WO2024186819A1 - Variants de subtilisine et procédés d'utilisation - Google Patents

Variants de subtilisine et procédés d'utilisation Download PDF

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WO2024186819A1
WO2024186819A1 PCT/US2024/018525 US2024018525W WO2024186819A1 WO 2024186819 A1 WO2024186819 A1 WO 2024186819A1 US 2024018525 W US2024018525 W US 2024018525W WO 2024186819 A1 WO2024186819 A1 WO 2024186819A1
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Prior art keywords
variant
composition
subtilisin
amino acid
seq
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PCT/US2024/018525
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English (en)
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Viktor Yuryevich Alekseyev
Lilia BABE
Frits Goedegebuur
Thijs Kaper
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Danisco Us Inc.
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Publication of WO2024186819A1 publication Critical patent/WO2024186819A1/fr

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    • 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)
    • 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
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38672—Granulated or coated enzymes
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38681—Chemically modified or immobilised enzymes
    • 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

Definitions

  • subtilisin variant Disclosed herein is one or more subtilisin variant, nucleic acid encoding same, and compositions and methods related to the production and use thereof, including one or more subtilisin variant that has improved stability and/or soil removal compared to one or more reference subtilisin.
  • sequence listing is submitted electronically via Patent Center as an XML document formatted sequence listing with a file named “20240301_NB42051WOPCT_SequenceListing” created on March 1, 2024 and having a size of 13,124 bytes and is filed concurrently with the specification.
  • the sequence listing contained in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • a protease (also known as a proteinase) is an enzyme that has the abi li ty to break down other proteins.
  • a protease has the ability to conduct proteolysis, which begins protein catabolism by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein.
  • proteolytic activity' This activity' of a protease as a protein-digesting enzyme is termed a proteolytic activity'.
  • Many well-known procedures exist for measuring proteolytic activity Kalisz, "Microbial Proteinases," In: Fiechter (ed.), Advances in Biochemical Engineering/Biotechnology. (1988)).
  • proteolytic activity may be ascertained by comparative assays which analyze the respective protease’s ability' to hydrolyze a commercial substrate.
  • Exemplary' substrates useful in the analysis of protease or proteolytic activity include, but are not limited to, di-methyl casein (Sigma C-9801). bovine collagen (Sigma C-9879), bovine elastin (Sigma E-1625). and Keratin Azure (Sigma- Aldrich K8500). Colorimetric assays utilizing these substrates are well known in the art (see, e.g., WO 99/34011 and U.S. Pat. No. 6,376,450, both of which are incorporated herein by reference).
  • Serine proteases are enzymes (EC No. 3.4.21) possessing an active site serine that initiates hydrolysis of peptide bonds of proteins. Serine proteases comprise a diverse class of enzymes having a wide range of specificities and biological functions that are further divided based on their structure into chymotrypsin-like (trypsin-like) and subtilisin-like. The prototypical subtilisin (EC No. 3.4.21.62) was initially obtained from Bacillus subtilis. Subtilisins and their homologues are members of the S8 peptidase family of the MEROPS classification scheme (Rawlings, N.D. et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors.
  • One embodiment is directed to a subtilisin variant comprising two or more amino acid substitutions at positions selected from the group consisting of 99, 1 14, 151, 154, 156, 224, 253, where the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1.
  • subtilisin variants have at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8 and comprise two or more amino acid substitutions selected from the group consisting of X099R, X114L, XI 5 IT, X154N, X156S, X224V, X253D, where the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1.
  • Such variants may further comprise one or more additional substitutions selected from the group consisting of XI 16V, X126L, X127Q, and X128A.
  • subtilisin variants comprising two or more amino acid substitutions selected from the group consisting of S099R, N114L, S151T, S154N, A156S, A224V, S253D, and N253D are provided, where the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, and where the subtilisin variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.
  • subtilisin variants comprising two or more amino acid substitutions further comprise: (i) two or more amino acid substitutions at positions selected from the group consisting of 99, 114, 151, 154, 156, 224, 253 and one or more amino acid substitutions at a position selected from the group consisting of 116, 126, 127, 128 and one or more additional amino acid substitutions at a position selected from the group consisting of 39, 198, 209.
  • subtilisin variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8; (ii) two or more amino acid substitutions selected from the group consisting of X099R, XI 14L, X151T, X154N, X156S, X224V, X253D, and one or more amino acid substitutions selected from the group consisting of XI 16V, X126L, X127Q, X128A, and one or more additional amino acid substitutions selected from the group consisting of X039E, X198G, X209V, X21 IQ, X212Q, and X242D; (iii) two or more amino acid substitutions selected from the group consisting of S099R, N114L, S151T, S154N, A156S, A224V, S253D, and N253D and one or more amino acid substitutions selected from the group consisting of G
  • S 128A and one or more additional amino acid substitutions selected from the group consisting of P039E, N198G, A209V, L21 IQ, N212Q, and N242D; where the positions are numbered according to SEQ ID NO: 1, and where the subtilisin variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.
  • Still other embodiments are directed to a method for producing a variant described herein, comprising stably transforming a host cell with an expression vector or an expression cassette comprising a polynucleotide encoding one or more subtilisin variant described herein. Still further embodiments are directed to a polynucleotide comprising a nucleic acid sequence encoding one or more subtilisin variant described herein.
  • the present disclosure provides one or more subtilisin variant comprising one or more amino acid substitutions as described in more detail below.
  • the variants provided herein demonstrate one or more improved properties, such as an improved cleaning performance, or improved stability, or both an improved cleaning performance and an improved stability when compared to a subtilisin having the amino acid sequence of SEQ ID NO: 1.
  • the subtilisin variants provided herein find use in the preparation of cleaning compositions (e.g. automatic dishwashing compositions).
  • the subtilisin variants provided herein also find use in methods of cleaning (e.g. laundry, dish washing methods) using such variants or compositions comprising such subtilisin variants.
  • subtilisin variant described herein can be made and used by a variety of techniques used in molecular biology, microbiology, protein purification, protein engineering, protein and DNA sequencing, recombinant DNA fields, and industrial enzyme use and development. Terms and abbreviations not defined should be accorded their ordinary' meaning as used in the art. Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any definitions provided herein are to be interpreted in the context of the specification as a whole. As used herein, the singular “a,” “an” and “the” includes the plural unless the context clearly indicates otherwise.
  • nucleic acid sequences are written left to right in 5' to 3' orientation; and amino acid sequences are written left to right in amino to carboxy orientation.
  • Each numerical range used herein includes every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
  • amino acid substitutions of the one or more subtilisin variants described herein uses one or more of the following: position; position: amino acid substitution(s); or starting amino acid(s):position:amino acid substitution(s).
  • Reference to a “position” e g. 5, 8, 17, 22, etc) encompasses any starting amino acid that may be present at such position, and any substitution that may be present at such position.
  • Reference to a “position: amino acid substitution(s)” e g. 1S/T/G, 3G, 17T, etc) encompasses any starting amino acid that may be present at such position and the one or more amino acid(s) with which such starting amino acid may be substituted.
  • Reference to a position can be recited in several forms, for example, position 003 can also be referred to as position 03 or 3.
  • Reference to a starting or a substituted amino acid may be further expressed as several starting or substituted amino acids separated by a foreslash (“/”).
  • D275S/K indicates position 275 is substituted with serine (S) or lysine (K)
  • P/S197K indicates that starting amino acid proline (P) or serine (S) at position 197 is substituted with lysine (K).
  • Reference to an X, as the amino acid in a position refers to any amino acid at the recited position.
  • the position of an amino acid residue in a given amino acid sequence is numbered by correspondence with the amino acid sequence of SEQ ID NO: 1. That is, the amino acid sequence of SEQ ID NO: 1 serves as a reference sequence for numbering of positions of an amino acid residue.
  • the amino acid sequence of one or more subtilisin variant described herein is aligned with the amino acid sequence of SEQ ID NO:1 using an alignment algorithm as described herein, and each amino acid residue in the given amino acid sequence that aligns (preferably optimally aligns) with an amino acid residue in SEQ ID NO: 1 is conveniently numbered by reference to the numerical position of that corresponding amino acid residue.
  • Sequence alignment algorithms such as, for example, described herein, will identify the location or locations where insertions or deletions occur in a subject sequence when compared to a query sequence (also sometimes referred to as a '‘reference sequence”). Sequence alignment with other subtilisin amino acid sequences can be determined using an amino acid alignment, for example, as provided in Figure 1 of PCT Application No. PCT/US2018/062768, filed November 28, 2018, claiming priority to U.S. Provisional Application No. 62/591.976, filed November 29, 2017. entitled “Highly Stable Subtilisin Enzymes”.
  • protease refers to an enzyme that has the ability to break down proteins and peptides.
  • a protease has the ability to conduct “proteolysis,” by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain forming the protein.
  • proteolytic activity This activity of a protease as a protein-digesting enzyme is referred to as “proteolytic activity.”
  • proteolytic activity may be ascertained by comparative assays that analyze the respective protease’s ability to hydrolyze a suitable substrate.
  • Exemplary substrates useful in the analysis of protease or proteolytic activity include, but are not limited to.
  • This assay measures the rate at which p-nitroaniline is released as the enzyme hydrolyzes a soluble synthetic substrate, such as succinyl-alanine-alanine- proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA).
  • a soluble synthetic substrate such as succinyl-alanine-alanine- proline-phenylalanine-p-nitroanilide (suc-AAPF-pNA).
  • the rate of production of yellow color from the hydrolysis reaction is measured at 405 or 410 nm on a spectrophotometer and is proportional to the active enzyme concentration.
  • absorbance measurements at 280 nanometers (nm) can be used to determine the total protein concentration in a sample of purified protein. The activity on substrate divided by protein concentration gives the enzyme specific activity.
  • the genus Bacillus includes all species within the genus “Bacillus,” as know n to those of skill in the art, including but not limited to B. subtilis. B. licheniformis.
  • B. lentus B. brevis, B. stearothermophilus, B. alkalophilus, B. amylo
  • stearothermophilus which is now named “Geobacillus stearothermophilus”, or B. polymyxa, which is now “Paenibacillus polymyxa”.
  • the production of resistant endospores under stressful environmental conditions is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacilhis , Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermob acillus. Ureibacillus, Alkalihalobacillus, Peribacillus.
  • a “B. lentus subtilisin’’ includes any subtilisin obtained from, or derived from, a B. lentus (e.g. Lederbergia lentus) source, including P29600.
  • the present invention provides “GG36 variants” (or “P29600 variants” or “GG36 subtilisin variants”) wherein the mutations are present in the mature GG36 amino acid sequence set forth in SEQ ID NO: 1.
  • the B. lentus subtilisins and variants thereof include those polypeptides having an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1.
  • the subtilisin variants herein include any variant subtilisin obtained from or derived from a B. patagoniensis (Alkalihalobacillus patagoniensis) source, including Bpan01744 (described in WO201669563, W02020112559).
  • the subtilisin variants provided herein include those polypeptides having at least 80% sequence identity to SEQ ID NO: 8.
  • vector refers to a nucleic acid construct used to introduce or transfer nucleic acid(s) into a target cell or tissue.
  • a vector is ty pically used to introduce foreign DNA into a cell or tissue.
  • Vectors include plasmids, cloning vectors, bacteriophages, viruses (e.g., viral vector), cosmids, expression vectors, shuttle vectors, and the like.
  • a vector typically includes an origin of replication, a multicloning site, and a selectable marker. The process of inserting a vector into a target cell is typically referred to as transformation.
  • the term ‘'introduced’’ refers to any method suitable for transferring the nucleic acid sequence into the cell. Such methods for introduction include but are not limited to protoplast fusion, transfection, transformation, electroporation, conjugation, and transduction. Transformation refers to the genetic alteration of a cell which results from the uptake, optional genomic incorporation, and expression of genetic material (e.g., DNA).
  • '‘expression” refers to the transcription and stable accumulation of sense (mRNA) or anti-sense RNA, derived from a nucleic acid molecule of the disclosure.
  • Expression may also refer to translation of mRNA into a polypeptide.
  • expression includes any step involved in the “production of the polypeptide” including, but not limited to, transcription, post-transcriptional modifications, translation, post-translational modifications, secretion and the like.
  • expression cassette refers to a nucleic acid construct or vector generated recombinantly or synthetically for the expression of a nucleic acid of interest (e.g.. a foreign nucleic acid or transgene) in a target cell.
  • the nucleic acid of interest typically expresses a protein of interest.
  • An expression vector or expression cassette typically comprises a promoter nucleotide sequence that drives or promotes expression of the nucleic acid of interest (e.g. a foreign nucleic acid).
  • the expression vector or cassette also typically includes other specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell.
  • a recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
  • Some expression vectors have the ability to incorporate and express heterologous DNA fragments in a host cell or genome of the host cell.
  • Many prokaryotic and eukaryotic expression vectors are commercially available. Selection of appropriate expression vectors and cassette for expression of a protein from a nucleic acid sequence incorporated into the expression vector is within the knowledge of those of skill in the art.
  • a nucleic acid is “operably linked” with another nucleic acid sequence when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a nucleotide coding sequence if the promoter affects the transcription of the coding sequence.
  • a ribosome binding site may be operably linked to a coding sequence if it is positioned so as to facilitate translation of the coding sequence.
  • “operably linked” DNA sequences are contiguous. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers may be used in accordance with conventional practice.
  • '‘gene” refers to a polynucleotide (e.g., a DNA segment), that encodes a polypeptide and includes regions preceding and following the coding regions. In some instances, a gene includes intervening sequences (introns) between individual coding segments (exons).
  • recombinant when used with reference to a cell, typically indicates that the cell has been modified by the introduction of a foreign nucleic acid sequence or that the cell is derived from a cell so modified.
  • a recombinant cell may comprise a gene not found in identical form within the native (non-recombinant) form of the cell, or a recombinant cell may comprise a native gene (found in the native form of the cell) that has been modified and re-introduced into the cell.
  • a recombinant cell may comprise a nucleic acid endogenous to the cell that has been modified without removing the nucleic acid from the cell; such modifications include those obtained by gene replacement, site-specific mutation, and related techniques known to those of ordinary skill in the art.
  • Recombinant DNA technology includes techniques for the production of recombinant DNA in vitro and transfer of the recombinant DNA into cells where it may be expressed or propagated, thereby producing a recombinant polypeptide. “Recombination” and “recombining” of polynucleotides or nucleic acids refer generally to the assembly or combining of two or more nucleic acid or polynucleotide strands or fragments to generate a new polynucleotide or nucleic acid.
  • a nucleic acid or polynucleotide is said to “encode” a polypeptide if, in its native state or w hen manipulated by methods know n to those of skill in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof.
  • the anti-sense strand of such a nucleic acid is also said to encode the sequence.
  • host strain and “host cell” refer to a suitable host for an expression vector or expression cassette comprising a DNA sequence of interest.
  • a “protein” or “polypeptide” comprises a polymeric sequence of amino acid residues.
  • the terms “protein” and “polypeptide” are used interchangeably herein.
  • the single and 3-letter code for amino acids as defined in conformity with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) is used throughout this disclosure.
  • the single letter X refers to any of the tw enty' amino acids. It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
  • propeptide sequence refers to an amino acid sequence between the signal peptide sequence and mature protease sequence that is necessary for the proper folding and secretion of the protease; they are sometimes referred to as intramolecular chaperones. Cleavage of the prosequence or propeptide sequence results in a mature active protease. Bacterial serine proteases are often expressed as pro-enzymes. Examples of modified propeptides are provided, for example, in WO 2016/205710.
  • signal sequence and “signal peptide” refer to a sequence of amino acid residues that may participate in the secretion or direct transport of the mature or precursor form of a protein.
  • the signal sequence is typically located N-terminal to the precursor or mature protein sequence.
  • the signal sequence may be endogenous or exogenous.
  • a signal sequence is normally absent from the mature protein.
  • a signal sequence is typically cleaved from the protein by a signal peptidase after the protein is transported.
  • mature form of a protein, polypeptide, or peptide refers to the functional form of the protein, polypeptide, or peptide without the signal peptide sequence and propeptide sequence.
  • precursor form of a protein or peptide refers to a immature form of the protein having a prosequence operably linked to the amino or carboxyl terminus of the protein.
  • the precursor may also have a “signal” sequence operably linked to the amino terminus of the prosequence.
  • the precursor may comprise additional amino acids between the signal and propeptide regions to facilitate protein production.
  • the precursor may also have additional polypeptides that are involved in post-translational activity (e.g., polypeptides cleaved therefrom to leave the mature form of a protein or peptide).
  • wildtype refers to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions.
  • wildtype refers to a naturally-occurring polynucleotide that does not include a man-made substitution, insertion, or deletion at one or more nucleotides.
  • a polynucleotide encoding a wildtype polypeptide is, however, not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wildtype or parental polypeptide.
  • parent includes reference to a naturally- occurring, or wildtype, polypeptide or to a naturally -occurring polypeptide in which a manmade substitution, insertion, or deletion at one or more amino acid positions has been made.
  • the term “parent” with respect to a polypeptide also includes any polypeptide that has protease activity that serves as the starting polypeptide for alteration, such as substitutions, additions, and/or deletions, to result in a variant having one or more alterations in comparison to the starting polypeptide. That is, a parental, or reference polypeptide is not limited to a naturally -occurring wildtype polypeptide, and encompasses any wildtype, parental, or reference polypeptide.
  • the term “parent,” with respect to a polynucleotide can refer to a naturally -occurring polynucleotide or to a polynucleotide that does include a manmade substitution, insertion, or deletion at one or more nucleotides.
  • the term “parent” with respect to a polynucleotide also includes any polynucleotide that encodes a polypeptide having protease activity that serves as the starting polynucleotide for alteration to result in a variant protease having a modification, such as substitutions, additions, and/or deletions, in comparison to the starting polynucleotide.
  • a polynucleotide encoding a wildtype, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wildtype, parental, or reference polypeptide.
  • the parent polypeptide herein comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.
  • Naturally-occurring refers to. for example, a sequence and residues contained therein (e g., polypeptide sequence and amino acids contained therein or nucleotide sequence and nucleotides contained therein) that are found in nature.
  • non-naturally occurring refers to, for example, a sequence and residues contained therein (e.g, polypeptide sequences and amino acids contained therein or nucleotide sequence and nucleic acids contained therein) that are not found in nature.
  • corresponding to or “corresponds to” or “corresponds” refers to an amino acid residue at the enumerated position in a protein or peptide, or an amino acid residue that is analogous, homologous, or equivalent to an enumerated residue in a protein or peptide.
  • corresponding region generally refers to an analogous position in a related protein or a reference protein.
  • the terms “derived from” and “obtained from” refer to not only a protein produced or producible by a strain of the organism in question, but also a protein encoded by a DNA sequence isolated from such strain and produced in a host organism containing such DNA sequence. Additionally, the term refers to a protein which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protein in question.
  • proteases derived from Bacillus refers to those enzymes having proteolytic activity that are naturally produced by Bacillus, as well as to serine proteases like those produced by Bacillus sources but which through the use of genetic engineering techniques are produced by other host cells transformed with a nucleic acid encoding the serine proteases.
  • nucleotide or polypeptide sequences refers to the nucleotides or amino acids in the two sequences that are the same when aligned for maximum correspondence, as measured using sequence comparison or analysis algorithms described below and known in the art.
  • % identity or “percent identity” or “PID” refer to protein sequence identity. Percent identity may be determined using standard techniques known in the art. The percent amino acid identity shared by sequences of interest can be determined by aligning the sequences to directly compare the sequence information, e.g., by using a program such as BLAST, MUSCLE, or CLUSTAL.
  • the BLAST algorithm is described, for example, in Altschul et al., J Mol Biol, 215:403-410 (1990) and Karlin et al., Proc Natl Acad Sci USA, 90:5873-5787 (1993).
  • a percent (%) amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the “reference” sequence including any gaps created by the program for optimal/maximum alignment.
  • BLAST algorithms refer to the “reference” sequence as the “query” sequence.
  • homologous proteins or “homologous proteases” refers to proteins that have distinct similarity in primary, secondary', and/or tertiary' structure. Protein homology can refer to the similarity in linear amino acid sequence when proteins are aligned. Homology can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, MUSCLE, or CLUSTAL. Homologous search of protein sequences can be done using BLASTP and PSI-BLAST from NCBI BLAST with threshold (E-value cut-off) at 0.001.
  • protein clan is commonly used for protease superfamilies based on the MEROPS protease classification system.
  • subtilisin includes any member of the S8 serine protease family as described in MEROPS - The Peptidase Data base (Rawlings, N.D., et al (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res 44, D343-D350).
  • the CLUSTAL W algorithm is another example of a sequence alignment algorithm (See, Thompson et al., Nucleic Acids Res, 22:4673-4680, 1994).
  • deletions occurring at either terminus are included.
  • a variant with a five amino acid deletion at either terminus (or within the polypeptide) of a polypeptide of 500 amino acids would have a percent sequence identity of 99% (495/500 identical residues x 100) relative to the 'reference" polypeptide.
  • Such a variant would be encompassed by a variant having “at least 99% sequence identity” to the polypeptide.
  • a nucleic acid or polynucleotide is “isolated” when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, cells, etc.
  • a polypeptide, protein or peptide is “isolated” when it is at least partially or completely separated from other components, including but not limited to, for example, other proteins, nucleic acids, cells, etc.
  • an isolated species is more abundant than are other species in a composition.
  • an isolated species may comprise at least about 60%, about 65%, about 70%.
  • the species of interest is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods).
  • Purity and homogeneity can be determined using a number of techniques well known in the art, such as agarose or polyacrylamide gel electrophoresis of a nucleic acid or a protein sample, respectively, followed by visualization upon staining. If desired, a high-resolution technique, such as high performance liquid chromatography (HPLC) or a similar means can be utilized for purification of the material.
  • HPLC high performance liquid chromatography
  • nucleic acids or polypeptides generally denotes a nucleic acid or polypeptide that is essentially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or polynucleotide forms a discrete band in an electrophoretic gel, chromatographic eluate, and/or a media subjected to density gradient centrifugation).
  • a nucleic acid or polypeptide that gives rise to essentially one band in an electrophoretic gel is “purified.”
  • a purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%.
  • 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.
  • enriched refers to a compound, polypeptide, 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 in a starting composition.
  • '‘dish” refers to all forms of dishware, including all forms of crockery such as plates, cups, glasses, bowls, etc; all forms of cutlery such as spoons, knives, forks, and serving utensils; all forms of ceramics; all forms of plastics, such as melamine; and all metals, china, glass, and acrylics.
  • cleaning activity refers to a cleaning performance achieved by a serine protease polypeptide, variant, or reference subtilisin under conditions prevailing during the proteolytic, hydrolyzing, cleaning, or other process of the disclosure.
  • cleaning performance of a serine protease or reference subtilisin may be determined by using various assays for cleaning one or more enzyme sensitive stain on an item or surface (e.g., a stain resulting from food, grass, blood, ink, milk, oil, and/or egg protein).
  • Cleaning performance of one or more subtilisin variant described herein or reference subtilisin can be determined by subjecting the stain on the item or surface to standard wash condition(s) and assessing the degree to which the stain is removed by using various chromatographic, spectrophotometric, or other quantitative methodologies.
  • Exemplary cleaning assays and methods are known in the art and include but are not limited to those described in WO99/34011 and US 6,605,458, as well as those cleaning assays and methods included in the Examples provided below.
  • the term “effective amount” of one or more subtilisin variant described herein or reference subtilisin refers to the amount of protease that achieves a desired level of enzymatic activity in a specific cleaning composition. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular protease used, the cleaning application, the specific composition of the cleaning composition, and whether a liquid or dry (e.g., granular, tablet, bar) composition is required, etc.
  • the term “adjunct material” refers to any liquid, solid, or gaseous material included in cleaning composition other than one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof.
  • the cleaning compositions of the present disclosure include one or more cleaning adjunct materials.
  • Each cleaning adjunct material is typically selected depending on the particular type and form of cleaning composition (e.g., liquid, granule, powder, bar, paste, spray, tablet, gel, foam, or other composition).
  • each cleaning adjunct material is compatible with the protease enzyme used in the composition.
  • Cleaning compositions and cleaning formulations include any composition that is suited for cleaning, bleaching, disinfecting, and/or sterilizing any obj ect, item, and/or surface.
  • Such compositions and formulations include, but are not limited to. for example, liquid and/or solid compositions, including cleaning or detergent compositions (e.g., liquid, tablet, gel, bar, granule, and/or solid laundry cleaning or detergent compositions and fine fabric detergent compositions); hard surface cleaning compositions and formulations, such as for glass, wood, ceramic and metal counter tops and windows; carpet cleaners; oven cleaners; fabric fresheners; fabric softeners; and textile, laundry booster cleaning or detergent compositions, laundry additive cleaning compositions, and laundry pre-spotter cleaning compositions; dishwashing compositions, including hand or manual dishwashing compositions (e.g., “hand’' or “manual” dishwashing detergents) and automatic dishwashing compositions (e.g.. “automatic dishwashing detergents”).
  • Single dosage unit forms also find use with the
  • Cleaning composition or cleaning formulations include, unless otherwise indicated, granular or powder-form all-purpose or heavy-duty’ washing agents, especially cleaning detergents; liquid, granular, gel. solid, tablet, paste, or unit dosage form all-purpose washing agents, especially the so-called heavy-duty liquid (HDL) detergent or heavy-duty dry’ (HDD) detergent types; liquid fine-fabric detergents; hand or manual dishwashing agents, including those of the high-foaming type; hand or manual dishwashing, automatic dishwashing, or dishware or tableware washing agents, including the various tablet, powder, solid, granular, liquid, gel, and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, including antibacterial hand-wash types, cleaning bars, mouthwashes, denture cleaners, car shampoos, carpet shampoos, bathroom cleaners; hair shampoos and/or hair-rinses for humans and other animals; shower gels and foam baths and metal cleaners; as well as cleaning auxiliaries, such as bleach additive
  • HDL heavy-duty liquid
  • detergent composition or “detergent formulation” is used in reference to a composition intended for use in a wash medium for the cleaning of soiled or dirty objects, including particular fabric and/or non-fabric objects or items.
  • the detergents of the disclosure comprise one or more subtilisin variant described herein and, in addition, one or more surfactants, transferase(s), hydrolytic enzymes, oxido reductases, builders (e.g., a builder salt), bleaching agents, bleach activators, bluing agents, fluorescent dyes, caking inhibitors, masking agents, enzyme stabilizers, calcium, enzyme activators.
  • Microorganisms may be used as the only biologically active ingredient, but they may also be used in conjunction with one or more of the enzymes described herein.
  • a Bacillus strain having the deposit accession number PTA- 7543, for example, may be used to reduce malodor as described in WO 2012/112718 and WO2017/157775.
  • Exemplary commercial microbial products include but are not limited to MicroviaTM (Novozymes).
  • a builder salt is a mixture of a silicate salt and a phosphate salt, preferably with more silicate (e.g.. sodium metasilicate) than phosphate (e.g.. sodium tripolyphosphate).
  • silicate e.g.. sodium metasilicate
  • phosphate e.g. sodium tripolyphosphate
  • Some embodiments are directed to cleaning compositions or detergent compositions that do not contain any phosphate (e.g., phosphate salt or phosphate builder).
  • composition(s) substantially -free of boron or “detergent(s) substantially -free of boron 7 ’ refers to composition(s) or detergent(s), respectively, that contain trace amounts of boron, for example, less than about 1000 ppm (Img/kg or liter equals 1 ppm), less than about 100 ppm, less than about 50 ppm, less than about 10 ppm, or less than about 5 ppm, or less than about 1 ppm, perhaps from other compositions or detergent constituents.
  • ppm Img/kg or liter equals 1 ppm
  • bleaching refers to the treatment of a material (e.g., fabric, laundry, pulp, etc.) or surface for a sufficient length of time and/or under appropriate pH and/or temperature conditions to effect a brightening (i.e., whitening) and/or cleaning of the material.
  • chemicals suitable for bleaching include, but are not limited to, for example, CIO2, H2O2, peracids, NO2, etc.
  • Bleaching agents also include enzymatic bleaching agents such as perhydrolase and arylesterases.
  • Another embodiment is directed to a composition comprising one or more subtilisin variant described herein, and one or more perhydrolase, such as, for example, is described in W02005/056782, W02007/106293, WO 2008/063400, W02008/106214. and W02008/106215.
  • wash performance of a protease (e.g., one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof) refers to the contribution of one or more subtilisin variant described herein to washing that provides additional cleaning performance to the detergent as compared to the detergent without the addition of the one or more subtilisin variant described herein to the composition. Wash performance is compared under relevant washing conditions.
  • a protease e.g., one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof
  • condition(s) typical for household application in a certain market segment e.g., hand or manual dishwashing, automatic dishwashing, dishware cleaning, tableware cleaning, fabric cleaning, etc.
  • condition(s) typical for household application in a certain market segment e.g., hand or manual dishwashing, automatic dishwashing, dishware cleaning, tableware cleaning, fabric cleaning, etc.
  • relevant washing conditions 7 is used herein to indicate the conditions, particularly washing temperature, time, washing mechanics, sud concentration, type of detergent and water hardness, actually used in households in a hand dishw ashing, automatic dishw ashing, or laundry detergent market segment.
  • the term “dish wash” refers to both household and industrial dish w ashing and relates to both automatic dish washing (e.g. in a dishwashing machine) and manual dishwashing (e.g. by hand).
  • defects refers to the removal of contaminants from the surfaces, as well as the inhibition or killing of microbes on the surfaces of items.
  • inorganic filler salts are conventional ingredients of detergent compositions in powder form.
  • the filler salts are present in substantial amounts, typically about 17 to about 35% by weight of the total composition.
  • the filler salt is present in amounts not exceeding about 15% of the total composition.
  • the filler salt is present in amounts that do not exceed about 10%, or more preferably, about 5%, by w eight of the composition.
  • the inorganic filler salts are selected from the alkali and alkaline-earth-metal salts of sulfates and chlorides.
  • the filler salt is sodium sulfate.
  • subtilisin variant useful for cleaning applications and in methods of cleaning, as well as in a variety of industrial applications. Also disclosed herein is one or more isolated, recombinant, substantially pure, or non-naturally occurring subtilisin variant. In some embodiments, one or more subtilisin variant described herein is useful in cleaning applications and can be incorporated into cleaning compositions that are useful in methods of cleaning an item or a surface in need thereof.
  • subtilisin variants are provided, where the variant comprises two, three or more amino acid substitutions at a position selected from the group consisting of 99, 114, 151, 154, 156, 224, and 253, where the positions are numbered according to SEQ ID NO: 1, and where the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.
  • the subtilisin variants may further comprise one or more additional substitutions at a position selected from the group consisting of 116, 126, 127, and 128, where the positions are numbered according to SEQ ID NO: 1.
  • subtilisin variants are provided, where the variant comprises two or more substitutions selected from the group consisting of X099R, XI 14L, X151T, X154N, X156S, X224V, and X253D wherein the positions are numbered according to SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or 8.
  • subtilisin variants are provided, where the variant comprises two, three, four, five, or more amino acid substitutions at a position selected from the group consisting of-S099R.
  • subtilisin variants further comprise one or more additional substitutions selected from the group consisting of: (i) a substitution at position 116, preferably XI 16V; (li) a substitution at position 126. preferably X126L; (iii) a substitution at position 127, preferably X127Q; (iv) a substitution at position 128, preferably X128A; and wherein the positions are numbered according to SEQ ID NO: 1, and wherein the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or 8, where the amino acid positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1.
  • subtilisin variants are provided, where the variant further comprises one or more additional substitutions selected from X039E, X198G, X209V, X21 IQ, X212Q, X242D wherein the positions are numbered according to SEQ ID NO: 1, and wherein the variant has at least 80% identity to the ammo acid sequence of SEQ ID NO: 1 or 8.
  • subtilisin variants are provided, where the variant comprises combinations of amino acid substitutions selected from: X099R-X114L. X099R- X151T, X099R-X154N, X099R-X156S, X099R-X224V, X099R-X253D, X114L-X151T, X114L-X154N, X114L-X156S, X114L-X224V, X114L-X253D, X151T-X154N, X151T- X156S, X151T-X224V, X151T-X253D, X154N-X156S, X154N-X224V, X154N-X253D, X156S-X224V, X156S-X253D, and X224V-X253D, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO: 1, wherein the positions are numbered by
  • subtilisin variants are provided, where the variant comprises combinations of amino acid substitutions selected from: S099R-N114L, S099R- S151T, S099R-S154N, S099R-A156S, S099R-A224V, S099R-S253D, S099R-N253D, N114L-S 15 IT.
  • the variants further comprise one of more substitutions selected from XI 16V, X126L, X127Q, and X128A.
  • subtilisin variants are provided, where the variant comprises combinations of amino acid substitutions selected from: S099R-G116V-S 126L- P127Q-S 128 A-S 151 T-Y203 W-A209V-L211 Q-N212Q; S099R-N114L-G116V-S 126L- P 127Q-S 128 A-A209V-L211 Q-N212Q-N242D; S099R-G116V-S 126L-P 127Q-S 128A- S 151 T-L211 Q-N212Q; P039E-S099R-G116V-S 126L-P 127Q-S 128A-N 198G-N212Q- A224V, P039E-S099R-G1 16V-S126L-P127Q-S128A-N198G-A224V, S099R-G116V- S 126L-P 127Q-S
  • Another embodiment is directed to one or more subtilisin variant described herein with the proviso that one or more substitutions is non-naturally occurring.
  • Yet an even still further embodiment is directed to one or more subtilisin variant described herein wherein said variant (i) is derived from a B. lentus subtilisin; (ii) is isolated; (iii) has proteolytic activity; or (iv) comprises a combination of (i) to (iii).
  • Still yet another embodiment is directed to one or more subtilisin variant described herein, wherein said variant is derived from a parent or reference polypeptide with (i) 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8; or (ii) 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.
  • the parent comprises the amino acid sequence of SEQ ID NO: 1.
  • An even further embodiment is directed to one or more subtilisin variant described herein, wherein said variant comprises an amino acid sequence with (i) 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8; (ii) 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%.
  • the term “enhanced stability” or “improved stabi 1 i ty” in the context of an oxidation, chelator, denaturant, surfactant, thermal and/or pH stable protease refers to a higher retained proteolytic activity over time as compared to a reference protease, for example, a wild-type protease or parent protease.
  • Autolysis has been identified as one mode of subtilisin activity loss in liquid detergents. (Stoner et al., 2004 Protease autolysis in heavy-duty liquid detergent formulations: effects of thermodynamic stabilizers and protease inhibitors, Enzyme and Microbial Technology 34:114-125.).
  • thermoally stable and “thermostable” and “thermostability” with regard to a protease variant refer to a protease that retains a greater amount of residual activity when compared to the parent or reference protease after exposure to altered temperatures over a given period of time under conditions (or “stress conditions”) prevailing during proteolytic, hydrolysing, cleaning or other process. Residual activity' is the amount of activity remaining after the test compared to the initial activity' of the sample and can be reported as a percentage, e g. % remaining activity. “Altered temperatures” encompass increased or decreased temperatures.
  • the variant proteases provided herein retain at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 92%, about 95%, about 96%, about 97%. about 98%, or about 99% proteolytic activity after exposure to temperatures of 40°C to 80°C.
  • subtilisin variants may be used in the production of various compositions, such as enzyme compositions and cleaning or detergent compositions.
  • An enzyme composition comprises a subtilisin variant as provided herein.
  • the enzyme composition can be in any form, such as granule, liquid formulations, or enzyme slurries.
  • Enzyme granules may be made by, e.g., rotary' atomization, wet granulation, dry' granulation, spray drying, disc granulation, extrusion, pan coating, spheronization, drum granulation, fluid-bed agglomeration, high-shear granulation, fluid-bed spray coating, crystallization, precipitation, emulsion gelation, spinning disc atomization and other casting approaches, and prilling processes.
  • the core of the granule may be the granule itself or the inner nucleus of a layered granule.
  • the core may comprise one or more water soluble or dispersible agent(s). including but not limited to, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, sugars (e.g., sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose), plasticizers (e.g, polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous material (e.g..
  • cellulose and cellulose derivatives such as hydroxyl-propyl-methyl cellulose, carboxy -methyl cellulose, and hydroxyl-ethyl cellulose), phosphate, calcium, a protease inhibitor and combinations thereof.
  • Suitable dispersible agents include, but are not limited to, clays, nonpareils (combinations of sugar and starch; e.g., starch-sucrose non-pareils - ASNP), talc, silicates, carboxymethyl cellulose, starch, and combinations thereof.
  • the core comprises mainly sodium sulfate. In some embodiments, the core consists essentially of sodium sulfate. In a particular embodiment, the core consists of only sodium sulfate.
  • the core comprises a subtilisin variant as provided herein.
  • the core comprises one or more enzymes in addition to protease.
  • the core is inert and does not comprise enzymes.
  • the core is an enzy me powder, including UFC containing an enzy me.
  • the enzyme powder may be spray dried and may optionally be admixed with any of the water soluble or dispersible agents listed, herein.
  • the enzyme may be, or may include, the protease to be stabilized, in which case the enzyme powder should further include a stabilizer.
  • the core is coated with at least one coating layer.
  • the core is coated with at least two coating layers.
  • the core is coated with at least three coating layers.
  • the materials used in the coating layer(s) can be suitable for use in cleaning and/or detergent compositions (see, e.g. US20100124586, WO9932595 and US5324649).
  • a coating layer comprises one of more of the following materials: an inorganic salt (e.g, sodium sulfate, sodium chloride, magnesium sulfate, zinc sulfate, and ammonium sulfate), citric acid, a sugar (e.g., sucrose, lactose, glucose, and fructose), a plasticizer (e.g, polyols, urea, dibutyl phthalate, and dimethyl phthalate), fibrous material (e.g., cellulose and cellulose derivatives such as hydroxy 1-propyl-methyl cellulose, carboxy-methyl cellulose, and hydroxyl -ethyl cellulose), clay, nonpareil (a combination of sugar and starch), silicate, carboxymethyl cellulose, phosphate, starch (e.g , com starch), fats, oils (e.g, rapeseed oil, and paraffin oil), lipids, vinyl polymers, vinyl copolymers, poly
  • plasticizers e.g , polyols, urea, dibutyl phthalate, dimethyl phthalate, and water
  • anti-agglomeration agents e.g, talc, clays, amorphous silica, and titanium dioxide
  • anti-foam agents such as FOAMBLAST 882® and EROL 6000K®
  • talc e.g., polyols, urea, dibutyl phthalate, dimethyl phthalate, and water
  • anti-agglomeration agents e.g, talc, clays, amorphous silica, and titanium dioxide
  • anti-foam agents such as FOAMBLAST 882® and EROL 6000K®
  • the coating layer comprises sugars (e.g, sucrose, lactose, glucose, granulated sucrose, maltodextrin and fructose).
  • the coating layer comprises a polymer such as polyvinyl alcohol (PVA). Suitable PVA for incorporation in the coating layer(s) of the multi-layered granule include partially hydrolyzed, fully hydrolyzed and intermediately hydrolyzed having low to high degrees of viscosity.
  • the coating layer comprises an inorganic salt, such as sodium sulfate.
  • At least one coating layer is an enzyme coating layer.
  • the core is coated with at least two enzy me layers. In another embodiment, the core is coated with at least three or more enzyme layers.
  • the enzymes are protease in combination with one or more additional enzy mes selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispersins, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannana
  • additional enzy mes
  • DNases and/or RNases DNases and/or RNases
  • oxidases oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases.
  • At least one enzyme coating layer comprises at least one protease.
  • Another embodiment is directed to a method of cleaning a surface, where the method comprises contacting a surface or an item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein, or composition containing one or more subtilisin variants, as provided herein.
  • the surface or item in need of cleaning comprises a proteinaceous stain on the surface.
  • the surface or item in need of cleaning comprises a proteinaceous or creme brulee, or egg (e.g. egg yolk, or scrambled eggs, or full egg), or gratin, or baked cheese stain.
  • the term “stain'’ comprises any type of soil on the surface of an item, such as a hard-surface item (e g. a dish) or a textile (e.g. fabric).
  • the stain is a proteinaceous stain.
  • a “proteinaceous stain” is a stain or soil that contains protein.
  • a further embodiment is directed to a method of cleaning a proteinaceous stain comprising contacting a surface or an item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or composition containing one or more subtilisin variants as provided herein.
  • Another embodiment is directed to a method of cleaning a creme brulee stain comprising contacting a surface or an item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or composition containing one or more subtilisin variants as provided herein.
  • Another embodiment is directed to a method of cleaning an egg or egg yolk stain comprising contacting a surface or an item in need of cleaning with an effective amount of one or more subtilisin variants as provided herein or composition containing one or more such subtilisin variants.
  • Still yet another embodiment is directed to the method of cleaning a creme brulee stain described herein, with the proviso that the one or more subtilisin used in said method comprises one or more non-naturally occurring substitutions.
  • the one or more subtilisin variant used in the method of cleaning a baked cheese stain described herein has a baked cheese stain cleaning PI >1. 1 when compared to SEQ ID NO: 1.
  • the one or more subtilisin variant used in the method of cleaning a baked cheese stain described herein has a baked cheese stain cleaning PI >1. 1 when compared to SEQ ID NO: 1, where the baked cheese stain cleaning performance of the variant is measured in accordance with the baked cheese assay described in Example 2.
  • Still yet another embodiment is directed to the method of cleaning a baked cheese stain described herein, with the proviso that the one or more subtilisin used in said method comprises one or more non-naturally occurring substitutions.
  • the one or more subtilisin variant used in the method of cleaning an egg yolk stain described herein has an egg yolk stain cleaning PI >1. 1 when compared to SEQ ID NO: 1.
  • the one or more subtilisin variant used in the method of cleaning an egg yolk stain described herein has an egg yolk stain cleaning PI >1. 1 when compared to SEQ ID NO: 1, where the egg yolk stain cleaning performance of the variant is measured in accordance with the egg yolk assay described in Example 2.
  • Still yet another embodiment is directed to the method of cleaning an egg yolk stain described herein, with the proviso that the one or more subtilisin used in said method comprises one or more non- naturally occurring substitutions.
  • variants provided herein comprise one or more variants having amino acids substitutions selected from the group consisting of those listed in Table 2 having a PI >1.1 in one or more of the cleaning assays, including dish cleaning assays such as, egg. creme brulee, baked cheese assays compared to a parent subtilisin having the amino acid sequence of SEQ ID NO: 1.
  • variants provided herein comprise one or more variants having amino acids substitutions selected from the group consisting of those listed in Table 4 having a PI >1.1 in one or more of the cleaning assays, including laundry' cleaning assays such as, egg assays compared to a parent subtilisin having the amino acid sequence of SEQ ID NO: 1.
  • subtilisin variant described herein can be subject to various changes, such as one or more amino acid insertion, deletion, and/or substitution, either conservative or non-conservative, including where such changes do not substantially alter the enzymatic activity of the variant.
  • a nucleic acid of the invention can also be subject to various changes, such as one or more substitution of one or more nucleotide in one or more codon such that a particular codon encodes the same or a different amino acid, resulting in either a silent variation (e.g., when the encoded amino acid is not altered by the nucleotide mutation) or non-silent variation; one or more deletion of one or more nucleotides (or codons) in the sequence; one or more addition or insertion of one or more nucleotides (or codons) in the sequence; and/or cleavage of, or one or more truncation of one or more nucleotides (or codons) in the sequence.
  • nucleic acid sequence described herein can also be modified to include one or more codon that provides for optimum expression in an expression system (e.g., bacterial expression system), while, if desired, said one or more codon still encodes the same amino acid(s).
  • an expression system e.g., bacterial expression system
  • Described herein is one or more isolated, non-naturally occurring, or recombinant polynucleotide comprising a nucleic acid sequence that encodes one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof.
  • One or more nucleic acid sequence described herein is useful in recombinant production (e.g., expression) of one or more subtilisin variant described herein, ty pically through expression of an expression cassette comprising a sequence encoding the one or more subtilisin variant described herein or fragment thereof.
  • One embodiment provides nucleic acids encoding one or more subtilisin variant described herein, wherein the variant is a mature form having proteolytic activity.
  • one or more subtilisin variant described herein is expressed recombinantly with a homologous pro-peptide sequence. In other embodiments, one or more subtilisin variant described herein is expressed recombinantly with a heterologous pro-peptide sequence (e.g.. pro-peptide sequence from /?, lentus or variant thereof (e.g. SEQ ID NO:4).
  • a heterologous pro-peptide sequence e.g.. pro-peptide sequence from /?, lentus or variant thereof (e.g. SEQ ID NO:4).
  • One or more nucleic acid sequence described herein can be generated by using any suitable synthesis, manipulation, and/or isolation techniques, or combinations thereof.
  • one or more polynucleotide described herein may be produced using standard nucleic acid synthesis techniques, such as solid-phase synthesis techniques that are well- known to those skilled in the art. In such techniques, fragments of up to 50 or more nucleotide bases are typically synthesized, then joined (e.g., by enzymatic or chemical ligation methods) to form essentially any desired continuous nucleic acid sequence.
  • the synthesis of the one or more polynucleotide described herein can be also facilitated by any suitable method known in the art, including but not limited to chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al. Tetrahedron Letters 22: 1859-69 (1981)), or the method described in Matthes et al., EMBO J. 3:801-805 (1984) as is typically practiced in automated synthetic methods.
  • One or more polynucleotide described herein can also be produced by using an automatic DNA synthesizer. Customized nucleic acids can be ordered from a variety of commercial sources (e.g., ATUM (DNA 2.0). Newark.
  • Recombinant DNA techniques useful in modification of nucleic acids are well known in the art, such as, for example, restriction endonuclease digestion, ligation, reverse transcription and cDNA production, and polymerase chain reaction (e.g., PCR).
  • One or more polynucleotide described herein may also be obtained by screening cDNA libraries using one or more oligonucleotide probes that can hybridize to or PCR-amplify polynucleotides which encode one or more subtilisin variant described herein, or recombinant polypeptide or active fragment thereof.
  • One or more polynucleotide described herein can be obtained by altering a naturally occurring polynucleotide backbone (e.g., that encodes one or more subtilisin variant described herein or reference subtilisin) by. for example, a known mutagenesis procedure (e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination).
  • a known mutagenesis procedure e.g., site-directed mutagenesis, site saturation mutagenesis, and in vitro recombination.
  • a variety of methods are known in the art that are suitable for generating modified polynucleotides described herein that encode one or more subtilisin variant described herein, including, but not limited to.
  • site-saturation mutagenesis for example, site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site-directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.
  • a further embodiment is directed to one or more vector comprising one or more subtilisin variant described herein (e.g., a polynucleotide encoding one or more subtilisin variant described herein); expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequence described herein; isolated, substantially pure, or recombinant DNA constructs comprising one or more nucleic acid or polynucleotide sequence described herein; isolated or recombinant cells comprising one or more polynucleotide sequence described herein; and compositions comprising one or more such vector, nucleic acid, expression vector, expression cassette, DNA construct, cell, cell culture, or any combination or mixtures thereof.
  • subtilisin variant described herein e.g., a polynucleotide encoding one or more subtilisin variant described herein
  • expression vectors or expression cassettes comprising one or more nucleic acid or polynucleotide sequence described herein
  • Some embodiments are directed to one or more recombinant cell comprising one or more vector (e.g., expression vector, cassette or DNA construct) described herein which comprises one or more nucleic acid or polynucleotide sequence described herein.
  • Some such recombinant cells are transformed or transfected with such at least one vector, although other methods are available and known in the art.
  • Such cells are typically referred to as host cells.
  • Some such cells comprise bacterial cells, including, but not limited to Bacillus sp. cells, such as B. subtilis cells.
  • Other embodiments are directed to recombinant cells (e.g., recombinant host cells) comprising one or more subtilisin described herein.
  • one or more vector described herein is an expression vector or expression cassette comprising one or more polynucleotide sequence described herein operably linked to one or more additional nucleic acid segments required for efficient gene expression (e.g., a promoter operably linked to one or more polynucleotide sequence described herein).
  • a vector may include a transcription terminator and/or a selection gene (e.g., an antibiotic resistant gene) that enables continuous cultural maintenance of plasmid- infected host cells by growth in antimicrobial-containing media.
  • An expression vector may be derived from plasmid or viral DNA, or in alternative embodiments, contains elements of both.
  • Exemplary vectors include, but are not limited to pC194, pJHIOl, pE194, pHP13 (See, Harwood and Cutting [eds.] , Chapter 3, Molecular Biological Methods for Bacillus. John Wiley & Sons (1990); suitable replicating plasmids for B. subtilis include those listed on p. 92).
  • one or more expression vector or expression cassette comprising one or more copy of a polynucleotide encoding one or more subtilisin variant described herein, and in some instances comprising multiple copies, is transformed into the cell under conditions suitable for expression of the variant.
  • a polynucleotide sequence encoding one or more subtilisin variant described herein (as well as other sequences included in the vector) is integrated into the genome of the host cell, while in other embodiments, a plasmid vector comprising a polynucleotide sequence encoding one or more subtilisin variant described herein remains as autonomous extra-chromosomal element within the cell. Some embodiments provide both extrachromosomal nucleic acid elements as well as incoming nucleotide sequences that are integrated into the host cell genome. The vectors described herein are useful for production of the one or more subtilisin variant described herein.
  • a polynucleotide construct encoding one or more subtilisin variant described herein is present on an integrating vector that enables the integration and optionally the amplification of the polynucleotide encoding the variant into the host chromosome. Examples of sites for integration are well known to those skilled in the art.
  • transcription of a polynucleotide encoding one or more subtilisin variant described herein is effectuated by a promoter that is the wild-type promoter for the parent subtilisin.
  • the promoter is heterologous to the one or more subtilisin variant described herein, but is functional in the host cell.
  • Exemplary- promoters for use in bacterial host cells include, but are not limited to the amyE, amyQ, amyL, pstS, sacB, pSPAC, pAprE, pVeg, pHpall promoters; the promoter of the B. stearothermophilus maltogenic amylase gene; the B. amyloliquefaciens (BAN) amylase gene; the B. subtilis alkaline protease gene; the B. clausii alkaline protease gene; the B. pumilis xylosidase gene; the B. thuringiensis crylllA; and the B. licheniformis alpha-amylase gene.
  • BAN amyloliquefaciens
  • Additional promoters include, but are not limited to the A4 promoter, as well as phage Lambda PR or PL promoters and the E. coli lac, trp or tac promoters, and the Bacillus rm promoters such as rmB, rmL and rmE ribosomal RNA promoters and variants thereof.
  • subtilisin variant described herein can be produced in host cells of any suitable microorganism, including bacteria and fungi.
  • one or more subtilisin variant described herein can be produced in Gram-positive bacteria.
  • the host cells are Bacillus spp., Streptomyces spp., Escherichia spp., Aspergillus spp., Trichoderma spp. , Pseudomonas spp., Corynebacterium spp., Saccharomyces spp., or Pichia spp.
  • one or more subtilisin variant described herein is produced by Bacillus sp. host cells.
  • Bacillus sp. host cells that find use in the production of the one or more subtilisin variant described herein include, but are not limited to B. licheniformis, B. gibsonii. B. lentus, B. subtilis, B. amyloliquefaciens, B. brevis. B. stearothermophilus, B. alkalophilus , B. coagulans. B. circulans, B. pumilis, B. thuringiensis , B. clausii, and B. megaterium, as well as other organisms within the genus Bacillus.
  • B. subtilis host cells are used to produce the variants described herein.
  • USPNs 5,264,366 and 4,760,025 describe various Bacillus host strains that can be used to produce one or more subtilisin variant described herein, although other suitable strains can be used.
  • subtilisin variants described herein include non-recombinant (i.e., wild-ty pe) Bacillus sp. strains, as well as variants of naturally-occurring strains and/or recombinant strains.
  • the host strain is a recombinant strain, wherein a polynucleotide encoding one or more subtilisin variant described herein has been introduced into the host.
  • the host strain is a B. subtilis host strain and particularly a recombinant B. subtilis host strain. Numerous B.
  • subtilis strains are known, including, but not limited to for example, 1 A6 (ATCC 39085), 168 (1A01). SB19, W23, Ts85. B637, PB1753 through PB1758. PB3360, JH642, 1A243 (ATCC 39,087), ATCC 21332, ATCC 6051, Mil 13, DE100 (ATCC 39,094), GX4931, PBT 110, and PEP 211strain (See e.g., Hoch et al., Genetics 73:215-228 (1973); See also, US 4,450,235; US 4,302.544; and EP 0134048). The use of B.
  • subtilis as an expression host cell is well known in the art (See e.g, Palva et al., Gene 19:81-87 (1982); Fahnestock and Fischer, J. Bacteriol., 165:796-804 (1986); and Wang et al., Gene 69:39-47 (1988)).
  • the Bacillus host cell is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes: degU, degS. degR and degQ.
  • the mutation is in a degU gene, and in some embodiments the mutation is degU(Hy)32 (See e.g., Msadek et al., J. Bacteriol. 172:824-834 (1990); and Olmos et al.. Mol. Gen. Genet. 253:562-567 (1997)).
  • the Bacillus host comprises a mutation or deletion in scoC4 (See e.g., Caldwell et al., J.
  • an altered Bacillus host cell strain that can be used to produce one or more subtilisin variant described herein is Bacillus host strain that already includes a mutation in one or more of the above- mentioned genes.
  • Bacillus sp. host cells that comprise mutation(s) and/or deletion(s) of endogenous protease genes find use.
  • the Bacillus host cell comprises a deletion of the aprE and the nprE genes.
  • the Bacillus sp. host cell comprises a deletion of 5 protease genes, while in other embodiments the Bacillus sp. host cell comprises a deletion of 9 protease genes See e.g.. US 2005/0202535).
  • Host cells are transformed with one or more nucleic acid sequence encoding one or more subtilisin variant described herein using any suitable method known in the art.
  • Methods for introducing a nucleic acid (e.g., DNA) into Bacillus cells or E. coli cells utilizing plasmid DNA constructs or vectors and transforming such plasmid DNA constructs or vectors into such cells are well known.
  • the plasmids are subsequently isolated from E. coli cells and transformed into Bacillus cells.
  • it is not essential to use intervening microorganisms such as E. coli and in some embodiments, a DNA construct or vector is directly introduced into a Bacillus host.
  • transformation including protoplast transformation and transfection, transduction, and protoplast fusion
  • Methods known in the art to transform Bacillus cells include such methods as plasmid marker rescue transformation, which involves the uptake of a donor plasmid by competent cells cartying a partially homologous resident plasmid (See, Contente et al., Plasmid 2:555-571 (1979);
  • host cells are directly transformed with a DNA construct or vector comprising a nucleic acid encoding one or more subtilisin variant described herein (i.e., an intermediate cell is not used to amplify, or otherwise process, the DNA construct or vector prior to introduction into the host cell).
  • DNA constructs or vector described herein into the host cell includes those physical and chemical methods known in the art to introduce a nucleic acid sequence (e g., DNA sequence) into a host cell without insertion into the host genome. Such methods include, but are not limited to calcium chloride precipitation, electroporation, naked DNA, and liposomes.
  • DNA constructs or vector are co-transformed with a plasmid, without being inserted into the plasmid.
  • a selective marker is deleted from the altered Bacillus strain by methods known in the art (See, Stahl et al., J. Bacteriol. 158:411-418 (1984); and Palmeros et al., Gene 247:255 -264 (2000)).
  • the transformed cells are cultured in conventional nutrient media.
  • suitable specific culture conditions such as temperature. pH and the like are known to those skilled in the art and are well described in the scientific literature.
  • Some embodiments provide a culture (e.g., cell culture) comprising one or more subtilisin variant or nucleic acid sequence described herein.
  • host cells transformed with one or more polynucleotide sequence encoding one or more subtilisin variant described herein are cultured in a suitable nutrient medium under conditions permitting the expression of the variant, after which the resulting variant is recovered from the culture.
  • the variant produced by the cells is recovered from the culture medium by conventional procedures, including, but not limited to, for example, separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt (e.g., ammonium sulfate), and chromatographic purification (e.g., ion exchange, gel filtration, affinity, etc.).
  • a salt e.g., ammonium sulfate
  • chromatographic purification e.g., ion exchange, gel filtration, affinity, etc.
  • one or more subtilisin variant produced by a recombinant host cell is secreted into the culture medium.
  • a nucleic acid sequence that encodes a purification facilitating domain may be used to facilitate purification of the variant.
  • a vector or DNA construct comprising a polynucleotide sequence encoding one or more subtilisin variant described herein may further comprise a nucleic acid sequence encoding a purification facilitating domain to facilitate purification of the variant (See e.g., Kroll et al., DNA Cell Biol. 12:441-53 (1993)).
  • Such purification facilitating domains include, but are not limited to, for example, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (See, Porath, Protein Expr. Purif 3:263-281 [1992]), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/ affinity purification system.
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (See, Porath, Protein Expr. Purif 3:263-281 [1992]
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/ affinity purification system The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (e g., sequences available from Invitrogen. San Diego. CA) between the purification domain and the heterologous protein also find use to
  • a variety of methods can be used to determine the level of production of one or more mature subtilisin variant described herein in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the protease. Exemplary methods include, but are not limited to enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See e.g., Maddox et al., J. Exp. Med. 158: 1211 (1983)).
  • Some other embodiments provide methods for making or producing one or more mature subtilisin variant described herein.
  • a mature subtilisin variant does not include a signal peptide or a propeptide sequence.
  • Some methods comprise making or producing one or more subtilisin variant described herein in a recombinant bacterial host cell, such as for example, a Bacillus sp. cell (e.g., a B. subtilis cell).
  • Other embodiments provide a method of producing one or more subtilisin variant described herein, wherein the method comprises cultivating a recombinant host cell comprising a recombinant expression vector comprising a nucleic acid sequence encoding one or more subtilisin variant described herein under conditions conducive to the production of the variant.
  • Some such methods further comprise recovering the variant from the culture.
  • FIG. 1 A further embodiment is directed to a method of improving the cleaning performance or stability of a subtilisin comprising modifying a subtilisin to include one or more substitutions, or combination of substitutions, as provided herein.
  • compositions described herein include cleaning compositions, such as detergent compositions.
  • the enzyme levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.
  • subtilisin variant described herein is useful in cleaning applications, such as, for example, but not limited to, cleaning dishware or tableware items, fabrics, medical instruments and items having hard surfaces (e.g., the hard surface of a table, table top, wall, furniture item, floor, and ceiling).
  • cleaning applications such as, for example, but not limited to, cleaning dishware or tableware items, fabrics, medical instruments and items having hard surfaces (e.g., the hard surface of a table, table top, wall, furniture item, floor, and ceiling).
  • one or more subtilisin variant described herein is useful in disinfecting applications, such as, for example, but not limited to, disinfecting an automatic dishwashing or laundry' machine.
  • the cleaning composition is boron-free. In other embodiments, the cleaning composition is phosphate-free. In still other embodiments, the composition comprises one or more subtilisin variant described herein and one or more of an excipient, adjunct material, and/or additional enzyme.
  • the disclosure provides detergent compositions (e.g. ADW compositions) comprising a surfactant and at least one subtilisin variant as provided herein. Such compositions may further comprise one or more of an excipient, adjunct material, and/or additional enzyme.
  • the composition described herein contains phosphate, is phosphate-free, contains boron, is boron-free, or combinations thereof.
  • the composition is a boron-free composition.
  • a boron-free composition is a composition to which a borate stabilizer has not been added.
  • a boron-free composition is a composition that contains less than 5.5% boron.
  • a boron-free composition is a composition that contains less than 4.5% boron.
  • a boron-free composition is a composition that contains less than 3.5% boron.
  • a boron-free composition is a composition that contains less than 2.5% boron. In even further embodiments, a boron-free composition is a composition that contains less than 1.5% boron. In another embodiment, a boron-free composition is a composition that contains less than 1.0% boron. In still further embodiments, a boron-free composition is a composition that contains less than 0.5% boron. In other embodiments, the composition is a composition free or substantially-free of enzyme stabilizers or peptide inhibitors.
  • one or more composition described herein is in a form selected from gel, tablet, powder, granular, solid, liquid, unit dose, and combinations thereof.
  • one or more composition described herein is in a form selected from a low water compact formula, low water HDL or Unit Dose (UD), or high water formula or HDL.
  • the cleaning composition described herein is in a unit dose form.
  • the unit dose form is selected from pills, tablets, capsules, gelcaps, sachets, pouches, multi-compartment pouches, and pre-measured powders or liquids.
  • the unit dose format is designed to provide controlled release of the ingredients within a multi -compartment pouch (or other unit dose format). Suitable unit dose and controlled release formats are described, for example, in EP 2100949; WO 02/102955; US 4.765.916; US 4,972.017; and WO 04/111178.
  • the unit dose form is a tablet or powder contained in a water-soluble film or pouch.
  • Exemplary laundry detergent compositions include, but are not limited to, for example, liquid and powder laundry detergent compositions.
  • Exemplary hard surface cleaning compositions include, but not limited to, for example, compositions used to clean the hard surface of a non-dishware item, non-tableware item, table, table top, furniture item. wall, floor, and ceiling.
  • Exemplary' hard surface cleaning compositions are described, for example, in USPNs 6,610,642, 6,376,450. and 6,376,450.
  • Exemplary’ personal care compositions include, but are not limited to, compositions used to clean dentures, teeth, hair, contact lenses, and skin.
  • Exemplary' components of such oral care composition include those described in, for example, US 6,376,450.
  • one or more subtilisin variant described herein cleans at low temperatures. In other embodiments, one or more composition described herein cleans at low temperatures. In other embodiments, one or more composition described herein comprises an effective amount of one or more subtilisin variant described herein as useful or effective for cleaning a surface or a fabric in need of proteinaceous stain removal.
  • adjunct materials are incorporated, for example, to assist or enhance cleaning performance; for treatment of the substrate to be cleaned; or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dy'es or the like.
  • One embodiment is directed to a composition comprising one or more adjunct material and one or more subtilisin variant described herein.
  • adjunct material is selected from a bleach catalyst, an additional enzy me, an enzyme stabilizer (including, for example, an enzy me stabilizing system), a chelant, an optical brightener, a soil release polymer, a dye transfer agent, a dispersant, a suds suppressor, a dye, a perfume, a colorant, a filler, a photoactivator, a fluorescer, a fabric conditioner, a hydrolyzable surfactant, a preservative, an anti-oxidant, an anti-shrinkage agent, an anti-wrinkle agent, a germicide, a fungicide, a color speckle, a silvercare agent, an anti-tarnish agent, an anti-corrosion agent, an alkalinity source, a solubilizing agent, a carrier, a processing aid, a pigment, a pH control agent, a surfactant
  • Some embodiments are directed to cleaning additive products comprising one or more subtilisin variant described herein.
  • the additive is packaged in a dosage form for addition to a cleaning process.
  • the additive is packaged in a dosage form for addition to a cleaning process where a source of peroxide is employed and increased bleaching effectiveness is desired.
  • Exemplary fillers or carriers for granular compositions include, but are not limited to, for example, various salts of sulfate, carbonate and silicate; talc; and clay.
  • Exemplary' fillers or carriers for liquid compositions include, but are not limited to, for example, water or low molecular weight primary and secondary alcohols including polyols and diols (e.g., methanol, ethanol, propanol and isopropanol). In some embodiments, the compositions contain from about 5% to about 90% of such filler or carrier. Acidic fillers may be included in such compositions to reduce the pH of the resulting solution in the cleaning method or application.
  • one or more cleaning composition described herein comprises an effective amount of one or more subtilisin variant described herein, alone or in combination with one or more additional enzy me.
  • a cleaning composition comprises at least about 0.0001 to about 20 wt %, from about 0.0001 to about 10 wt %, from about 0.0001 to about 1 wt %, from about 0.001 to about 1 wt %, or from about 0.01 to about 0.2 wt % of one or more protease.
  • one or more cleaning composition described herein comprises from about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.01 to about 2 mg, about 0.01 to about 1 mg.
  • the cleaning compositions described herein are typically formulated such that during use in aqueous cleaning operations, the wash water will have a pH of from about 4.0 to about 11.5, or even from about 5.0 to about 11.5, or even from about 5.0 to about 8.0, or even from about 7.5 to about 10.5.
  • Liquid product formulations are ty pically formulated to have a pH from about 3.0 to about 9.0 or even from about 3 to about 5.
  • Granular laundry’ products are typically formulated to have a pH from about 8 to about 11.
  • the cleaning compositions of the present invention can be formulated to have an alkaline pH under wash conditions, such as a pH of from about 8.0 to about 12.0, or from about 8.5 to about 11.0, or from about 9.0 to about 11.0.
  • the cleaning compositions of the present invention can be formulated to have a neutral pH under wash conditions, such as a pH of from about 5.0 to about 8.0, or from about 5.5 to about 8.0, or from about 6.0 to about 8.0, or from about 6.0 to about 7.5.
  • the neutral pH conditions can be measured when the cleaning composition is dissolved 1: 100 (wt:wt) in de-ionised water at 20°C, measured using a conventional pH meter. 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.
  • one or more subtilisin variant described herein is encapsulated to protect it during storage from the other components in the composition and/or control the availability of the variant during cleaning.
  • encapsulation enhances the performance of the variant and/or additional enzyme.
  • the encapsulating material typically encapsulates at least part of the subtilisin variant described herein.
  • the encapsulating material is water-soluble and/or water- dispersible.
  • the encapsulating material has a glass transition temperature (Tg) of 0°C or higher.
  • Exemplary' encapsulating materials include, but are not limited to, carbohydrates, natural or synthetic gums, chitin, chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes, and combinations thereof.
  • the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof.
  • the encapsulating material is a starch (See e.g., EP0922499, US 4,977.252, US 5,354,559. and US 5,935,826).
  • the encapsulating material is a microsphere made from plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof.
  • plastic such as thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof.
  • Exemplary' commercial microspheres include, but are not limited to EXPANCEL® (Stockviksverken, Sweden); and PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL®, and SPHERICEL® (PQ Corp., Valley Forge, PA).
  • w ash conditions including varying detergent formulations, wash water volumes, w ash w ater temperatures, and lengths of w ash time to which one or more subtilisin variant described herein may be exposed.
  • a low detergent concentration system is directed to wash water containing less than about 800 ppm detergent components.
  • a medium detergent concentration system is directed to wash containing between about 800 ppm and about 2000 ppm detergent components.
  • a high detergent concentration system is directed to wash water containing greater than about 2000 ppm detergent components.
  • the “cold water washing” of the present invention utilizes “cold water detergent” suitable for washing at temperatures from about 10°C to about 40°C, from about 20°C to about 30°C, or from about 15°C to about 25°C, as well as all other combinations within the range of about 15°C to about 35°C or 10°C to 40°C.
  • Hardness is a measure of the amount of calcium (Ca 2+ ) and magnesium (Mg 2+ ) in the water. Water hardness is usually described in terms of the grains per gallon (gpg) mixed Ca 2 Mg 2+ . Most w ater in the United States is hard, but the degree of hardness varies. Moderately hard (60-120 ppm) to hard (121- 181 ppm) water has 60 to 181 ppm (ppm can be converted to grains per U.S. gallon by dividing ppm by 17.1) of hardness minerals.
  • cleaning composition comprising from about 0.00001 % to about 10% by weight composition of one or more subtihsin variant described herein and from about 99.999% to about 90.0% by weight composition of one or more adjunct material.
  • the cleaning composition comprises from about 0.0001 % to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% by weight composition of one or more subtilisin variant and from about 99.9999% to about 90.0%, about 99.999 % to about 98%, about 99.995% to about 99.5% by weight composition of one or more adjunct material.
  • the composition described herein comprises one or more subtihsin variant described herein and one or more additional enzyme.
  • the one or more additional enzyme is selected from acyl transferases, alpha-amylases, beta-amylases, alphagalactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispersins, endo-beta-1, 4- glucanases.
  • endo-beta-mannanases esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidase, hyaluronidases, keratinases, laccases, lactases, ligninases.
  • DNases and/or RNases DNases and/or RNases
  • oxidases oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases, polyesterases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetyl-esterases, xylanases, xyloglucanases.
  • xylosidases and any combination or mixture thereof.
  • Some embodiments are directed to a combination of enzymes (i.e., a ‘'cocktail”) comprising conventional enzymes like amylase, lipase, cutinase, mannanase and/or cellulase in conjunction with one or more subtilisin variant described herein and/or one or more additional protease.
  • a ‘'cocktail” comprising conventional enzymes like amylase, lipase, cutinase, mannanase and/or cellulase in conjunction with one or more subtilisin variant described herein and/or one or more additional protease.
  • one or more composition described herein comprises one or more subtilisin variant described herein and one or more additional protease.
  • the additional protease is a serine protease.
  • the additional protease is a metalloprotease, a fungal subtilisin, or an alkaline microbial protease or a trypsin-like protease.
  • Suitable additional proteases include those of animal, vegetable or microbial origin.
  • the additional protease is a microbial protease.
  • the additional protease is a chemically or genetically modified mutant.
  • the additional protease is an alkaline microbial protease or a tr psinlike protease.
  • the additional protease does not contain cross-reactive epitopes with the subtilisin variant as measured by antibody binding or other assays available in the art.
  • Exemplary alkaline proteases include subtilisins derived from, for example, Bacillus (e.g., BPN’, Carlsberg, subtilisin 309, subtilisin 147, B. gibsonii, B. pumilus, B sp.TY145, and subtilisin 168), or fungal origin, such as, for example, those described in US Patent No. 8,362,222.
  • Exemplary additional proteases include but are not limited to those descnbed in WO92/21760, WO95/23221, W02008/010925, W009/149200, WO09/149144, WO09/149145, WO 10/056640, W010/056653, W02010/0566356, WO11/072099, W02011/13022, WO11/140364, WO 12/151534, WO2015/038792, WO2015/089447, WO2015/089441, WO 2017/215925, US Publ. No.
  • PCT/US2015/057497 PCT/US2015/057492, PCT/US2015/057512, PCT/US2015/057526, PCT/US2015/057520, PCT/US2015/057502, PCT/US2016/022282, and PCT/US 16/32514, as well as metalloproteases described in WO1999014341, WO1999033960, WO1999014342, W01999034003.
  • W02007044993 W02009058303, WO 2009058661, W02014071410, WO2014194032, WO2014194034, WO 2014194054.
  • Exemplary additional proteases include, but are not limited to trypsin (e.g., of porcine or bovine origin) and the Fusarium protease described in W089/06270.
  • Exemplary 7 commercial proteases include, but are not limited to MAXATASE®, MAXACALTM, MAXAPEMTM, OPTICLEAN®, OPTIMASE®, PROPERASE®, PURAFECT®, PURAFECT® OXP, PURAMAXTM, EXCELLASETM.
  • PREFERENZTM proteases e.g. Pl 00, Pl 10, P280, P300
  • EFFECTENZTM proteases e.g. P1000, P1050, P2000
  • EXCELLENZTM proteases e.g.
  • compositions comprising one or more subtilisin variant described herein and one or more lipase.
  • the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% lipase by weight composition.
  • An exemplary lipase can be a chemically or genetically modified mutant.
  • Exemplary lipases include, but are not limited to, e.g., those of bacterial or fungal origin, such as, e g., H. lanuginosa lipase (see, e.g., EP 258068 and EP 305216), T.
  • lanuginosa lipase see, e.g., WO 2014/059360 and WO2015/010009
  • Rhizomucor miehei lipase see. e.g., EP 238023
  • Candida lipase such as C. antarctica lipase (e.g., C. antarctica lipase A or B) (see, e.g., EP 214761)
  • Pseudomonas lipases such as P. alcaligenes and P. pseudoalcaligenes lipase (see, e.g., EP 218272), P. cepacia lipase (see, e.g., EP 331376), P.
  • stutzeri lipase see, e.g., GB 1,372,034
  • P. fluorescens lipase Bacillus lipase (e.g., B. subtilis lipase (Dartois et al.. Biochem. Biophys. Acta 1131 :253-260 (1993)).
  • B. stearothermophilus lipase see, e.g., JP 64/744992
  • B. pumilus lipase see, e.g., WO 91/16422)
  • Exemplary cloned lipases include, but are not limited to Penicillium camember tii lipase (See, Yamaguchi et al., Gene 103:61-67 (1991)), Geotricum candidum lipase (See, Schimada et al., J.
  • Rhizopus lipases such as, R. delemar lipase (See, Hass et al.. Gene 109: 117-113 (1991)), R. niveus lipase (Kugimiya et al., Biosci. Biotech. Biochem. 56:716-719 (1992)) and R. oryzae lipase.
  • lipolytic enzy mes such as cutinases
  • cutinases may also find use in one or more composition described herein, including, but not limited to, e.g., cutinase derived from Pseudomonas mendocina (see, WO 88/09367) and/or Fusarium solani pisi (see, W090/09446).
  • Exemplary commercial lipases include, but are not limited to Ml LIPASETM, LUMA FASTTM, LIPOMAXTM, and PREFERENZTM LI 00 (IFF); LIPEX®, LIPOCLEAN®. LIPOLASE® and LIPOLASE® ULTRA (Novozymes); and LIPASE PTM (Amano Pharmaceutical Co. Ltd).
  • a still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more amylase.
  • the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% amylase by weight composition.
  • Any amylase e.g., alpha and/or beta
  • suitable for use in alkaline solutions may be useful to include in such composition.
  • An exemplary' amylase can be a chemically or genetically modified mutant.
  • amylases include, but are not limited to those of bacterial or fungal origin, such as, for example, amylases described in GB 1,296,839, W09100353. WO9402597, WO94183314, WO9510603, WO9526397.
  • WO9535382 WO9605295, WO9623873, WO9623874, WO 9630481, WO9710342, WO9741213, WO9743424, WO9813481, WO 9826078, W09902702, WO 9909183, WO9919467, WO9923211, WO9929876, WO9942567.
  • Exemplary commercial amy lases include, but are not limited to AMPLIFY®, DURAMYL®, TERMAMYL®, FUNGAMYL®, STAINZYME®, STAINZYME PLUS®, STAINZYME PLUS®, STAINZYME ULTRA® EVITY®, and BANTM (Novozymes); EFFECTENZTM S 1000. POWERASETM, PREFERENZTM S 100, PREFERENZTM S 110, PREFERENZTM S 210, EXCELLENZTM S 2000, RAPID ASE® and MAXAMYL® P (IFF). In some embodiments, the B.
  • gibsonii variants provided herein may be combined with one or more amylases selected from the group consisting of AA707, AA560, AAI10, BspAmy24, SP722, and CspAmyl, and variants thereof, and combinations thereof.
  • compositions comprising one or more subtilisin variant described herein and one or more cellulase.
  • the composition comprises from about 0.00001 % to about 10%, 0.0001 % to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% cellulase by weight of composition.
  • Any suitable cellulase may find use in a composition described herein.
  • An exemplary cellulase can be a chemically or genetically modified mutant.
  • Exemplary cellulases include but are not limited, to those of bacterial or fungal origin, such as, for example, those described in W02005054475, W02005056787, US 7,449,318, US 7,833,773, US 4,435,307; EP 0495257; and US Provisional Appl. No. 62/296,678.
  • Exemplary commercial cellulases include, but are not limited to, CELLUCLEAN®, CELLUZYME®. CAREZYME®, ENDOLASE®, RENOZYME®.
  • cellulases are incorporated as portions or fragments of mature wild-type or variant cellulases, wherein a portion of the N-terminus is deleted (see, e.g., US 5,874,276).
  • An even still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more mannanase.
  • the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% mannanase by weight composition.
  • An exemplary mannanase can be a chemically or genetically modified mutant.
  • Exemplary mannanases include, but are not limited to, those of bacterial or fungal origin, such as, for example, those described in WO 2016/007929; USPNs 6,566,114; 6,602,842; and 6,440,991 : and US Provisional Appl.
  • a still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more nuclease, such as a DNase or RNase.
  • the composition comprises from about 0.00001% to about 10%, about 0.0001% to about 10%, about 0.001% to about 5%, about 0.001% to about 2%, or about 0.005% to about 0.5% nuclease by weight composition.
  • Exemplary’ nucleases include, but are not limited to, those described in WO2015181287, WO2015155350, WO2016162556, WO2017162836, W02017060475 (e.g.
  • nucleases which can be used in combination with the substilisin variants provided herein in the compositions and methods provided herein include those described in Nijland R, Hall MJ, Burgess JG (2010) Dispersal of Biofilms by Secreted, Matrix Degrading, Bacterial DNase.
  • a yet even still further embodiment is directed to a composition comprising one or more subtilisin variant described herein and one or more peroxidase and/or oxidase enzyme.
  • the composition comprises from about 0.00001% to about 10%. about 0.0001% to about 10%, about 0.001% to about 5%. about 0.001% to about 2%, or about 0.005% to about 0.5% peroxidase or oxidase by’ weight composition.
  • a peroxidase may be used in combination with hydrogen peroxide or a source thereof (e.g., a percarbonate, perborate or persulfate) and an oxidase may be used in combination with oxygen.
  • Peroxidases and oxidases are used for “solution bleaching” (i.e., to prevent transfer of a textile dye from a dyed fabric to another fabric when the fabrics are washed together in a wash liquor), alone or in combination with an enhancing agent (see, e.g., WO94/12621 and WO95/01426).
  • An exemplary peroxidase and/or oxidase can be a chemically or genetically modified mutant.
  • Exemplary’ peroxidases/oxidases include, but are not limited to those of plant, bacterial, or fungal origin.
  • Another embodiment is directed to a composition comprising one or more subtilisin variant described herein, and one or more perhydrolase, such as, for example, is described in W02005/056782. W02007/106293, WO 2008/063400, W02008/106214, and W02008/ 106215.
  • the one or more subtilisin variant described herein and one or more additional enzyme contained in one or more composition described herein may each independently range to about 10% by weight composition, wherein the balance of the cleaning composition is one or more adjunct material.
  • one or more composition described herein finds use as a detergent additive, wherein said additive is in a solid or liquid form.
  • Such additive products are intended to supplement and/or boost the performance of conventional detergent compositions and can be added at any stage of the cleaning process.
  • the density of the laundry detergent composition ranges from about 400 to about 1200 g/liter, while in other embodiments it ranges from about 500 to about 950 g/liter of composition measured at 20° C.
  • Some embodiments are directed to a laundry detergent composition
  • a laundry detergent composition comprising one or more subtilisin variant described herein and one or more adjunct material selected from surfactants, enzyme stabilizers, builder compounds, polymeric compounds, bleaching agents, additional enzymes, suds suppressors, dispersants, lime-soap dispersants, soil suspension agents, anti-redeposition agents, corrosion inhibitors, and combinations thereof.
  • the laundry compositions also contain softening agents.
  • Further embodiments are directed to manual dishwashing composition
  • Exemplary builders include, but are not limited to alkali metal silicates; alkaline earth and alkali metal carbonates; aluminosilicates; polycarboxylate compounds; ether hydroxypolycarboxylates; copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6- trisulphonic acid, and carboxymethyloxysuccinic acid; ammonium and substituted ammonium salts of poly acetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid; polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid; and soluble salts thereof.
  • the builders form water-soluble hardness ion complexes (e.g., sequestering builders), such as sodium citrate.
  • exemplary builders are described in, e.g., EP 2100949.
  • Other suitable nonphosphate builders include homopolymers and copolymers of poly carboxylic acids and their partially or completely neutralized salts, monomeric poly carboxylic acids and hydroxy carboxylic acids and their salts.
  • salts of the above-mentioned compounds include the ammonium and/or alkali metal salts, i.e. the lithium, sodium, and potassium salts, including sodium salts.
  • the cleaning compositions comprise an amino carboxylic builder.
  • an amino carboxylic builder include aminocarboxylic acids, salts and derivatives thereof.
  • the amino carboxylic builder is an aminopolycarboxylic builder, such as glycine-N,N-diacetic acid or derivative of general formula MOOC-CHR-N(CH2COOM)2 where R is Ci-nalkyl and M is alkali metal.
  • the amino carboxylic builder can be methylglycine diacetic acid (MGDA), GLDA (glutamic-N,N-diacetic acid), iminodisuccinic acid (IDS), carboxymethyl inulin and salts and derivatives thereof, aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N- diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), N-(2-sulfomethyl) aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl) glutamic acid (SEGL), IDA (iminodiacetic acid) and salts and derivatives thereof such as N-methyliminodiacetic acid (MIDA) , alpha-alanine-N,N-diacetic acid (alpha-
  • ASMA
  • the cleaning compositions comprise an acidifying particle.
  • the acidifying particle can comprise any acid, including organic acids and mineral acids.
  • Organic acids can have one or two carboxyls and in some instances up to 15 carbons, especially up to 10 carbons, such as formic, acetic, propionic, capric, oxalic, succinic, adipic, maleic, fumaric, sebacic, malic, lactic, glycolic, tartaric and glyoxylic acids.
  • the acid is citric acid.
  • Mineral acids include hydrochloric and sulphuric acid.
  • the acidifying particle is a highly active particle comprising a high level of amino carboxylic builder.
  • the acidifying particle has a weight geometric mean particle size of from about 400p to about 1200p and a bulk density of at least 550 g/L. In some embodiments, the acidifying particle comprises at least about 5% of the builder.
  • Additional embodiments are directed to a cleaning composition comprising one or more subtilisin variant and one or more surfactant and/or surfactant system, wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants, and mixtures thereof.
  • the surfactant is present at a level of from about 0. 1 to about 60%, while in alternative embodiments the level is from about 1 to about 50%, while in still further embodiments the level is from about 5 to about 40%. by weight of the cleaning composition.
  • one or more composition described herein comprises one or more deposition aid.
  • deposition aids include, but are not limited to, e.g., polyethylene glycol; polypropylene glycol; polycarboxylate; soil release polymers, such as, e.g., poly terephthalic acid; clays such as, e.g., kaolinite, montmorillonite, attapulgite, illite, bentonite, and halloysite; and mixtures thereof.
  • one or more composition described herein comprises one or more anti-redeposition agent or non-ionic surfactant (which can prevent the re-deposition of soils) see, e.g., EP 2100949).
  • non-ionic surfactants find use for surface modification purposes, in particular for sheeting, to avoid filming and spotting and to improve shine. These non-ionic surfactants also find use in preventing the redeposition of soils.
  • the non-ionic surfactant can be ethoxylated nonionic surfactants, epoxy-capped poly(oxyalkylated) alcohols and amine oxides surfactants.
  • one or more composition described herein comprises one or more dye transfer inhibiting agent.
  • exemplary 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, polyvinylimidazoles, and mixtures thereof.
  • the composition comprises from about 0.0001% to about 10%, about 0.01% to about 5%, or about 0.1% to about 3% dye transfer inhibiting agent by weight composition.
  • one or more composition described herein comprises one or more silicate.
  • silicates include, but are not limited to, sodium silicates, e.g., sodium disilicate, sodium metasilicate, and cry stalline phyllosilicates.
  • silicates are present at a level of from about 1% to about 20% or about 5% to about 15% byweight of the composition.
  • one or more composition described herein comprises one or more dispersant.
  • exemplary- water-soluble organic materials include, but are not limited to, e.g., homo- or co-polymeric acids or their salts, in which the poly carboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
  • Other examples of dispersants for use in the methods and compositions herein include polysaccharides and polysaccharide derivatives, such as those disclosed in WO2022178073.
  • one or more composition described herein comprises one or more enzyme stabilizer.
  • the enzyme stabilizer is water-soluble sources of calcium and/or magnesium ions.
  • the enzyme stabilizers include oligosaccharides, polysaccharides, and inorganic divalent metal salts, including alkaline earth metals, such as calcium salts.
  • 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)). Chlorides and sulfates also find use in some embodiments.
  • 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),
  • oligosaccharides and polysaccharides are described, for example, in WO 07/145964.
  • reversible protease inhibitors also find use, such as boron-containing compounds (e.g., borate, 4-formyl phenyl boronic acid, and phenyl-boronic acid derivatives (such as for example, those described in WO96/41859)) and/or a peptide aldehyde, such as, for example, is further described in W02009/118375 and W02013004636.
  • Peptide aldehydes may be used as protease stabilizers in detergent formulations as previously described (WO199813458, WO2011036153, US20140228274).
  • peptide aldehyde stabilizers are peptide aldehydes, ketones, or halomethyl ketones and might be ‘N-capped’ with for instance a ureido, a carbamate, or a urea moiety, or ‘doubly N- capped’ with for instance a carbonyl, a ureido, an oxiamide, a thioureido, a dithiooxamide, or a thiooxamide moiety (EP2358857B1).
  • the molar ratio of these inhibitors to the protease may be 0.1: 1 to 100: 1, e.g. 0.5: 1-50: 1. 1: 1-25: 1 or 2: 1-10: 1.
  • Other examples of protease stabilizers are benzophenone or benzoic acid anilide derivatives, which might contain carboxyl groups (US 7,968,508 B2).
  • the molar ratio of these stabilizers to protease is preferably in the range of 1 : 1 to 1000: 1 in particular 1 : 1 to 500: 1 especially preferably from 1: 1 to 100: 1, most especially preferably from 1: 1 to 20: 1.
  • one or more composition described herein comprises one or more bleach, bleach activator, and/or bleach catalyst.
  • one or more composition described herein comprises one or more inorganic and/or organic bleaching compound.
  • Exemplary inorganic bleaches include, but are not limited to perhydrate salts, e.g., perborate, percarbonate, perphosphate, persulfate, and persilicate salts.
  • inorganic perhydrate salts are alkali metal salts.
  • inorganic perhydrate salts are included as the crystalline solid, without additional protection, although in some other embodiments, the salt is coated.
  • Bleach activators are typically organic peracid precursors that enhance the bleaching action in the course of cleaning at temperatures of 60°C and below.
  • Exemplary bleach activators include compounds which, under perhydrolysis conditions, give aliphatic peroxy carboxylic acids having from about 1 to about 10 carbon atoms or about 2 to about 4 carbon atoms, and/or optionally substituted perbenzoic acid.
  • Exemplary bleach activators as described, for example, in EP 2100949.
  • Exemplary bleach catalysts include, but are not limited to, manganese triazacyclononane and related complexes, as well as cobalt, copper, manganese, and iron complexes. Additional exemplary bleach catalysts are described, for example, in US 4,246,612; US 5,227,084; US 4,810,410; WO 99/06521; and EP 2100949.
  • one or more composition described herein comprises one or more catalytic metal complexes.
  • a metal-containing bleach catalyst finds use.
  • the metal bleach catalyst comprises a catalyst system comprising a transition metal cation of defined bleach catalytic activity' (e.g., copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations), an auxiliary metal cation having little or no bleach catalytic activity (e.g..
  • one or more composition described herein is catalyzed by means of a manganese compound.
  • a manganese compound Such compounds and levels of use are described, for example, in US 5,576,282.
  • cobalt bleach catalysts find use and are included in one or more composition described herein.
  • Various cobalt bleach catalysts are described, for example, in USPNs 5,597,936 and 5,595,967.
  • one or more composition described herein includes a transition metal complex of a macropolycyclic rigid ligand (MRL).
  • MRL macropolycyclic rigid ligand
  • the compositions and cleaning processes described herein are adjusted to provide on the order of at least one part per hundred million, from about 0.005 ppm to about 25 ppm, about 0.05 ppm to about 10 ppm, or about 0. 1 ppm to about 5 ppm of active MRL in the wash liquor.
  • Exemplary 7 MRLs include, but are not limited to special ultra-rigid ligands that are cross-bridged, such as, e.g., 5,12- diethyl-l,5,8,12-tetraazabicyclo(6.6.2)hexadecane.
  • Exemplary metal MRLs are described, for example, in WO 2000/32601 and US 6,225,464.
  • one or more composition described herein comprises one or more metal care agent.
  • the composition comprises from about 0.1% to about 5% metal care agent by weight composition.
  • Exemplary metal care agents include, for example, aluminum, stainless steel, and non-ferrous metals (e.g.. silver and copper). Additional exemplary metal care agents are described, for example, in EP 2100949, WO 94/26860, and WO 94/26859.
  • the metal care agent is a zinc salt.
  • the cleaning composition is a heavy-duty liquid (HDL) composition comprising one or more subtilisin variant described herein.
  • the HDL liquid laundry detergent can comprise a detersive surfactant (10-40%) comprising anionic detersive surfactant selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl sulphates, alkyl sulphonates, alky l alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates, and/or mixtures thereof; and optionally non-ionic surfactant selected from a group of linear or branched or random chain, substituted or unsubstituted alkyl alkoxylated alcohol, for example, a Cs-Cisalkyl ethoxylated alcohol and/or C6-Ci2alkyl phenol alkoxylates, optionally wherein the weight ratio of anionic detersive surfactant (with a hydrophilic index (HIc) of from 6.0 to 9) to non-ionic detersive surfactant is greater than 1: 1.
  • Suitable detersive surfactants also include cationic detersive surfactants (selected from alkyd pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary phosphonium compounds, alkyl ternary 7 sulphonium compounds, and/or mixtures thereof); zwitterionic and/or amphoteric detersive surfactants (selected from alkanolamine sulpho-betaines); ampholytic surfactants; semi-polar non-ionic surfactants; and mixtures thereof.
  • the cleaning composition is a liquid or gel detergent, which is not unit dosed, that may be aqueous, typically containing at least 20% and up to 95% water by weight, such as up to about 70% water by weight, up to about 65% water by weight, up to about 55% water by weight, up to about 45% water by weight, or up to about 35% water by weight.
  • aqueous liquid or gel detergent may contain from 0-30% organic solvent.
  • a liquid or gel detergent may be non-aqueous.
  • the composition can comprise optionally, a surfactancy boosting polymer consisting of amphiphilic alkoxylated grease cleaning polymers selected from a group of alkoxylated polymers having branched hydrophilic and hydrophobic properties, such as alkoxylated polyalkylenimines in the range of 0.05wt%-10wt% and/or random graft polymers ty pically comprising a hydrophilic backbone comprising monomers selected from the group consisting of: unsaturated Ci-Cecarboxylic acids, ethers, alcohols, aldehydes, ketones, esters, sugar units, alkoxy units, maleic anhydride, saturated polyalcohols such as glycerol, and mixtures thereof; and hydrophobic side chain(s) selected from the group consisting of: C4-C25 alkyl group, polypropylene, polybutylene, vinyl ester of a saturated C2-C6 mono-carboxylic acid, Ci-Ce alkyl ester of
  • the composition can comprise additional polymers such as soil release polymers including, for example, anionically end-capped polyesters, for example SRP1; polymers comprising at least one monomer unit selected from saccharide, di carboxylic acid, polyol and combinations thereof, in random or block configuration; ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example, Repel-o- tex SF, SF-2 and SRP6, Texcare SRA100. SRA300.
  • soil release polymers including, for example, anionically end-capped polyesters, for example SRP1; polymers comprising at least one monomer unit selected from saccharide, di carboxylic acid, polyol and combinations thereof, in random or block configuration; ethylene terephthalate-based polymers and co-polymers thereof in random or block configuration, for example, Repel-o- tex SF, SF-2 and SRP6, Texcare SRA100. SRA300.
  • anti-redeposition polymers (0.1 wt% to 10wt%, including, for example, carboxylate polymers, such as polymers comprising at least one monomer selected from acrylic acid, maleic acid (or maleic anhydride), fumaric acid, itaconic acid, aconitic acid, mesaconic acid, citraconic acid, methylenemalonic acid, and any mixture thereof; vinylpyrrolidone homopolymer; and/or polyethylene glycol with a molecular weight in the range of from 500 to 100,000 Da); cellulosic polymer (including, for example, alky l cellulose; alkyl alkoxyalkyl cellulose; carboxyalkyl cellulose; alkyl carboxyalkyl cellulose, examples of which include carboxymethyl cellulose, methyl cellulose, methyl hydroxyethyl cellulose, methyl carboxymethyl cellulose; and mixtures thereof); and polymeric
  • the composition can further comprise saturated or unsaturated fatty acid, preferably saturated or unsaturated C12-C24 fatty acid (0-10 wt%); deposition aids (including, for example, polysaccharides, cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DADMAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides, and mixtures thereof, in random or block configuration; cationic guar gum; cationic cellulose such as cationic hydroxyethyl cellulose; cationic starch; cationic poly acylamides; and mixtures thereof.
  • deposition aids including, for example, polysaccharides, cellulosic polymers, poly diallyl dimethyl ammonium halides (DADMAC), and co-polymers of DADMAC with vinyl pyrrolidone, acrylamides, imidazoles, imidazolinium halides,
  • the composition can further comprise dye transfer inhibiting agents examples of which include manganese phthalocyanine, peroxidases, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles and/or mixtures thereof; chelating agents examples of which include ethylene-diamine-tetraacetic acid (EDTA); diethylene triamine penta methylene phosphonic acid (DTPMP); hydroxy-ethane diphosphonic acid (HEDP); ethylenediamine N,N'-disuccinic acid (EDDS); methyl glycine diacetic acid (MGDA); diethylene triamine penta acetic acid (DTP A); propylene diamine tetracetic acid (PDT A); 2- hydroxypyridine-N-oxide (HPNO); or methyl glycine diacetic acid (MGDA)
  • the composition can further comprise silicone or fatty -acid based suds suppressors; an enzyme stabilizer; hueing dyes, calcium and magnesium cations, visual signaling ingredients, anti-foam (0.001 to about 4.0 wt%), and/or structurant/thickener (0.01- 5 wt%) selected from the group consisting of diglycerides, triglycerides, ethylene glycol distearate, microcrystalline cellulose, cellulose based materials, microfiber cellulose, biopolymers, xanthan gum, gellan gum, and mixtures thereof.
  • the cleaning composition is a heavy duty powder (HDD) composition comprising one or more subtilisin variant described herein.
  • the HDD powder laundry 7 detergent can comprise a detersive surfactant including anionic detersive surfactants (selected from linear or branched or random chain, substituted or unsubstituted alky l sulphates, alkyl sulphonates, alkyl alkoxylated sulphate, alkyl phosphates, alkyl phosphonates, alkyl carboxylates and/or mixtures thereof), non-ionic detersive surfactant (selected from linear or branched or random chain, substituted or unsubstituted Cs-Cis alky l ethoxylates, and/or C6-C12 alkyd phenol alkoxylates), cationic detersive surfactants (selected from alky l pyridinium compounds, alkyl quaternary ammonium compounds, alkyl quaternary
  • zeolite builders examples of which include zeolite A, zeolite X, zeolite P and zeolite MAP in the range of 0 to less than 10 wt%); phosphate builders, e.g., sodium tri-polyphosphate in the range of 0 to less than 10 wt%; citric acid, citrate salts and nitrilotriacetic acid or salt thereof in the range of less than 15 wt%; silicate salt (sodium or potassium silicate or sodium meta-silicate in the range of 0 to less than 10 wt% or layered silicate (SKS-6)); carbonate salt (sodium carbonate and/or sodium bicarbonate in the range of 0 to less than 10 wt%); and bleaching agents (photobleaches, e.g., sulfonated zinc phthalocyanines, sulfonated aluminum phthalocyanines, xanthenes dyes, and mixtures thereof); hydrophobic or hydrophilic bleach activators
  • hydrogen peroxide sources of hydrogen peroxide (inorganic perhydrate salts, e.g., mono or tetra hydrate sodium salt of perborate, percarbonate, persulfate, perphosphate, or persilicate); preformed hydrophilic and/or hydrophobic peracids (selected from percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxy monosulfuric acids and salts, and mixtures thereof); and/or bleach catalyst (e.g., imine bleach boosters, such as iminium cations and polyions; iminium zwitterions; modified amines; modified amine oxides; N-sulphonyl
  • sources of hydrogen peroxide inorganic perhydrate salts, e.g., mono or tetra hydrate sodium salt of perborate, percarbonate, persulfate, perphosphate, or persilicate
  • preformed hydrophilic and/or hydrophobic peracids selected from per
  • composition can further comprise additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, an enzyme stabilizer, hueing agents, additional polymers including fabric integrity and cationic polymers, dye lock ingredients, fabric-softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.
  • additional detergent ingredients including perfume microcapsules, starch encapsulated perfume accord, an enzyme stabilizer, hueing agents, additional polymers including fabric integrity and cationic polymers, dye lock ingredients, fabric-softening agents, brighteners (for example C.I. Fluorescent brighteners), flocculating agents, chelating agents, alkoxylated polyamines, fabric deposition aids, and/or cyclodextrin.
  • the cleaning composition is a “phosphate-free’’ automatic dishwashing (ADW) detergent composition comprising one or more subtilisin variants described herein.
  • ADW automatic dishwashing
  • phosphate-free is herein understood that the composition comprises less than 1%. preferably less than 0. 1% by weight of the composition of phosphate.
  • the ADW detergent composition can comprise two or more non-ionic surfactants selected from ethoxylated non-ionic surfactants, alcohol alkoxylated surfactants, epoxy-capped poly(oxyalkylated) alcohols, and amine oxide surfactants present in amounts from 0-10% by weight: builders in the range of 5-60% by weight, comprising phosphate-free builders (amino acid based compounds, e.g., MGDA (methyl-glycine-diacetic acid) and salts and derivatives thereof, GLDA (glutamic-N,N-diacetic acid) and salts and derivatives thereof, IDS (iminodisuccinic acid) and salts and derivatives thereof, ethylenediaminetetraacetic acid (EDTA), ethylenediaminedisuccinic acid (EDDS), carboxymethy l inulin and salts and derivatives thereof and mixtures thereof, nitrilotriacetic acid (NTA), diethylene triamine penta
  • perhydrate salts such as perborate, percarbonate, perphosphate, persulfate and persilicate salts
  • organic peroxyacids including diacyl and tetraacylperoxides, especially diperoxydodecanedioic acid, diperoxytetradecanedioic acid, and diperoxyhexadecanedioic acid
  • bleach activator-organic peracid precursors in the range from about 0.1 to about 10% by weight
  • bleach catalysts selected from manganese triazacyclononane and related complexes, Co, Cu, Mn and Fe bispyridylamine and related complexes, and pentamine acetate cobalt(III) and related complexes
  • metal care agents in the range from about 0.
  • crystal growth inhibitors selected from the group comprising 1 -hydroxy ethylidene 1,1-diphosphonic, carboxymethyl inulin, tricarballylic acid, cyclic carboxylates, and partially decarboxylated polyitaconic acid homopolymers
  • glass care agents preferably a zinc containing material, e.g., hydrozincite, or polyethyleneimine, or bismuth citrate
  • enzymes in the range from about 0.01-5.0 mg of active enzy me per gram of ADW detergent composition (acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, betagalactosidases.
  • carrageenases catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, dispersins, endo-beta-1, 4-glucanases, endo-beta-mannanases, esterases, exo- mannanases, galactanases, glucoamylases, hemicellulases, hexosaminidase, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, nucleases, oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases.
  • peroxidases peroxidases, phenoloxidases, phosphodiesterases, phosphatases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xanthan lyases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, and mixtures thereof); and enzyme stabilizer components (selected from oligosaccharides, polysaccharides and inorganic divalent metal salts).
  • Exemplary ADW compositions are provided in the Table below.
  • More embodiments are directed to compositions and methods of treating fabrics (e.g, to desize a textile) using one or more subtilisin variant described herein.
  • Fabric-treating methods are well known in the art (see, e.g.. US 6,077,316).
  • the feel and appearance of a fabric can be improved by a method comprising contacting the fabric with a variant described herein in a solution.
  • the fabric can be treated with the solution under pressure.
  • One or more subtilisin variant described herein can be applied during or after weaving a textile, during the desizing stage, or one or more additional fabric processing steps. During the w eaving of textiles, the threads are exposed to considerable mechanical strain. Prior to w eaving on mechanical looms, warp yams are often coated with sizing starch or starch derivatives to increase their tensile strength and to prevent breaking. One or more subtilisin variant described herein can be applied during or after weaving to remove the sizing starch or starch derivatives. After weaving, the variant can be used to remove the size coating before further processing the fabric to ensure a homogeneous and wash-proof result.
  • subtilisin variant described herein can be used alone or with other desizing chemical reagents and/or desizing enzymes to desize fabrics, including cotton-containing fabrics, as detergent additives, e.g., in aqueous compositions.
  • An amylase also can be used in combination with the subtilisin variant in compositions and methods for producing a stonewashed look on indigo-dyed denim fabric and garments.
  • the fabric can be cut and sewn into clothes or garments, which are afterwards finished.
  • different enzymatic finishing methods have been developed.
  • the finishing of denim garment normally is initiated with an enzymatic desizing step, during which garments are subjected to the action of proteolytic enzymes to provide softness to the fabric and make the cotton more accessible to the subsequent enzymatic finishing steps.
  • One or more subtilisin variant described herein can be used in methods of finishing denim garments (e.g, a “bio-stoning process”), enzymatic desizing and providing softness to fabrics, and/or finishing process.
  • the present disclosure also provides methods for cleaning a surface of an article, the method comprising contacting the article with at least one subtilisin variants provided herein (or a composition comprising such subtilisin variant).
  • the article may have a proteinaceous stain, for example, on its surface.
  • the proteinaceous stain may comprise egg or an egg-based stain, such as creme brulee, baked cheese, or other protein-containing substance.
  • Embodiment 1 A subtilisin variant comprising two or more substitutions selected from the group consisting of X099R, XI 14L, X151T, X154N, X156S, X224V, and X253D where the positions are numbered according to SEQ ID NO: 1, and where the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or 8.
  • Embodiment 2 The subtilisin variant according to Embodiment 1, where the variant further comprises one or more substitutions selected from the group consisting of:
  • Embodiment 3 The subtilisin variant according to Embodiment 1 or 2, where the variant comprises one or more additional substitutions selected from X039E, X198G, X209V, X21 IQ, X212Q, X242D where the positions are numbered according to SEQ ID NO: 1, and where the variant has at least 80% identity to the amino acid sequence of SEQ ID NO: 1 or 8.
  • Embodiment 5 The subtilisin variant of Embodiment 2, comprising two mutations selected from the group consisting of S099R-N114L, S099R-S151T.
  • Embodiment 6 The subtilisin variant according to any of the preceding Embodiments, wherein the variant
  • (i) is derived from a Lederbergia lentus subtilisin
  • (hi) comprises a combination of (i) and (ii).
  • Embodiment 7 The subtilisin variant according to any preceding Embodiment, where the variant is derived from a parent or reference polypeptide with 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 8.
  • Embodiment 8 The subtilisin variant of any preceding Embodiment, where the variant comprises an amino acid sequence with 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or less than 100% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 8.
  • Embodiment 9 The subtilisin variant of any preceding Embodiment, where the variant has one or more improved properties when compared to a parent or reference subtilisin; where the improved property is improved cleaning performance in detergent.
  • Embodiment 12 The enzyme composition according to Embodiment 1 1, where the composition is an enzyme granule.
  • Embodiment 13 The enzy me composition according to any one of Embodiments 11 or 12, where the composition further comprises one or more ions selected from calcium and/or zinc; one or more enzyme stabilizer; from about 0.001% to about 1 .0 weight % of the subtilisin variant; one or more bleaching agent; one or more adjunct material; one or more microbe and/or one or more additional enzy mes or enzyme derivatives selected from the group consisting of acyl transferases, alphaamylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, betagalactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, DNases, dispersins, endo-beta-1, 4-glucanases, endo-beta- mannanases, esterases, exo-mannanases
  • oxidases pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, RNases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, xylosidases, metalloproteases, nucleases, additional serine proteases, and combinations thereof.
  • Embodiment 14 The enzyme composition of Embodiment 13, where the one or more enzymes comprises an amylase selected from the group consisting of AA707, AA560, AAI10, BspAmy24, SP722. and CspAmyl. and variants thereof, and combinations thereof.
  • Embodiment 15 A polynucleotide comprising a nucleic acid sequence encoding a variant of any one of Embodiments 1-10, where the polynucleotide is, optionally, isolated.
  • Embodiment 16 The polynucleotide of Embodiment 15, where the nucleic acid sequence is operably linked to a promoter.
  • Embodiment 17 An expression vector or cassette comprising the polynucleotide of Embodiment 15 or 16.
  • Embodiment 18 A recombinant host cell comprising the polynucleotide of
  • Embodiment 19 A recombinant host cell comprising the polynucleotide of Embodiment 15 or 16 or the vector or cassette of Embodiment 17 comprising SEQ ID NO:4.
  • Embodiment 20 A composition comprising one or more subtilisin variant according to any of Embodiments 1-10.
  • Embodiment 21 The composition according to Embodiment 20, where the composition is selected from an enzyme composition and a detergent composition.
  • Embodiment 22 The composition according to Embodiment 21 , wherein the detergent composition is selected from a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning product.
  • Embodiment 23 The composition of any one of Embodiments 20-22, where the composition further comprises one or more ions selected from calcium and/or zinc; one or more enzyme stabilizer; from about 0.001% to about 1.0 weight % of the subtilisin variant; one or more bleaching agent; one or more adjunct material; one or more microbe and/or one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alginate lyases, alpha-amylases, betaamylases, beta glucanases, alpha-galactosidases, arabinosidases, aryl esterases, betagalactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, DNases, dispersins, endo-beta-1.
  • one or more ions selected from calcium and/or zinc one or more enzyme stabilizer; from about 0.001% to about
  • DNAses and/or RNAses DNAses and/or RNAses), oxidases, oxidoreductases, pectate lyases, pectin acetyl esterases, pectin methyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phosphodiesterases, phospholipases, phytases, polyesterases, polygalacturonases, additional proteases, pullulanases, RNases, reductases, rhamnogalacturonases, tannases, transglutaminases, xanthanases, xanthan lyases, xylan acetyl-esterases, xylanases, xyloglucanases. xylosidases, and combinations thereof.
  • Embodiment 24 The composition of any one of Embodiments 20-23, wherein the composition is a granular, regular or compact powder, solid, bar, regular or compact or concentrated liquid, homogeneous tablet, tablet having two or more layers, pouch having one or more compartments, gel, paste or unit dose composition.
  • the Bacillus lentus P29600 subtilisin (GG36) mature protein sequence is provided in SEQ ID NO: 1.
  • This GG36 subtilisin wild type was used as the starting point in the engineering of further substituted variants.
  • All GG36 subtilisin variants were expressed using a DNA fragment comprising: a 5’ AprE flanking region that contains a variant of the B. subtilis rrnlp2 promoter sequence (SEQ ID NO:2) (the B. subtilis rrnl 2 promoter and engineered variant are more fully described in patent application W02020112609), the nucleotide sequence encoding the aprE signal peptide sequence (SEQ ID NO:3), the nucleotide sequence encoding a variant of the B.
  • lentus propeptide SEQ ID NO:4
  • sequence corresponding to the gene encoding the mature GG36 subtilisin SEQ ID NO: 5
  • BPN’ terminator SEQ ID NO:6
  • 3’AprE flanking sequences including a kanamycin gene expression cassette SEQ ID NO:7
  • the transformation mixtures were plated onto LA plates containing 1.6% skim milk and 1.8 ppm kanamycin and incubated overnight at 37°C. Single colonies were picked and grown in Luria broth at 37°C under antibiotic selection.
  • transformed cells were grown in 96-well microtiter plates (MTPs) in cultivation medium (enriched semi-defined media based on MOPS buffer, with urea as major nitrogen source, glucose as the main carbon source, supplemented with 1% soytone for robust cell growth, containing antibiotic selection) for 3 days at 32°C, 300 rpm, w ith 80% humidity' in a shaking incubator. After centrifugation and filtration, clarified culture supernatants containing the proteases of interest were used for assays.
  • MTPs 96-well microtiter plates
  • the concentration of the GG36 subtilisin variants in culture supernatant was determined by UHPLC using a Zorbax 300 SB-C3 column and linear gradient of 0.1% Trifl uoroacetic acid (Solution A) and 0.07% Trifluoroacetic acid in Acetonitrile (Solution B) and detection at 220nm.
  • Culture supernatants were diluted in 10 mM NaCl, 0. ImM CaCL, 0.005% Tween®-80 for loading onto column.
  • the protein concentration of the samples was calculated using a standard curve of the parent enzy me.
  • protease activity of GG36 subtilisin variants was tested by measuring the hydrolysis of AAPF-pNA synthetic peptidic substrate.
  • the reagent solutions used were: 100 mM Tris pH 8.6, 10 mM CalCh, 0.005% Tween®-80 (Tris/Ca buffer) and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) (Sigma: S-7388).
  • 100 mM Tris pH 8.6, 10 mM CalCh, 0.005% Tween®-80 (Tris/Ca buffer) and 160 mM suc-AAPF-pNA in DMSO (suc-AAPF-pNA stock solution) Sigma: S-7388.
  • suc- AAPF-pNA stock solution 1 mL suc- AAPF-pNA stock solution was added to 100 mL Tris/Ca buffer and mixed.
  • Creme Brulee stain The cleaning performance of GG36 subtilisin variants on creme brulee stain was tested by using custom ordered melamine dishwasher monitors (tiles) prepared by CFT (Center for Testmaterials BV, Vlaardingen, the Netherlands) as set forth herein, and labeled DM1 lOGs.
  • the DM1 lOGs tiles used in this study are prepared using the same stain used to prepare the commercially available DM10 monitors (creme brulee Debic.com product), but baked at 140°C for 2 hours, instead of 150°C.
  • the DM1 lOGs melamine tiles were used as a lid and tightly pressed onto a microtiter plate (MTP).
  • MTP microtiter plate
  • 3.8 g/L of MGDA-Citrate detergent solution (composition shown in Tablet) adjusted to 374ppm water hardness and each enzyme sample were added to the MTP prior to attaching the melamine tile lid to the MTP.
  • the volume capacity of the MTP, and therefore the volume of solution added thereto, may vary, wherein a minimal volume of solution that enables contact between solution and stain surface should be added to the MTP.
  • a volume of 300pL of detergent solution containing enzy me was added to each well of an aluminum 96-well MTP.
  • the MTPs were incubated in an Infors thermal shaker for 45 min at 40°C, unless otherwise specified, at 250 rpm. After incubation, the tiles were removed from the MTP, briefly rinsed with tap water, and air-dried.
  • Baked Cheese stain The cleaning performance of GG36 subtilisin variants on baked cheese was tested by using custom ordered melamine dishwasher monitors (tiles) prepared by CFT (Vlaardingen, the Netherlands) as set forth herein, and labeled DM06Gs.
  • the DM06Gs tiles used in this study are prepared using the same stain used to prepare the commercially available DM06 monitors.
  • the DM06Gs melamine tiles were used as a lid and tightly pressed onto a microtiter plate (MTP).
  • MTP microtiter plate
  • 3.8 g/L of MGDA-Citrate detergent solution (composition shown in Table 1) adjusted to 374ppm water hardness and each enzyme sample were added to the MTP prior to attaching the melamine tile lid to the MTP.
  • the volume capacity of the MTP. and therefore the volume of solution added thereto, may vary, wherein a minimal volume of solution that enables contact between solution and stain surface should be added to the MTP.
  • a volume of 300pL of detergent solution containing enzyme was added to each well of an aluminum 96-well MTP.
  • the MTPs were incubated in an Infors thermal shaker for 45 mm at 40°C. unless otherwise specified, at 250 rpm. After incubation, the tiles were removed from the MTP, briefly rinsed with tap water, and air-dried.
  • % SRI (AE/AE initial ) * 100 [00219] Cleaning performance was obtained by subtracting the value of a blank control (no enzyme) from each sample value (hereinafter ’‘blank subtracted cleaning”). For each condition and GG36 subtilisin variant, a performance index (PI) was calculated by dividing the blank subtracted cleaning by that of the parent protease at the same concentration. The value for the parent protease PI was determined from a standard curve of the parent protease which was included in the test and which was fitted to a Langmuir fit or Hill Sigmoidal fit, as appropriate.
  • Egg yolk stain The cleaning performance of GG36 subtilisin variants on egg yolk microswatches (PAS-38. Center for Testmaterials BV, Vlaardingen, Netherlands) was measured on pre-rinsed swatches. To prepare rinsed PAS38 swatches, 180pl of lOmM CAPS buffer, pH 11, was added to MTPs containing PAS38 microswatches. The plates were sealed and incubated in an iEMS incubator for 30 min at 60°C with 1100 rpm shaking. After this incubation, the buffer was removed, and the swatches were rinsed with deionized water to remove any residual buffer. The plates were then air dried prior to use in the performance assay. The microswatch plates, containing PAS-38 swatches, were filled with ADW detergent solution prior to enzyme addition with a final enzyme concentration betw een 0.05 and lOppm.
  • Full Egg stain The cleaning performance of GG36 subtilisin variants on full egg cotton microswatches was measured using C-S-39 sw atches (order code: C-S-39, full egg with carbon black, aged; Center for Testmaterials BV, Vlaardingen, Netherlands).
  • ADW cleaning assays were performed using the MGDA-Citrate detergent at a final concentrations of 3.8 g/L (composition shown on Table 1), with water hardness adjusted to 374 ppm.
  • Laundry cleaning assay was performed using the ECE-2 HDD detergent solution.
  • the composition of ECE-2 powder detergent purchased from WFT Testgewebe is described in Table 3 set forth in Example 4 below. Furthermore. 150 g of TAED and 25 g of sodium percarbonate were added to 825 g of ECE-2 detergent (from WFT Testgewebe) and mixed. An aqueous solution of this mixture (6.5 g/L final concentration) was prepared, adjusted to 171 ppm water hardness, and used as the ECE-2 HDD detergent solution in the laundry' cleaning assay.
  • Bacillus lentus P29600 subtilisin (GG36) wildtype parent (SEQ ID NO: 1) served as the reference enzy me.
  • the expression of the proteins used in the assays is described in Example 1.
  • the ADW cleaning performance on Egg Yolk (PAS -38), Baked Cheese (DM06Gs) and Creme Brulee (DM1 lOGs) technical stains of these GG36 subtilisin variants were measured using detergents and assays described in Example 2. The results are reported in Table 2.
  • the cleaning is expressed as PI values versus the GG36 wildtype parent enzyme.
  • the GG36 variant PB92 049 also served as a reference, and all newly disclosed variants showed significant cleaning benefits across stains when compared to GG36 WT parent and to PB92 049 variant.
  • Bacillus lentus P29600 subtilisin (GG36) wildtype parent served as the reference enzyme.
  • the expression of the proteins used in the assay is described in Example 1.
  • the laundry cleaning performance on full egg technical stain (C-S-39, full egg with carbon black, aged; Center for Testmaterials BV, Vlaardingen, Netherlands) of these GG36 subtilisin variants was measured using a modified ECE-2 HDD detergent solution prepared as shown below.
  • the composition of ECE-2 powder detergent purchased from WFT Testgewebe is shown in Table 3 below .
  • 150 g of TAED and 25 g of sodium percarbonate were added to 825 g of ECE-2 detergent (from WFT Testgewebe) and mixed.
  • An aqueous solution of this mixture (6.5 g/L final concentration) was prepared, adjusted to 171 ppm water hardness, and used as the ECE-2 HDD detergent solution in the laundry cleaning assay described in Example 2.
  • the cleaning assay test results are reported in Table 4.
  • the cleaning is expressed as PI values versus the GG36 wildtype parent enzyme.
  • the GG36 variant PB92 049 also served as a reference, and the variants in Table 4 showed cleaning benefits when compared to GG36 WT parent and to PB92 049 variant.

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Abstract

L'invention concerne un ou plusieurs variants de subtilisine, un acide nucléique codant pour ceux-ci, et des compositions et des procédés associés à la production et à l'utilisation de ceux-ci, comprenant un ou plusieurs variants de subtilisine qui ont une stabilité et/ou une élimination des sols améliorées par rapport à une ou plusieurs subtilisines de référence.
PCT/US2024/018525 2023-03-06 2024-03-05 Variants de subtilisine et procédés d'utilisation WO2024186819A1 (fr)

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