L metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial top-loader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker was filled with 1 L detergent solution, and test swatches, ballast and enzymes are added to the requisite levels. After a timed wash period, the swatches are promptly removed from the beakers and rinsed thoroughly with tap water.
  • the swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a Macbeth Color Eye 7000 reflectance spectrophotometer with large aperture. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.
  • REM incident light and remission
  • the effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without enzyme (blank) from the swatch washed together with enzyme.
  • the performance of a new protease e.g., a protease variant
  • RP relative performance
  • Full-scale washing machines (Panasonic XQB65-Q680U, Top Loader) are used to evaluate protease performance in large-scale. Detergent, swatches, ballast, water, and enzyme are added together and washed at a defined temperature, in this case for 15 minutes, followed by rinse. The level of detergent, water, enzymes, and ballast used may be adjusted based on different factors, such as regional habits. The swatches are then removed from the wash, and dried flat overnight at room temperature. Evaluation of FSW swatches is carried out in a similar manner as for TOM swatches.
  • Mini-o-tometer Wash Assay and determination of relative strain removal rate
  • Mini-TOM is a medium scale wash assay that can be applied to simultaneously test up to 16 different conditions at the same time. Briefly, it consists of 16 x 0.2 L metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial top-loader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker are filled with 0.15 L detergent solution, and test swatches and enzymes are added to the requisite levels. The swatches are promptly removed from the beakers at the desired time points and rinsed thoroughly with ice water to stop the hydrolysis of soil.
  • the swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a Macbeth Color Eye 7000 reflectance spectrophotometer with large aperture. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.
  • REM incident light and remission
  • the effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without enzyme (blank) from the swatch washed together with protease.
  • the stain removal rate (SRR) of a new protease may be compared to the stain removal rate of a reference protease by calculating the relative stain removal rate (RSRR) at at given time point:
  • SRR stain removal rate
  • RSRR relative stain removal rate
  • Proteases were included in wash tests carried out in Asia Pacific top loader washing ma- chines using a model detergent and a range of stain types. At least six different swatches, representing at least five different stain categories (e.g., blood, blood milk ink, chocolate, grass, and egg) showed improvements in wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at low washing temperature (15 °C; see Table 8).
  • SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at low washing temperature (15 °C; see Table 8).
  • Proteases were included in wash tests carried out in Asia Pacific top loader washing machines using a model detergent and a range of stain types. At least eight different swatches, representing at least four different stain categories (e.g., blood milk ink, chocolate, grass, and egg) showed improvements in wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at normal washing temperature (25 °C; see Table 10).
  • SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at normal washing temperature (25 °C; see Table 10).
  • Protease formulations were incorporated into a basic laundry soap bar matrix and incubated at 37°C. After 4 weeks, the soap bars were evaluated for performance in a terg-o-tometer wash assay. In this assay, the soap samples were grated and washed together with seven differ- ent stain monitors, representing at least 4 different stain categories (e.g., blood milk ink, chocolate, grass, and egg). SEQ ID NO:9 showed improved wash performance compared to SEQ ID NO:1 and SEQ ID NO:8 for six out of seven stains tested (see Table 12).
  • Example 4 TOM Wash Performance Evaluation after storage in a Low pH European Detergent
  • SEQ ID NO:8 and SEQ ID NO:9 were evaluated in a terg-o-tometer wash assay after storage for 24 hours at 25°C in a low pH European detergent. A total of seven swatches were used to evaluate the wash performance, representing at least four different stain categories (e.g., blood milk ink, chocolate, grass, and egg). SEQ ID NO:9 showed improved wash performance compared to SEQ ID NO:8 across all stains tested with SEQ ID NO:9 (see Table 14).
  • SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 were tested using an AMSA wash assay, with results expressed in terms of relative performance to SEQ ID NO:1. Results showed improved wash performance of SEQ ID NO:9 across both stains tested (see Table 18).
  • SEQ ID N0:8 and SEQ ID N0:9 were incubated in a high-water liquid model detergent for 4 - 8 weeks at 30 or 37 °C, and then analyzed for protease activity. Residual protease activity was determined relative to protease activity present in the original non-incubated samples stored at -18 °C, expressed in %. The results show that SEQ ID NO:9 has improved storage stability in high water liquid detergent compared to SEQ ID NO:8 (see Table 20).
  • a commercially available alpha-amylase (Amplify Prime 100L, Novozymes A/S) was incubated together with either SEQ ID NO:8 orSEQ ID NO:9 for2 -4 weeks at 37 °C, and the samples were subsequently analyzed for protease and alpha-amylase activity. Residual protease activity was determined relative to protease activity present in the original non-incubated samples stored at -18 °C, expressed in %. The results show that SEQ ID NO:9 has improved storage stability in liquid detergent compared to SEQ ID NO:8 (see Table 22).
  • Residual alpha-amylase activity was determined relative to amylase activity present in the original non-incubated samples stored at -18 °C, expressed in %.
  • the results show that the alphaamylase has improved residual activity in presence of SEQ ID NO:9 compared to SEQ ID NO:8 (see Table 23).
  • SEQ ID NO:9 has improved mildness towards companion enzymes, in particular alpha-amylases, compared to SEQ ID NO:8.
  • SEQ ID NO:1 (see Table 25). SEQ ID NO:9 is relatively faster at soil removal compared to SEQ ID NO:8 as well as SEQ ID NO:1.
  • SEQ ID NO: 14 exhibits a better wash performance than SEQ ID NO:13 as well as SEQ ID NO:5.
  • Purified protease samples were diluted with 0.01% Triton X-100 to suitable concentrations based on absorbance at 280 nm (0.1 -0.4 mg/ml for variants of SEQ ID NO:1 and SEQ ID NO:3, 1 .3-5 mg/ml for variants of SEQ ID NO:5).
  • 30 l of the protease dilutions were mixed with 270 pl concentrated Model O detergent (pH 8 or pH 10) in the wells of a microtiter plate using a magnetic bar. After mixing and sealing with a plate seal, the detergent plate was incubated at 45°C or 55°C in a Biosan PST-100HL thermomixer. Every sample was tested in two or three concentrations.
  • T% were found from linear regression of logarithm of activity versus incubation time.
  • Half-life improvement factors (T1 IF) were calculated as the ratio of the half-life of a protease variant relative to the half-life of the parent protease.
  • SEQ ID NO:9 has improved storage stability compared to SEQ ID NO:8 as well as SEQ ID NO:1.
  • SEQ ID NO: 12 has improved storage stability compared to SEQ ID NO:11 as well as SEQ ID NO:3.
  • SEQ ID NQ:10 has improved storage stability compared to SEQ ID NO:3.
  • SEQ ID NO: 14 has improved storage stability compared to SEQ ID NO:13 as well as SEQ ID NO:5.
  • a variant of a parent protease wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variant has protease activity.
  • parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:1.
  • the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2.
  • the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:3.
  • the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:5.
  • parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:7.
  • variants corresponding to G95D and A209K of SEQ ID NO:1 and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
  • variant according to any of the preceding paragraphs which further comprises one or more substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.
  • a granule which comprises (a) a core comprising the variant or fusion polypeptide of any of paragraphs 1-35; and, optionally, (b) a coating consisting of one or more layer(s) surrounding the core.
  • a granule which comprises: (a) a core; and (b)a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the variant or fusion polypeptide of any one of paragraphs 1-35.
  • a liquid composition comprising the variant or fusion polypeptide of any of paragraphs 1- 35 and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • an enzyme stabilizer e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
  • a composition comprising the variant or fusion polypeptide of any of paragraphs 1-35, the granule of paragraphs 36 or 37, or the liquid compositions of any of paragraphs 38-40.
  • a nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 42-44.
  • the recombinant host cell of paragraph 46 which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of any one of paragraphs 42-44.
  • the recombinant host cell of paragraph 46 or 47 which is a yeast recombinant host cell, e.g., 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.
  • yeast recombinant host cell e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomy
  • the recombinant host cell of paragraph 46 or 47 which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Mag- naporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phan- erochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus
  • the recombinant host cell of paragraph 46 or 47 which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E.
  • a prokaryotic recombinant host cell e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E.
  • coli Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amylo- liquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fir- mus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp.
  • Bacillus alkalophilus Bacillus amylo- liquefaciens
  • Bacillus brevis
  • a method of producing a protease variant or fusion polypeptide comprising (a) cultivating the host cell of any one of paragraphs 46-52 under conditions suitable for expression of the variant; and (b) recovering the variant.
  • a whole broth formulation or cell culture composition comprising the variant or fusion polypeptide of any of paragraphs 1-35.
  • a detergent composition comprising the variant of fusion polypeptide of any of paragraphs 1-35.
  • the detergent composition of paragraph 55 in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.
  • the detergent composition of paragraph 55 in the form of a liquid detergent, a powder detergent, or a laundry soap bar; preferably wherein the detergent composition is a liquid detergent or a laundry soap bar.
  • a method of cleaning an object comprising contacting the object with a detergent composition according to any of paragraphs 55-57 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, dishware, or a hard surface; most preferably wherein the object is a fabric.

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Abstract

The present invention relates to protease variants. The present invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, detergent compositions comprising the variants, and use of the variants in a cleaning process.

Description

PROTEASE VARIANTS AND POLYNUCLEOTIDES ENCODING SAME
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to protease variants. The present invention also relates to polynucleotides encoding the variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, detergent compositions comprising the variants, and use of the variants in a cleaning process.
BACKGROUND OF THE INVENTION
In the detergent industry, enzymes have been implemented in detergent compositions for many decades. Enzymes used in such compositions include proteases, lipases, amylases, cellulases, mannanases as well as other enzymes or mixtures thereof. Commercially, the most important enzymes are proteases.
An increasing number of commercially available proteases used for, e.g., laundry and dishwashing detergents are engineered variants of naturally occurring wild type proteases. Protease variants have been described in the art with alterations relative to a parent protease resulting in improvements such as better wash performance, thermal stability, storage stability, and catalytic activity.
However, various factors make further improvement of proteases advantageous. For example, washing conditions such as temperature and pH tend to change over time and are also different in different countries or regions of the world. In addition, many stains are still difficult to completely remove under conventional washing conditions. Thus, new protease variants having improved wash performance under various conditions remain commercially relevant. In addition, new protease variants with improved storage stability are warranted to maintain sufficient proteolytic activity and thus wash performance of the protease variant after a period of storage in a detergent composition, either during manufacturing and distribution of the detergent composition and/or following storage of the detergent composition by the end consumer before use. Furthermore, new protease variants displaying improved mildness towards companion enzymes (/.e., other enzymes present in a detergent composition) are warranted to avoid unnecessary proteolytic degradation and preserve enzymatic activity of such enzymes. Lastly, new protease variants with improved stain removal rate are desirable as they are faster acting and thus particularly suitable for cleaning methods, such as, e.g., laundering and dishwashing methods, involving shorter wash cycles.
SUMMARY OF THE INVENTION
The present invention relates to protease variants with improved properties. The variants of the invention exhibit improved wash performance as well as improved storage stability under several different temperature and pH conditions. The variants of the invention also provide improved mildness towards companion enzymes, which expands the compatibility of the variants of the invention with other enzymes and reduces the need for co-formulation with protease inhibitors. The variants of the invention also display improved stain removal rate, making them particularly suitable for cleaning methods with short wash cycles.
The variants of the invention are particularly suitable for high pH liquid detergents having pH 10 or above and for laundry soap bars. In high pH liquid detergents, the variants of the invention display improved wash performance and improved storage stability. When included in laundry soap bars, the variants of the invention provide improved proteinaceous stain removal and reduce the need for stabilizing agents, which further decreases production costs associated with manufacturing of laundry soap bars.
In a first aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants has a TM-score of at least 0.80 but less than 1.0 compared to the three-dimensional structure of the parent protease, wherein the three- dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.
In a second aspect, the present invention relates to a variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60% but less than 100% sequence identity to the parent protease; and wherein the variant has protease activity.
In a third aspect, the present invention relates to a polynucleotide encoding a variant of the first aspect or the second aspect. In a fourth aspect, the present invention relates to a nucleic acid construct or expression vector comprising a polynucleotide of the third aspect.
In a fifth aspect, the present invention relates to a recombinant host cell comprising in its genome a nucleic acid construct or expression vector according to the fourth aspect.
In a sixth aspect, the present invention relates to a method for obtaining a variant according to the first aspect or the second aspect, comprising (a) introducing into a parent protease substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further introducing substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has protease activity; and (b) recovering the variant.
In a seventh aspect, the present invention relates to a method of producing a variant of the first aspect or the second aspect, comprising (a) cultivating a recombinant host cell of the fifth aspect under conditions suitable for expression of the variant; and (b) recovering the variant.
In an eighth aspect, the present invention relates to a detergent composition comprising a variant of the first aspect or the second aspect.
In a ninth aspect, the present invention relates to a method of cleaning an object, comprising contacting the object with a detergent composition of the eighth aspect under conditions suitable for cleaning the object.
In a tenth aspect, the present invention relates to use of a variant of the first aspect or the second aspect or a detergent composition of the eighth aspect in a cleaning process.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:2.
Figure 2 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:3.
Figure 3 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:4.
Figure 4 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:5.
Figure 5 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:6.
Figure 6 is an alignment of the polypeptides of SEQ ID NO:1 and SEQ ID NO:7.
Figure 7 is an overview of corresponding substitutions in SEQ ID NOs:1-7.
SEQUENCE OVERVIEW
SEQ ID NO:1 is Subtilisin Savinase (Savinase®) from Bacillus lentus.
SEQ ID NO:2 is Subtilisin BPN’ from Bacillus amyloliquefaciens. SEQ ID N0:3 is Subtilisin Carlsberg (Alcalase®) from Bacillus licheniformis.
SEQ ID NO:4 is a protease from Bacillus gibsonii.
SEQ ID NO:5 is a protease from Bacillus gibsonii.
SEQ ID NO:6 is a protease from Bacillus sp. TY-145.
SEQ ID NO:7 is a protease from Actinomadura keratinilytica.
SEQ ID NO:8 is a stabilized variant of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E.
SEQ ID NO:9 is SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.
SEQ ID NQ:10 is SEQ ID NO:3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P.
SEQ ID NO:11 is SEQ ID NO:3 with the substitutions P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E.
SEQ ID NO:12 is SEQ ID NO:3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
SEQ ID NO:13 is SEQ ID NO:5 with the substitutions T9E, N42R, N74D, V199I, Q200L, Y203W, N253D, S255W, and Q256E.
SEQ ID NO:14 is SEQ ID NO:5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, and S255W.
DEFINITIONS
In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Protease: The term “protease” means an enzyme having peptidase activity (EC 3.4; also known as proteolytic activity or protease activity) that catalyzes the hydrolysis of peptide bonds. The EC 3.4 group includes several sub-groups, including EC 3.4.21 (serine endopeptidase), which further contains several sub-groups, including EC. 3.4.21.62 (subtilisin). For purpose of the present invention, protease activity may be determined according to Protease Activity Assay I or Protease Activity Assay II described in the Examples herein.
AlphaFold structure prediction: AlphaFold is a computational method for predicting the three-dimensional structure of a polypeptide from its amino acid sequence (Jumper et al., Highly accurate protein structure prediction with AlphaFold. Nature, 2021). Predicted structures for millions of polypeptides deposited in the UniProt database have been deposited in the AlphaFold Protein Structure Database, using the AlphaFold Monomer v2.0 model (Varadi et al. AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, 2021). In the AlphaFold Protein Structure Database, the three-dimensional structure of a polypeptide can be obtained by searching for the UniProt accession number of the polypeptide.
In addition to the many three-dimensional structures that are already publicly available, code is available for reproducing and predicting structures of new polypeptides at source code repositories such as Github.com under deepmind/alphafold/, using notebooks/AlphaFold.ipynb, which uses AlphaFold v2.3.1 or newer. Additionally, it can be found in Github.com under sokrypton/ColabFold using v1.5.2 or newer, using AlphaFold2.ipynb. For technical details, please see Jumper et al. (vide supra).
AlphaFold produces a per-residue estimate of its confidence on a scale from 0 to 100. This confidence measure is called pLDDT and corresponds to the model’s predicted score on the IDDT-Ca metric. It is stored in the B-factor fields of the mmCIF and PDB files available for download (although unlike a B-factor, higher pLDDT is better). Regions with pLDDT score of more than 90 are expected to be modelled to high accuracy. These should be suitable for any application that benefits from high accuracy (e.g., characterization of binding sites). Regions with a pLDDT score between 70 and 90 are expected to be modelled well, corresponding to a generally good backbone prediction. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native (/.e., from the same gene) or heterologous (/.e., from a different gene) to the polynucleotide encoding the variant, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a variant.
Expression: The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
Extension: The term “extension” means an addition of one or more amino acids to the amino and/or carboxyl terminus of a variant, wherein the “extended” variant has protease activity.
Fragment: The term “fragment” means a variant having one or more amino acids absent from the amino and/or carboxyl terminus of the variant; wherein the fragment has protease activity.
Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one polypeptide is fused at the N-terminus and/or the C-terminus of a variant of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and include 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 al., 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. Examples of 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 al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982- 987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.
Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.
Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a variant has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides. The term "host cell" includes protoplasts created from cells.
Improved property: The term “improved property” means a characteristic associated with a variant that is improved compared to the parent. Such improved properties include, but are not limited to, catalytic efficiency, catalytic rate, chemical stability, mildness, oxidation stability, pH activity, pH stability, specific activity, stability under storage conditions, substrate binding, substrate cleavage, substrate specificity, substrate stability, surface properties, thermal activity, thermostability, and wash performance.
Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection", "transformation" or "transduction," as known in the art.
Isolated: The term “isolated” means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell.
Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing and/or C-terminal processing (e.g., removal of signal peptide).
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having protease activity.
Mutant: The term “mutant” means a polynucleotide encoding a variant.
Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.
Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a variant. Nucleic acids may be single stranded or double stranded and may be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence.
Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.
Parent or parent protease: The term “parent” or “parent protease” means a protease to which an alteration is made to produce the protease variants of the present invention.
Purified: The term “purified” means a nucleic acid, variant or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.
In one aspect, the term "purified" as used herein refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (/.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.
Accordingly, the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The variant may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1 %, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.
It is, therefore, preferred that the substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The variant of the present invention is preferably in a substantially pure form i.e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.
Recombinant: The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.
Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheet or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hydro cyclones or similar), or by precipitating the polypeptide and using relevant solidliquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and/or purification of the polypeptide.
Sequence Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.
For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment- Total Number of Gaps in Alignment)
Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the secretion process.
Structural Similarity: The relatedness between two amino acid sequences has conventionally been described by the parameter “sequence identity”. However, since the biological function of a polypeptide is defined by its three-dimensional structure rather than its amino acid sequence, a better way of assessing a functional relationship between polypeptides is by comparing their three-dimensional structures. Thus, for the purposes of the present invention, the relatedness between the three-dimensional structure of two polypeptides is described by the parameter “structural similarity”.
A three-dimensional structure of any polypeptide may be obtained experimentally via, e.g.,
X-ray crystallography or using in silico methods such as AlphaFold (vide supra). The structural similarity between three-dimensional structures may then be determined by the TM-score, which is calculated using the following general formula (Zhang & Skolnick, Proteins 57:702-710, 2004):
TM-score where LN is the length of the native structure, LT is the length of the aligned residues to the template structure, d, is the distance between the /th pair of aligned residues and do is a scale to normalize the match difference. ‘Max’ denotes the maximum value after optimal spatial superposition.
For the purposes of the present invention, LN is always the length of the reference protein, indicating the use of a fixed reference length L to prevent artificially large TM-scores from alignment of substructures:
TM-score
A structural alignment of the three-dimensional structures of two polypeptides is necessary before the TM-score can be calculated. This is achieved via algorithms that optimize the structural overlap, and several methods are available, such as CEalign (Shindyalov and Bourne, Protein Eng., 11 , 739-747, 1998), DALI (Holm and Sander, Trends Biochem. Sci., 20, 478-480, 1995), or TM-align (Nucleic Acids Res. 33:2302-2309, 2005).
For the purposes of the present invention, TM-align is applied. For convenience, TM-score is integrated in the TM-align software, which is available from the author’s website. The version of TM-align is preferably updated 2019-08-22 or later, and the TM-score between a reference and a query protein is determined by running this command:
TMalign <query . pdb> <ref erence . pdb> -L <length of reference>
Where <query.pdb> is the name of the PDB file containing coordinates of the query polypeptide, <reference.pdb> is the name of the PDB file containing coordinates of the reference polypeptide. The TM-score is calculated and reported in the output, along with several other parameters from the alignment.
The maximal TM-score is 1 , e.g., 1.0, corresponding to identical three-dimensional structures.
Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having protease activity.
Variant: The term “variant” means a polypeptide having protease activity comprising a substitution, an insertion (including extension), and/or a deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.
Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence).
Conventions for Designation of Variants
For purposes of the present invention, the polypeptide disclosed in SEQ ID NO:1 is used to determine the corresponding amino acid positions in another protease. The amino acid sequence of another protease is aligned with the polypeptide disclosed in SEQ ID NO:1 , and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO:1 is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
Alignments of SEQ ID NO:1 and SEQ ID NOs: 2, 3, 4, 5, 6, and 7 are provided as Figures 1-6. An overview of corresponding substitutions in SEQ ID NOs: 1-7 is provided in Figure 7.
In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IIIPAC single letter or three letter amino acid abbreviation is employed.
Substitutions: For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of Thr at position 226 with Ala is designated as “T226A”. Multiple mutations are separated by addition marks (“+”) or by commas, e.g., “G205R+S411 F” or “G205R,S411 F”, representing substitutions at positions 205 and 411 of Gly (G) with Arg (R) and Ser (S) with Phe (F), respectively. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted may be indicated with X, e.g., “X226A”.
Deletions: For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of the amino acid Gly at position 195 is designated as “Gly195*”. Multiple deletions are separated by addition marks (“+”) or by commas, e.g., “G195*+S411*” or “G195*,S411*”. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be deleted may be indicated with X, e.g., “X195*”.
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 Lys after the amino acid Gly at position 195 is designated “G195GK”. Because the amino acid residue at a given position varies from parent to parent, the insertion of lysine after the amino acid at position 195 may be indicated with “X195*”.
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 Lys and Ala after the amino acid Gly at position 195 is indicated as “G195GKA”. In such cases, the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:
Alternatively, an insertion of an amino acid residue such as lysine after the amino acid at position 195 may be indicated by “195aK”, and the insertion of two or more additional amino acid residues such as Lys and Ala after the amino acid at position 195 may be indicated by “195aK,195bA”.
Multiple alterations: Variants comprising multiple alterations are separated by addition marks (“+”), e.g., “R170Y+G195E” representing a substitution of Arg and Gly at positions 170 and 195 with Tyr and Glu, respectively.
Different alterations: Where different alterations can be introduced at a position, the different alterations are separated by a comma, e.g., “R170Y.E” represents a substitution of Arg at position 170 with Tyr or Glu. Thus, “Y167G,A+R170G,A” designates the following variants: “Y167G+R170G”, Y167G+R170A”, “Y167A+R170G”, and “Y167A+R170A”.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to protease variants with improved properties. The variants of the invention exhibit improved wash performance as well as improved storage stability under several different temperature and pH conditions. The variants of the invention also provide improved mildness towards companion enzymes, which expands the compatibility of the variants of the invention with other enzymes and reduces the need for co-formulation with protease inhibitors. The variants of the invention also display improved stain removal rate, making them particularly suitable for cleaning methods with short wash cycles.
The variants of the invention are particularly suitable for high pH liquid detergents having pH 10 or above and for laundry soap bars. In high pH liquid detergents, the variants of the invention display improved wash performance and improved storage stability. When included in laundry soap bars, the variants of the invention provide improved proteinaceous stain removal and reduce the need for stabilizing agents, which further decreases production costs associated with manufacturing of laundry soap bars.
The present invention also relates to polynucleotides encoding variants of the invention, nucleic acid constructs and expression vectors comprising such polynucleotides, recombinant host cells expression variants of the invention, methods for obtaining variants of the invention, methods of producing variants of the invention, detergent compositions comprising variants of the invention, and uses of variants of the invention.
Variants
In a first aspect, the present invention relates to variants of a parent protease, wherein the variants comprise substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants have a TM-score of at least 0.80, e.g., at least
0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least
0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least
0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three- dimensional structure is calculated using AlphaFold; and wherein the variants have protease activity.
In an embodiment, the variant has a TM-score of at least 0.90, e.g., at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
In an embodiment, the variant has a TM-score of at least 0.95, e.g., at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three- dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold. In an embodiment, the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
In an embodiment, the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold.
In an embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.
In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1.
In another embodiment, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:1 , wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:2, wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:3, wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:4 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:4, wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:5 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:5, wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:6 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:6, wherein the three-dimensional structure is calculated using AlphaFold.
In another embodiment, the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Preferably, the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. Most preferably, the parent is SEQ ID NO:7 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of SEQ ID NO:7, wherein the three-dimensional structure is calculated using AlphaFold. For some parent proteases, a three-dimensional structure is publicly available. A three- dimensional structure of SEQ ID NO:1 (Savinase®) is available under UniProt accession number P29600 or, alternatively, PDB accession number 1SVN. A three-dimensional structure of SEQ ID NO:2 (BPN’) is available under UniProt accession number P00782. A three-dimensional structure of SEQ ID NO:3 (Alcalase®) is available under UniProt accession number P00780.
In one embodiment, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Preferably, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600. Most preferably, the parent is SEQ ID NO:1 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P29600.
In one embodiment, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Preferably, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782. Most preferably, the parent is SEQ ID NO:2 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00782.
In one embodiment, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Preferably, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.980, e.g., at least 0.985, at least 0.990, at least 0.995, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780. Most preferably, the parent is SEQ ID NO:3 and the variant has a TM-score of at least 0.990, e.g., at least 0.991 , at least 0.992, at least 0.993, at least 0.994, at least 0.995, at least 0.996, at least 0.997, at least 0.998, at least 0.999, but less than 1.0, compared to the three-dimensional structure of the polypeptide having UniProt accession number P00780.
In a second aspect, the present invention relates to variants of a parent protease, wherein the variants comprise substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprise substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variants have protease activity.
In an embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7. In a preferred embodiment, the parent protease is SEQ ID NO:1.
In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
In another embodiment, the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1 .
In another embodiment, the parent is SEQ ID NO:1 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:1.
In another embodiment, the parent is SEQ ID NO:2 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:2.
In another embodiment, the parent is SEQ ID NO:3 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3.
In another embodiment, the parent is SEQ ID NO:4 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:4.
In another embodiment, the parent is SEQ ID NO:5 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:5.
In another embodiment, the parent is SEQ ID NO:6 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:6.
In another embodiment, the parent is SEQ ID NO:7 and the variant has at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO:7.
In one aspect, the number of substitutions in the variants of the present invention is 5-30, e.g., 5-25, 5-20, 5-15 and 5-10, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30, substitutions. In a preferred embodiment, the number of substitutions in the variants of the present invention is 5-11 , such as 5, 6, 7, 8, 9, 10, or 11 substitutions.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
In another aspect, a variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises nine substitutions at positions corresponding to each of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1.
The variants of the invention comprise a substitution at a position corresponding to position 95 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 95 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp.
The variants of the invention comprise a substitution at a position corresponding to position 209 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 209 of SEQ ID NO:1 is substituted with Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.
The variants of the invention may comprise a substitution at a position corresponding to position 9 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 9 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Trp, Tyr, or Vai, preferably with Glu.
The variants of the invention may comprise a substitution at a position corresponding to position 42 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 42 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, His, lie, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Arg.
The variants of the invention may comprise a substitution at a position corresponding to position 74 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 74 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.
The variants of the invention may comprise a substitution at a position corresponding to position 199 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position of SEQ ID NO:199 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with lie.
The variants of the invention may comprise a substitution at a position corresponding to position 200 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 200 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Vai, preferably with Leu.
The variants of the invention may comprise a substitution at a position corresponding to position 203 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 203 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Trp.
The variants of the invention may comprise a substitution at a position corresponding to position 253 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position 253 of SEQ ID NO:1 is substituted with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asp.
The variants of the invention may comprise a substitution at a position corresponding to position 255 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position
255 of SEQ ID NO:1 is substituted with Ala, Arg, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Vai, preferably with Trp.
The variants of the invention may comprise a substitution at a position corresponding to position 256 of SEQ ID NO:1. In one aspect, the amino acid at a position corresponding to position
256 of SEQ ID NO:1 is substituted with Ala, Arg, Asn, Cys, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Glu.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least four substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least five substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ I D NO: 1 and further comprise at least six substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to substitutions G95D and A209K of SEQ ID NO:1 and further comprise at least seven substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprise at least eight substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the variants of the invention comprise substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprising nine substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
In one aspect, the present invention relates to variants of SEQ ID NO:1 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., S9E), X42R (e.g., N42R), X74D (e.g., N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., S253D), X255W (e.g., N255W), and X256E (e.g., L256E); wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:1 ; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:1 with the substitutions S9E, N42R, N74D, G95D, V199I, Q200L, Y203W, A209K, S253D, N255W, and L256E.
In one aspect, the present invention relates to variants of SEQ ID NO:2 comprising the substitutions X97D (e.g., G97D) and X215K (e.g., G215K) and further comprising at least three, e.g., at least four, at least five, at least six, or seven, substitutions selected from the group consisting of X9E (e.g., S9E), X43R (e.g., K43R), X76D (e.g., N76D), X206L (e.g., Q206L), X209W (e.g., L209W), X261W (e.g., F261W), and X262E (e.g., Y262E); wherein position numbering is based on the numbering of SEQ ID NO:2; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:2; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:2 with the substitutions S9E, K43R, N76D, G97D, Q206L, L209W, G215K, F261W, and Y262E.
In one aspect, the present invention relates to variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) and X214K (e.g., A214K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g.,Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, N96D, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
In one aspect, the present invention relates to variants of SEQ ID NO:4 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., A209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., T42R), X74D (e.g., N74D), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein position numbering is based on the numbering of SEQ ID NO:4; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:4; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:4 with the substitutions T9E, T42R, N74D, G95D, Q200L, Y203W, A209K, N253D, S255W, and Q256E.
In one aspect, the present invention relates to variants of SEQ ID NO:5 comprising the substitutions X95D (e.g., G95D) and X209K (e.g., V209K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of X9E (e.g., T9E), X42R (e.g., N42R), X74D (e.g. N74D), X199I (e.g., V199I), X200L (e.g., Q200L), X203W (e.g., Y203W), X253D (e.g., N253D), X255W (e.g., S255W), and X256E (e.g., Q256E); wherein position numbering is based on the numbering of SEQ ID NO:5; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:5; and wherein the variant have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:5 with the substitutions T9E, N42R, N74D, G95D, V199I, Q200L, Y203W, V209K, N253D, S255W, and Q256E.
In one aspect, the present invention relates to variants of SEQ ID NO:6 comprising the substitutions X107D (e.g., G107D) and X245K (e.g., N245K) and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X12E (e.g., K12E), X44R (e.g., D44R), X86D (e.g., S86D), X235I (e.g., V235I), X236L (e.g., E236L), X297D (e.g., T297D), X299W (e.g., D299W), and X300E (e.g., D300E); wherein position numbering is based on the numbering of SEQ ID NO:6; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:6; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:6 with the substitutions K12E, D44R, S86D, G107D, V235I, E236L, N245K, T297D, D299W, and D300E.
In one aspect, the present invention relates to variants of SEQ ID NO:7 comprising the substitutions X99D (e.g., N99D) and X215K (e.g., N215K) and further comprising at least three, e.g., at least four, at least five, or six, substitutions selected from the group consisting of X12E (e.g., D12E), X51 R (e.g., G51 R), X206L (e.g., T206L), X266D (e.g., T266D), X268W (e.g., N268W), and X269E (e.g., L269E); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:7; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:7 with the substitutions D12E, G51 R, N99D, T206L, N215K, T266D, N268W, and L269E.
In one aspect, the present invention relates to variants of SEQ ID NO:7 comprising the substitutions X99D (e.g. , N99D) and X215K (e.g. , N215K) and further comprising the substitutions X12E (e.g., D12E), X51 R (e.g., G51 R), and X206L (e.g., T206L); wherein position numbering is based on the numbering of SEQ ID NO:7; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:7; and wherein the variants have protease activity. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:7 with the substitutions D12E, G51 R, N99D, T206L, and N215K.
In an alternative aspect, the present invention relates to variants of SEQ ID NO:3 comprising the substitutions X96D (e.g., N96D) or X214K (e.g., A214K), preferably X96D (e.g., N96D) and X214K (e.g., A214K), and further comprising at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting of X68S (e.g., A68S), X77N (e.g., T77N), X78I (e.g., T78I), X127S (e.g., G127S), X128P (e.g., A128P), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X217S (e.g., N217S), and X258P (e.g., S258P); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the variants have protease activity. In one embodiment, the variant comprises the substitutions N96D and A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or ten, substitutions selected from the group consisting ofA68S, T77N, T78I, G127S, A128P, G165Q, N184Q, A202V, N217S, and S258P. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, A214K, N217S, and S258P. In a more preferred embodiment, the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E). In an even more preferred embodiment, the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258P, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, N43R, A68S, T77N, T78I, N96D, G127S, A128P, G165Q, N184Q, A202V, V204I, Y205I, Y208W, A214K, N217S, S258D, F260W, and Y261 E.
In an alternative aspect, the present invention relates to variants of SEQ ID NO:3 comprising the substitution X214K (e.g., A214K) and further comprising at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of X9T, (e.g., P9T), X17H, (e.g., Q17H), X77N (e.g., T77N), X78I (e.g., T78I), X96D (e.g., N96D), X103F (e.g., Y103F), X127T (e.g., G127T), X128K (e.g., A128K), X129Q, (e.g., S129Q), X165Q (e.g., G165Q), X184Q (e.g., N184Q), X202V (e.g., A202V), X203E (e.g., G203E), and X258P (e.g., S258P); wherein position numbering is based on the numbering of SEQ ID NO:3; wherein the variants have a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100%, to SEQ ID NO:3; and wherein the variants have protease activity. In one embodiment, the variant comprises the substitution A214K and further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least elleven, at least twelve, at least thirteen, or fourteen, substitutions selected from the group consisting of P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, and S258P. In a preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, A214K, and S258P. In a more preferred embodiment, the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of X9E (e.g., P9E), X43R (e.g., N43R), X204I (e.g., V204I), X205L (e.g., Y205L), X208W (e.g., Y208W), X258D (e.g., S258D), X260W (e.g., F260W), and X261 E (e.g., Y261 E). In an even more preferred embodiment, the variant further comprises at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or eight, substitutions selected from the group consisting of P9E, N43R, V204I, Y205L, Y208W, S258D, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9T, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258P, F260W, and Y261 E. In a most preferred embodiment, the variant comprises or consists of SEQ ID NO:3 with the substitutions P9E, Q17H, N43R, T77N, T78I, N96D, Y103F, G127T, A128K, S129Q, G165Q, N184Q, A202V, G203E, V204I, Y205L, Y208W, A214K, S258D, F260W, and Y261 E.
The variants of the invention may comprise further substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97E), 99 (e.g., S99E), 116 (e.g., G116N) , and 246 (e.g., N246L) of SEQ ID NO:1. Preferably, the variants comprise one or more further substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L) of SEQ ID NO:1.
The variants of the invention may further comprise an extension of one or more amino acids at the N-terminal and/or C-terminal ends.
Alternatively, the variants of the invention may further comprise a truncation of one or more amino acids at the N-terminal and/or C-terminal ends.
The amino acid changes introduced into parent proteases to provide variants according to the present invention 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 aminoterminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain. Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, LeuA/al, Ala/Glu, and Asp/Gly.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for protease 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 a!., 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, and/or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides/proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity. Additionally, or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and/or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.
In one aspect, the variants of the invention have improved stability under storage conditions (/.e., improved storage stability) compared to a reference protease. In one embodiment, storage stability is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more. In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:7.
In some embodiments, the variant has improved storage stability compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1. In one embodiment, storage stability is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved storage stability compared to SEQ ID NO: 13.
In one embodiment, the variant has improved storage stability in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
In one embodiment, the variant has improved storage stability in a liquid detergent having pH 7-11 , preferably pH 7-10, most preferably pH 8-10.
In one embodiment, the variant has improved storage stability at a temperature of IQ- 40 °C, preferably 10-30 °C, most preferably 15-25 °C.
In one embodiment, the variant has improved improved storage stability at a temperature of 10-60 °C, more preferably 30-55 °C, most preferably 45-55 °C.
In a preferred embodiment, the variant has improved storage stability in liquid detergent as determined according to Example 7 herein.
In a preferred embodiment, the variant has improved storage stability in liquid detergent as determined according to Example 11 herein, preferably in Model O detergent having pH 8-10 or preferably in Model B detergent having pH 7-8. In one aspect, the variants of the invention have improved wash performance compared to a reference parent protease. In one embodiment, wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:7.
In some embodiments, the variant has improved wash performance compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1. In one embodiment, wash performance is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved wash performance compared to SEQ ID NO: 13.
In one embodiment, the variant has improved wash performance in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
In one embodiment, the variant has improved wash performance in a liquid detergent having pH 7-11 , preferably pH 7-10, most preferably pH 8-10.
In one embodiment, the variant has improved wash performance at a washing temperature of 10-60 °C, preferably 10-40 °C, more preferably 10-30 °C, most preferably 15- 25 °C. In one embodiment, the variant has improved wash performance as determined in Example 1 , Example 2, Example 3, Example 4, Example 5, Example 6, or Example 10 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 1 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 2 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 3 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 4 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 5 herein. In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 6 herein.
In a preferred embodiment, the variant has improved wash performance in liquid detergent as determined according to Example 10 herein, preferably in Model O detergent having pH 8-10 or preferably in Model B detergent having pH 7-8.
In one aspect, the variants of the invention have improved mildness compared to a reference protease. In the context of the present invention, improved mildness means that a protease variant is less aggressive towards other enzymes in the detergent matrix (also referred to as companion enzymes or secondary enzymes), thereby providing improved residual activity of a companion enzyme after storage with a protease variant of the invention in a detergent composition. In one embodiment, mildness is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:6. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:7.
In some embodiments, the variant has improved mildness compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1. In one embodiment, mildness is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved mildness compared to SEQ ID NO: 13.
In some embodiments, the variant provides improved residual activity of a companion enzyme. In one prefered embodiment, the residual activity of the companion enzyme is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more. In one embodiment, the companion enzyme is selected from the group consisting of amylase (e.g., alpha-amylase), arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, DNase, galactanase, haloperoxygenase, lipase, mannanase, oxidase (e.g., laccase or peroxidase), pectinase, pectin lyase, protease, xylanase, xanthanase or xyloglucanase. In a preferred embodiment, the companion enzyme is an alpha-amylase. In a preferred embodiment, the residual activity is determined according to Example 8 herein.
In one embodiment, the variant provides improved residual activity of a companion enzyme, preferably an alpha-amylase, after storage in a liquid detergent having pH 8-14, preferably pH 9-13, most preferably pH 10-12.
In one embodiment, the variant provides improved residual activity of a companion enzyme, preferably an alpha-amylase, after storage with a protease variant of the invention when stored in a liquid detergent at a temperature of 10-60 °C, preferably 15-50 °C, most preferably 20-40 °C.
In a preferred embodiment, the variant has improved mildness in liquid detergent as determined according to Example 8 herein.
In a preferred embodiment, the variant provides improved residual activity of an alphaamylase in liquid detergent as determined according to Example 8 herein.
In a preferred embodiment, the variant has improved mildness in Model O liquid detergent.
In a preferred embodiment, the variant provides improved residual activity of an alphaamylase in Model O liquid detergent. In one aspect, the variants of the invention have improved strain removal rate compared to a reference protease. In the context of the present invention, improved stain removal rate means that the variant is relatively faster at removing proteinaceous soil compared to the parent protease within a given timeframe. In one embodiment, stain removal rate is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%, or more.
In some embodiments, the reference protease is a parent protease. In one embodiment, the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In one embodiment, the parent is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:1. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:2. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:3. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:4. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:5. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:6. In a preferred embodiment, the variant has stain removal rate mildness compared to SEQ ID NO:7.
In some embodiments, the variant has improved stain removal rate compared to an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 . In one embodiment, stain removal rate is improved by at least 5%, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 250%, at least 300%, at least 400%, at least 500%. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:8. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO:11. In a preferred embodiment, the variant has improved stain removal rate compared to SEQ ID NO: 13.
In one embodiment, the variant has improved improved stain removal rate at a washing temperature of 10-60 °C, more preferably 15-50 °C, most preferably 20-40 °C.
In a preferred embodiment, the variant has improved stain removal rate in liquid detergent as determined according to Example 9 herein. The variant of the invention may be a fusion polypeptide comprising a variant of the invention.
In an aspect, the variant of the invention is isolated.
In another aspect, the variant of the invention is purified.
Parent Proteases
The parent protease may be a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.
In an aspect, the parent protease is a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
In an aspect, the parent protease is a polypeptide having at least 60%%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the polypeptide of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:1. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:1.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:2 of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:2. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:2.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:3 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:3. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:3.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:4 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:4. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:4.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:5 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:5.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:5 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:5. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:5.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:6 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:6. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:6.
In an aspect, the parent has a sequence identity to the polypeptide of SEQ ID NO:7 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 protease activity. In one aspect, the amino acid sequence of the parent differs by up to 20 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20, from the polypeptide of SEQ ID NO:7. In another aspect, the parent comprises or consists of the polypeptide of SEQ ID NO:7.
The parent may be a fusion polypeptide or cleavable fusion polypeptide. 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 al., 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. Examples of 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 al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.
The parent may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the parent encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the parent is secreted extracellularly.
In one aspect, the parent is a Bacillus lentus protease, e.g., the protease of SEQ ID NO:1 . In one aspect, the parent is a Bacillus amyloliquefaciens protease, e.g., the protease of SEQ ID NO:2. In one aspect, the parent is a Bacillus licheniformis protease, e.g., the protease of SEQ ID NO:3. In one aspect, the parent is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:4. In one aspect, the parent is a Bacillus gibsonii protease, e.g., the protease of SEQ ID NO:5. In one aspect, the parent is a Bacillus sp. TY145 protease, e.g., the protease of SEQ ID NO:6. In one aspect, the parent is a Actinomadura keratinilytica protease, e.g., the protease of SEQ ID NO:7.
Preparation of Variants
The present invention also relates to methods for obtaining a variant having protease activity, comprising: (a) introducing into a parent protease substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further introducing substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 , wherein the variant has protease activity; and (b) recovering the variant.
The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.
Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.
Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually, the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 7Q: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.
Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004/0171154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.
Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.
Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing several techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127). Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.
Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and/or site-directed mutagenesis, and/or random mutagenesis, and/or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.
Polynucleotides
The present invention also relates to polynucleotides encoding a variant of the present invention.
The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.
In an aspect, the polynucleotide is isolated.
In another aspect, the polynucleotide is purified.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
Promoters
The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a bacterial host cell are described in Sambrook et al. , 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., NY, Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier, and Song et al., 2016, PLOS One 11(7): e0158447.
Examples of suitable promoters for directing transcription of the polynucleotide of the present invention in a filamentous fungal host cell are promoters obtained from Aspergillus, Fusarium, Rhizomucor and Trichoderma cells, such as the promoters described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
For expression in a yeast host, examples of useful promoters are described by Smolke et al., 2018, “Synthetic Biology: Parts, Devices and Applications” (Chapter 6: Constitutive and Regulated Promoters in Yeast: How to Design and Make Use of Promoters in S. cerevisiae), and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
Terminators
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells may be 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 may be obtained from Aspergillus or Trichoderma species, such as obtained from the genes for Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, “Trichoderma-. Biology and Applications”, and by Schmoll and Dattenbdck, 2016, “Gene Expression Systems in Fungi: Advancements and Applications”, Fungal Biology.
Preferred terminators for yeast host cells may be 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, Yeast 8: 423-488. mRNA Stabilizers
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue etal., 1995, J. Bacterid. 177: 3465-3471).
Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce// 5(11): 1838-1846.
Leader Sequences
The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.
Suitable leaders for bacterial host cells are described by Hambraeus et al., 2000, Microbiology 146(12): 3051-3059, and by Kaberdin and Blasi, 2006, FEMS Microbiol. Rev. 30(6): 967-979.
Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells may be 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).
Polyadenylation Sequences
The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.
Signal Peptides
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptide described by Xu etal., 2018, Biotechnology Letters 40: 949-955
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.
Propeptides
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
Regulatory Sequences
It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, 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.
Transcription Factors
The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and/or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-binding domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, /.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, /.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017/144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7- 23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, /.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. Host Cells
The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.
A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.
The host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.
The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.
The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Grampositive 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. In an embodiment, the Bacillus cell is a Bacillus amyloliquefaciens, Bacillus licheniformis and Bacillus subtilis cell.
For purposes of this invention, Bacillus classes/genera/species shall be defined as described in Patel and Gupta, 2020, Int. J. Syst. Evol. Microbiol. 70: 406-438.
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.
Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.
The host cell may be a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi (as defined by Hawksworth et al., In, Ainsworth and Bisby’s Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).
Fungal cells may be transformed by a process involving protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation as reviewed by Li et al., 2017, Microbial Cell Factories 16: 168 and procedures described in EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81 : 1470-1474, Christensen et al., 1988, Bio/TechnologyQ: 1419-1422, and Lubertozzi and Keasling, 2009, Biotechn. Advances 27: 53-75. However, any method known in the art for introducing DNA into a fungal host cell can be used, and the DNA can be introduced as linearized or as circular polynucleotide.
The fungal host cell may be a 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. In a preferred embodiment, the yeast host cell is a Pichia or Komagataella cell, e.g., a Pichia pastoris cell (Komagataella phaffii).
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, Fili basidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell. In a preferred embodiment, the filamentous fungal host cell is an Aspergillus, Trichoderma or Fusarium cell. In a further preferred embodiment, the filamentous fungal host cell is an Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum cell.
For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
In an aspect, the host cell is isolated.
In another aspect, the host cell is purified.
Methods of Production
The present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.
The host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and/or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.
The variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant.
The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, a cell- free fermentation broth comprising the polypeptide is recovered.
The variant may be purified by a variety of procedures known in the art to obtain substantially pure variants and/or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).
In an alternative aspect, the variant is not recovered.
Protease Granules
The present invention also relates to enzyme granules/particles comprising a variant of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm. The core diameter, measured as equivalent spherical diameter, can be determined using laser diffraction, such as using a Malvern Mastersizer and/or the method described under ISO13320 (2020).
In an embodiment, the core comprises a variant of the present invention.
The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
The core may include an inert particle with the variant absorbed into it, or applied onto the surface, e.g., by fluid bed coating. The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.
The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.
The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.
A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.
The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
The salt in the coating may have a constant humidity at 20°C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00/01793 or WO 2006/034710.
Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO3 (CH2o°c=92%), NaNO3 (CH2O C=73%), Na2HPO4 (CH2o°c=95%), Na3PO4 (CH25°c=92%), NH4CI (CH2o°c = 79.5%), (NH4)2HPO4 (CH2O C = 93,0%), NH4H2PO4 (CH2Q C = 93.1%), (NH4)2SO4 (CH2o°c=81 .1%), KOI (CH2O C=85%), K2HPO4 (CH2O C=92%), KH2PO4 (CH2O°C=96.5%), KNO3 (CH2O°C=93.5%), Na2SO4 (CH2O C=93%), K2SO4 (CH2O C=98%), KHSO4 (CH2O C=86%), MgSO4 (CH2o°c=9O%), ZnSO4 (CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2 and magnesium acetate.
The salt may be in anhydrous form, or it may be a hydrated salt, /.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO47H2O), zinc sulfate heptahydrate (ZnSO47H2O), sodium phosphate dibasic heptahydrate (Na2HPO47H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.
The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,
(a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker).
(b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606.
(c) Absorbed core particles, wherein rather than coating the variant as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116.
(d) Extrusion or pelletized products, wherein a variant-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140- 142; Marcel Dekker).
(e) Prilled products, wherein a variant-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents’, Surfactant Science Series; 1998; Vol. 71 ; pages 140-142; Marcel Dekker). The product obtained is one wherein the variant is uniformly distributed throughout an inert material instead of being concentrated on its surface. US 4,016,040 and US 4,713,245 describe this technique.
(f) Mixer granulation products, wherein a variant-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in US 4,106,991 , EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular aspect of this process, various high-shear mixers can be used as granulators. Granulates consisting of variant, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so- called T-granulate. Reinforced particles are more robust and release less enzymatic dust.
(g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.
(h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule.
(i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the variant contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, the excessive water will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w/w water.
Non-dusting granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661 ,452 and may optionally be coated by methods known in the art.
The granulate may further comprise one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.
The present invention also relates to protected enzymes prepared according to the method disclosed in EP 238216.
In an embodiment, the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta- mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.
Liquid Formulations
The present invention also relates to liquid compositions comprising a variant of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
In some embodiments, filler(s) or carrier material(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.
In an aspect, the liquid formulation comprises 20-80% w/w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative.
In another embodiment, the invention relates to liquid formulations comprising:
(A) 0.001-25% w/w of a variant of the present invention;
(B) 20-80% w/w of polyol;
(C) optionally 0.001-2% w/w preservative; and
(D) water.
In another embodiment, the invention relates to liquid formulations comprising:
(A) 0.001-25% w/w of a variant of the present invention;
(B) 0.001-2% w/w preservative;
(C) optionally 20-80% w/w of polyol; and
(D) water.
In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulphate, potassium sulphate, magnesium sulphate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulphate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.
In another embodiment, the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20-80% polyol (/.e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).
In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w/w preservative, e.g., 0.05-1% w/w preservative or 0.1-0.5% w/w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w/w preservative (/.e., total amount of preservative), e.g., 0.02- 1.5% w/w preservative, 0.05-1% w/w preservative, or 0.1-0.5% w/w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.
In another embodiment, the liquid formulation further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, betagalactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha- mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta- xylosidase or any combination thereof.
Fermentation Broth Formulations or Cell Compositions
The present invention also relates to a fermentation broth formulation or a cell composition comprising a variant of the present invention. The fermentation broth formulation or the cell composition further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding the variant of the present invention which are used to produce the variant of interest), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.
The term "fermentation broth" as used herein refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.
In some embodiments, the fermentation broth formulation or the cell composition comprises a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. In some embodiments, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing.
In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In some embodiments, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.
The fermentation broth formulation or cell composition may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.
The cell-killed whole broth or cell composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or cell composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon- limiting conditions to allow protein synthesis. In some embodiments, the cell-killed whole broth or cell composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or cell composition can be permeabilized and/or lysed using methods known in the art.
A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.
The whole broth formulations and cell compositions of the present invention may be produced by a method described in WO 90/15861 or WO 2010/096673.
Detergent Compositions
The invention also relates to a composition comprising a variant of the invention, e.g., a detergent or cleaning composition.
The invention also relates to a composition comprising a variant of the invention and further comprising: one or more detergent components; and/or one or more additional enzymes. In a preferred embodiment, the composition is a detergent composition comprising one or more detergent components, in particular one or more non-naturally occurring detergent components.
The present invention also relates to a composition comprising a variant of the present invention and further comprising one or more additional enzymes selected from the group consisting of amylases (e.g., alpha-amylases), catalases, cellulases (e.g., endoglucanases), cutinases, DNases, haloperoxygenases, lipases, mannanases, pectinases, pectin lyases, peroxidases, proteases, xanthanases, lichenases and xyloglucanases, or any mixture thereof.
A detergent composition may, e.g., be in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.
In one preferred embodiment, the detergent composition is a liquid composition.
In one preferred embodiment, the detergent composition is a powder composition.
In one preferred embodiment, the detergent composition is a laundry soap bar.
The invention also relates to use of a composition of the present in a cleaning process, such as laundry or hard surface cleaning such as dishwashing.
The choice of additional components for a detergent composition is within the skill of the artisan and includes conventional ingredients, including the exemplary non-limiting components set forth below. The choice of components may include, for fabric care, the consideration of the type of fabric to be cleaned, the type and/or degree of soiling, the temperature at which cleaning is to take place, and the formulation of the detergent product.
In a particular embodiment, a detergent composition comprises a variant of the invention and one or more non-naturally occurring detergent components, such as surfactants, hydrotropes, builders, co-builders, chelators or chelating agents, bleaching system or bleach components, polymers, fabric hueing agents, fabric conditioners, foam boosters, suds suppressors, dispersants, dye transfer inhibitors, fluorescent whitening agents, perfume, optical brighteners, bactericides, fungicides, soil suspending agents, soil release polymers, anti-redeposition agents, enzyme inhibitors or stabilizers, enzyme activators, antioxidants, and solubilizers. In one embodiment, the variant of the invention may be added to a detergent composition in an amount corresponding to 0.01-200 mg of enzyme protein per liter of wash liquor, preferably 0.05-50 mg of enzyme protein per liter of wash liquor, in particular 0.1-10 mg of enzyme protein per liter of wash liquor.
An automatic dish wash (ADW) composition may for example include 0.001 %-30%, such as 0.01 %-20%, such as 0.1-15%, such as 0.5-10% of enzyme protein by weight of the composition.
A granulated composition for laundry may for example include 0.001 %-20%, such as 0.01 %-10%, such as 0.05%-5% of enzyme protein by weight of the composition.
A liquid composition for laundry may for example include 0.0001 %-10%, such as 0.001- 7%, such as 0.1 %-5% of enzyme protein by weight of the composition.
The enzymes such as the variant of the invention may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, and the composition may be formulated as described in, for example, WO 92/19709 and WO 92/19708 or the variants according to the invention may be stabilized using peptide aldehydes or ketones such as described in WO 2005/105826 and WO 2009/118375.
The variants of the invention may be formulated in liquid laundry compositions such as a liquid laundry compositions composition comprising: a) at least 0.01 mg of active variant per liter detergent, b) 2 wt% to 60 wt% of at least one surfactant c) 5 wt% to 50 wt% of at least one builder
The detergent composition may be formulated into a granular detergent for laundry. Such detergent may comprise; a) at least 0.01 mg of active protease variant per gram of composition b) anionic surfactant, preferably 5 wt % to 50 wt % c) nonionic surfactant, preferably 1 wt % to 8 wt % d) builder, preferably 5 wt % to 40 wt %, such as carbonates, zeolites, phosphate builder, calcium sequestering builders or complexing agents.
Although components mentioned below are categorized by general header according to a particular functionality, this is not to be construed as a limitation, as a component may comprise additional functionalities as will be appreciated by the person skilled in the art.
Surfactants
The detergent composition may comprise one or more surfactants, which may be anionic and/or cationic and/or non-ionic and/or semi-polar and/or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. The surfactant(s) is typically present at a level of from about 0.1 % to 60% by weight, such as about 1 % to about 40%, or about 3% to about 20%, or about 3% to about 10%. The surfactant(s) is chosen based on the desired cleaning application, and includes any conventional surfactant(s) known in the art. Any surfactant known in the art for use in detergents may be utilized. Surfactants lower the surface tension in the detergent, which allows the stain being cleaned to be lifted and dispersed and then washed away.
When included therein, the detergent will usually contain from about 1% to about 40% by weight, such as from about 5% to about 30%, including from about 5% to about 15%, or from about 20% to about 25% of an anionic surfactant. Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ethersulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates), secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap, and combinations thereof.
When included therein, the detergent will usually contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alklydimethylethanolamine quat (ADMEAQ), cetyltrimethylammonium bromide (CTAB), dimethyldistearylammonium chloride (DSDMAC), and alkylbenzyldimethylammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.
When included therein, the detergent will usually contain from about 0.2% to about 40% by weight of a non-ionic surfactant, for example from about 0.5% to about 30%, in particular from about 1% to about 20%, from about 3% to about 10%, such as from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of non-ionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.
When included therein, the detergent will usually contain from about 0% to about 10% by weight of a semipolar surfactant. Non-limiting examples of semipolar surfactants include amine oxides (AO) such as alkyldimethylamineoxide, N-(coco alkyl)-N,N-dimethylamine oxide and N- (tallow-alkyl)-N,N-bis(2-hydroxyethyl)amine oxide, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.
When included therein, the detergent will usually contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.
Builders and Co-Builders
The detergent composition may contain about 0-65% by weight, such as about 5% to about 45% of a detergent builder or co-builder, or a mixture thereof. In a dish wash detergent, the level of builder is typically 40-65%, particularly 50-65%. Builders and chelators soften, e.g., the wash water by removing the metal ions form the liquid. 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 laundry 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 iminodiethanol), triethanolamine (TEA, also known as 2,2’,2”-nitrilotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.
The detergent composition may also contain 0-20% by weight, such as about 5% to about 10%, of a detergent co-builder, or a mixture thereof. The detergent composition may include a co-builder alone, or in combination with a builder, for example a zeolite builder. Non-limiting examples of co-builders include 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. Additional specific examples include 2, 2’, 2”- nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N’-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane- 1 ,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepentakis (methylenephosphonic acid) (DTPMPA or DTMPA), N-(2- hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid- N, N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(2-sulfomethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2- sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), a-alanine-N, N-diacetic acid (a-ALDA), serine-N, N-diacetic acid (SEDA), isoserine- N, N-diacetic acid (ISDA), phenylalanine-N, N-diacetic acid (PHDA), anthranilic acid-N, N-diacetic acid (ANDA), sulfanilic acid-N, N-diacetic acid (SLDA), taurine-N, N-diacetic acid (TLIDA) and sulfomethyl-N, N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylidenediamine-N, N’, N’-triacetate (HEDTA), diethanolglycine (DEG), diethylenetriamine penta(methylenephosphonic acid) (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations and salts thereof. Further exemplary builders and/or co-builders are described in, e.g., WO 2009/102854 and US 5,977,053.
The variants of the invention may also be formulated into a dish wash composition, preferably an automatic dish wash composition (ADW), comprising: a) at least 0.01 mg of active protease variant according to the invention, and b) 10-50 wt % builder preferably selected from citric acid, methylglycine-N, N-diacetic acid (MGDA) and/or glutamic acid-N, N-diacetic acid (GLDA) and mixtures thereof, and c) at least one bleach component.
Bleaching Systems
The detergent may contain 0-50% by weight, such as about 0.1 % to about 25%, of a bleaching system. Bleach systems remove discolor often by oxidation, and many bleaches also have strong bactericidal properties, and are used for disinfecting and sterilizing. Any bleaching system known in the art for use in laundry detergents may be utilized. Suitable bleaching system components include bleaching catalysts, photobleaches, bleach activators, sources of hydrogen peroxide such as sodium percarbonate and sodium perborates, preformed peracids and mixtures thereof. Suitable preformed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone (R), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium salts of perborate (usually mono- or tetra- hydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a peracid-forming bleach activator.
The term bleach activator is meant herein as a compound which reacts with peroxygen bleach like hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated bleach. Suitable bleach activators to be used herein include those belonging to the class of esters amides, imides or anhydrides. Suitable examples are tetracetylethylene diamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzene sulfonate (ISONOBS), diperoxy dodecanoic acid, 4- (dodecanoyloxy) benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4- (decanoyloxy)benzoate (DOBS), 4-(nonanoyloxy)-benzenesulfonate (NOBS), and/or those disclosed in WO 98/17767. A particular family of bleach activators of interest was disclosed in EP 624154 and particularly preferred in that family is acetyl triethyl citrate (ATC). ATC or a short chain triglyceride like triacetin has the advantage that it is environmentally friendly as it eventually degrades into citric acid and alcohol. Furthermore, acetyl triethyl citrate and triacetin have good hydrolytic stability in the product upon storage and are efficient bleach activators. Finally, ATC provides a good building capacity to the laundry additive. Alternatively, the bleaching system may comprise peroxyacids of, for example, the amide, imide, or sulfone type. The bleaching system may also comprise peracids such as 6-(phthalimido)peroxyhexanoic acid (PAP). The bleaching system may also include a bleach catalyst or a booster.
Some non-limiting examples of bleach catalysts that may be used in the compositions of the present invention include manganese oxalate, manganese acetate, manganese-collagen, cobalt-amine catalysts and manganese triazacyclononane (MnTACN) catalysts; particularly preferred are complexes of manganese with 1 ,4,7-trimethyl-1 ,4,7-triazacyclononane (Me3-TACN) or 1 ,2,4,7-tetramethyl-1 ,4,7-triazacyclononane (Me4-TACN), in particular Me3-TACN, such as the dinuclear manganese complex [(Me3-TACN)Mn(O)3Mn(Me3-TACN)](PF6)2, and [2, 2', 2"- nitrilotris(ethane-1 ,2-diylazanylylidene-KN-methanylylidene)triphenolato-K3O]manganese(lll). The bleach catalysts may also be other metal compounds, such as iron or cobalt complexes.
In some embodiments, the bleach component may be an organic catalyst selected from the group consisting of organic catalysts having the following formula:
(iii) and mixtures thereof; wherein each R1 is independently a branched alkyl group containing from 9 to 24 carbons or linear alkyl group containing from 11 to 24 carbons, preferably each R1 is independently a branched alkyl group containing from 9 to 18 carbons or linear alkyl group containing from 11 to 18 carbons, more preferably each R1 is independently selected from the group consisting of 2-propyl heptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, iso-nonyl, iso-decyl, iso-tridecyl and isopentadecyl. Other exemplary bleaching systems are described, e.g., in WO 2007/087258, WO 2007/087244, WO 2007/087259 and WO 2007/087242. Suitable photobleaches may for example be sulfonated zinc phthalocyanine. Hydrotropes
A hydrotrope is a compound that solubilizes hydrophobic compounds in aqueous solutions (or oppositely, polar substances in a non-polar environment). Typically, hydrotropes have both hydrophilic and hydrophobic characters (so-called amphiphilic properties as known from surfactants); however, the molecular structures of hydrotropes generally do not favour spontaneous self-aggregation, see, e.g., review by Hodgdon and Kaier, 2007, Current Opinion in Colloid & Interface Science 12: 121-128. Hydrotropes do not display a critical concentration above which self-aggregation occurs as found for surfactants and lipids forming micellar, lamellar or other well defined meso-phases. Instead, many hydrotropes show a continuous-type aggregation process where the sizes of aggregates grow as concentration increases. However, many hydrotropes alter the phase behavior, stability, and colloidal properties of systems containing substances of polar and non-polar character, including mixtures of water, oil, surfactants, and polymers. Hydrotropes are classically used across industries from pharma, personal care and food to technical applications. Use of hydrotropes in detergent compositions allows for example more concentrated formulations of surfactants (as in the process of compacting liquid detergents by removing water) without inducing undesired phenomena such as phase separation or high viscosity.
The detergent may contain 0-5% by weight, such as about 0.5 to about 5%, or about 3% to about 5%, of a hydrotrope. Any hydrotrope known in the art for use in detergents may be utilized. Non-limiting examples of hydrotropes include sodium benzene sulfonate, sodium p-toluene sulfonate (STS), sodium xylene sulfonate (SXS), sodium cumene sulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyglycol ethers, sodium hydroxynaphthoate, sodium hydroxynaphthalene sulfonate, sodium ethylhexyl sulfate, and combinations thereof.
Polymers
The detergent may contain 0-10% by weight, such as 0.5-5%, 2-5%, 0.5-2% or 0.2-1% of a polymer. Any polymer known in the art for use in detergents may be utilized. The polymer may function as a co-builder as mentioned above, or may provide antiredeposition, fiber protection, soil release, dye transfer inhibition, grease cleaning and/or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and/or more than one of the below-mentioned motifs. Exemplary polymers include (carboxymethyl)cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA/PMA, poly-aspartic acid, and lauryl methacrylate/acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric glycols, copolymers of poly(ethylene terephthalate) and poly(oxyethene terephthalate) (PET-POET), PVP, poly(vinylimidazole) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO) and polyvinylpyrrolidone-vinylimidazole (PVPVI). Further exemplary polymers include sulfonated polycarboxylates, polyethylene oxide and polypropylene oxide (PEO-PPO) and diquaternium ethoxy sulfate. Other exemplary polymers are disclosed in, e.g., WO 2006/130575. Salts of the above-mentioned polymers are also contemplated.
Fabric hueinq agents
The detergent compositions of the present invention may also include fabric hueing agents such as dyes or pigments, which when formulated in detergent compositions can deposit onto a fabric when the fabric is contacted with a wash liquor comprising the detergent compositions and thus altering the tint of the fabric through absorption/reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric hueing agents alter the tint of a surface as they absorb at least a portion of the visible light spectrum. Suitable fabric hueing agents include dyes and dye-clay conjugates and may also include pigments. Suitable dyes include small molecule dyes and polymeric dyes. Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Color Index (C.l.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example as described in WO 2005/003274, WO 2005/003275, WO 2005/003276 and EP 1876226 (hereby incorporated by reference). The detergent composition preferably comprises from about 0.00003 wt. % to about 0.2 wt. %, from about 0.00008 wt. % to about 0.05 wt. %, or even from about 0.0001 wt. % to about 0.04 wt. % fabric hueing agent. The composition may comprise from 0.0001 wt % to 0.2 wt. % fabric hueing agent, this may be especially preferred when the composition is in the form of a unit dose pouch. Suitable hueing agents are also disclosed in, e.g., WO 2007/087257 and WO 2007/087243.
Additional Enzymes
A detergent additive or detergent composition comprising the variant of the invention may comprise one or more enzymes such as an amylase (e.g., alpha-amylase), arabinase, carbohydrase, cellulase (e.g., endoglucanase), cutinase, DNase, galactanase, haloperoxygenase, lipase, mannanase, oxidase, e.g., laccase and/or peroxidase, pectinase, pectin lyase, protease, xylanase, xanthanase or xyloglucanase.
The properties of the selected enzyme(s) should be compatible with the selected detergent (e.g., pH-optimum, compatibility with other enzymatic and non-enzymatic ingredients, etc.).
Cellulases
The term “cellulase” means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. The terms “cellulase” and the expression “polypeptide having cellulase activity” are used interchangeably. Cellulases may be selected from the group consisting of cellulases belonging to GH5, GH44, GH45, EC 3.2.1.4, EC 3.2.1.21 , EC 3.2.1.91 and EC 3.2.1.172. Such enzymes include endoglucanase(s) (e.g., EC 3.2.1.4), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof.
Suitable cellulases include mono-component and mixtures of enzymes of bacterial or fungal origin. Chemically modified or protein engineered mutants are also contemplated. The cellulase may for example be a mono-component or a mixture of mono-component endo-1 ,4-beta- glucanase also referred to as endoglucanase.
Suitable cellulases include those from the genera Bacillus, Pseudomonas, Humicola, My- celiophthora, Fusarium, Thielavia, Trichoderma, and Acremonium. Exemplary cellulases include a fungal cellulase from Humicola insolens (US 4,435,307) or from Trichoderma, e.g., T. reesei or T. viride. Other suitable cellulases are from Thielavia, e.g., Thielavia terrestris as described inWO 96/29397, or the fungal cellulases produced from Myceliophthora thermophila and Fusarium ox- ysporum disclosed in US 5,648,263, US 5,691 ,178, US 5,776,757, WO 89/09259 and WO 91/17244. Also relevant are cellulases from Bacillus as described in WO 02/099091 and JP 2000210081. Suitable cellulases are alkaline or neutral cellulases having care benefits. Examples of cellulases are described in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples are cellulase variants such as those described in WO 94/07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95/24471 , WO 98/12307.
Other cellulases are endo-beta-1 , 4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2 of WO 2002/099091 or a family 44 xyloglucanase, which a xyloglucanase enzyme having a sequence of at least 60% identity to positions 40-559 of SEQ ID NO: 2 of WO 2001/062903.
Yet another group of suitable cellulases comprise a stabilized linker between the core and the CBM. Particularly useful are such cellulase having at least 80% identity to SEQ ID NO: 397, SEQ ID NO: 398 or SEQ ID NO: 399 of WO 2023/061928.
Commercially available cellulases include Carezyme®, Carezyme® Premium, Cel- luzyme®, Carezyme Elite®, Celluclean®, Celluclast®, Endolase®, Renozyme®, Whitezyme® Celluclean® Classic, and Cellusoft® (Novozymes A/S); Puradax®, Puradax HA, Puradax EG, Revitalenz 1000, Revitalenz 200, and Revitalenz 2000 (Dupont Industrial Biosciences); KAC- 500(B)™ (Kao Corporation); and Biotouch DCL and Biotouch FLX1 (AB Enzymes).
The two basic approaches for measuring cellulolytic enzyme activity include: (1) measuring the total cellulolytic enzyme activity, and (2) measuring the individual cellulolytic enzyme activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., 2006, Biotechnology Advances 24: 452-481. Total cellulolytic enzyme activity can be measured using insoluble substrates, including Whatman N°1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman N°1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appt. Chem. 59: 257-68).
Proteases
The composition may comprise one or more additional proteases including 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 metalloprotease may for example be a thermolysin from, e.g., family M4 or other metalloprotease such as those from M5, M7 or M8 families.
Examples of metalloproteases are the neutral metalloproteases as described in WO 2007/044993 (Genencor I nt.) such as those derived from Bacillus amyloliquefaciens.
Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase®, Esperase®, Progress® Excel, Progress® Key, and Progress® Uno (Novozymes A/S), those sold under the tradename Maxatase®, Maxacai®, Maxapem®, Purafect®, Purafect Prime®, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Opticlean®, Optimase®, Preferenz® P200, Preferenz® P300, and Preferenz® P400 (DuPont/IFF), Axapem™ (Gist-Brocades N.V.), BLAP (sequence shown in Figure 29 of US5352604) and variants hereof (Henkel AG) and KAP (Bacillus alkalophilus subtilisin) from Kao.
Lipases and Cutinases
Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified or protein engineered mutant enzymes are included. Examples include lipase from Thermomyces, e.g., from T. lanuginosus (previously named Humicola lanuginosa) as described in EP 258068 and EP 305216, cutinase from Humicola, e.g., H. insolens (WO 96/13580), lipase from strains of Pseudomonas (some of these now renamed to Burkholderia), e.g., P. alcaligenes or P. pseudoalcaligenes (EP 218272), P. cepacia (EP 331376), P. sp. strain SD705 (WO 95/06720 & WO 96/27002), P. wisconsinensis (WO 96/12012), GDSL-type Streptomyces lipases (WO 2010/065455), cutinase from Magnaporthe grisea (WO 2010/107560), cutinase from Pseudomonas mendocina (US 5,389,536), lipase from Thermobifida fusca (WO 2011/084412), Geobacillus stearothermophilus lipase (WO 2011/084417), lipase from Bacillus subtilis (WO 2011/084599), and lipase from Streptomyces griseus (WO 2011/150157) and S. pristinaespiralis (WO 2012/137147).
Other examples are lipase variants such as those described in EP 407225, WO 92/05249, WO 94/01541 , WO 94/25578, WO 95/14783, WO 95/30744, WO 95/35381 , WO 95/22615, WO 96/00292, WO 97/04079, WO 97/07202, WO 00/34450, WO 00/60063, WO 01/92502, WO 2007/87508 and WO 2009/109500.
Preferred commercial lipase products include Lipolase™, Lipex™; Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (originally from Gist-Brocades).
Still other examples are lipases sometimes referred to as acyltransferases or perhydrolases, e.g., acyltransferases with homology to Candida antarctica lipase A (WO 2010/111143), acyltransferase from Mycobacterium smegmatis (WO 2005/056782), perhydrolases from the CE 7 family (WO 2009/067279), and variants of the M. smegmatis perhydrolase in particular the S54V variant used in the commercial product Gentle Power Bleach from Huntsman Textile Effects Pte Ltd (WO 2010/100028).
Amylases
Suitable amylases which can be used together with the variants 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 99/19467, such as variants with substitutions in one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 178, 179, 181 , 188, 190, 197, 201 , 202, 207, 208, 209, 211 , 243, 264, 304, 305, 391 , 408, and 444.
Different suitable amylases include amylases having SEQ ID NO:6 in WO 02/10355 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of SEQ ID NO:6 are those having a deletion in positions 181 and 182 and a substitution in position 193.
Other amylases which are suitable are hybrid alpha-amylases comprising residues 1-33 of the alpha-amylase derived from B. amyloliquefaciens shown in SEQ ID NO:6 of WO 2006/066594 and residues 36-483 of the B. licheniformis alpha-amylase shown in SEQ ID NO:4 of WO 2006/066594 or variants having 90% sequence identity thereof. 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. Most preferred variants of the 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:
M197T;
H 156Y+A 181 T+ N 190F+A209V+Q264S; or
G48A+T49I +G 107A+ H 156Y+A 181 T+ N 190F+ 1201 F+A209V+Q264S.
Other suitable amylases are amylases having the sequence of SEQ ID NO:6 in WO 99/19467 or variants thereof having 90% sequence identity to SEQ ID NO:6. Preferred variants of 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 96/23873 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, 206, 212, 243, 260, 269, 304 and 476, using SEQ ID 2 of WO 96/23873 for numbering. More preferred variants are those having a deletion in two positions selected from 181 , 182, 183 and 184, such as 181 and 182, 182 and 183, or positions 183 and 184. Most preferred amylase variants of SEQ ID NO:1 , SEQ ID NO:2 or SEQ ID NO:7 are those having a deletion in positions 183 and 184 and a substitution in one or more of positions 140, 195, 206, 243, 260, 304 and 476.
Other amylases which can be used are amylases having SEQ ID NO:2 of WO 2008/153815, SEQ ID NO: 10 in WO 01/66712 or variants thereof having 90% sequence identity to SEQ ID NO:2 of WO 2008/153815 or 90% sequence identity to SEQ ID NO: 10 in WO 01/66712. Preferred variants of SEQ ID NQ: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 , 207, 211 and 264.
Further suitable amylases are amylases having SEQ ID NO:2 of WO 2009/061380 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:
N 128C+K178L+T182G+Y305R+G475K;
N128C+K178L+T182G+F202Y+Y305R+D319T+G475K;
S125A+N 128C+K178L+T182G+Y305R+G475K; or
S125A+N128C+T131 I+T165I+K178L+T182G+Y305R+G475K, wherein the variants are C-terminally truncated and optionally further comprise a substitution at position 243 and/or a deletion at position 180 and/or position 181.
Further suitable amylases are amylases having SEQ ID NO:1 of WO 2013/184577 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: 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, N192FYH, M199L, I203YF, S241QADN, R458N, T459S, D460T, G476K and G477K and/or a deletion in position R178 and/or S179 or of T180 and/or G181. Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions: E187P+I203Y+G476K E187P+I203Y+R458N+T459S+D460T+G476K and optionally further comprise a substitution at position 241 and/or a deletion at position 178 and/or position 179.
Further suitable amylases are amylases having SEQ ID NO:1 of WO 2010/104675 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.
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 a deletion in position R179 and/or S180 or of 1181 and/or G182. Most preferred amylase variants of SEQ ID NO:1 comprise the substitutions N21 D+D97N+V128I, and optionally further comprise a substitution at position 200 and/or a deletion at position 180 and/or position 181.
Other suitable amylases are the alpha-amylase having SEQ ID NO: 12 in WO 01/66712 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 WO 01/66712: R28, R118, N174; R181 , G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 , N484. Particularly preferred amylases include variants having a deletion of D183 and G184 and having the substitutions R118K, 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.
Other examples are amylase variants such as those described in WO 2011/098531 , WO 2013/001078 and WO 2013/001087. Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™/Effectenz™, Powerase, Preferenz S1000, Preferenz S100 and Preferenz S110 (from Genencor International Inc./DuPont).
Peroxidases/Oxidases
Suitable peroxidases/oxidases include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257.
Commercially available peroxidases include Guardzyme™ (Novozymes A/S).
Adjunct materials
Any detergent components known in the art for use in laundry detergents may also be utilized. Other optional detergent components include anti-corrosion agents, anti-shrink agents, anti-soil redeposition agents, anti-wrinkling agents, bactericides, binders, corrosion inhibitors, disintegrants/disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and/or polyols such as propylene glycol), fabric conditioners including clays, fillers/processing aids, fluorescent whitening agents/optical brighteners, foam boosters, foam (suds) regulators, perfumes, soil-suspending agents, softeners, suds suppressors, tarnish inhibitors, and wicking agents, either alone or in combination. Any ingredient known in the art for use in laundry detergents may be utilized. The choice of such ingredients is well within the skill of the artisan.
Dispersants: The detergent compositions of the present invention can also contain dispersants. In particular powdered detergents may comprise dispersants. Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms. Suitable dispersants are for example described in Powdered Detergents, Surfactant Science Series, volume 71 , Marcel Dekker, Inc., 1997.
Dye Transfer Inhibiting Agents: The detergent compositions of the present invention may also include one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof. When present in a subject composition, the dye transfer inhibiting agents may be present at levels from about 0.0001 % to about 10%, from about 0.01% to about 5% or even from about 0.1 % to about 3% by weight of the composition.
Fluorescent whitening agent: The detergent compositions of the present invention will preferably also contain additional components that may tint articles being cleaned, such as fluorescent whitening agent or optical brighteners. Where present the brightener is preferably at a level of about 0.01 % to about 05%. Any fluorescent whitening agent suitable for use in a laundry detergent composition may be used in the composition of the present invention. The most commonly used fluorescent whitening agents are those belonging to the classes of diaminostilbene-sulphonic acid derivatives, diarylpyrazoline derivatives and bisphenyl-distyryl derivatives. Examples of the diaminostilbene-sulphonic acid derivative type of fluorescent whitening agents include the sodium salts of: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6- ylamino) stilbene-2,2'-disulphonate; 4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino) stilbene-2.2'- disulphonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate, 4,4'-bis-(4-phenyl-2, 1 ,3-triazol-2-yl)stilbene-2,2'-disulphonate; 4,4'- bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazin-6-ylamino) stilbene-2,2'-disulphonate and 2-(stilbyl-4"-naptho-1.,2':4,5)-1 ,2,3-trizole-2"-sulphonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4 anilino-s-triazin-6-ylamino) stilbene disulphonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styryl) disulphonate. Also preferred are fluorescent whitening agents is the commercially available Parawhite KX, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescers suitable for use in the invention include the 1 -3-diaryl pyrazolines and the 7-alkylaminocoumarins. Suitable fluorescent brightener levels include lower levels of from about 0.01 , from 0.05, from about 0.1 or even from about 0.2 wt. % to upper levels of 0.5 or even 0.75 wt. %.
Soil release polymers: The detergent compositions of the present invention may also include one or more soil release polymers which aid the removal of soils from fabrics such as cotton and polyester based fabrics, in particular the removal of hydrophobic soils from polyester based fabrics. The soil release polymers may for example be nonionic or anionic terephthalate based polymers, polyvinyl caprolactam and related copolymers, vinyl graft copolymers, polyester polyamides see for example Chapter 7 in Powdered Detergents, Surfactant science series volume 71 , Marcel Dekker, Inc. Another type of soil release polymers is amphiphilic alkoxylated grease cleaning polymers comprising a core structure and a plurality of alkoxylate groups attached to that core structure. The core structure may comprise a polyalkylenimine structure or a polyalkanolamine structure as described in detail in WO 2009/087523 (hereby incorporated by reference). Furthermore, random graft co-polymers are suitable soil release polymers Suitable graft co-polymers are described in more detail in WO 2007/138054, WO 2006/108856 and WO 2006/113314 (hereby incorporated by reference). Other soil release polymers are substituted polysaccharide structures especially substituted cellulosic structures such as modified cellulose derivatives such as those described in EP 1867808 or WO 03/040279 (both are hereby incorporated by reference). Suitable cellulosic polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulosic polymers include anionically modified cellulose, nonionically modified cellulose, cationically modified cellulose, zwitterionically modified cellulose, and mixtures thereof. Suitable cellulosic polymers include methyl cellulose, carboxy methyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, hydroxyl propyl methyl cellulose, ester carboxy methyl cellulose, and mixtures thereof.
Anti-redeposition agents: The detergent compositions of the present invention may also include one or more anti-redeposition agents such as carboxymethylcellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyoxyethylene and/or polyethyleneglycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimines. The cellulose based polymers described under soil release polymers above may also function as anti-redeposition agents.
Other suitable adjunct materials include, but are not limited to, anti-shrink agents, antiwrinkling agents, bactericides, binders, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam regulators, hydrotropes, perfumes, pigments, sod suppressors, solvents, and structurants for liquid detergents and/or structure elasticizing agents.
Formulation of Detergent Products
The detergent enzyme(s), /.e., a variant of the invention and optionally one or more additional enzymes, 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 comprising one or more enzymes can be formulated, for example, as a granulate, liquid, slurry, etc. Preferred detergent additive formulations include granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids, or slurries.
The detergent composition of the invention may be in any convenient form, e.g., a bar, a homogenous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact or concentrated liquid. There are a number of detergent formulation forms such as layers (same or different phases), pouches, as well as forms for machine dosing unit.
Pouches can be configured as single or multiple compartments. It can be of any form, shape and material which is suitable for hold the composition, e.g., without allowing the release of the composition from the pouch prior to water contact. The pouch is made from water soluble film which encloses an inner volume. The inner volume can be divided into compartments of the pouch. Preferred films are polymeric materials, preferably polymers which are formed into a film or sheet. Preferred polymers, copolymers or derivates thereof are selected from polyacrylates, and water-soluble acrylate copolymers, methyl cellulose, carboxy methyl cellulose, sodium dextrin, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, maltodextrin, polymethacrylates, most preferably polyvinyl alcohol copolymers and hydroxypropyl methyl cellulose (HPMC). Preferably the level of polymer in the film for example PVA is at least about 60%. The preferred average molecular weight will typically be about 20,000 to about 150,000. Films can also be of blend compositions comprising hydrolytically degradable and water-soluble polymer blends such as polylactide and polyvinyl alcohol (known under the Trade reference M8630 as sold by Chris Craft In. Prod, of Gary, Indiana, US) plus plasticizers like glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The pouches can comprise a solid laundry detergent composition or part components and/or a liquid cleaning composition or part components separated by the water-soluble film. The compartment for liquid components can be different in composition than compartments containing solids. See, e.g., US 2009/0011970.
Detergent ingredients can be separated physically from each other by compartments in water dissolvable pouches or in different layers of tablets. Thereby negative storage interaction between components can be avoided. Different dissolution profiles of each of the compartments can also give rise to delayed dissolution of selected components in the wash solution.
A liquid or gel detergent which is not unit dosed may be aqueous, typically containing at least 20% by weight and up to 95% water, such as up to about 70% water, up to about 65% water, up to about 55% water, up to about 45% water, up to about 35% water. Other types of liquids, including without limitation, alkanols, amines, diols, ethers and polyols may be included in an aqueous liquid or gel. An aqueous liquid or gel detergent may contain from 0-30% organic solvent. A liquid or gel detergent may be non-aqueous.
Laundry Soap Bars
The enzymes of the invention may be added to laundry soap bars and used for hand washing laundry, fabrics and/or textiles. The term laundry soap bar includes laundry bars, soap bars, combo bars, syndet bars and detergent bars. The types of bar usually differ in the type of surfactant they contain, and the term laundry soap bar includes those containing soaps from fatty acids and/or synthetic soaps. The laundry soap bar has a physical form which is solid and thus not a liquid, gel or powder at room temperature.
The laundry soap bar may contain one or more additional enzymes, protease inhibitors such as peptide aldehydes (or hydrosulfite adduct or hemiacetal adduct), boric acid, borate, borax and/or phenylboronic acid derivatives such as 4-formylphenylboronic acid, one or more soaps or synthetic surfactants, polyols such as glycerin, pH controlling compounds such as fatty acids, citric acid, acetic acid and/or formic acid, and/or a salt of a monovalent cation and an organic anion wherein the monovalent cation may be for example Na+, K+, or NH4+ and the organic anion may be for example formate, acetate, citrate, or lactate such that the salt of a monovalent cation and an organic anion may be, for example, sodium formate.
The laundry soap bar may also contain complexing agents such as EDTA and HEDP, perfumes and/or different type of fillers, surfactants, e.g., anionic synthetic surfactants, builders, polymeric soil release agents, detergent chelators, stabilizing agents, fillers, dyes, colorants, dye transfer inhibitors, alkoxylated polycarbonates, suds suppressers, structurants, binders, leaching agents, bleaching activators, clay soil removal agents, anti-redeposition agents, polymeric dispersing agents, brighteners, fabric softeners, perfumes and/or other compounds known in the art.
The laundry soap bar may be processed in conventional laundry soap bar making equipment such as, but not limited to, mixers, plodders, e.g., a two-stage vacuum plodder, extruders, cutters, logo-stampers, cooling tunnels and wrappers. A premix containing a soap, the enzyme of the invention, optionally one or more additional enzymes, a protease inhibitor, and a salt of a monovalent cation and an organic anion may be prepared and the mixture is then plodded. The enzyme and optional additional enzymes may be added at the same time as the protease inhibitor for example in liquid form. Besides the mixing step and the plodding step, the process may further comprise the steps of milling, extruding, cutting, stamping, cooling and/or wrapping.
Granular detergent formulations
Enzymes such as variants of the present invention in the form of granules, comprising an enzyme-containing core and optionally one or more coatings, are commonly used in granular (powder) detergents. Various methods for preparing the core are well-known in the art and include, for example, a) spray drying of a liquid enzyme-containing solution, b) production of layered products with an enzyme coated as a layer around a pre-formed inert core particle, e.g. using a fluid bed apparatus, c) absorbing an enzyme onto and/or into the surface of a pre-formed core, d) extrusion of an enzyme-containing paste, e) suspending an enzyme-containing powder in molten wax and atomization to result in prilled products, f) mixer granulation by adding an enzymecontaining liquid to a dry powder composition of granulation components, g) size reduction of enzyme-containing cores by milling or crushing of larger particles, pellets, etc., and h) fluid bed granulation. The enzyme-containing cores may be dried, e.g., using a fluid bed drier or other known methods for drying granules in the feed or enzyme industry, to result in a water content of typically 0.1 -10% w/w water.
The enzyme-containing cores are optionally provided with a coating to improve storage stability and/or to reduce dust formation. One type of coating that is often used for enzyme granulates for detergents is a salt coating, typically an inorganic salt coating, which may, e.g., be applied as a solution of the salt using a fluid bed. Other coating materials that may be used are, for example, polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). The granules may contain more than one coating, for example a salt coating followed by an additional coating of a material such as PEG, MHPC or PVA.
The present invention thus also relates to enzyme granules/particles comprising the variant of the invention. In an embodiment, the granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core.
The core may have a diameter, measured as equivalent spherical diameter (volume based average particle size), of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
In an embodiment, the core comprises one or more polypeptides having protease activity of the present invention.
The core may include additional materials such as fillers, fiber materials (cellulose or synthetic fibers), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances.
The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate.
The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and/or an acidic buffer component, typically as a homogenous blend.
The core may include an inert particle with the enzyme absorbed into it, or applied onto the surface, e.g., by fluid bed coating.
The core may have a diameter of 20-2000 pm, particularly 50-1500 pm, 100-1500 pm or 250-1200 pm.
The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for coloring the granule. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA).
The coating may be applied in an amount of at least 0.1% by weight of the core, e.g., at least 0.5%, at least 1 %, at least 5%, at least 10%, or at least 15%. The amount may be at most 100%, 70%, 50%, 40% or 30%.
The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In some embodiments, the thickness of the coating is below 100 pm, such as below 60 pm, or below 40 pm.
The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit it is encapsulating/enclosing has few or none uncoated areas. The layer or coating should, in particular, be homogeneous in thickness.
The coating can further contain other materials as known in the art, e.g., fillers, antisticking agents, pigments, dyes, plasticizers and/or binders, such as titanium dioxide, kaolin, calcium carbonate or talc. A salt coating may comprise at least 60% by weight of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% by weight.
To provide acceptable protection, the salt coating is preferably at least 0.1 pm thick, e.g., at least 0.5 pm, at least 1 pm, at least 2 pm, at least 4 pm, at least 5 pm, or at least 8 pm. In a particular embodiment, the thickness of the salt coating is below 100 pm, such as below 60 pm, or below 40 pm.
The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles are less than 50 pm, such as less than 10 pm or less than 5 pm.
The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular, having a solubility at least 0.1 g in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.
The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular, alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.
The salt in the coating may have a constant humidity at 20 °C above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00/01793 or WO 2006/034710.
Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO3 (CH2o°c=92%), NaNO3 (CH2O C=73%), Na2HPO4 (CH2o°c=95%), Na3PO4 (CH25°c=92%), NH4CI (CH2o°c = 79.5%), (NH4)2HPO4 (CH2O C = 93,0%), NH4H2PO4 (CH2Q C = 93.1%), (NH4)2SO4 (CH2o°c=81 .1%), KOI (CH2O C=85%), K2HPO4 (CH2O C=92%), KH2PO4 (CH2O°C=96.5%), KNO3 (CH2O°C=93.5%), Na2SO4 (CH2O C=93%), K2SO4 (CH2O C=98%), KHSO4 (CH2O C=86%), MgSO4 (CH2o°c=9O%), ZnSO4 (CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2 and magnesium acetate.
The salt may be in anhydrous form, or it may be a hydrated salt, i.e., a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99/32595. Specific examples include anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSC ), magnesium sulfate heptahydrate (MgSC - tW), zinc sulfate heptahydrate (ZnSC - tW), sodium phosphate dibasic heptahydrate (Na2HPO4'7H2O), magnesium nitrate hexahydrate (Mg(NC>3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.
Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.
The coating materials can be waxy coating materials and film-forming coating materials. Examples of waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluid bed techniques are given in GB 1483591.
The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Examples of enzyme granules with multiple coatings are described in WO 93/07263 and WO 97/23606.
The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and/or high shear granulation.
Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1 ; 1980; Elsevier. Preparation methods include known feed and granule formulation technologies, e.g.,
(a) Spray dried products, wherein a liquid enzyme-containing solution is atomized in a spray drying tower to form small droplets which during their way down the drying tower dry to form an enzyme-containing particulate material. Very small particles can be produced this way (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker).
(b) Layered products, wherein the enzyme is coated as a layer around a pre-formed inert core particle, wherein an enzyme-containing solution is atomized, typically in a fluid bed apparatus wherein the pre-formed core particles are fluidized, and the enzyme-containing solution adheres to the core particles and dries up to leave a layer of dry enzyme on the surface of the core particle. Particles of a desired size can be obtained this way if a useful core particle of the desired size can be found. This type of product is described in, e.g., WO 97/23606. (c) Absorbed core particles, wherein rather than coating the enzyme as a layer around the core, the enzyme is absorbed onto and/or into the surface of the core. Such a process is described in WO 97/39116.
(d) Extrusion or pelletized products, wherein an enzyme-containing paste is pressed to pellets or under pressure is extruded through a small opening and cut into particles which are subsequently dried. Such particles usually have a considerable size because of the material in which the extrusion opening is made (usually a plate with bore holes) sets a limit on the allowable pressure drop over the extrusion opening. Also, very high extrusion pressures when using a small opening increase heat generation in the enzyme paste, which is harmful to the enzyme (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71 ; pages 140- 142; Marcel Dekker).
(e) Prilled products, wherein an enzyme-containing powder is suspended in molten wax and the suspension is sprayed, e.g., through a rotating disk atomizer, into a cooling chamber where the droplets quickly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; vol. 71 ; page 140-142; Marcel Dekker). The product obtained is one wherein the enzyme is uniformly distributed throughout an inert material instead of being concentrated on its surface. U.S. Patent Nos. 4,016,040 and 4,713,245 describe this technique.
(f) Mixer granulation products, wherein an enzyme-containing liquid is added to a dry powder composition of conventional granulating components. The liquid and the powder in a suitable proportion are mixed and as the moisture of the liquid is absorbed in the dry powder, the components of the dry powder will start to adhere and agglomerate and particles will build up, forming granulates comprising the enzyme. Such a process is described in U.S. Patent No. 4,106,991 and related documents EP 170360, EP 304332, EP 304331 , WO 90/09440 and WO 90/09428. In a particular product of this process, various high-shear mixers can be used as granulators. Granulates consisting of enzyme, fillers and binders etc. are mixed with cellulose fibers to reinforce the particles to produce a so-called T-granulate. Reinforced particles are more robust, and release less enzymatic dust.
(g) Size reduction, wherein the cores are produced by milling or crushing of larger particles, pellets, tablets, briquettes etc. containing the enzyme. The wanted core particle fraction is obtained by sieving the milled or crushed product. Over and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.
(h) Fluid bed granulation. Fluid bed granulation involves suspending particulates in an air stream and spraying a liquid onto the fluidized particles via nozzles. Particles hit by spray droplets get wetted and become tacky. The tacky particles collide with other particles and adhere to them to form a granule. (i) The cores may be subjected to drying, such as in a fluid bed drier. Other known methods for drying granules in the feed or enzyme industry can be used by the skilled person. The drying preferably takes place at a product temperature of from 25 to 90°C. For some enzymes, it is important the cores comprising the enzyme contain a low amount of water before coating with the salt. If water sensitive enzymes are coated with a salt before excessive water is removed, it will be trapped within the core and may affect the activity of the enzyme negatively. After drying, the cores preferably contain 0.1-10% w/w water.
Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661 ,452 and may optionally be coated by methods known in the art.
The granulate may further one or more additional enzymes. Each enzyme will then be present in more granules securing a more uniform distribution of the enzymes, and also reduces the physical segregation of different enzymes due to different particle sizes. Methods for producing multi-enzyme co-granulates is disclosed in the ip.com disclosure IPCOM000200739D.
Another example of formulation of enzymes by the use of co-granulates is disclosed in WO 2013/188331.
The enzyme may also be a protected enzyme prepared according to the method disclosed in EP 238,216.
In an embodiment, the granule further comprises one or more additional enzymes, e.g., hydrolase, isomerase, ligase, lyase, oxidoreductase, and transferase. The one or more additional enzymes are preferably selected from the group consisting of acetylxylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolases, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta- glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta- mannosidase (mannanase), phytase, phospholipase A1 , phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase or any combination thereof.
For further information on enzyme granules and production thereof, see WO 2013/007594 as well as, e.g., WO 2009/092699, EP 1705241 , EP 1382668, WO 2007/001262, US 6,472,364, WO 2004/074419, and WO 2009/102854.
Uses
The present invention is also directed to methods for using the variants of the invention or compositions comprising said variants in laundering of textile and fabrics, such as household laundry washing and industrial laundry washing.
The invention is also directed to methods for using the variants according to the invention or compositions thereof in cleaning hard surfaces such as floors, tables, walls, roofs etc. as well as surfaces of hard objects such as cars (car wash) and dishes (dishwashing). The variants of the present invention may be added to and thus become a component of a detergent composition. Thus, one aspect of the invention relates to the use of a variant of the invention in a cleaning process such as laundering and/or hard surface cleaning.
A detergent composition of the present invention may be formulated, for example, as a hand or machine laundry detergent composition including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition or be formulated as a detergent composition for use in general household hard surface cleaning operations or be formulated for hand or machine dishwashing operations.
The cleaning process or the textile care process may for example be a laundry process, a dishwashing process, or cleaning of hard surfaces such as bathroom tiles, floors, tabletops, drains, sinks and washbasins. Laundry processes can for example be household laundering but may also be industrial laundering. Furthermore, the invention relates to a process for laundering of fabrics and/or garments, where the process comprises treating fabrics with a washing solution containing a detergent composition and at least one protease variant of the invention. The cleaning process or a textile care process can for example be carried out in a machine washing or manually. The washing solution can for example be an aqueous washing solution containing a detergent composition.
In one aspect, the variants of the invention are used in a cleaning process, e.g., a laundry process, that comprises a short wash cycle, typically a wash cycle of not more than about 30 minutes, such as not more than about 20 minutes, e.g., not more than about 15 minutes or not more than about 10 minutes. It has surprisingly been found that the subtilase variants of the invention are remarkably effective in short wash cycles lasting, for example, only about 10-20 minutes. This may be useful in, e.g., top-loading washing machines that often have short wash cycles or for hand-washing of laundry.
In another aspect, the variants of the invention are used in a cleaning process, e.g., a laundry process, where the wash water is used for more than one portion of laundry. In this case, the wash water containing a detergent with a variant of the invention may be used in a first wash cycle for a first portion of laundry, and then reused one or more times for additional wash cycles with new portions of laundry. It has been found that detergents containing a variant of the invention are able to substantially maintain cleaning performance on protease-sensitive stains even after three wash cycles or more. This may for example be useful for laundry washed by hand and/or in regions with water scarcity.
The last few years there has been an increasing interest in replacing components in detergents that are derived from petrochemicals with renewable biological components such as enzymes and polypeptides without compromising the wash performance. When the components of detergent compositions change, new enzyme activities or new enzymes having alternative and/or improved properties compared to the previously used detergent enzymes such as proteases, lipases and amylases may be needed to achieve a similar or improved wash performance when compared to the traditional detergent compositions.
The invention further concerns the use of variants of the invention in a proteinaceous stain removing process. The proteinaceous stains may be stains such as food stains, e.g., baby food, cocoa, egg or milk, or other stains such as sebum, blood, ink or grass, or a combination hereof.
Washing Method
The present invention provides a method of cleaning a fabric, dishware or a hard surface with a detergent composition comprising a variant of the invention.
The method of cleaning comprises contacting an object with a detergent composition comprising a protease variant of the invention under conditions suitable for cleaning the object. In a preferred embodiment the detergent composition is used in a laundry or a dish wash process.
Another embodiment relates to a method for removing stains from fabric or dishware which comprises contacting the fabric or dishware with a composition comprising a protease of the invention under conditions suitable for cleaning the object. In the method of cleaning of the invention, the object being cleaned may be any suitable object such as a textile or a hard surface such as dishware or a floor, table, wall, etc.
Also contemplated are compositions and methods of treating fabrics (e.g., to desize a textile) using a protease variant of the invention. The protease variant can be used in any fabrictreating method which is well known in the art (see, e.g., US 6,077,316). For example, in one aspect, the feel and appearance of a fabric is improved by a method comprising contacting the fabric with a protease variant in a solution. In one aspect, the fabric is treated with the solution under pressure.
The detergent compositions of the present invention are suited for use in laundry and hard surface applications, including dishwashing. Accordingly, the present invention includes a method for laundering a fabric or washing dishware, comprising contacting the fabric/dishware to be cleaned with a solution comprising the detergent composition according to the invention. The fabric may comprise any fabric capable of being laundered in normal consumer use conditions. The dishware may comprise any dishware such as crockery, cutlery, ceramics, plastics such as melamine, metals, china, glass and acrylics. The solution preferably has a pH from about 5.5 to about 11.5. The compositions may be employed at concentrations from about 100 ppm, preferably 500 ppm to about 15,000 ppm in solution. The water temperatures typically range from about 5°C to about 95°C, including about 10°C, about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C and about 90°C. The water to fabric ratio is typically from about 1 :1 to about 30:1. The enzyme(s) of the detergent composition of the invention may be stabilized using conventional stabilizing agents and protease inhibitors, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, different salts such as NaCI; KCI; lactic acid, formic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid, or a peptide aldehyde such as di-, tri- or tetrapeptide aldehydes or aldehyde analogues (either of the form B1-B0-R wherein, R is H, CH3, CX3, CHX2, or CH2X (X=halogen), BO is a single amino acid residue (preferably with an optionally substituted aliphatic or aromatic side chain); and B1 consists of one or more amino acid residues (preferably one, two or three), optionally comprising an N-terminal protection group, or as described in WO 2009/118375, WO 98/13459) or a protease inhibitor of the protein type such as RASI, BASI, WASI (bifunctional alpha-amylase/subtilisin inhibitors of rice, barley and wheat) or CI2 or SSI. The composition may be formulated as described in, e.g., \NQ) 92/19709, WO 92/19708 and US 6,472,364. In some embodiments, the enzymes employed herein are stabilized by the presence of water-soluble sources of zinc (II), calcium (II) and/or magnesium (II) ions in the finished compositions that provide such ions to the enzymes, as well as other metal ions (e.g., barium (II), scandium (II), iron (II), manganese (II), aluminum (III), Tin (II), cobalt (II), copper (II), Nickel (II), and oxovanadium (IV)).
The detergent compositions provided herein are typically formulated such that, during use in aqueous cleaning operations, the wash water has a pH of from about 5.0 to about 12.5, such as from about 5.0 to about 11.5, or from about 6.0 to about 10.5. In some embodiments, granular or liquid laundry products are formulated to have a pH from about 6 to about 8. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
The present invention relates to novel protease variants exhibiting increased stability and/or improved wash performance (exemplified by improved removal of proteinaceous stains) in liquid detergent and soap bar compositions compared with the parent protease. Materials
Textiles
Standard textiles or swatches were obtained from Center for Testmaterials BV (P.O. Box 120, 3133 KT Vlaardingen, The Netherlands), Warwick Equest Ltd (Consett, DH8 6BN, United Kingdom) and EMPA (Ueberlandstrasse 129, 8600 Dubendorf, Switzerland).
Detergent formats
The following detergents used can be composed according to the description herein. The protease(s) to be tested with the swatches can then be added to the model detergent base for testing.
Methods
Preparation and purification of polypeptides
Mutation and introduction of expression cassettes into Bacillus subtilis was performed by standard methods known in the art. All DNA manipulations were performed by PCR (e.g., as described by Sambrook et al., 2001) using standard methods known to the skilled person.
Recombinant B. subtilis constructs encoding protease polypeptides were inoculated into and cultivated in a complex medium (TBgly) under antibiotic selection for 24 h at 37 °C. Shake flasks containing a rich media (PS-1 : 100 g/L sucrose (Danisco cat.no. 109-0429), 40 g/L crust soy (soybean flour), 10 g/L Na2HPO4 l2H2O (Merck cat.no. 106579), 0.1 ml/L Dowfax63N10 (Dow) were inoculated in a ratio of 1 :100 with the overnight culture. Shake flask cultivation was performed for 4 days at 30 °C shaking at 270 rpm.
Purification of culture supernatants was performed as follows: The culture broth is centrifuged at 26,000 x g for 20 minutes and the supernatant is carefully decanted from the precipitate. The supernatant is filtered through a Nalgene 0.2 pm filtration unit to remove the remains of the host cells. The pH in the 0.2 pm filtrate is adjusted to pH 8 with 3 M Tris base and the pH-adjusted filtrate is applied to a M EP Hypercel column (Pall Corporation) equilibrated in 20 mM Tris/HCI, 1 mM CaCh, pH 8.0. After washing the column with the equilibration buffer, the column is step-eluted with 20 mM CHsCOOH/NaOH, 1 mM CaCh, pH 4.5. Fractions from the column are analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 and peak fractions are pooled. The pH of the pool from the MEP Hypercel column is adjusted to pH 6 with 20% (v/v) CH3COOH or 3 M Tris base and the pH-adjusted pool is diluted with deionized water to the same conductivity as 20 mM MES/NaOH, 2 mM CaCh, pH 6.0. The diluted pool is applied to an SP-Sepharose® Fast Flow column (GE Healthcare) equilibrated in 20 mM MES/NaOH, 2 mM CaCh, pH 6.0. After washing the column with the equilibration buffer, the protease variant is eluted with a linear NaCI gradient (0 0.5 M) in the same buffer over five column volumes. Fractions from the column are analyzed for protease activity using the Suc-AAPF-pNA assay at pH 9 and active fractions are analyzed by SDS-PAGE. Fractions in which only one band is observed on the Coomassie stained SDS-PAGE gel are pooled as the purified preparation and used for further experiments.
Protease Activity Assay I
The proteolytic activity of a variant of the invention can be determined by a method employing the Suc-AAPF-pNA substrate. Suc-AAPF-pNA is an abbreviation for N-Succinyl-Alanine- Alanine-Proline-Phenylalanine-p-Nitroanilide, and it is a blocked peptide which can be cleaved by endo-proteases. Following proteolytic cleavage, a free pNA molecule having a yellow color is liberated and can be measured by visible spectrophotometry at wavelength 405 nm. The Suc- AAPF-PNA substrate may be purchased from Bachem.
A sample containing the variant to be analyzed is diluted in residual activity buffer (100 mM Tris, pH 8.6). The assay is performed by transferring 30 pl of diluted enzyme samples to 96 well microtiter plate and adding 70 pl substrate working solution (0.72 mg/ml in 100 mM Tris, pH 8.6). The solution is mixed at room temperature and absorption at 405 nm is measured over time, e.g., every 20 sec. over 5 minutes. The slope (absorbance per minute) of the time-dependent absorption curve is directly proportional to proteolytic activity.
Protease Activity Assay II
The proteolytic activity of a detergent composition comprising a variant of the invention can be determined by a method employing N,N-dimethyl casein (DMC) as substrate. By hydrolysis of peptide bonds, carboxylic acids and primary amines are produced. The produced amines then react under alkaline conditions with 2,4,6-tri-nitrobenzene-sulphonic acid (TNBS, Sigma) to form a colored complex which can be measured at 405 nm.
A detergent sample containing a variant of the invention is dissolved in 0.08 M sodium sulfite buffer and stirred for 10 minutes, after which the sample is filtered (Whatman filter no. 54 or similar). Sample dilutions are made using buffer (0.05 M boric acid + 0.16 M sodium sulfite + 0.15 M potassium chloride + 0.0225% (w/v) Brij® L23, pH 9.00). Reagents, including 1) 3.2 g/L DMC substrate + 0.1 M sodium dihydrogen phosphate monohydrate + 0.07 M Borax + 0.02% (w/v) Brij® L23, pH 8.00, 2) 0.1% TNBS and 3) 0.1 % TNBS + 0.4% DSAA are employed in running the analysis using a Konelab 30 Analyzer (ThermoFisher Scientific) according to the assay parameters outlined in Table 19. Activity values may then be calculated based on a standard curve.
Automatic Mechanical Stress Assay (AMSA)
To assess wash performance in laundry, washing experiments are performed using the Automatic Mechanical Stress Assay (AMSA). With AMSA, the wash performance of a large quantity of small volume enzyme-detergent solutions can be examined. The AMSA plate has several slots for test solutions and a lid firmly squeezing the laundry sample, the textile to be washed (or melamine tile in the case of dishwashing detergents) against all the slot openings. During the washing time, the plate, test solutions, textile (or else tile for dishwash) and lid are vigorously shaken to bring the test solution into contact with the soiled test sample and to apply mechanical stress in a regular, periodic oscillating manner. For further description see WO 2002/42740, in particular the paragraph "Special method embodiments" at page 23-24.
The wash performance is measured as the brightness of the color of the textile washed. The brightness can be expressed as the intensity of the light reflected from the textile sample when illuminated with white light. When the textile is stained, the intensity of the reflected light is lower than that of a clean textile. Therefore, the intensity of the reflected light can be used to measure wash performance of the proteases of interest.
Color measurements are made with a professional flatbed scanner (Epson Expression 10000XL, Atea A/S, Lautrupvang 6, 2750 Ballerup, Denmark), which is used to capture an image of the washed textile sample. To extract a value for the light intensity from the scanned images, a specially designed software application is used {Novozymes Color Vector Analyzer). The program retrieves the values from the image and converts them into values for red, green, and blue (RGB). The intensity value (Int) may be calculated by adding the RGB values together as vectors and then taking the length of the resulting vector:
Int= r2 +g2 +b2
Terq-o-tometer (TOM) Wash Assay
The Terg-o-tometer (TOM) is a medium scale wash assay that can be applied to simultaneously test up to 16 different conditions at the same time. Briefly, it consists of 16 x 2 L metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial top-loader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker was filled with 1 L detergent solution, and test swatches, ballast and enzymes are added to the requisite levels. After a timed wash period, the swatches are promptly removed from the beakers and rinsed thoroughly with tap water.
The swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a Macbeth Color Eye 7000 reflectance spectrophotometer with large aperture. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.
The effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without enzyme (blank) from the swatch washed together with enzyme. The performance of a new protease (e.g., a protease variant) may be compared to the performance of a reference protease by calculating the relative performance (RP):
Full-Scale Wash
Full-scale washing machines (Panasonic XQB65-Q680U, Top Loader) are used to evaluate protease performance in large-scale. Detergent, swatches, ballast, water, and enzyme are added together and washed at a defined temperature, in this case for 15 minutes, followed by rinse. The level of detergent, water, enzymes, and ballast used may be adjusted based on different factors, such as regional habits. The swatches are then removed from the wash, and dried flat overnight at room temperature. Evaluation of FSW swatches is carried out in a similar manner as for TOM swatches.
Mini Terg-o-tometer (mini-TOM) Wash Assay and determination of relative strain removal rate
The Mini Terg-o-tometer (mini-TOM) is a medium scale wash assay that can be applied to simultaneously test up to 16 different conditions at the same time. Briefly, it consists of 16 x 0.2 L metal beakers, each fitted with an agitator, which rotate in a back-and-forth manner at a controlled speed to simulate the agitation occurring in commercial top-loader washing machines. The beakers are partly submerged in thermostatic water baths where the temperature can be controlled. Each beaker are filled with 0.15 L detergent solution, and test swatches and enzymes are added to the requisite levels. The swatches are promptly removed from the beakers at the desired time points and rinsed thoroughly with ice water to stop the hydrolysis of soil.
The swatches are then spread out flat on a rack covered with filter paper, covered, and allowed to dry overnight at room temperature. All washes are evaluated the day after the wash. Light reflectance evaluations of the swatches are done using a Macbeth Color Eye 7000 reflectance spectrophotometer with large aperture. The measurements are made without UV in the incident light and remission (REM) at 460 nm is extracted. Measurements are made on unwashed and washed swatches. The test swatch to be measured is placed on top of another swatch of the same type and color.
The effect of a protease on each swatch is calculated by subtracting the remission value of the swatch washed without enzyme (blank) from the swatch washed together with protease. The stain removal rate (SRR) of a new protease (e.g., a protease variant) may be compared to the stain removal rate of a reference protease by calculating the relative stain removal rate (RSRR) at at given time point: Example 1 : FSW Testing in an Asia Pacific Detergent at Low Temperature (15°C)
Wash performance of SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 was evaluated under the following conditions (Table 7):
Proteases were included in wash tests carried out in Asia Pacific top loader washing ma- chines using a model detergent and a range of stain types. At least six different swatches, representing at least five different stain categories (e.g., blood, blood milk ink, chocolate, grass, and egg) showed improvements in wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at low washing temperature (15 °C; see Table 8).
Example 2: FSW Testing in an Asia Pacific Detergent at Normal Temperature (25°C)
Wash performance of SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 was evaluated under the following conditions (Table 19):
Proteases were included in wash tests carried out in Asia Pacific top loader washing machines using a model detergent and a range of stain types. At least eight different swatches, representing at least four different stain categories (e.g., blood milk ink, chocolate, grass, and egg) showed improvements in wash performance with SEQ ID NO:9 compared to SEQ ID NO:1 and SEQ ID NO:8 at normal washing temperature (25 °C; see Table 10).
Example 3: TOM Wash Performance Evaluation after storage in a Laundry Soap Bar Matrix
Wash performance of SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 was evaluated under the following conditions (Table 11):
Protease formulations were incorporated into a basic laundry soap bar matrix and incubated at 37°C. After 4 weeks, the soap bars were evaluated for performance in a terg-o-tometer wash assay. In this assay, the soap samples were grated and washed together with seven differ- ent stain monitors, representing at least 4 different stain categories (e.g., blood milk ink, chocolate, grass, and egg). SEQ ID NO:9 showed improved wash performance compared to SEQ ID NO:1 and SEQ ID NO:8 for six out of seven stains tested (see Table 12). Example 4: TOM Wash Performance Evaluation after storage in a Low pH European Detergent
Wash performance of SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 13):
SEQ ID NO:8 and SEQ ID NO:9 were evaluated in a terg-o-tometer wash assay after storage for 24 hours at 25°C in a low pH European detergent. A total of seven swatches were used to evaluate the wash performance, representing at least four different stain categories (e.g., blood milk ink, chocolate, grass, and egg). SEQ ID NO:9 showed improved wash performance compared to SEQ ID NO:8 across all stains tested with SEQ ID NO:9 (see Table 14).
Example 5: TOM Wash Performance Evaluation in a High pH European Liquid Detergent
Wash performance of, SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 15):
Proteases were evaluated in a terg-o-tometer wash assay using a high pH European detergent. Results showed improved wash performance on selected stains with SEQ ID NO:9 compared to SEQ ID NO:8 (see Table 16). Example 6: AMSA Evaluation in a Model European Liquid Detergent
Wash performance of SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 was evaluated under the following conditions (Table 17):
SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 were tested using an AMSA wash assay, with results expressed in terms of relative performance to SEQ ID NO:1. Results showed improved wash performance of SEQ ID NO:9 across both stains tested (see Table 18).
Example 7: Storage Stability in High Water Liquid Detergent
Storage stability SEQ ID NO:8 and SEQ ID NO:9 was evaluated under the following conditions (Table 19):
SEQ ID N0:8 and SEQ ID N0:9 were incubated in a high-water liquid model detergent for 4 - 8 weeks at 30 or 37 °C, and then analyzed for protease activity. Residual protease activity was determined relative to protease activity present in the original non-incubated samples stored at -18 °C, expressed in %. The results show that SEQ ID NO:9 has improved storage stability in high water liquid detergent compared to SEQ ID NO:8 (see Table 20).
Example 8: Storage Stability and Mildness towards Companion Enzymes in Liquid Detergent
Storage stability and mildness of SEQ ID NO:8 and SEQ ID NO:9 was evaluated in com- mercially available liquid detergent (Bluemoon, China) under the following conditions (Table 21):
A commercially available alpha-amylase (Amplify Prime 100L, Novozymes A/S) was incubated together with either SEQ ID NO:8 orSEQ ID NO:9 for2 -4 weeks at 37 °C, and the samples were subsequently analyzed for protease and alpha-amylase activity. Residual protease activity was determined relative to protease activity present in the original non-incubated samples stored at -18 °C, expressed in %. The results show that SEQ ID NO:9 has improved storage stability in liquid detergent compared to SEQ ID NO:8 (see Table 22). Residual alpha-amylase activity was determined relative to amylase activity present in the original non-incubated samples stored at -18 °C, expressed in %. The results show that the alphaamylase has improved residual activity in presence of SEQ ID NO:9 compared to SEQ ID NO:8 (see Table 23). Thus, SEQ ID NO:9 has improved mildness towards companion enzymes, in particular alpha-amylases, compared to SEQ ID NO:8.
Example 9: Stain Removal Rate in Liquid Detergent
The difference in stain removal rate between by SEQ ID NO:1 , SEQ ID NO:8, and SEQ ID NO:9 was evaluated under the following conditions (Table 24): The stain removal rate of SEQ ID NO:9 is improved compared to SEQ ID NO:8 as well
SEQ ID NO:1 (see Table 25). SEQ ID NO:9 is relatively faster at soil removal compared to SEQ ID NO:8 as well as SEQ ID NO:1.
Example 10: AMSA Evaluation in Liquid Detergents
Wash performance of variants of SEQ ID NO:3 and SEQ ID NO:5 was evaluated under the conditions described in Table 26. The results are expressed in terms of relative performance compared to the parent protease. As can be seen from Table 27, SEQ ID NO: 10 exhibits a better wash performance than
SEQ ID NO:3.
As can be seen from Table 28, SEQ ID NO: 14 exhibits a better wash performance than SEQ ID NO:13 as well as SEQ ID NO:5.
Example 11 : Storage Stability in Liquid Detergent
Purified protease samples were diluted with 0.01% Triton X-100 to suitable concentrations based on absorbance at 280 nm (0.1 -0.4 mg/ml for variants of SEQ ID NO:1 and SEQ ID NO:3, 1 .3-5 mg/ml for variants of SEQ ID NO:5). 30 l of the protease dilutions were mixed with 270 pl concentrated Model O detergent (pH 8 or pH 10) in the wells of a microtiter plate using a magnetic bar. After mixing and sealing with a plate seal, the detergent plate was incubated at 45°C or 55°C in a Biosan PST-100HL thermomixer. Every sample was tested in two or three concentrations.
After various incubation times (e.g., 0, 1 , 4, 24, 48, 96, 192 and 264 hours), residual protease activity was measured. 20 pl sample from the detergent plate was mixed with 150 pl 0.1 M Tris buffer pH 8.6. 30 pl of this dilution was transferred to a microtiter plate followed by addition of 70 pl substrate solution (0.72 mg/ml Suc-Ala-Ala-Pro-Phe-pNA in 0.1 M Tris pH 8.6) and mixing, and absorbance at 405 nm was subsequently read every 10 seconds for 5 min on a BioTek Synergy H1 plate reader. Activity was determined from the slope of initial absorbance increase by linear regression.
The decrease in activity during incubation with detergent is assumed to be exponential. Half-lives (T%) were found from linear regression of logarithm of activity versus incubation time. Half-life improvement factors (T1 IF) were calculated as the ratio of the half-life of a protease variant relative to the half-life of the parent protease.
As seen in Table 29, SEQ ID NO:9 has improved storage stability compared to SEQ ID NO:8 as well as SEQ ID NO:1. As seen in Table 30, SEQ ID NO: 12 has improved storage stability compared to SEQ ID NO:11 as well as SEQ ID NO:3. In addition, SEQ ID NQ:10 has improved storage stability compared to SEQ ID NO:3.
As seen in Table 31 , SEQ ID NO: 14 has improved storage stability compared to SEQ ID NO:13 as well as SEQ ID NO:5.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control. The invention is further defined by the following numbered paragraphs:
1. A variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variant has protease activity.
2. The variant of paragraph 1 which comprises a substitution of the amino acid residue at a position corresponding to position 95 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Vai, preferably with Asp.
3. The variant according to any of the preceding paragraphs which comprises a substitution of the amino acid residue at a position corresponding to position 209 of SEQ ID NO:1 with Arg, Asp, Cys, Gin, Glu, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with Lys.
4. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 9 of SEQ ID NO:1 with Ala, Arg, Asn, Cys, Gin, Glu, Gly, His, lie, Leu, Met, Phe, Trp, Tyr, or Vai, preferably with Glu.
5. The variant according to any one of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 42 of SEQ ID NO:1 with Ala, Arg, Cys, Gin, Glu, His, lie, Leu, Met, Phe, Pro, Ser, Trp, Tyr, or Vai, preferably with Arg.
6. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 74 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Vai, preferably with Asp.
7. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 199 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably with lie.
8. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 200 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Trp, or Vai, preferably with Leu. 9. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 203 of SEQ ID NO:1 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Trp.
10. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 253 of SEQ ID NO:1 with Ala, Arg, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Trp, Tyr, or Vai, preferably with Asp.
11. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 255 of SEQ ID NO:1 with Ala, Arg, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Pro, Thr, Trp, Tyr, or Vai, preferably with Trp.
12. The variant according to any of the preceding paragraphs, which comprises a substitution of the amino acid residue at a position corresponding to position 256 of SEQ ID NO:1 with Ala, Arg, Asn, Cys, Glu, Gly, His, lie, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Vai, preferably with Glu.
13. The variant according to any of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least three substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1.
14. The variant according to any of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least four substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1.
15. The variant according to any of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least five substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1.
16. The variant according to any of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least six substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1.
17. The variant according to any of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least seven substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1. 18. The variant according to any one of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises at least eight substitutions at positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ I D NO: 1 .
19. The variant according to any one of the preceding paragraphs, which comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises nine substitutions at positions corresponding to each of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO: 1.
20. The variant according to any of the preceding paragraphs, wherein the total number of substitutions compared to the parent is 5-20, e.g., 5-15 or 5-10, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 substitutions.
21. The variant according to any of the preceding paragraphs, wherein the total number of substitutions compared to the parent is 5-11 , such as 5, 6, 7, 8, 9, 10, or 11 substitutions.
21a. The variant according to any of paragraphs 1-21 , wherein the parent protease is an otherwise identical protease without the substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and without the substitutions at at least three positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 .
22. The variant according to any of paragraphs 1-21 , wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:1.
23. The variant according to any of paragraphs 1-21 , wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:2.
24. The variant according to any of paragraphs 1-21 , wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:3.
25. The variant according to any of paragraphs 1-21 , wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:4. 26. The variant according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:5.
27. The variant according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:6.
28. The variant according to any of paragraphs 1-21, wherein the parent protease has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, to the amino acid sequence of SEQ ID NO:7.
29. The variant according to any of the preceding paragraphs, wherein the variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
30. The variant according to any of the preceding paragraphs, which comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
30. The variant according to any of the preceding paragraphs, which comprises further substitutions at one or more other positions corresponding to positions 60 (e.g., N60D), 97 (e.g., S97D), 99 (e.g., S99E), 116 (e.g., G116N), and 246 (e.g., N246L) of SEQ ID NO:1.
31. The variant according to any of the preceding paragraphs, which further comprises one or more substitutions selected from the group consisting of substitutions corresponding to N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.
32. The variant according to any of the preceding paragraphs, which has improved storage stability and/or improved wash performance compared to the parent protease.
32a. The variant according to any of the preceding paragraphs, which has improved mildness compared to the parent protease.
32b. The variant according to any of the precedings paragraphs, which has improved stain removal rate compared to the parent protease. 33. A fusion polypeptide comprising the variant of any of the preceding paragraphs and a second polypeptide.
34. The variant or fusion polypeptide of any of the preceding paragraphs, which is isolated.
35. The variant or fusion polypeptide of any of the preceding paragraphs, which is purified.
36. A granule, which comprises (a) a core comprising the variant or fusion polypeptide of any of paragraphs 1-35; and, optionally, (b) a coating consisting of one or more layer(s) surrounding the core.
37. A granule, which comprises: (a) a core; and (b)a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises the variant or fusion polypeptide of any one of paragraphs 1-35.
38. A liquid composition comprising the variant or fusion polypeptide of any of paragraphs 1- 35 and an enzyme stabilizer, e.g., a polyol such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid).
39. The liquid composition of paragraph 38, further comprising a filler or carrier material.
40. The liquid composition of paragraph 38 or 39, further comprising a preservative.
41. A composition comprising the variant or fusion polypeptide of any of paragraphs 1-35, the granule of paragraphs 36 or 37, or the liquid compositions of any of paragraphs 38-40.
42. A polynucleotide encoding the variant or fusion polypeptide of any one of paragraphs 1- 35.
43. The polynucleotide of paragraph 42, which is isolated.
44. The polynucleotide of paragraph 42 or 43, which is purified.
45. A nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 42-44.
46. A recombinant host cell transformed with the polynucleotide of any one of paragraphs 42- 44.
47. The recombinant host cell of paragraph 46, which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of any one of paragraphs 42-44.
48. The recombinant host cell of paragraph 46 or 47, which is a yeast recombinant host cell, e.g., 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.
49. The recombinant host cell of paragraph 46 or 47, which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Mag- naporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phan- erochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium luck- nowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queensland- icum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
50. The recombinant host cell of paragraph 46 or 47, which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amylo- liquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fir- mus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.
51 . The recombinant host cell of any one of paragraphs 46-50, which is isolated. 52. The recombinant host cell of any one of paragraphs 46-50, which is purified.
53. A method of producing a protease variant or fusion polypeptide, comprising (a) cultivating the host cell of any one of paragraphs 46-52 under conditions suitable for expression of the variant; and (b) recovering the variant.
54. A whole broth formulation or cell culture composition comprising the variant or fusion polypeptide of any of paragraphs 1-35.
55. A detergent composition comprising the variant of fusion polypeptide of any of paragraphs 1-35.
56. The detergent composition of paragraph 55 in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.
57. The detergent composition of paragraph 55 in the form of a liquid detergent, a powder detergent, or a laundry soap bar; preferably wherein the detergent composition is a liquid detergent or a laundry soap bar.
58. A method of cleaning an object, comprising contacting the object with a detergent composition according to any of paragraphs 55-57 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, dishware, or a hard surface; most preferably wherein the object is a fabric.
59. Use of a variant according to any of paragraphs 1 -35 or a detergent composition according to any of paragraphs 55-57 in a cleaning process, preferably laundry or hard surface cleaning such as automatic dishwashing (ADW).

Claims

1. A variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a TM-score of at least 0.80, e.g., at least 0.81 , at least 0.82, at least 0.83, at least 0.84, at least 0.85, at least 0.86, at least 0.87, at least 0.88, at least 0.89, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, at least 0.995, at least 0.999, but less than 1 .0, compared to the three-dimensional structure of the parent protease, wherein the three-dimensional structure is calculated using AlphaFold; and wherein the variant has protease activity.
2. A variant of a parent protease, wherein the variant comprises substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further comprises substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; wherein the variant has a sequence identity of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the parent protease; and wherein the variant has protease activity.
3. The variant according to any of claims 1-2, wherein the parent protease is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; preferably wherein the parent protease is SEQ ID NO:1.
4. The variant according to any of the preceding claims, wherein the variant comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprising at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, substitutions selected from the group consisting of substitutions corresponding to S9E, N42R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
5. The variant according to any of the preceding claims, which comprises substitutions corresponding to G95D and A209K of SEQ ID NO:1 and further comprises substitutions corresponding to S9E, N43R, N74D, V199I, Q200L, Y203W, S253D, N255W, and L256E of SEQ ID NO:1.
6. The variant according to any of the preceding claims, which further comprises a substitution at one or more positions selected from the group consisting of positions 60, 97, 99, 116, and 246 of SEQ ID NO:1.
7. The variant according to claim 5, which comprises one or more further substitutions selected from the group consisting of N60D, S97D, S97E, S99D, S99E, S99H, G116N, and N246L of SEQ ID NO:1.
8. The variant according any of the preceding claims, wherein the variant exhibits improved wash performance and/or improved storage stability compared to the parent protease.
9. A polynucleotide encoding a protease variant according to any of claims 1-8.
10. A nucleic acid construct or expression vector comprising a polynucleotide of claim 9.
11. A recombinant host cell comprising in its genome a nucleic acid construct or expression vector according to claim 10.
12. A method for obtaining a variant according to any of claims 1-8, comprising (a) introducing into a parent protease substitutions at positions corresponding to positions 95 and 209 of SEQ ID NO:1 and further introducing substitutions at at least three, e.g., at least four, at least five, at least six, at least seven, at least eight, or nine, positions corresponding to any of positions 9, 42, 74, 199, 200, 203, 253, 255, and 256 of SEQ ID NO:1 ; wherein position numbering is based on the numbering of SEQ ID NO:1 ; and (b) recovering the variant.
13. A method of producing a variant according to any of claims 1-8, comprising (a) cultivating the recombinant host cell of claim 11 under conditions suitable for expression of the variant; and (b) recovering the variant.
14. A detergent composition comprising a variant according to any of claims 1-8; preferably wherein the detergent composition is in the form of a bar, a homogeneous tablet, a tablet having two or more layers, a pouch having one or more compartments, a regular or compact powder, a granule, a paste, a gel, or a regular, compact, or concentrated liquid.
15. A method of cleaning an object, comprising contacting the object with a detergent composition according to claim 14 under conditions suitable for cleaning the object; preferably wherein the object is a fabric, dishware, or a hard surface; most preferably wherein the object is a fabric.
16. Use of a variant according to any of claims 1-8 or a detergent composition according to claim 14 in a cleaning process, preferably laundry or hard surface cleaning such as automatic dishwashing (ADW).
EP23817746.3A 2022-12-05 2023-12-04 Protease variants and polynucleotides encoding same Pending EP4630528A1 (en)

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