WO2011072117A1 - Produits d'entretien du linge et de la maison - Google Patents

Produits d'entretien du linge et de la maison Download PDF

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Publication number
WO2011072117A1
WO2011072117A1 PCT/US2010/059669 US2010059669W WO2011072117A1 WO 2011072117 A1 WO2011072117 A1 WO 2011072117A1 US 2010059669 W US2010059669 W US 2010059669W WO 2011072117 A1 WO2011072117 A1 WO 2011072117A1
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WO
WIPO (PCT)
Prior art keywords
protease
variant
group
mutations
seq
Prior art date
Application number
PCT/US2010/059669
Other languages
English (en)
Inventor
Philip Frank Souter
Glenn Steven Ward
Jashua Roy Basler
Luis Gustavo Cascao-Pereira
David A. Estell
James T. Kellis, Jr.
Alexander Pisarchik
Ayrookaran J. Poulouse
Daniel Torres-Pazmino
Viktor Yuryevich ALEKSEYEV
Original Assignee
The Procter & Gamble Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Procter & Gamble Company filed Critical The Procter & Gamble Company
Priority to EP18181021.9A priority Critical patent/EP3434764A3/fr
Priority to EP22182416.2A priority patent/EP4159833A3/fr
Priority to CA2782613A priority patent/CA2782613C/fr
Priority to CN201080055885.5A priority patent/CN102652175B/zh
Priority to MX2012006616A priority patent/MX2012006616A/es
Priority to EP10796231A priority patent/EP2510092A1/fr
Priority to JP2012543280A priority patent/JP5882904B2/ja
Publication of WO2011072117A1 publication Critical patent/WO2011072117A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38618—Protease or amylase in liquid compositions only
    • 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
    • C11D3/38609—Protease or amylase in solid compositions only
    • 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)

Definitions

  • This invention relates to fabric and home care products comprising cold water proteases as well as methods of making and using such fabric and home care products.
  • a laundry detergent composition that comprises a hueing agent, a cleaning polymer and/or a perfume capsule, improves the whiteness, whiteness perception, and/or freshness.
  • This invention relates to fabric and home care products comprising one or more cold water proteases and processes for making and using such products.
  • Such compositions provide improved cleaning and freshness.
  • Such cold water proteases may be derived from parent enzymes, including BPN' subtilisin and subtilisin derived from Bacillus lentus, by substitution, insertion and/or deletion of one or more of the parent enzymes' amino acids.
  • Figure 1 provides a plasmid map of pHPLT-BPN'-v3.
  • Figure 2 provides a plasmid map of pHPLT-BPN'-v3+S78N.
  • Figure 3 provides a plasmid map of pHPLT-BPN' partial opt.
  • Figure 4 provides a plasmid map of pHPLT-BPN'-v36.
  • Figure 5 provides an alignment of the mature reference subtilisin proteases including: BPN' (SEQ ID NO:2) and GG36 (SEQ ID NO:755).
  • BPN' SEQ ID NO:2
  • GG36 SEQ ID NO:755
  • Each amino acid position of each protease variant described herein, including each cold water protease variant, is numbered according to the numbering of the corresponding amino acid position in the amino acid sequence of Bacillus amyloliquejaciens subtilisin protease BPN' (SEQ ID NO:2), as shown in Figure 5, as determined by alignment of the protease variant amino acid sequence with the Bacillus amyloliquejaciens subtilisin protease BPN' amino acid sequence.
  • substitution positions are given in relationship to BPN'.
  • Figure 6 provides map of pHPLT-GG36.
  • Figure 7 provides a map of pRA68.
  • Figure 8 provides a map of pRA96.
  • Figure 9 provides a map of pAC-FNAre.
  • Figure 10 is a schematic representation of method or targeted ISD library construction.
  • Figure 11 provides a map of pAC-FNAlO.
  • Figure 12 provides a map of pHPLT- FN A
  • fabric and home products means products or devices generally intended to be used or consumed in the form in which they are sold and that are for treating fabrics, hard surfaces and any other surfaces in the area of fabric and home care, including: air care including air fresheners and scent delivery systems, car care, dishwashing, fabric conditioning (including softening and/or freshening), laundry detergency, laundry and rinse additive and/or care, hard surface cleaning and/or treatment including floor and toilet bowl cleaners, and other cleaning for consumer or institutional use.
  • air care including air fresheners and scent delivery systems
  • car care dishwashing
  • fabric conditioning including softening and/or freshening
  • laundry detergency including softening and/or freshening
  • laundry and rinse additive and/or care including floor and toilet bowl cleaners, and other cleaning for consumer or institutional use.
  • cleaning and/or treatment composition is a subset of fabric and home care products that includes, unless otherwise indicated fabric & home care products.
  • Such products include, but are not limited to, products for treating fabrics, hard surfaces and any other surfaces in the area of fabric and home care, including: air care including air fresheners and scent delivery systems, car care, dishwashing, fabric conditioning (including softening and/or freshening), laundry detergency, laundry and rinse additive and/or care, hard surface cleaning and/or treatment including floor and toilet bowl cleaners, granular or powder-form all- purpose or "heavy-duty” washing agents, especially cleaning detergents; liquid, gel or paste- form all-purpose washing agents, especially the so-called heavy-duty liquid types; liquid fine- fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use: car or carpet shampoos, bathroom cleaners including toilet
  • the term "fabric and/or hard surface cleaning and/or treatment composition” is a subset of cleaning and treatment compositions that includes, unless otherwise indicated, granular or powder-form all-purpose or "heavy-duty” washing agents, especially cleaning detergents; liquid, gel or paste-form all-purpose washing agents, especially the so- called heavy-duty liquid types; liquid fine-fabric detergents; hand dishwashing agents or light duty dishwashing agents, especially those of the high-foaming type; machine dishwashing agents, including the various tablet, granular, liquid and rinse-aid types for household and institutional use; liquid cleaning and disinfecting agents, car or carpet shampoos, bathroom cleaners including toilet bowl cleaners; fabric conditioning products including softening and/or freshening that may be in liquid, solid and/or dryer sheet form ; as well as cleaning auxiliaries such as bleach additives and "stain-stick" or pre-treat types, substrate-laden products such as dryer added sheets. All of such products which are applicable may be in standard, concentrated or even highly concentrated form even
  • solid includes granular, powder, bar and tablet product forms.
  • fluid includes liquid, gel, paste and gas product forms.
  • situs includes fabrics, garments, and/or hard surfaces.
  • component or composition levels are in reference to the active portion of that component or composition, and are exclusive of impurities, for example, residual solvents or by-products, which may be present in commercially available sources of such components or compositions.
  • a composition comprising a protease, said protease being selected from the group consisting of:
  • a cold water protease having a performance index at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 from 1 to about 10, from 1 to about 8 or even from 1 to about 5 on BMI at pH 8 and 60°F when compared to an enzyme having SEQ ID NO:6, as defined in Test Method 2 and/or Test Method 3;
  • cold water protease being selected from the group consisting of:
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:4, said variant comprising a group of mutations selected from the following groups of mutations: P210S, P210S-N218A, S063T-S078N-S101A-S183T-T244N, N061A- S078N-S224A, S053G-S078N-P129T-Q185T, S063T-S078N-S101A, S078N-P129T, S063T-S078N-S101A-S183T and S063T-S078N-S101A-T244I;
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising a group of mutations selected from the following groups of mutations:
  • G097A-I111 V-M124V-Y217Q G097A-I111 V-Y167A-Y217Q
  • S024G-N025G-N061P- G097A-S101N-G128S-Y217Q S024G-N025G-S053G-N061P-G097A-S101N-G128A- V203Y-Y217Q
  • V068A-A092G-Y217Q N061P-G097A-S101N-G128A-P210S-Y217Q
  • S024G- N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q S024G- N0
  • variants of a parent protease said parent having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising three, four, five or six of the following mutations X24G/R, X53G, X78N, X101N, X128A/S andX217L/Q; said variant optionally comprising at least one group of mutations selected from the following groups of mutations:
  • cold water protease being selected from the group consisting of:
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:4, said variant comprising a group of mutations selected from the following groups of mutations: E156S-P210S, P210S-N218S, Y104F-E156S-P210I, E156A-P210S- N218S, N218A, N061A-S078N-S087E-S224A, Q059V-S078N-G211A, Q059V-S078N- V147Q, Q059V-S078N, Q059V-S078N-I108V-N252Q, S053G-S078N-P129T, S078N- G211A, S078N-Q185T, S078N-V147Q, S078N, S078N-P129T
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising a group of mutations selected from the following groups of mutations: G097A-I111V-Y217Q, I111V-G128A-Y217Q, I111V-M124V-Y167A, I111V-M124V-Y217Q, L096T-G097A-Y217Q, N062Q-G097A-I111V, S053G-N061P- G097A-S101N-G128S-V203Y-Y217Q, S089Y-M124V-Y217Q, V068A-I111V-Y217Q, G097A-I111V-M124V, G097A-L126A-Y217Q, G097A
  • a variant of a parent protease said parent protease, having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising three, four, five or six of the following mutations
  • said variant optionally comprising at least one group of mutations selected from the following groups of mutations: A001G, A001Y, A013G, A013V, A015F, A015G, A015K, A015M, A015P, A015T, A015W, A015Y, A029G, A073S, A088C, A088I, A088L, A088T, A088V, A098D, A098K, A098P, A098R, A098W, A116D, A116E, A116R, A128S, A133L, A133M, A133S, A134G, A134S, A137N, A137V, A144M, A144Q, A144S, A144T, A144V, A151C, A176S, A176T, A
  • protease being selected from the group consisting of
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:4, said variant comprising a group of mutations selected from the following groups of mutations: Q059V-S078N-I108V-V147Q-G211A-N252Q, S078N-I108V-V147Q- G211A, S078N-S087E-M124I-S224A, S078N-I108V-V147Q-N252Q, S078N-I108V- V147Q-G211A-N252Q, and Q059V-S078N-I108V-V147Q-G211A-N252Q;
  • a variant of a parent protease said parent protease, having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising a group of mutations selected from the following groups of mutations: G097A-M124V-Y167A-Y217Q, V068A-Y167A-Y217Q, G097A-I111V- M124V-Y167A, I111V-M124V-Y167A-Y217Q, V068A-I111V-Y167A-Y217Q, G097A-I111 V-M124V-Y167A-Y217Q, P052L-V068A-I11 IV, G097A-N123A-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, N061P-S101N-G102A-G1
  • a variant of a parent protease said parent protease having at least 60%, or 80%, or 85% or 90% or 95% or 96% or 97% or 98% or 99% or even 100% identity to SEQ ID NO:2, said variant comprising three, four, five or six of the following mutations:
  • said variant optionally comprising at least one group of mutations selected from the following groups of mutations: A001D, A001H, A001N, A015C, A048C, A048E, A085T, A133R, A137R, A142C, A144D, A144R, A152S, A153G, A187P, A187Q, A187T, A187V, A216R, A230S, A272R, A273H, A273T, A274H, D036N, D036S, D181H, D181T, D259N, D259P, D259S, E156G, E156H, E156L, E156Q, E156V, E251C, F189S, F189T, F189W, F189Y, F261E, G020C, G024D, G
  • a variant of a parent protease comprising an amino acid sequence comprising from three to six amino acid substitutions selected from the group consisting of X024G/R, X053G, X078N, X101N, X128A/S, and X217L/Q, wherein the variant has proteolytic activity;
  • a variant of a protease the variant having proteolytic activity and comprising an amino acid sequence which comprises from two to seven alterations at the amino acid positions corresponding to amino acid positions of SEQ ID NO:2 selected from the group consisting of positions 24, 53, 78, 97, 101, 128, and 217, wherein each alteration is independently
  • the variant comprises an amino acid sequence having no more than 15 alterations relative to the parent protease, wherein the alterations are independently selected from an insertion, a deletion, or a substitution, and the alterations include a substitution of glycine at positions 24 and 53, a substitution of asparagine at positions 78 and 101, a substitution of alanine or serine at position 128, and a substitution of glutamine at position 217;
  • the parent protease has at least 90% sequence identity to SEQ ID NO:2; and the variant has increased proteolytic activity relative to the parent protease
  • a variant of a parent protease said parent protease at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to SEQ ID NO:2, said variant having an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% identity to the sequence of SEQ IDNO:2; said variant comprising an a substitution of glycine at positions 24 and 53, a substitution of asparagine at positions 78 and 101, a substitution of alanine or serine at position 128, and a substitution of glutamine at position 217, and said variant having increased proteolytic activity relative to the parent protease;
  • composition being a fabric and home care product is disclosed.
  • said cold water protease has either a performance index greater than 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 from at least 1 to about 10, from at least 1 to about 8 or even from at least 1 to about 5 on BMI at pH 8 and 60°F when compared to an enzyme having SEQ ID NO:4, as defined in Test Method 2 or Test Method 3 or said cold water protease has a performance index at least 1, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2 from 1.0 to about 10, from 1.0 to about 8 or even from 1.0 to about 5 on BMI at pH 8 and 60°F when compared to an enzyme having SEQ ID NO:6, as defined in Test Method 2 or Test Method 3and said cold water protease is selected from the group
  • a variant of a parent protease having SEQ ID NO: 4 said variant comprising a group of mutations selected from the following groups of mutations: P210S, P210S-N218A, S063T- S078N-S101A-S183T-T244N, N061A-S078N-S224A, S053G-S078N-P129T-Q185T, S063T- S078N-S101A, S078N-P129T, S063T-S078N-S101A-S183T and S063T-S078N-S101A-T244I; b) a variant of a parent protease having SEQ ID NO: 2, said variant comprising a group of mutations selected from the following groups of mutations: G097A-I111V-M124V-Y217Q, G097A-I111V-Y167A-Y217Q, S024G-N025G-N
  • said variant of a parent protease comprises the amino acid substitutions: S024G+S053G+S078N+S101N+G128A/S+Y217Q/L/D; and optionally one or more groups of subsitutions selected from the following groups of mutations:
  • said protease is a variant of a parent protease having SEQ ID NO: 6, said variant comprising one or more of the following groups of amino acid substitutions: A088T+N109G+A116T+G131H+N243V+L257G, S033T+N076D,
  • said parent protease is a subtilisin protease.
  • said parent protease is a subtilisin protease selected from the group consisting of B. amyloliquefaciens subtilisin protease BPN' (SEQ ID NO:2), Geobacillus stearothermophilus (formerly classified as B. stearothermophilus), B. subtilis, B. licheniformis, B. lentus, B. brevis, B. alkalophilus, B. amyloliquefaciens, B. clausii, B.
  • said cold water protease is a variant of a parent protease, said cold water protease comprising a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below, wherein at least one mutation is selected from group (a):
  • said variant of a parent protease comprises a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below, wherein at least one mutation is selected from group (a):
  • said variant of a parent protease has a total net charge of -1, 0 or +1 relative to the BPN' wild-type.
  • said composition comprises an adjunct ingredient selected from the group consisting of: a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzymes, an enzyme stabilizer, a catalytic material, a bleach activator, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a brightener, a suds suppressor, a dye, a perfume, a perfume delivery system, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.
  • a surfactant e.g., a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzymes, an enzyme stabilizer, a catalytic material, a bleach activator, a hydrogen peroxid
  • said composition comprises a material selected from the group consisting of an encapsulate comprising a perfume, a hueing agent, an amphiphilic cleaning polymer, a brightener, a chelating agent and mixtures thereof.
  • said composition comprises a second non- immunoequivalent protease selected from the group comprising:
  • said composition comprises a second non- immunoequivalent protease that is a subtilisin (EC 3.4.21.62) protease said subtilisin (EC 3.4.21.62) protease being a cold water GG36 variant protease.
  • said composition comprises an additional enzyme selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, cellobiose dehydrogenases, xyloglucanases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, or mixtures thereof.
  • an additional enzyme selected from the group consisting of hemicellulases, peroxidases, proteases, cellulases, cellobiose dehydrogenases, xylogluc
  • said composition comprises an additional enzyme is selected from the group consisting of:
  • alpha-amylases that are greater than 90% identical to the wild-type alkaline alpha- amylases derived from Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649, KSM AP1378, KSM K36 or KSM K38;
  • said composition comprises a material selected from the group consisting of: a) an alcohol ethoxysulphate surfactant;
  • a chelant selected from the group consisting of DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), ethylenediaminedisuccinic acid (EDDS), 1,2- Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and derivatives of such chelants; and
  • said composition comprises a material selected from the group consisting of:
  • an alcohol ethoxysulphate surfactant having an alkyl chain length of from 10 to 14 and a degree of ethoxylation from 1 to 4;
  • a chelant selected from the group consisting of DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), ethylenediaminedisuccinic acid (EDDS), 1,2- Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and derivatives of such chelants; and
  • said composition comprises, based on total composition weight, a chelant level of from about 1% to about 5%.
  • said composition comprises a fabric hueing agent selected from the group consisting of
  • dye-clay conjugates comprising at least one cationic-basic dye and a smectite clay; and c) mixtures thereof.
  • said composition comprises a fabric hueing agent selected from the group consisting of
  • dye-clay conjugates comprising at least one cationic-basic dye and a smectite clay; and c) mixtures thereof.
  • said composition comprises, based on total composition weight: a) from about 0.0005 wt% to about 0.1 wt% of said cold water protease; and
  • said composition has a single or multi-compartment unit dose form.
  • said composition is in the form of a multicompartment unit dose, wherein at least one protease is in a different compartment to any additional enzymes and/or chelant.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof said composition being a fabric and home care product is disclosed.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof
  • said cold water protease is a variant of the protease having SEQ ID NO:2 (subtilisin BPN') said variant comprising one or more of the following sets of mutations, insertions or deletions: S182E, N109I, N109D-Y217L-S248R, N109D-S188R-Y217L, S87D-Y217L-S248R, S87R-N109D- Y217L-S248R, S87R-N109D-S188D-Y217L-S248R, G128A-Y217Q, I111V-M124V, M124V- Y217Q, N62Q-G97A, S89Y
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations within the group of positions comprising positions 24, 25, 40, 52.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from groups (a) and (b) below:
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said variant of the protease having SEQ ID NO:2 comprises one or more mutations, and having a total net charge of -1, 0 or +1 relative to the BPN' wild-type.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof
  • said composition comprises an adjunct ingredient selected from the group consisting of: a surfactant, a builder, a chelating agent, a dye transfer inhibiting agent, a dispersant, one or more additional enzyme, an enzyme stabilizer, a catalytic material, a bleach activators, a hydrogen peroxide, a source of hydrogen peroxide, a preformed peracid, a polymeric dispersing agent, a clay soil removal/anti-redeposition agent, a brightener, a suds suppressor, a dye, a perfume, a perfume delivery system, a structure elasticizing agent, a fabric softener, a carrier, a hydrotrope, a processing aid, a solvent, a pigment and mixtures thereof.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof
  • said composition comprises a third non-immunoequivalent protease that is a subtilisin (EC 3.4.21.62) protease said subtilisin (EC 3.4.21.62) protease being a cold water GG36 variant protease.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises an additional enzyme selected from the group consisting of
  • hemicellulases peroxidases, proteases, cellulases, cellobiose dehydrogenases, xyloglucanases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, mannanases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, lichenases glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, amylases, or mixtures thereof.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises an additional enzyme is selected from the group consisting of:
  • alpha-amylases that are greater than 90% identical to the wild-type alkaline alpha- amylases derived from Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 DSM 12368, DSMZ no. 12649, KSM AP1378, KSM K36 or KSM K38;
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a material selected from the group consisting of:
  • a) an alcohol ethoxysulphate surfactant b) a chelant selected from the group consisting of DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), ethylenediaminedisuccinic acid (EDDS), 1,2- Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and derivatives of such chelants; and
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a material selected from the group consisting of:
  • an alcohol ethoxy sulphate surfactant having an alkyl chain length of from 10 to 14 and a degree of ethoxylation from 1 to 4;
  • a chelant selected from the group consisting of DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), ethylenediaminedisuccinic acid (EDDS), 1,2- Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, and derivatives of such chelants; and
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises, based on total composition weight, a chelant level of from about 1% to about 5%.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a fabric hueing agent selected from the group consisting of
  • dye-clay conjugates comprising at least one cationic-basic dye and a smectite clay; and c) mixtures thereof.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises a fabric hueing agent selected from the group consisting of
  • dye-clay conjugates comprising at least one cationic-basic dye and a smectite clay; and c) mixtures thereof.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition comprises based on total composition weight:
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition has a single or multi-compartment unit dose form.
  • composition comprising a cold water protease and a material selected from the group consisting of: an encapsulate comprising a perfume, a GG36 variant protease, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, said composition is in the form of a multi-compartment unit dose, wherein at least one protease is in a different compartment to any additional enzymes and/or chelant.
  • a fabric and home product that may comprise one or more cold water proteases and a material selected from the group consisting of: an encapsulate comprising a perfume, a hueing agent, an amphiphilic cleaning polymer and mixtures thereof, with the balance of any aspects of the aforementioned composition is made up of one or more adjunct materials, is disclosed.
  • said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.0005 wt% to about 0.1 wt%, from about 0.001 wt% to about 0.05 wt%, or even from about 0.002wt% to about 0.03wt% of said cold water protease.
  • said fabric and home care product may comprise, based on total fabric and home care product weight, about
  • said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.001wt% to about 5wt%, from about 0.01wt% to about 2wt%, or even from about 0.03 wt% to about 0.5wt%, perfume capsules.
  • said fabric and home care product may comprise, based on total fabric and home care product weight, from about 0.1 wt% to about 5 wt%, from about 0.25 wt% to about 2.5 wt%, or even from about 0.3 wt% to about 1.5 wt% amphiphilic cleaning polymer.
  • suitable cold water protease variants are enzymes that exhibit one or more of the following four criteria:
  • GG36 cold water proteases may be Series 1 GG36 cold water protesaes.
  • Suitable Series 1 GG36 cold water protease variants include enzymes derived from a parent protease, said parent protease 's sequence being at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% identical to the amino acid sequence of SEQ ID NO:755, said variant having one or more of the following characteristics:
  • Suitable Series 1 GG36 cold water proteases can be derived from subtilisins, particularly those derived from subtilisin Bacillus Lentus of SEQ ID NO:755 and in one aspect may comprise one or more of the following mutations:
  • suitable Series 1 GG36 cold water proteases include subtlisins, particularly Bacillus Lentus of SEQ ID NO:755, that may comprise one or more of the following sets of mutations, insertions or deletions:
  • suitable Series 1 GG36 cold water proteases include variants of subtlisins, particularly Bacillus Lentus of SEQ ID NO:755, said variants comprising three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations within the group of positions comprising positions 1, 2, 4, 9, 10, 14, 16, 17, 18, 20, 22, 24, 25, 26, 42, 43, 46, 52, 57, 59, 62, 68, 71, 72, 74, 75, 76, 78, 82, 86, 89, 91, 94, 100, 101, 103, 104, 106, 108, 111, 112, 115, 117, 118, 121, 128, 129, 144, 148, 158, 159, 160, 166, 185, 186, 188, 197, 203, 209, 210, 212, 214, 215, 217, 224, 230, 231, 236, 238, 239, 241, 242, 243, 244, 248,
  • suitable Series 1 GG36 cold water proteases include variants of subtlisins, particularly Bacillus Lentus of SEQ ID NO:755, said variants comprising a total of three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14 or 15 mutations selected from the group comprising: AIR, Q2S, V4R, V4S, S9A, R10S, P14K, A16S, H17R, N18R, G20R, T22A, T22R, S24R, S24W, G25R, G25V, V26F, L42I, N43R, N43A, G46R, P52F, P52E, P52N, T57R, Q59A, N62E, N62Q, V68A, V68C, T71G, I72C, A74C.
  • said Series 1 GG36 cold water protease is variant of subtilisin GG36 having SEQ ID NO:755, said variant comprising one or more mutations, and having a total net charge of -5, -4, -3, -2, -1 or 0 relative to subtilisin GG36 wild-type, is disclosed.
  • said Series 1 GG36 cold water proteases are low ionic strength cold water proteases.
  • Such low ionic strength proteases are variants of subtilisin GG36 having SEQ ID NO:755, said variants comprising one or more mutations, and having a total net charge of -5, -4, - 3, -2, -1 or 0 relative to subtilisin GG36 wild-type, is disclosed. These mutations are selected from:
  • the above low ionic strength Series 1 GG36 cold water proteases form part of a detergent composition that is diluted in water, typically within a washing machine, to form a wash liquor, whose conductivity is from about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5 mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm
  • said Series 1 GG36 cold water proteases are high ionic strength cold water proteases.
  • Such high ionic strength proteases are variants of subtilisin GG36 having SEQ ID NO:755, said variants comprising two or more mutations, and having a total net charge of +5, +4, +3, +2, +1 or 0 relative to subtilisin GG36 wild-type. These mutations are selected from:
  • the above high ionic strength Series 1 GG36 cold water proteases form part of a detergent composition that is diluted in water, typically within a washing machine, to form a wash liquor, whose conductivity is from about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20 mS/cm, or even from about 4mS/cm to about 10 mS/cm.
  • the charge of the Series 1 GG36 cold water protease variants is expressed relative to subtilisin GG36 protease wild-type having the amino acid sequence of SEQ ID NO:755.
  • the amino acids that impart a single negative charge are D and E and those that impart a single positive charge are R, H and K. Any amino acid change versus SEQ ID NO:755 that changes a charge is used to calculate the charge of the Series 1 GG36 cold water protease variant.
  • introducing a negative charge mutation from a wild-type neutral position will add a net charge of -1 to the Series 1 GG36 cold water protease variant
  • introducing a negative charge mutation (D or E) from a wild-type positive amino acid residue (R, H or K) will add a net charge of -2.
  • Summing the charge changes from all the amino acid residues that are different for the Series 1 GG36 cold water protease variant versus subtilisin GG36 protease wild-type having the amino acid sequence of SEQ ID NO:755 gives the charge change of the Series 1 GG36 cold water protease variant.
  • Low conductivity solutions are defined as having a conductivity of from about 0.1 mS/cm to about 3 mS/cm, from about 0.3 mS/cm to about 2.5 mS/cm, or even from about 0.5 mS/cm to about 2 mS/cm.
  • High conductivity having conductivity solutions are defined as having a conductivity of from about 3 mS/cm to about 30 mS/cm, from about 3.5 mS/cm to about 20 mS/cm, or even from about 4 mS/cm to about 10 mS/cm
  • the enzyme charge can also be balanced by mutations in further positions.
  • amyloliquejaciens subtilisin BPN' numbering system Each amino acid position of each protease variant, including each Series 1 GG36 cold water protease variant, is numbered according to the numbering of corresponding amino acid position in the amino acid sequence of Bacillus amyloliquejaciens subtilisin BPN' as determined by alignment of the variant protease amino acid sequence with the Bacillus amyloliquejaciens subtilisin BPN' amino acid sequence.
  • An alternative numbering scheme is numbering the specific amino acid sequence of the B. lentus subtilisin GG36 protease, having the amino acid sequence of SEQ ID NO:755. None of the amino acid positions of the protease variants, including the Series 1 GG36 cold water protease variants, described herein are numbered using this alternative numbering scheme.
  • Mutations are named by the one letter code for the parent amino acid, followed by a three digit position number and then the one letter code for the variant amino acid.
  • mutating glycine (G) at position 87 to serine (S) is represented as “G087S” or “G87S”.
  • Multiple mutations are indicated by inserting a "-" between the mutations.
  • Mutations at positions 87 and 90 are represented as either "G087S-A090Y” or "G87S-A90Y” or "G87S + A90Y” or "G087S + A090Y”.
  • the one letter code "Z” is used.
  • the one letter code "Z" is on the left side of the position number.
  • the one letter code "Z" is on the right side of the position number.
  • the position number is the position number before the inserted amino acid(s), plus 0.01 for each amino acid.
  • an insertion of three amino acids alanine (A), serine (S) and tyrosine (Y) between position 87 and 88 is shown as "Z087.01A-Z087.02S-Z087.03Y.”
  • the accepted IUPAC single letter or triple letter amino acid abbreviation is employed.
  • the single letter X refers to any of the twenty amino acids.
  • wild-type in reference to an amino acid sequence or nucleic acid sequence indicates that the amino acid sequence or nucleic acid sequence is native or naturally occurring sequence.
  • naturally-occurring refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that are found in nature (i.e., have not been manipulated by means of recombinant methods).
  • non-naturally occurring refers to anything that is not found in nature (e.g., recombinant nucleic acids produced in the laboratory).
  • 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 along related proteins or a reference protein.
  • the terms "derived from” and “obtained from” refer to not only a protease produced or producible by a strain of the organism in question, but also a protease 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 protease which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the protease in question.
  • proteases derived from Bacillus refers to those enzymes having proteolytic activity which 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 non-Bacillus organisms transformed with a nucleic acid encoding the serine proteases.
  • nucleic acids or polypeptide sequences refers to the residues in the two sequences that are the same when aligned for maximum
  • homologous genes refers to a pair of genes from different, but usually related species, which correspond to each other and which are identical or very similar to each other.
  • the term encompasses genes that are separated by speciation (i.e., the development of new species) (e.g., orthologous genes), as well as genes that have been separated by genetic duplication (e.g., paralogous genes).
  • 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.
  • homology refers to sequence similarity or identity, with identity being preferred. Homology may be determined using standard techniques known in the art (See e.g., Smith and Waterman, Adv. Appl. Math. 2:482 [1981]; Needleman and Wunsch, J. Mol. Biol. 48:443 [1970 ⁇ ; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 [1988]; software programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al., Nucl. Acid Res. 12:387-395 [1984]).
  • PILEUP One example of a useful algorithm is PILEUP.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (See, Feng and Doolittle, J. Mol. Evol. 35:351-360 [1987]). The method is similar to that described by Higgins and Sharp (See, Higgins and Sharp, CABIOS 5: 151-153 [1989]). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST BLAST algorithm
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. However, the values may be adjusted to increase sensitivity.
  • the percent sequence identity between a reference sequence and a test sequence of interest may be readily determined by one skilled in the art.
  • the percent identity shared by polynucleotide or polypeptide sequences is determined by direct comparison of the sequence information between the molecules by aligning the sequences and determining the identity by methods known in the art.
  • An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, (See, Altschul, et al, J. Mol. Biol., 215:403-410 [1990]).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pairs
  • These initial neighborhood word hits act as starting points to find longer HSPs containing them.
  • the word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased.
  • Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 [1992]) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.
  • the BLAST algorithm then performs a statistical analysis of the similarity between two sequences (See e.g., Karlin and Altschul, supra).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a serine protease nucleic acid of this invention if the smallest sum probability in a comparison of the test nucleic acid to a serine protease nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • test nucleic acid encodes a serine protease polypeptide
  • it is considered similar to a specified serine protease nucleic acid if the comparison results in a smallest sum probability of less than about 0.5, and more preferably less than about 0.2.
  • Percent “identical” or “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection.
  • "Percent sequence identity” or “% identity” or “% sequence identity or “% amino acid sequence identity” of a subject amino acid sequence to a reference (i.e., query) amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by-amino acid basis) by a specified percentage to the query amino acid sequence over a comparison length when the sequences are optimally aligned.
  • 80% amino acid sequence identity or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.
  • Percent sequence identity or “% identity” or “% sequence identity or “% nucleotide sequence identity” of a subject nucleic acid sequence to a reference (i.e. query) nucleic acid sequence means that the subject nucleic acid sequence is identical (i.e., on a nucleotide-by- nucleotide basis for a polynucleotide sequence) by a specified percentage to the query sequence over a comparison length when the sequences are optimally aligned.
  • 80% nucleotide sequence identity or 80% identity with respect to two nucleic acid sequences means that 80% of the nucleotide residues in two optimally aligned nucleic acid sequences are identical.
  • the percent sequence identity or % sequence identity" or "% identity" of a subject sequence to a query sequence can be calculated by optimally aligning the two sequences and comparing the two optimally aligned sequences over the comparison length. The number of positions in the optimal alignment at which identical residues occur in both sequences are determined, thereby providing the number of matched positions, and the number of matched positions is then divided by the total number of positions of the comparison length (which, unless otherwise specified, is the length of the query sequence). The resulting number is multiplied by 100 to yield the percent sequence identity of the subject sequence to the query sequence.
  • Optimal alignment or “optimally aligned” refers to the alignment of two (or more) sequences giving the highest percent identity score.
  • optimal alignment of two protein sequences can be achieved by manually aligning the sequences such that the maximum number of identical amino acid residues in each sequence are aligned together or by using software programs or procedures described herein or known in the art.
  • Optimal alignment of two nucleic acid sequences can be achieved by manually aligning the sequences such that the maximum number of identical nucleotide residues in each sequence are aligned together or by using software programs or procedures described herein or known in the art.
  • two polypeptide sequences are deemed "optimally aligned" when they are aligned using defined parameters, such as a defined amino acid substitution matrix, gap existence penalty (also termed gap open penalty), and gap extension penalty, so as to achieve the highest similarity score possible for that pair of sequences.
  • a defined amino acid substitution matrix such as a defined amino acid substitution matrix, gap existence penalty (also termed gap open penalty), and gap extension penalty, so as to achieve the highest similarity score possible for that pair of sequences.
  • gap existence penalty also termed gap open penalty
  • gap extension penalty e.g., BLOSUM62 scoring matrix (See, Henikoff and Henikoff, supra) is often used as a default scoring substitution matrix in polypeptide sequence alignment algorithms (e.g., BLASTP).
  • the gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each residue position in the gap.
  • the alignment score is defined by the amino acid positions of each sequence at which the alignment begins and ends (e.g., the alignment window), and optionally by the insertion of a gap or multiple gaps into one or both sequences, so as to achieve the highest possible similarity score.
  • Optimal alignment between two or more sequences can be determined manually by visual inspection or by using a computer, such as, but not limited to for example, the BLASTP program for amino acid sequences and the BLASTN program for nucleic acid sequences (See e.g., Altschul et al, Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the National Center for Biotechnology Information (NCBI) website).
  • a computer such as, but not limited to for example, the BLASTP program for amino acid sequences and the BLASTN program for nucleic acid sequences (See e.g., Altschul et al, Nucleic Acids Res. 25(17):3389-3402 (1997); See also, the National Center for Biotechnology Information (NCBI) website).
  • Fluorescent optical brighteners emit at least some visible light.
  • fabric hueing agents can alter the tint of a surface as they absorb at least a portion of the visible light spectrum.
  • Suitable fabric hueing agents include dyes, dye-clay conjugates, and pigments that satisfy the requirements of Test Method 1 in the Test Method Section of the present specification.
  • Suitable dyes include small molecule dyes and polymeric dyes.
  • Suitable small molecule dyes include small molecule dyes selected from the group consisting of dyes falling into the Colour Index (C.I.) classifications of Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet and Basic Red, or mixtures thereof, for example:
  • the C ring may be substituted at the 5 position by an NH 2 or NHPh group
  • X is a benzyl or naphthyl ring substituted with up to 2 sulfonate groups and may be substituted at the 2 position with an OH group and may also be substituted with an N3 ⁇ 4 or NHPh group.
  • the A ring is preferably substituted by a methyl and methoxy group at the positions indicated by arrows, the A ring may also be a naphthyl ring, the Y group is a benzyl or naphthyl ring, which is substituted by sulfate group and may be mono or disubstituted by methyl groups.
  • both the aromatic groups may be a substituted benzyl or naphthyl group, which may be substituted with non water- solubilising groups such as alkyl or alkyloxy or aryloxy groups, X and Y may not be substituted with water solubilising groups such as sulfonates or carboxylates.
  • X is a nitro substituted benzyl group and Y is a benzyl group
  • B is a naphthyl or benzyl group that may be substituted with non water solubilising groups such as alkyl or alkyloxy or aryloxy groups, B may not be substituted with water solubilising groups such as sulfonates or carboxylates.
  • X and Y independently of one another, are each hydrogen, Ci-C 4 alkyl or C1-C4- alkoxy, Ra is hydrogen or aryl, Z is C1-C4 alkyl; Ci-C4-alkoxy; halogen; hydroxyl or carboxyl, n is 1 or 2 and m is 0, 1 or 2, as well as corresponding salts thereof and mixtures thereof
  • suitable small molecule dyes include small molecule dyes selected from the group consisting of Colour Index (Society of Dyers and Colourists, Bradford, UK) numbers 1 ,4-Naphthalenedione, 1 - [2- [4- [[4-(acetyloxy)butyl]ethylamino] -2- methylphenyl]diazenyl]-5-nitro-3-thienyl]-Ethanone, l-hydroxy-2-(l-naphthalenylazo)- Naphthalenedisulfonic acid, ion(2-), l-hydroxy-2-[[4-(phenylazo)phenyl]azo]- Naphthalenedisulfonic acid, ion(2-), 2-[(lE)-[4-[bis(3-methoxy-3-oxopropyl)amino]-2- methylphenyl]azo]-5-nitro-3-Thiophenecarboxylic acid, ethyl ester, 2-[
  • Acid black 1 C.I. Acid Blue 10, C.I. Acid Blue 113, C.I. Acid Blue 25, C.I. Acid Blue 29, CI. Acid Blue 290 C.I. Acid Red 103, C.I. Acid red 150, C.I. Acid red 52, C.I. Acid red 73, C.I. Acid red 88, C.I. Acid red 91, C.I. Acid violet 17, C.I. Acid violet 43, C.I. Direct Blue 1, C.I. Direct Blue 120, C.I. Direct Blue 34, C.I. Direct Blue 70, C.I. Direct Blue 71, C.I. Direct Blue 72, C.I. Direct Blue 82, C.I. Direct violet 51, C.I. Disperse Blue 10, C.I.
  • Disperse Blue 100 C.I. Disperse Blue 101, C.I. Disperse Blue 102, C.I. Disperse Blue 106:1, C.I. Disperse Blue 11, C.I. Disperse Blue 12, C.I. Disperse Blue 121, C.I. Disperse Blue 122, C.I. Disperse Blue 124, C.I. Disperse Blue 125, C.I. Disperse Blue 128, C.I. Disperse Blue 130, C.I. Disperse Blue 133, C.I. Disperse Blue 137, C.I. Disperse Blue 138, C.I. Disperse Blue 139, C.I. Disperse Blue 142, C.I. Disperse Blue 146, C.I.
  • Disperse Blue 148 C.I. Disperse Blue 149, C.I. Disperse Blue 165, C.I. Disperse Blue 165:1, C.I. Disperse Blue 165:2, C.I. Disperse Blue 165:3, C.I. Disperse Blue 171, C.I. Disperse Blue 173, C.I. Disperse Blue 174, C.I. Disperse Blue 175, C.I. Disperse Blue 177, C.I. Disperse Blue 183, C.I. Disperse Blue 187, C.I. Disperse Blue 189, C.I. Disperse Blue 193, C.I. Disperse Blue 194, C.I. Disperse Blue 200, C.I.
  • Disperse Blue 201 C.I. Disperse Blue 202, C.I. Disperse Blue 205, C.I. Disperse Blue 206, C.I. Disperse Blue 207, C.I. Disperse Blue 209, C.I. Disperse Blue 21, C.I. Disperse Blue 210, C.I. Disperse Blue 211, C.I. Disperse Blue 212, C.I. Disperse Blue 219, C.I. Disperse Blue 220, C.I. Disperse Blue 222, C.I. Disperse Blue 224, C.I. Disperse Blue 225, C.I. Disperse Blue 248, C.I. Disperse Blue 252, C.I.
  • Disperse Blue 346 C.I. Disperse Blue 351, C.I. Disperse Blue 352, C.I. Disperse Blue 353, C.I. Disperse Blue 355, C.I. Disperse Blue 356, C.I. Disperse Blue 357C.I. Disperse Blue 358, C.I. Disperse Blue 36, C.I. Disperse Blue 360, C.I. Disperse Blue 366, C.I. Disperse Blue 368, C.I. Disperse Blue 369, C.I. Disperse Blue 371, C.I. Disperse Blue 373, C.I. Disperse Blue 374, C.I. Disperse Blue 375, C.I. Disperse Blue 376, C.I.
  • Disperse Blue 378 C.I. Disperse Blue 38, C.I. Disperse Blue 42, C.I. Disperse Blue 43, C.I. Disperse Blue 44, C.I. Disperse Blue 47, C.I. Disperse Blue 79, C.I. Disperse Blue 79:1, C.I. Disperse Blue 79:2, C.I. Disperse Blue 79:3, C.I. Disperse Blue 82, C.I. Disperse Blue 85, C.I. Disperse Blue 88, C.I. Disperse Blue 90, C.I. Disperse Blue 94, C.I. Disperse Blue 96, C.I. Disperse Violet 10, C.I. Disperse Violet 100, C.I.
  • Disperse Violet 102 C.I. Disperse Violet 103, C.I. Disperse Violet 104, C.I. Disperse Violet 106, C.I. Disperse Violet 107, C.I. Disperse Violet 12, C.I. Disperse Violet 13, C.I. Disperse Violet 16, C.I. Disperse Violet 2, C.I. Disperse Violet 24, C.I. Disperse Violet 25, C.I. Disperse Violet 3, C.I. Disperse Violet 33, C.I. Disperse Violet 39, C.I. Disperse Violet 42, C.I. Disperse Violet 43, C.I. Disperse Violet 45, C.I. Disperse Violet 48, C.I. Disperse Violet 49, C.I.
  • Disperse Violet 5 C.I. Disperse Violet 50, C.I. Disperse Violet 53, C.I. Disperse Violet 54, C.I. Disperse Violet 55, C.I. Disperse Violet 58, C.I. Disperse Violet 6, C.I. Disperse Violet 60, C.I. Disperse Violet 63, C.I. Disperse Violet 66, C.I. Disperse Violet 69, C.I. Disperse Violet 7, C.I. Disperse Violet 75, C.I. Disperse Violet 76, C.I. Disperse Violet 77, C.I. Disperse Violet 82, C.I. Disperse Violet 86, C.I. Disperse Violet 88, C.I.
  • Disperse Violet 9 C.I. Disperse Violet 91, C.I. Disperse Violet 92, C.I. Disperse Violet 93, C.I. Disperse Violet 93:1, C.I. Disperse Violet 94, C.I. Disperse Violet 95, C.I. Disperse Violet 96, C.I. Disperse Violet 97, C.I. Disperse Violet 98, C.I. Disperse Violet 99, C.I. Reactive Black 5, C.I. Reactive Blue 19, C.I. Reactive Blue 4, C.I. Reactive Red 2, C.I. Solvent Blue 43, C.I. Solvent Blue 43, C.I.
  • Solvent Red 14 C.I.Acid black 24, C.I.Acid blue 113, C.I.Acid Blue 29, C.I.Direct violet 7, C.I.Food Red 14, Dianix Violet CC, Direct blue 1, Direct Blue 71, Direct blue 75, Direct blue 78, Direct blue 80, Direct blue 279, Direct violet 11, Direct violet 31, Direct violet 35, Direct violet 48, Direct violet 5, Direct Violet 51, Direct violet 66, Direct violet 9, Disperse Blue 106, Disperse blue 148, Disperse blue 165, Disperse Blue 3, Disperse Blue 354, Disperse Blue 364, Disperse blue 367, Disperse Blue 56, Disperse Blue 77, Disperse Blue 79, Disperse blue 79:1, Disperse Red 1, Disperse Red 15, Disperse Violet 26, Disperse Violet 27, Disperse Violet 28, Disperse violet 63, Disperse violet 77, Eosin Y, Ethanol, 2,2'-[[4-[(3,5-dinitro-2- thienyl)
  • Suitable polymeric dyes include polymeric dyes selected from the group consisting of polymers containing conjugated chromogens (dye-polymer conjugates) and polymers with chromogens co-polymerized into the backbone of the polymer and mixtures thereof.
  • suitable polymeric dyes include polymeric dyes selected from the group consisting of fabric-substantive colorants sold under the name of Liquitint® (Milliken, Spartanburg, South Carolina, USA), dye-polymer conjugates formed from at least one reactive dye and a polymer selected from the group consisting of polymers comprising a moiety selected from the group consisting of a hydroxyl moiety, a primary amine moiety, a secondary amine moiety, a thiol moiety and mixtures thereof.
  • suitable polymeric dyes include polymeric dyes selected from the group consisting of Liquitint® (Milliken, Spartanburg, South Carolina, USA) Violet CT, carboxymethyl cellulose (CMC) conjugated with a reactive blue, reactive violet or reactive red dye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE, product code S-ACMC, alkoxylated triphenyl-methane polymeric colourants, alkoxylated thiophene polymeric colourants, and mixtures thereof.
  • Liquitint® Moquitint®
  • CMC carboxymethyl cellulose
  • a reactive blue, reactive violet or reactive red dye such as CMC conjugated with C.I. Reactive Blue 19, sold by Megazyme, Wicklow, Ireland under the product name AZO-CM-CELLULOSE
  • product code S-ACMC alkoxylated triphenyl-methane polymeric colourants, alkoxylated
  • Suitable dye clay conjugates include dye clay conjugates selected from the group comprising at least one cationic/basic dye and a smectite clay, and mixtures thereof.
  • suitable dye clay conjugates include dye clay conjugates selected from the group consisting of one cationic/basic dye selected from the group consisting of C.I. Basic Yellow 1 through 108, C.I. Basic Orange 1 through 69, C.I. Basic Red 1 through 118, C.I. Basic Violet 1 through 51, C.I. Basic Blue 1 through 164, C.I. Basic Green 1 through 14, C.I. Basic Brown 1 through 23, CI Basic Black 1 through 11, and a clay selected from the group consisting of Montmorillonite clay, Hectorite clay, Saponite clay and mixtures thereof.
  • suitable dye clay conjugates include dye clay conjugates selected from the group consisting of: Montmorillonite Basic Blue B7 C.I. 42595 conjugate, Montmorillonite Basic Blue B9 C.I. 52015 conjugate, Montmorillonite Basic Violet V3 C.I. 42555 conjugate, Montmorillonite Basic Green Gl C.I. 42040 conjugate, Montmorillonite Basic Red Rl C.I. 45160 conjugate,
  • Suitable pigments include pigments selected from the group consisting of flavanthrone, indanthrone, chlorinated indanthrone containing from 1 to 4 chlorine atoms, pyranthrone, dichloropyranthrone, monobromodichloropyranthrone, dibromodichloropyranthrone, tetrabromopyranthrone, perylene-3,4,9,10-tetracarboxylic acid diimide, wherein the imide groups may be unsubstituted or substituted by C1-C3 -alkyl or a phenyl or heterocyclic radical, and wherein the phenyl and heterocyclic radicals may additionally carry substituents which do not confer solubility in water, anthrapyrimidinecarboxylic acid amides, violanthrone, isoviolanthrone, dioxazine pigments, copper phthalocyanine which may contain up to 2 chlorine atoms per molecule, polychloro
  • phthalocyanine containing up to 14 bromine atoms per molecule and mixtures thereof.
  • suitable pigments include pigments selected from the group consisting of Ultramarine Blue (C.I. Pigment Blue 29), Ultramarine Violet (C.I. Pigment Violet 15) and mixtures thereof.
  • the aforementioned fabric hueing agents can be used in combination (any mixture of fabric hueing agents can be used).
  • Suitable fabric hueing agents can be purchased from Aldrich, Milwaukee, Wisconsin, USA; Ciba Specialty Chemicals, Basel, Switzerland; BASF,
  • Suitable hueing agents are described in more detail in US 7,208,459 B2.
  • an encapsulate comprising a core, a shell having an inner and outer surface, said shell encapsulating said core.
  • said core may comprise a material selected from the group consisting of perfumes; brighteners; dyes; insect repellants; silicones; waxes; flavors; vitamins; fabric softening agents; skin care agents in one aspect, paraffins; enzymes; antibacterial agents; bleaches; sensates; and mixtures thereof; and said shell may comprise a material selected from the group consisting of polyethylenes; polyamides; polystyrenes;
  • aminoplasts in one aspect said aminoplast may comprise a polyureas, polyurethane, and/or polyureaurethane, in one aspect said polyurea may comprise polyoxymethyleneurea and/or melamine formaldehyde; poly olefins; polysaccharides, in one aspect said polysaccharide may comprise alginate and/or chitosan; gelatin; shellac; epoxy resins; vinyl polymers; water insoluble inorganics; silicone; and mixtures thereof.
  • said core may comprise perfume.
  • said shell may comprise melamine formaldehyde and/or cross linked melamine formaldehyde.
  • suitable encapsulates may comprise a core material and a shell, said shell at least partially surrounding said core material, is disclosed. At least 75%, 85% or even 90% of said encapsulates may have a fracture strength of from about 0.2 MPa to about 10 MPa, from about 0.4 MPa to about 5MPa, from about 0.6 MPa to about 3.5 MPa, or even from about 0.7 MPa to about 3MPa; and a benefit agent leakage of from 0% to about 30%, from 0% to about 20%, or even from 0% to about 5%.
  • At least 75%, 85% or even 90% of said encapsulates may have a particle size of from about 1 microns to about 80 microns, about 5 microns to 60 microns, from about 10 microns to about 50 microns, or even from about 15 microns to about 40 microns.
  • At least 75%, 85% or even 90% of said encapsulates may have a particle wall thickness of from about 30 nm to about 250 nm, from about 80 nm to about 180 nm, or even from about 100 nm to about 160 nm.
  • said encapsulates' core material may comprise a material selected from the group consisting of a perfume raw material and/or optionally a material selected from the group consisting of vegetable oil, including neat and/or blended vegetable oils including caster oil, coconut oil, cottonseed oil, grape oil, rapeseed, soybean oil, corn oil, palm oil, linseed oil, safflower oil, olive oil, peanut oil, coconut oil, palm kernel oil, castor oil, lemon oil and mixtures thereof; esters of vegetable oils, esters, including dibutyl adipate, dibutyl phthalate, butyl benzyl adipate, benzyl octyl adipate, tricresyl phosphate, trioctyl phosphate and mixtures thereof; straight or branched chain hydrocarbons, including those straight or branched chain hydrocarbons having a boiling point of greater than about 80 °C; partially hydrogenated terphenyls, dialkyl phthalates, alky
  • said encapsulates' wall material may comprise a suitable resin including the reaction product of an aldehyde and an amine
  • suitable aldehydes include, formaldehyde.
  • suitable amines include melamine, urea, benzoguanamine, glycoluril, and mixtures thereof.
  • Suitable melamines include, methylol melamine, methylated methylol melamine, imino melamine and mixtures thereof.
  • Suitable ureas include, dimethylol urea, methylated dimethylol urea, urea-resorcinol, and mixtures thereof.
  • suitable formaldehyde scavengers may be employed with the encapsulates, for example, in a capsule slurry and/or added to a fabric and home care product before, during or after the encapsulates are added to such fabric and home care product.
  • Suitable capsules that can be made by following the teaching of USPA 2008/0305982 Al; and/or USPA 2009/0247449 Al.
  • suitable capsules can be purchased from Appleton Papers Inc. of Appleton, Wisconsin USA.
  • the materials for making the aforementioned encapsulates can be obtained from Solutia Inc. (St Louis, Missouri U.S.A.), Cytec Industries (West Paterson, New Jersey U.S.A.), sigma-Aldrich (St. Louis, Missouri U.S.A.), CP Kelco Corp. of San Diego, California, USA; BASF AG of Ludwigshafen, Germany; Rhodia Corp. of Cranbury, New Jersey, USA; Hercules Corp. of Wilmington, Delaware, USA; Agrium Inc.
  • Amphiphilic alkoxylated grease cleaning polymers of the present invention refer to any alkoxylated polymers having balanced hydrophilic and hydrophobic properties such that they remove grease particles from fabrics and surfaces.
  • Specific embodiments of the amphiphilic alkoxylated grease cleaning polymers of the present invention comprise a core structure and a plurality of alkoxylate groups attached to that core structure.
  • the core structure may comprise a polyalkylenimine structure comprising, in condensed form, repeating units of formulae (I), (II), (III) and (IV):
  • the core structure may alternatively comprise a polyalkanolamine structure of the condensation products of at least one compound selected from N- (hy droxy alky 1) amines of formulae (I.a) and/or (I.b),
  • A are independently selected from Ci-C 6 -alkylene;
  • R 1 , R 1 *, R 2 , R 2 *, R 3 , R 3 *, R 4 , R 4 *, R 5 and R 5 * are independently selected from hydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentioned radicals may be optionally substituted;
  • R 6 is selected from hydrogen, alkyl, cycloalkyl or aryl, wherein the last three mentioned radicals may be optionally substituted.
  • the plurality of alkylenoxy groups attached to the core structure are independently selected from alkylenoxy units of the formula (V)
  • a 2 is in each case independently selected from 1,2-propylene, 1,2- butylene and 1,2-isobutylene;
  • a 3 is 1,2-propylene;
  • R is in each case independently selected from hydrogen and Ci-C4-alkyl;
  • m has an average value in the range of from 0 to about 2;
  • n has an average value in the range of from about 20 to about 50;
  • p has an average value in the range of from about 10 to about 50.
  • amphiphilic alkoxylated grease cleaning polymers may be selected from alkoxylated polyalkylenimines having an inner polyethylene oxide block and an outer polypropylene oxide block, the degree of ethoxylation and the degree of propoxylation not going above or below specific limiting values.
  • Specific embodiments of the alkoxylated polyalkylenimines according to the present invention have a minimum ratio of polyethylene blocks to polypropylene blocks (n/p) of about 0.6 and a maximum of about 1.5(x+2y+l) 1/2.
  • Alkoxykated polyalkyenimines having an n/p ratio of from about 0.8 to about 1.2(x+2y+l) 1 2 have been found to have especially beneficial properties.
  • the alkoxylated polyalkylenimines according to the present invention have a backbone which consists of primary, secondary and tertiary amine nitrogen atoms which are attached to one another by alkylene radicals A and are randomly arranged.
  • Primary amino moieties which start or terminate the main chain and the side chains of the polyalkylenimine backbone and whose remaining hydrogen atoms are subsequently replaced by alkylenoxy units are referred to as repeating units of formulae (I) or (IV), respectively.
  • Secondary amino moieties whose remaining hydrogen atom is subsequently replaced by alkylenoxy units are referred to as repeating units of formula (II).
  • Tertiary amino moieties which branch the main chain and the side chains are referred to as repeating units of formula (III).
  • cyclization can occur in the formation of the polyalkylenimine backbone, it is also possible for cyclic amino moieties to be present to a small extent in the backbone.
  • Such polyalkylenimines containing cyclic amino moieties are of course alkoxylated in the same way as those consisting of the noncyclic primary and secondary amino moieties.
  • the polyalkylenimine backbone consisting of the nitrogen atoms and the groups A 1 has an average molecular weight Mw of from about 60 to about 10,000 g/mole, preferably from about 100 to about 8,000 g/mole and more preferably from about 500 to about 6,000 g/mole.
  • the sum (x+2y+l) corresponds to the total number of alkylenimine units present in one individual polyalkylenimine backbone and thus is directly related to the molecular weight of the polyalkylenimine backbone.
  • the values given in the specification however relate to the number average of all polyalkylenimines present in the mixture.
  • the sum (x+2y+2) corresponds to the total number amino groups present in one individual polyalkylenimine backbone.
  • the radicals A connecting the amino nitrogen atoms may be identical or different, linear or branched C2-C6-alkylene radicals, such as 1 ,2-ethylene, 1 ,2-propylene, 1,2-butylene, 1,2- isobutylene,l,2-pentanediyl, 1 ,2-hexanediyl or hexamethylen.
  • a preferred branched alkylene is 1,2-propylene.
  • Preferred linear alkylene are ethylene and hexamethylene.
  • a more preferred alkylene is 1,2-ethylene.
  • a 2 in each case is selected from 1,2-propylene, 1,2-butylene and 1,2-isobutylene; preferably A 2 is 1,2-propylene.
  • a 3 is 1,2-propylene; R in each case is selected from hydrogen and Ci-C4-alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert.-butyl; preferably R is hydrogen.
  • the index m in each case has a value of 0 to about 2; preferably m is 0 or approximately 1; more preferably m is 0.
  • the index n has an average value in the range of from about 20 to about 50, preferably in the range of from about 22 to about 40, and more preferably in the range of from about 24 to about 30.
  • the index p has an average value in the range of from about 10 to about 50, preferably in the range of from about 11 to about 40, and more preferably in the range of from about 12 to about 30.
  • the alkylenoxy unit of formula (V) is a non-random sequence of alkoxylate blocks.
  • non-random sequence it is meant that the [-A 2 -0-] m is added first (i.e., closest to the bond to the nitrgen atom of the repeating unit of formula (I), (II), or (III)), the [- CH 2 -CH 2 -0-] n is added second, and the [-A 3 -0-] p is added third.
  • This orientation provides the alkoxylated polyalkylenimine with an inner polyethylene oxide block and an outer polypropylene oxide block.
  • alkylenoxy units of formula (V) The substantial part of these alkylenoxy units of formula (V) is formed by the ethylenoxy units -[CH 2 -CH 2 -0)] n - and the propylenoxy units -[CH 2 -CH 2 (CH 3 )-0] P -.
  • the alkylenoxy units may additionally also have a small proportion of propylenoxy or butylenoxy units -[A 2 -0] m -, i.e.
  • the polyalkylenimine backbone saturated with hydrogen atoms may be reacted initially with small amounts of up to about 2 mol, especially from about 0.5 to about 1.5 mol, in particular from about 0.8 to about 1.2 mol, of propylene oxide or butylene oxide per mole of NH- moieties present, i.e. incipiently alkoxylated.
  • the amphiphilic alkoxylated grease cleaning polymers are present in the fabric and home care products, including but not limited to detergents, of the present invention at levels ranging from about 0.05% to 10% by weight of the fabric and home care product.
  • Embodiments of the fabric and home care products may comprise from about 0.1% to about 5% by weight. More specifically, the embodiments may comprise from about 0.25 to about 2.5% of the grease cleaning polymer.
  • the fabric and home care products can comprise one or more enzymes which provide cleaning performance and/or fabric care benefits.
  • suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases,
  • a typical combination is an enzyme cocktail that may comprise, for example, a protease and lipase in conjunction with amylase.
  • the aforementioned additional enzymes may be present at levels from about 0.00001% to about 2%, from about 0.0001% to about 1% or even from about 0.001% to about 0.5% enzyme protein by weight of the fabric and home care product.
  • Suitable proteases include metalloproteases and serine proteases, including neutral or alkaline microbial serine proteases, such as subtilisins (EC 3.4.21.62).
  • Suitable proteases include those of animal, vegetable or microbial origin. In one aspect, such suitable protease may be of microbial origin.
  • the suitable proteases include chemically or genetically modified mutants of the aforementioned suitable proteases.
  • the suitable protease may be a serine protease, such as an alkaline microbial protease or/and a trypsin-type protease.
  • suitable neutral or alkaline proteases include:
  • subtilisins (EC 3.4.21.62), including those derived from Bacillus, such as Bacillus lentus, B. alkalophilus, B. subtilis, B. amyloliquefaciens, Bacillus pumilus and Bacillus gibsonii described in US 6,312,936 B l, US 5,679,630, US 4,760,025, US7,262,042 and WO09/021867.
  • trypsin-type or chymotrypsin-type proteases such as trypsin (e.g. , of porcine or bovine origin), including the Fusarium protease described in WO 89/06270 and the
  • chymotrypsin proteases derived from Cellumonas described in WO 05/052161 and WO 05/052146.
  • metalloproteases including those derived from Bacillus amyloliquefaciens described in WO 07/044993 A2.
  • Preferred proteases include those derived from Bacillus gibsonii or Bacillus Lentus.
  • Suitable commercially available protease enzymes include those sold under the trade names Alcalase®, Savinase®, Primase®, Durazym®, Polarzyme®, Kannase®, Liquanase®, Liquanase Ultra®, Savinase Ultra®, Ovozyme®, Neutrase®, Everlase® and Esperase® by Novozymes A/S (Denmark), those sold under the tradename Maxatase®, Maxacal®,
  • BLAP BLAP
  • BLAP R BLAP with S3T + V4I + V199M + V205I + L217D
  • BLAP X BLAP with S3T + V4I + V205I
  • BLAP F49 BLAP with S3T + V4I + A194P + V199M + V205I + L217D
  • the fabric and home care product may comprise a protease that is not immunoequivalent to the cold water protease of this invention.
  • an immunoequivalent protease will have a high degree of identity (>80%) with BPN' and will cross-react with the same antibody.
  • Suitable non-immunoequivalent enzymes will include those derived from Bacillus Lentus, Bacillus gibsonii and the metalloprotease derived from Bacillus amyloliquefaciens.
  • Suitable alpha- amylases include those of bacterial or fungal origin. Chemically or genetically modified mutants (variants) are included.
  • a preferred alkaline alpha-amylase is derived from a strain of Bacillus, such as Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus subtilis, or other Bacillus sp., such as Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513, DSM 9375 (USP 7,153,818) DSM 12368, DSMZ no. 12649, KSM AP1378 (WO 97/00324), KSM K36 or KSM K38 (EP 1,022,334).
  • Preferred amylases include:
  • WOOO/60060 and WO 06/002643 especially the variants with one or more substitutions in the following positions versus the AA560 enzyme listed as SEQ ID No. 12 in WO 06/002643:
  • variants exhibiting at least 90% identity with SEQ ID No. 4 in WO06/002643, the wild-type enzyme from Bacillus SP722, especially variants with deletions in the 183 and 184 positions and variants described in WO 00/60060, which is incorporated herein by reference.
  • variants exhibiting at least 95% identity with the wild- type enzyme from Bacillus sp.707 (SEQ ID NO:7 in US 6,093, 562), especially those comprising one or more of the following mutations M202, M208, S255, R172, and/or M261.
  • said amylase comprises one or more of M202L, M202V, M202S, M202T, M202I, M202Q, M202W, S255N and/or R172Q. Particularly preferred are those comprising the M202L or M202T mutations.
  • Suitable commercially available alpha-amylases include DURAMYL®,
  • suitable amylases include NATALASE®, STAINZYME® and STAINZYME PLUS® and mixtures thereof.
  • such additional enzyme may be selected from the group consisting of: lipases, including "first cycle lipases” such as those described in U.S. Patent 6,939,702 Bl and US PA 2009/0217464.
  • the lipase is a first-wash lipase, preferably a variant of the wild-type lipase from Thermomyces lanuginosus comprising T231R and N233R mutations.
  • the wild-type sequence is the 269 amino acids (amino acids 23 - 291) of the Swissprot accession number Swiss-Prot 059952 (derived from Thermomyces lanuginosus (Humicola lanuginosa)).
  • Preferred lipases would include those sold under the tradenames Lipex® and Lipolex®.
  • other preferred enzymes include microbial-derived endoglucanases exhibiting endo-beta-l,4-glucanase activity (E.C. 3.2.1.4), including a bacterial polypeptide endogenous to a member of the genus Bacillus which has a sequence of at least 90%, 94%, 97% and even 99% identity to the amino acid sequence SEQ ID NO:2 in 7,141,403B2) and mixtures thereof.
  • Suitable endoglucanases are sold under the tradenames Celluclean® and Whitezyme® (Novozymes A/S, Bagsvaerd, Denmark).
  • Pectate lyases sold under the tradenames Pectawash®, Pectaway® and mannanases sold under the tradenames Mannaway® (all from Novozymes A/S, Bagsvaerd, Denmark), and Purabrite® (Genencor International Inc., Palo Alto, California).
  • adjuncts illustrated hereinafter are suitable for use in the instant fabric and home care products and may be desirably incorporated in certain embodiments of the invention, for example to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the fabric and home care product as is the case with perfumes, colorants, dyes or the like.
  • the levels of any such adjuncts incorporated in any fabric and home care product are in addition to any materials previously recited for incorporation. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the fabric and home care product and the nature of the cleaning operation for which it is to be used.
  • Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments.
  • suitable examples of such other adjuncts and levels of use are found in U.S. Patent Nos. 5,576,282, 6,306,812 Bl and 6,326,348 Bl that are incorporated by reference.
  • adjunct ingredients are not essential to Applicants' fabric and home care products.
  • certain embodiments of Applicants' fabric and home care products do not contain one or more of the following adjuncts materials: surfactants, builders, chelating agents, dye transfer inhibiting agents, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, perfume delivery systems, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids, solvents and/or pigments.
  • one or more adjuncts may be present as detailed below:
  • compositions of the present invention may comprise a neat perfume and/or perfume technology systems that can be combined to yield the desired scent experience from the store shelf stage of a product, through its total performance cycle.
  • Suitable perfumes include those perfumes that are enduring perfumes and/or quadrant perfumes. Examples of such neat perfumes are disclosed USP Nos. 5,500,138; 5,500,154; 6,491,728; 5,500,137 and 5,780,404.
  • Suitable perfume delivery systems, methods of making certain perfume delivery systems and the uses of such perfume delivery systems are disclosed in USPA 2007/0275866 Al.
  • Such perfume delivery systems include:
  • PAD Polymer Assisted Delivery
  • This perfume delivery technology uses polymeric materials to deliver perfume materials.
  • Classical coacervation, water soluble or partly soluble to insoluble charged or neutral polymers, liquid crystals, hot melts, hydrogels, perfumed plastics, microcapsules, nano- and micro-latexes, polymeric film formers, and polymeric absorbents, polymeric adsorbents, etc. are some examples.
  • PAD includes but is not limited to:
  • the fragrance is dissolved or dispersed in a polymer matrix or particle.
  • Perfumes for example, may be 1) dispersed into the polymer prior to formulating into the product or 2) added separately from the polymer during or after formulation of the product. Diffusion of perfume from the polymer is a common trigger that allows or increases the rate of perfume release from a polymeric matrix system that is deposited or applied to the desired surface (situs), although many other triggers are know that may control perfume release.
  • Absorption and/or adsorption into or onto polymeric particles, films, solutions, and the like are aspects of this technology. Nano- or micro-particles composed of organic materials (e.g., latexes) are examples.
  • Suitable particles include a wide range of materials including, but not limited to polyacetal, polyacrylate, polyacrylic, polyacrylonitrile, polyamide, polyaryletherketone, polybutadiene, polybutylene, polybutylene terephthalate, polychloroprene, poly ethylene, polyethylene terephthalate, polycyclohexylene dimethylene terephthalate, polycarbonate, polychloroprene, polyhydroxyalkanoate, polyketone, polyester, polyethylene, polyetherimide, polyethersulfone, polyethylenechlorinates, polyimide, polyisoprene, polylactic acid, polymethylpentene, polyphenylene oxide, polyphenylene sulfide, polyphthalamide, polypropylene, polystyrene, polysulfone, polyvinyl acetate, polyvinyl chloride, as well as polymers or copolymers based on acrylonitrile-butadiene, cellulose acetate, ethylene-
  • “Standard” systems refer to those that are "pre-loaded” with the intent of keeping the pre-loaded perfume associated with the polymer until the moment or moments of perfume release. Such polymers may also suppress the neat product odor and provide a bloom and/or longevity benefit depending on the rate of perfume release.
  • One challenge with such systems is to achieve the ideal balance between 1) in- product stability (keeping perfume inside carrier until you need it) and 2) timely release (during use or from dry situs). Achieving such stability is particularly important during in-product storage and product aging. This challenge is particularly apparent for aqueous-based, surfactant-containing products, such as heavy duty liquid laundry detergents. Many "Standard" matrix systems available effectively become “Equilibrium” systems when formulated into aqueous-based products.
  • "Equilibrium” systems are those in which the perfume and polymer may be added separately to the product, and the equilibrium interaction between perfume and polymer leads to a benefit at one or more consumer touch points (versus a free perfume control that has no polymer-assisted delivery technology).
  • the polymer may also be pre-loaded with perfume; however, part or all of the perfume may diffuse during in-product storage reaching an equilibrium that includes having desired perfume raw materials (PRMs) associated with the polymer.
  • PRMs perfume raw materials
  • Matrix systems also include hot melt adhesives and perfume plastics.
  • hydrophobically modified polysaccharides may be formulated into the perfumed product to increase perfume deposition and/or modify perfume release. All such matrix systems, including for example polysaccharides and nanolatexes may be combined with other PDTs, including other PAD systems such as PAD reservoir systems in the form of a perfume microcapsule (PMC).
  • PMC perfume microcapsule
  • Polymer Assisted Delivery (PAD) matrix systems may include those described in the following references: US Patent Applications 2004/0110648 Al; 2004/0092414 Al; 2004/0091445 Al and 2004/0087476 Al; and US Patents 6,531,444; 6,024,943; 6,042,792; 6,051,540; 4,540,721 and 4,973,422.
  • Silicones are also examples of polymers that may be used as PDT, and can provide perfume benefits in a manner similar to the polymer-assisted delivery "matrix system". Such a PDT is referred to as silicone-assisted delivery (SAD).
  • SAD silicone-assisted delivery
  • Suitable silicones as well as making same may be found in WO 2005/102261; USPA 20050124530A1; USPA 20050143282A1; and WO 2003/015736.
  • Functionalized silicones may also be used as described in USPA 2006/003913 Al. Examples of silicones include polydimethylsiloxane and poly alky ldimethylsiloxanes.
  • Reservoir systems are also known as a core-shell type technology, or one in which the fragrance is surrounded by a perfume release controlling membrane, which may serve as a protective shell.
  • the material inside the microcapsule is referred to as the core, internal phase, or fill, whereas the wall is sometimes called a shell, coating, or membrane.
  • Microparticles or pressure sensitive capsules or microcapsules are examples of this technology.
  • Microcapsules of the current invention are formed by a variety of procedures that include, but are not limited to, coating, extrusion, spray-drying, interfacial, in-situ and matrix polymerization.
  • the possible shell materials vary widely in their stability toward water. Among the most stable are polyoxymethyleneurea (PMU)-based materials, which may hold certain PRMs for even long periods of time in aqueous solution (or product).
  • PMU polyoxymethyleneurea
  • Such systems include but are not limited to urea-formaldehyde and/or melamine-formaldehyde.
  • Gelatin-based microcapsules may be prepared so that they dissolve quickly or slowly in water, depending for example on the degree of cross- linking.
  • capsule wall materials are available and vary in the degree of perfume diffusion stability observed.
  • rate of release of perfume from a capsule for example, once deposited on a surface is typically in reverse order of in-product perfume diffusion stability.
  • urea- formaldehyde and melamine-formaldehyde microcapsules for example, typically require a release mechanism other than, or in addition to, diffusion for release, such as mechanical force (e.g., friction, pressure, shear stress) that serves to break the capsule and increase the rate of perfume (fragrance) release.
  • Other triggers include melting, dissolution, hydrolysis or other chemical reaction, electromagnetic radiation, and the like.
  • microcapsules that are based on urea-formaldehyde and/or melamine-formaldehyde are relatively stable, especially in near neutral aqueous- based solutions.
  • suitable microcapsules include microcapsules having shells that comprise urethane materials, acrylics and/or vinyl alcohols. These materials may require a friction trigger which may not be applicable to all product applications.
  • Other microcapsule materials e.g., gelatin
  • Perfume microcapsules may include those described in the following references: US Patent Applications: 2003/0125222 Al; 2003/215417 Al; 2003/216488 Al; 2003/158344 Al; 2003/165692 Al; 2004/071742 Al; 2004/071746 Al; 2004/072719 Al; 2004/072720 Al; 2006/0039934 Al; 2003/203829 Al; 2003/195133 Al; 2004/087477 Al; 2004/0106536 Al; and US Patents 6,645,479 Bl; 6,200,949 Bl; 4,882,220; 4,917,920; 4,514,461; 6,106,875 and 4,234,627, 3,594,328 and US RE 32713.
  • Non-polymer materials or molecules may also serve to improve the delivery of perfume.
  • perfume may non- covalently interact with organic materials, resulting in altered deposition and/or release.
  • organic materials include but are not limited to hydrophobic materials such as organic oils, waxes, mineral oils, petrolatum, fatty acids or esters, sugars, surfactants, liposomes and even other perfume raw material (perfume oils), as well as natural oils, including body and/or other soils.
  • Perfume fixatives are yet another example.
  • non- polymeric materials or molecules have a CLogP greater than about 2.
  • Molecule-Assisted Delivery (MAD) may also include those described in USP 7,119,060 and USP 5,506,201.
  • Fiber- Assisted Delivery The choice or use of a situs itself may serve to improve the delivery of perfume.
  • the situs itself may be a perfume delivery technology.
  • different fabric types such as cotton or polyester will have different properties with respect to ability to attract and/or retain and/or release perfume.
  • the amount of perfume deposited on or in fibers may be altered by the choice of fiber, and also by the history or treatment of the fiber, as well as by any fiber coatings or treatments. Fibers may be woven and non-woven as well as natural or synthetic.
  • Natural fibers include those produced by plants, animals, and geological processes, and include but are not limited to cellulose materials such as cotton, linen, hemp jute, flax, ramie, and sisal, and fibers used to manufacture paper and cloth.
  • Fiber-Assisted Delivery may consist of the use of wood fiber, such as thermomechanical pulp and bleached or unbleached kraft or sulfite pulps.
  • Animal fibers consist largely of particular proteins, such as silk, sinew, catgut and hair (including wool).
  • Polymer fibers based on synthetic chemicals include but are not limited to polyamide nylon, PET or PBT polyester, phenol-formaldehyde (PF), polyvinyl alcohol fiber (PVOH), polyvinyl chloride fiber (PVC), polyolefins (PP and PE), and acrylic polymers. All such fibers may be pre-loaded with a perfume, and then added to a product that may or may not contain free perfume and/or one or more perfume delivery technologies. In one aspect, the fibers may be added to a product prior to being loaded with a perfume, and then loaded with a perfume by adding a perfume that may diffuse into the fiber, to the product. Without wishing to be bound by theory, the perfume may absorb onto or be adsorbed into the fiber, for example, during product storage, and then be released at one or more moments of truth or consumer touch points.
  • Amine Assisted Delivery The amine-assisted delivery technology approach utilizes materials that contain an amine group to increase perfume deposition or modify perfume release during product use. There is no requirement in this approach to pre-complex or pre-react the perfume raw material(s) and amine prior to addition to the product.
  • amine- containing AAD materials suitable for use herein may be non-aromatic; for example, polyalkylimine, such as polyethyleneimine (PEI), or polyvinylamine (PVAm), or aromatic, for example, anthranilates. Such materials may also be polymeric or non-polymeric. In one aspect, such materials contain at least one primary amine.
  • This technology will allow increased longevity and controlled release also of low ODT perfume notes (e.g., aldehydes, ketones, enones) via amine functionality, and delivery of other PRMs, without being bound by theory, via polymer-assisted delivery for polymeric amines. Without technology, volatile top notes can be lost too quickly, leaving a higher ratio of middle and base notes to top notes.
  • the use of a polymeric amine allows higher levels of top notes and other PRMS to be used to obtain freshness longevity without causing neat product odor to be more intense than desired, or allows top notes and other PRMs to be used more efficiently.
  • AAD systems are effective at delivering PRMs at pH greater than about neutral.
  • conditions in which more of the amines of the AAD system are deprotonated may result in an increased affinity of the deprotonated amines for PRMs such as aldehydes and ketones, including unsaturated ketones and enones such as damascone.
  • PRMs such as aldehydes and ketones
  • polymeric amines are effective at delivering PRMs at pH less than about neutral.
  • conditions in which more of the amines of the AAD system are protonated may result in a decreased affinity of the protonated amines for PRMs such as aldehydes and ketones, and a strong affinity of the polymer framework for a broad range of PRMs.
  • polymer-assisted delivery may be delivering more of the perfume benefit; such systems are a subspecies of AAD and may be referred to as Amine- Polymer-Assisted Delivery or APAD.
  • APAD Amine- Polymer-Assisted Delivery
  • PAD Polymer-Assisted Delivery
  • AAD and PAD systems may interact with other materials, such as anionic surfactants or polymers to form coacervate and/or coacervates-like systems.
  • a material that contains a heteroatom other than nitrogen for example sulfur, phosphorus or selenium, may be used as an alternative to amine compounds.
  • the aforementioned alternative compounds can be used in combination with amine compounds.
  • a single molecule may comprise an amine moiety and one or more of the alternative heteroatom moieties, for example, thiols, phosphines and selenols.
  • Suitable AAD systems as well as methods of making same may be found in US Patent Applications 2005/0003980 Al; 2003/0199422 Al; 2003/0036489 Al; 2004/0220074 Al and USP 6,103,678.
  • Cyclodextrin Delivery System This technology approach uses a cyclic oligosaccharide or cyclodextrin to improve the delivery of perfume.
  • a perfume and cyclodextrin (CD) complex is formed.
  • Such complexes may be preformed, formed in-situ, or formed on or in the situs.
  • loss of water may serve to shift the equilibrium toward the CD-Perfume complex, especially if other adjunct ingredients (e.g., surfactant) are not present at high concentration to compete with the perfume for the cyclodextrin cavity.
  • a bloom benefit may be achieved if water exposure or an increase in moisture content occurs at a later time point.
  • cyclodextrin allows the perfume formulator increased flexibility in selection of PRMs. Cyclodextrin may be pre-loaded with perfume or added separately from perfume to obtain the desired perfume stability, deposition or release benefit. Suitable CDs as well as methods of making same may be found in USPA 2005/0003980 Al and 2006/0263313 Al and US Patents 5,552,378; 3,812,011; 4,317,881; 4,418,144 and 4,378,923.
  • Starch Encapsulated Accord The use of a starch encapsulated accord (SEA) technology allows one to modify the properties of the perfume, for example, by converting a liquid perfume into a solid by adding ingredients such as starch.
  • the benefit includes increased perfume retention during product storage, especially under non-aqueous conditions. Upon exposure to moisture, a perfume bloom may be triggered. Benefits at other moments of truth may also be achieved because the starch allows the product formulator to select PRMs or PRM concentrations that normally cannot be used without the presence of SEA.
  • Another technology example includes the use of other organic and inorganic materials, such as silica to convert perfume from liquid to solid. Suitable SEAs as well as methods of making same may be found in USPA 2005/0003980 Al and USP 6,458,754 B l.
  • SEA's may be made by preparing a mixture comprising starch, water, acid and a perfume, the acid being incorporated in the mixture in an amount sufficient to lower the pH of the starch- water mixture by at least 0.25 units; and atomising and drying the mixture thereby forming encapsulated perfume.
  • an aqueous mixture is prepared comprising starch, water, perfume and acid. These ingredients may be added in any order, but usually the starch-water mixture is prepared first and
  • the acid and perfume are added.
  • the acid may be added prior to the ingredient for encapsulation.
  • the acid is added after the ingredient for encapsulation.
  • concentration of starch in the aqueous mixture may be from as low as 5 or 10 wt% to as high as 60 or even 75 wt%. Generally the concentration of starch in the mixture is from 20 to 50 wt%, more usually around 25 to 40 wt% in the aqueous mixture.
  • Suitable starches can be made from raw starch, pregelatinized starch, modified starch derived from tubers, legumes, cereal and grains for example corn starch , wheat starch, rice starch, waxy corn starch, oat starch, cassava starch, waxy barley starch, waxy rice starch, sweet rice starch, amioca, potato starch, tapioca starch and mixtures thereof.
  • Modified starches may be particularly suitable for use in the present invention, and these include hydrolyzed starch, acid thinned starch, starch having hydrophobic groups, such as starch esters of long chain hydrocarbons (C 5 or greater), starch acetates, starch octenyl succinate and mixtures thereof.
  • starch esters such as starch octenyl succinates are employed .
  • hydrolyzed starch refers to oligosaccharide-type materials that are typically obtained by acid and/or enzymatic hydrolysis of starches, preferably corn starch. It may be preferred to include in the starch water-mixture, a starch ester. Particularly preferred are the modified starches comprising a starch derivative containing a hydrophobic group or both a hydrophobic and a hydrophilic group which has been degraded by at least one enzyme capable of cleaving the 1,4 linkages of the starch molecule from the non-reducing ends to produce short chained saccharides to provide high oxidation resistance while maintaining substantially high molecular weight portions of the starch base.
  • the aqueous starch mixture may also include a plasticizer for the starch.
  • Suitable examples include monosaccharides, disaccharides, oligosaccharides and maltodextrins, such as glucose, sucrose, sorbitol, gum arabic, guar gums and maltodextrin.
  • the acid used in the process of the invention may be any acid. Examples include sulphuric acid, nitric acid, hydrochloric acid, sulphamic acid and phosphonic acid.
  • carboxylic organic acids are employed.
  • organic acids comprising more than one carboxylic acid groups are employed.
  • suitable organic acids include citric acid, tartaric acid, maleic acid, malic acid, succinic acid, sebacic acid, adipic acid, itaconic acid, acetic acid and ascorbic acid, etc.
  • saturated acids such as citric acid, are employed.
  • Suitable perfumes for encapsulation include the HIA perfumes including those having a boiling point determined at the normal standard pressure of about 760 mmHg of 275 °C or lower, an octanol/water partition coefficient P of about 2000 or higher and an odour detection thresholdof less than or equal 50 parts per billion (ppb).
  • the perfume may have logP of 2 or higher.
  • Suitable perfumes may be selected from the group consisting of 3-(4-t-butylphenyl)-2- methyl propanal, 3-(4-t-butylphenyl)-propanal, 3-(4-isopropylphenyl)-2-methylpropanal, 3-(3,4- methylenedioxyphenyl)-2-methylpropanal, and 2,6-dimethyl-5-heptenal, Alpha -damascone, Delta -damascone, Iso-damascone, Beta-damascenone, 6,7-dihydro-l, 1,2,3, 3-pentamethyl- 4(5H)-indanone, methyl-7,3-dihydro-2H-l,5-benzodioxepine-3-one, 2-[2-(4-methyl-3- cyclohexenyl-l-yl)propyl]cyclopentan-2-one, 2-sec-butylcyclohexanone, and Alpha
  • the mixture is mixed under high shear to form an emulsion or dispersion of ingredient for encapsulation in the aqueous starch solution.
  • Suitable technique may then be used for the final stage of processing where the aqueous mixture including acid and perfumes is atomised and dried.
  • Suitable techniques include, but are not limited to those known in the art including spray drying, extrusion, spray chilling/crystallisation methods, fluid bed coating and the use of phase transfer catalysts to promote interfacial polymerization. Spray efficiencies may be increased by methods known in the art, such as by using high drying towers, lightly oiling the chamber walls, or using preconditioned air in which the moisture has been substantially removed.
  • ZIC Inorganic Carrier Delivery System
  • PZ perfume-loaded zeolite
  • Suitable zeolite and inorganic carriers as well as methods of making same may be found in USPA 2005/0003980 Al and US Patents 5,858,959; 6,245,732 Bl; 6,048,830 and 4,539,135.
  • Silica is another form of ZIC.
  • a suitable inorganic carrier includes inorganic tubules, where the perfume or other active material is contained within the lumen of the nano- or micro-tubules.
  • the perfume-loaded inorganic tubule (or Perfume- Loaded Tubule or PLT) is a mineral nano- or micro-tubule, such as halloysite or mixtures of halloysite with other inorganic materials, including other clays.
  • the PLT technology may also comprise additional ingredients on the inside and/or outside of the tubule for the purpose of improving in-product diffusion stability, deposition on the desired situs or for controlling the release rate of the loaded perfume.
  • Monomeric and/or polymeric materials may be used to coat, plug, cap, or otherwise encapsulate the PLT. Suitable PLT systems as well as methods of making same may be found in USP 5,651,976.
  • Pro-Perfume (PP) This technology refers to perfume technologies that result from the reaction of perfume materials with other substrates or chemicals to form materials that have a covalent bond between one or more PRMs and one or more carriers. The PRM is converted into a new material called a pro-PRM (i.e., pro-perfume), which then may release the original PRM upon exposure to a trigger such as water or light.
  • a pro-PRM i.e., pro-perfume
  • Pro-perfumes may provide enhanced perfume delivery properties such as increased perfume deposition, longevity, stability, retention, and the like.
  • Pro-perfumes include those that are monomeric (non-polymeric) or polymeric, and may be pre-formed or may be formed in-situ under equilibrium conditions, such as those that may be present during in-product storage or on the wet or dry situs.
  • Nonlimiting examples of pro- perfumes include Michael adducts (e.g., beta-amino ketones), aromatic or non-aromatic imines (Schiffs Bases), oxazolidines, beta-keto esters, and orthoesters.
  • Another aspect includes compounds comprising one or more beta-oxy or beta-thio carbonyl moieties capable of releasing a PRM, for example, an alpha, beta-unsaturated ketone, aldehyde or carboxylic ester.
  • the typical trigger for perfume release is exposure to water; although other triggers may include enzymes, heat, light, pH change, autoxidation, a shift of equilibrium, change in concentration or ionic strength and others.
  • light-triggered pro-perfumes are particularly suited.
  • Such photo-pro-perfumes (PPPs) include but are not limited to those that release coumarin derivatives and perfumes and/or pro-perfumes upon being triggered.
  • the released pro-perfume may release one or more PRMs by means of any of the above mentioned triggers.
  • the photo-pro-perfume releases a nitrogen-based pro-perfume when exposed to a light and/or moisture trigger.
  • the nitrogen-based pro-perfume, released from the photo-pro-perfume releases one or more PRMs selected, for example, from aldehydes, ketones (including enones) and alcohols.
  • the PPP releases a dihydroxy coumarin derivative.
  • the light-triggered pro-perfume may also be an ester that releases a coumarin derivative and a perfume alcohol.
  • the pro-perfume is a dimethoxybenzoin derivative as described in USPA 2006/0020459 Al.
  • the pro-perfume is a 3', 5 '-dimethoxybenzoin (DMB) derivative that releases an alcohol upon exposure to electromagnetic radiation.
  • the pro-perfume releases one or more low ODT PRMs, including tertiary alcohols such as linalool, tetrahydrolinalool, or dihydromyrcenol.
  • ARP Amine Reaction Product
  • ARP is a subclass or species of PP.
  • the reactive amines are primary and/or secondary amines, and may be part of a polymer or a monomer (non-polymer).
  • Such ARPs may also be mixed with additional PRMs to provide benefits of polymer-assisted delivery and/or amine-assisted delivery.
  • Nonlimiting examples of polymeric amines include polymers based on poly alky limines, such as polyethyleneimine (PEI), or polyvinylamine (PVAm).
  • Nonlimiting examples of monomeric (non-polymeric) amines include hydroxyl amines, such as 2-aminoethanol and its alkyl substituted derivatives, and aromatic amines such as anthranilates.
  • the ARPs may be premixed with perfume or added separately in leave-on or rinse-off applications.
  • a material that contains a heteroatom other than nitrogen, for example oxygen, sulfur, phosphorus or selenium may be used as an alternative to amine compounds.
  • the aforementioned alternative compounds can be used in combination with amine compounds.
  • a single molecule may comprise an amine moiety and one or more of the alternative heteroatom moieties, for example, thiols, phosphines and selenols.
  • the benefit may include improved delivery of perfume as well as controlled perfume release. Suitable ARPs as well as methods of making same can be found in USPA 2005/0003980 Al and USP 6,413,920 Bl.
  • the amine reaction product's perfume component which is reacted with the amine to form the amine reaction product, is selected from a perfume comprising a ketone moiety and/or an aldehyde moiety.
  • such perfumes comprise a chain containing at least 5 carbon atoms.
  • suitable perfumes comprising a ketone moiety may be selected from Alpha Damascone, Delta Damascone, Iso Damascone, Carvone, Gamma-Methyl-Ionone, Iso-E-Super, 2,4,4,7-Tetramethyl-oct-6-en-3-one, Benzyl Acetone, Beta Damascone, Damascenone, methyl dihydrojasmonate, methyl cedrylone, and mixtures thereof.
  • suitable perfumes comprising an aldehyde moiety may be selected from 1 -decanal, benzaldehyde, florhydral, 2,4-dimethyl-3-cyclohexen-l-carboxaldehyde; cis/trans-3,7-dimethyl- 2,6-octadien- 1-al; heliotropin; 2,4,6-trimethyl-3-cyclohexene- 1-carboxaldehyde; 2,6-nonadienal; alpha-n-amyl cinnamic aldehyde, alpha-n-hexyl cinnamic aldehyde, P.T.
  • the suitable perfume be selected from undecylenic aldehyde, undecalactone gamma, heliotropin, dodecalactone gamma, p-anisic aldehyde, para hydroxy-phenyl-butanone, cymal, benzyl acetone, ionone alpha, p.t.bucinal, damascenone, ionone beta and methyl-nonyl ketone, and/or mixtures thereof.
  • suitable amine reaction products are those resulting from the reaction of polethyleneimine polymer like Lupasol polymers, with one or more of the following Alpha Damascone, Delta Damascone, Carvone, Hedione, Florhydral, Lilial, Heliotropine, Gamma-Methyl-Ionone and 2,4-dimethyl-3- cyclohexen- 1-carboxaldehyde; amine reaction products are those resulting from the reaction of Astramol Dendrimers with Carvone and amine reaction products resulting from the reaction of ethyl-4-amino benzoate with 2,4-dimethyl-3-cyclohexen- 1-carboxaldehyde.
  • suitable amine reaction products are those resulting from the reaction of Lupasol HF with Delta Damascone; LupasolG35 with Alpha Damascone; LupasolGlOO with 2,4-dimethyl-3- cyclohexen- 1-carboxaldehyde, ethyl-4-amino benzoate with 2,4-dimethyl-3-cyclohexen-l- carboxaldehyde.
  • suitable primary and/or secondary amine containing compounds are characterized by an Odor Intensity Index of less than that of a 1% solution of methylanthranilate in dipropylene glycol.
  • a general structure for a suitable primary amine compound is as follows:
  • B is a carrier material
  • n is an index of value of at least 1.
  • Suitable compounds comprising a secondary amine group may have a structure similar to the above excepted that the compound comprises one or more -NH- moieties in addition to any - NH2 moieties.
  • an amine compound may have the formula:
  • B is a carrier material, and each n is independently an index of value of at least 1.
  • B carriers may be inorganic having non-or substantially non carbon based backbones, or organic carriers having essentially carbon bond backbones.
  • Suitable inorganic carriers include mono or polymers or organic-organosilicon copolymers of amino derivatised organo silane, siloxane, silazane, alumane, aluminum siloxane, or aluminum silicate compounds.
  • Typical examples of such carriers are: organosiloxanes with at least one primary amine moiety like the diaminoalkylsiloxane [ ⁇ 2 ⁇ 3 ⁇ 4( ⁇ 3 ⁇ 4) 2 8 ⁇ ]0, or the organoaminosilane (C 6 H 5 ) 3SiN3 ⁇ 4 (described in: Chemistry and Technology of Silicone, W. Noll, Academic Press Inc. 1998, London, pp 209, 106).
  • Mono or polymer or organic- organosilicon copolymers containing one or more organosilylhydrasine moiety are also suitable.
  • a typical example of such a carrier material is N,N'-bis(trimethylsilyl)hydrazine (Me 3 Si) 2 NNH 2 .
  • Typical suitable amines comprising an organic carrier include aminoaryl derivatives, polyamines, aminoacids and derivatives, substituted amines and amides, glucamines, dendrimers and amino-substitued mono-, di-, oligo-, poly-saccharides.
  • the amine compound may be interrupted or substituted by linkers or cellulose substantive group.
  • a general formula for this amine compound is as follows:
  • each m is an index of value 0 or at least 1
  • n is an index of value of at least 1 as defined herein before.
  • the amine group is linked to a carrier molecule as defined by classes hereinafter described.
  • the primary and/or secondary amine group is either directly linked to the carrier group or via a linker group L.
  • the carrier can also be substituted by a R* substituent, and R* can be linked to the carrier either directly or via a linker group L.
  • R* can also contain branching groups like e.g. tertiary amine and amide groups.
  • the amine compound comprises at least one primary and/or secondary amine group to react with the perfume aldehyde and/or ketone to form the reaction products.
  • Such reaction is typically known as a Schiff base reaction as a Schiff base is formed.
  • the amine compound is not limited to having only one amine function. Indeed, more preferably, the amine compound comprises more than one amine function, thereby enabling the amine compound to react with several aldehydes and/or ketones. Accordingly, reaction products carrying mixed aldehyde(s) and/or ketone(s) can be achieved, thereby resulting in a mixed release of such fragrances.
  • the fabric and home care products of the present invention may comprise one or more bleaching agents.
  • Suitable bleaching agents other than bleaching catalysts include photobleaches, bleach activators, hydrogen peroxide, sources of hydrogen peroxide, preformed peracids and mixtures thereof.
  • the fabric and home care products of the present invention may comprise from about 0.1% to about 50% or even from about 0.1% to about 25% bleaching agent by weight of the subject fabric and home care product.
  • suitable bleaching agents include:
  • photobleaches for example sulfonated zinc phthalocyanine sulfonated aluminium phthalocyanines, xanthene dyes and mixtures thereof;
  • Suitable preformed peracids include, but are not limited to, compounds selected from the group consisting of percarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxone ®, and mixtures thereof.
  • inorganic perhydrate salts including alkali metal salts such as sodium salts of perborate (usually mono- or tetra-hydrate), percarbonate, persulphate, perphosphate, persilicate salts and mixtures thereof.
  • the inorganic perhydrate salts are selected from the group consisting of sodium salts of perborate, percarbonate and mixtures thereof.
  • inorganic perhydrate salts are typically present in amounts of from 0.05 to 40 wt%, or 1 to 30 wt% of the overall fabric and home care product and are typically incorporated into such fabric and home care products as a crystalline solid that may be coated. Suitable coatings include, inorganic salts such as alkali metal silicate, carbonate or borate salts or mixtures thereof, or organic materials such as water-soluble or dispersible polymers, waxes, oils or fatty soaps; and
  • suitable leaving groups are benzoic acid and derivatives thereof - especially benzene sulphonate.
  • Suitable bleach activators include dodecanoyl oxybenzene sulphonate, decanoyl oxybenzene sulphonate, decanoyl oxybenzoic acid or salts thereof, 3,5,5-trimethyl hexanoyloxybenzene sulphonate, tetraacetyl ethylene diamine (TAED) and nonanoyloxybenzene sulphonate (NOBS).
  • TAED tetraacetyl ethylene diamine
  • NOBS nonanoyloxybenzene sulphonate
  • Suitable bleach activators are also disclosed in WO 98/17767. While any suitable bleach activator may be employed, in one aspect of the invention the subject fabric and home care product may comprise NOBS, TAED or mixtures thereof.
  • the peracid and/or bleach activator is generally present in the fabric and home care product in an amount of from about 0.1 to about 60 wt%, from about 0.5 to about 40 wt % or even from about 0.6 to about 10 wt% based on the fabric and home care product.
  • One or more hydrophobic peracids or precursors thereof may be used in combination with one or more hydrophilic peracid or precursor thereof.
  • the amounts of hydrogen peroxide source and peracid or bleach activator may be selected such that the molar ratio of available oxygen (from the peroxide source) to peracid is from 1:1 to 35:1, or even 2:1 to 10:1.
  • the fabric and home care products according to the present invention may comprise a surfactant or surfactant system wherein the surfactant can be selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.
  • surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject fabric and home care product.
  • the fabric and home care products of the present invention may comprise one or more detergent builders or builder systems.
  • the subject fabric and home care product will typically comprise at least about 1%, from about 5% to about 60% or even from about 10% to about 40% builder by weight of the subject fabric and home care product.
  • Builders include, but are not limited to, the alkali metal, ammonium and
  • hydroxypolycarboxylates copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1, 3, 5-trihydroxy benzene-2, 4, 6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as 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.
  • polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid
  • polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzen
  • the fabric and home care products herein may contain a chelating agent.
  • Suitable chelating agents include copper, iron and/or manganese chelating agents and mixtures thereof.
  • the subject fabric and home care product may comprise from about 0.005% to about 15% or even from about 3.0% to about 10% chelating agent by weight of the subject fabric and home care product.
  • Suitable chelants include DTPA (Diethylene triamine pentaacetic acid), HEDP (Hydroxyethane diphosphonic acid), DTPMP (Diethylene triamine penta(methylene phosphonic acid)), l,2-Dihydroxybenzene-3,5-disulfonic acid disodium salt hydrate, ethylenediamine, diethylene triamine, ethylenediaminedisuccinic acid (EDDS), N-hydroxyethylethylenediaminetri-acetic acid (HEDTA),
  • TTHA triethylenetetraaminehexaacetic acid
  • HEIDA N-hydroxyethyliminodiacetic acid
  • DHEG dihydroxyethylglycine
  • EDTP ethylenediaminetetrapropionic acid
  • the fabric and home care products of the present invention may also include one or more dye transfer inhibiting agents.
  • Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
  • the dye transfer inhibiting agents may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5% or even from about 0.1% to about 3% by weight of the fabric and home care product.
  • Brighteners - The fabric and home care products of the present invention can also contain additional components that may tint articles being cleaned, such as fluorescent brighteners.
  • Suitable fluorescent brightener levels include lower levels of from about 0.01, from about 0.05, from about 0.1 or even from about 0.2 wt % to upper levels of 0.5 or even 0.75 wt %.
  • Dispersants - The fabric and home care products of the present invention can also contain dispersants.
  • Suitable water-soluble organic materials include the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
  • Enzyme Stabilizers - Enzymes for use in detergents can be stabilized by various techniques.
  • the enzymes employed herein can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished fabric and home care products that provide such ions to the enzymes.
  • a reversible protease inhibitor such as a boron compound, or compounds such as calcium formate, sodium formate and 1,2-propane diol can be added to further improve stability.
  • Catalytic Metal Complexes - Applicants' compositions may include catalytic metal complexes.
  • One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly
  • compositions herein can be catalyzed by means of a manganese compound.
  • a manganese compound Such compounds and levels of use are well known in the art and include, for example, the manganese- based catalysts disclosed in U.S. 5,576,282.
  • Cobalt bleach catalysts useful herein are known, and are described, for example, in U.S.
  • compositions herein may also suitably include a transition metal complex of ligands such as bispidones (WO 05/042532 Al) and/or macropolycyclic rigid ligands - abbreviated as "MRLs".
  • ligands such as bispidones (WO 05/042532 Al) and/or macropolycyclic rigid ligands - abbreviated as "MRLs”.
  • MRLs macropolycyclic rigid ligands
  • Suitable transition-metals in the instant transition-metal bleach catalyst include, for example, manganese, iron and chromium.
  • Suitable MRLs include 5,12-diethyl-l,5,8,12- tetraazabicyclo[6.6.2]hexadecane.
  • Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/32601, and U.S. 6,225,464.
  • Solvents - Suitable solvents include water and other solvents such as lipophilic fluids.
  • suitable lipophilic fluids include siloxanes, other silicones, hydrocarbons, glycol ethers, glycerine derivatives such as glycerine ethers, perfluorinated amines, perfluorinated and hydrofluoroether solvents, low-volatility nonfluorinated organic solvents, diol solvents, other environmentally-friendly solvents and mixtures thereof.
  • the fabric and home care products of the present invention can be formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in Applicants' examples and in U.S. 4,990,280; U.S. 20030087791A1; U.S. 20030087790A1; U.S. 20050003983 Al; U.S. 20040048764A1; U.S. 4,762,636; U.S. 6,291,412; U.S. 20050227891 Al; EP 1070115A2; U.S. 5,879,584; U.S. 5,691,297; U.S. 5,574,005; U.S. 5,569,645; U.S. 5,565,422; U.S. 5,516,448; U.S. 5,489,392; U.S. 5,486,303 all of which are incorporated herein by reference.
  • the present invention includes a method for cleaning and/or treating a situs inter alia a surface or fabric.
  • such method comprises the steps of optionally washing and/or rinsing said surface or fabric, contacting said surface or fabric with any fabric and home care product disclosed in this specification then optionally washing and/or rinsing said surface or fabric is disclosed.
  • washing includes but is not limited to, scrubbing, and mechanical agitation. Drying of such surfaces or fabrics may be accomplished by any one of the common means employed either in domestic or industrial settings. Such means include but are not limited to forced air or still air drying at ambient or elevated temperatures at pressures between 5 and 0.01 atmospheres in the presence or absence of electromagnetic radiation, including sunlight, infrared, ultraviolet and microwave irradiation.
  • said drying may be accomplished at temperatures above ambient by employing an iron wherein, for example, said fabric may be in direct contact with said iron for relatively short or even extended periods of time and wherein pressure may be exerted beyond that otherwise normally present due to gravitational force.
  • said drying may be accomplished at temperatures above ambient by employing a dryer.
  • Apparatus for drying fabric is well known and it is frequently referred to as a clothes dryer.
  • clothes In addition to clothes such appliances are used to dry many other items including towels, sheets, pillowcases, diapers and so forth and such equipment has been accepted as a standard convenience in many countries of the world substantially replacing the use of clothes lines for drying of fabric.
  • Most dryers in use today use heated air which is passed over and or through the fabric as it is tumbled within the dryer.
  • the air may be heated, for example, either electronically, via gas flame, or even with microwave radiation.
  • the cleaning compositions of the present invention are ideally suited for use in laundry applications. Accordingly, the present invention includes a method for laundering a fabric. The method comprises the steps of contacting a fabric to be laundered with a said cleaning laundry solution comprising at least one embodiment of Applicants' cleaning composition, cleaning additive or mixture thereof.
  • the fabric may comprise most any fabric capable of being laundered in normal consumer or institutional use conditions.
  • the solution preferably has a pH of from about 8 to about 10.5.
  • the compositions may be employed at concentrations of from about 500 ppm to about 15,000 ppm in solution.
  • the water temperatures typically range from about 5 °C to about 90 °C.
  • the water to fabric ratio is typically from about 1:1 to about 30:1.
  • IEC-B detergent IEC 60456 Washing Machine Reference Base Detergent Type B, supplied by wfk, Briiggen-Bracht, Germany, to each pot.
  • TEST METHOD 2- For Test Method 2 the BMI microswatch assay is run using the detergent compositions 1, 2 or 4 from Table 1-3.
  • the detergent is dissolved in water that has a hardness of 6gpg and adjusted to a temperature of 16°C.
  • Performance of the variant enzymes is then determined as per the BMI microswatch assay described.
  • the performance index is determined by comparing the performance of the variant with that of the enzyme of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage being 1 ppm. This Method is described in further detail in Example 31.
  • TEST METHOD 3- For Test Method 3 the BMI microswatch assay is run using the detergent compositions 1, 2 or 4 from Table 1-3.
  • the detergent is dissolved in water that has a hardness of 6gpg and adjusted to a temperature of 16°C.
  • Performance of the variant enzymes is then determined as per the BMI microswatch assay described.
  • the performance index is determined by comparing the performance of the variant with that of the enzyme of SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, with in all cases the enzyme dosage being 0.4 ppm. This Method is described in further detail in Example 1.
  • TEST METHOD 4- For Test Method 4 the BMI microswatch assay is run using the detergent composition 10 from Table 19-4. The detergent is dissolved in water that has a hardness of 12gpg and adjusted to a temperature of 16°C. Performance of the variant enzymes is then determined as per the BMI microswatch assay described. The performance index is determined by comparing the performance of the variant with that of the enzyme of SEQ ID NO:755, with in all cases the enzyme dosage being 1.6 ppm. Enzymes having a performance index of 1.1 or greater are viewed to be Series 1 GG36 cold water proteases. This Method is described in further detail in Example 19.
  • TEST METHOD 5 - For Test Method 5 the BMI microswatch assay is run using the detergent composition 7 from Table 19-4. The detergent is dissolved in water that has a hardness of 6gpg and adjusted to a temperature of 16°C. Performance of the variant enzymes is then determined as per the BMI microswatch assay described. The performance index is determined by comparing the performance of the variant with that of the enzyme of SEQ ID NO:755, with in all cases the enzyme dosage being 4 ppm. Enzymes having a performance index of 1.1 or greater are viewed to be Series 1 GG36 cold water proteases. This Method is described in further detail in Example 19.
  • TEST METHOD 6 - For Test Method 6 the BMI microswatch assay is run using the detergent composition 7 from Table 19-4.
  • the detergent is dissolved in water that has a hardness of 6gpg and adjusted to a temperature of 16°C.
  • Performance of the variant enzymes is then determined as per the BMI microswatch assay described.
  • the performance index is determined by comparing the performance of the variant with that of a reference enzyme, said reference enzyme being the enzyme of SEQ ID NO:755 consisting the A158E mutation, with in all cases the enzyme dosage being 4 ppm.
  • Enzymes having a performance index of 1.0 or greater are viewed to be Series 1 GG36 cold water proteases. This Method is described in further detail in Example 19.
  • TEST METHOD 7 Electrical conductivity of an aqueous solution is assayed according to the standard method ASTM Dl 125 and reported in units of milliSiemens/cm, abbreviated to mS/cm in this patent.
  • RNA ribonucleic acid
  • MgCl 2 magnesium chloride
  • NaCl sodium chloride
  • w/v weight to volume
  • v/v volume to volume
  • w/w weight to weight
  • Tricine N- [tris-(hydroxymethyl)-methyl] - glycine
  • CHES (2-(N-cyclo-hexylamino) ethane- sulfonic acid)
  • TAPS (3- ⁇ [frisky droxymethyl) -methyl] -amino ⁇ -propanesulfonic acid);
  • CAPS (3-(cyclo-hexylamino)-propane- sulfonic acid;
  • DMSO dimethyl sulfoxide
  • DTT 1,4-dithio-DL-threitol
  • SA sinapinic acid (s,5-dimethoxy-4-hydroxy cinnamic acid
  • TCA trichloroacetic acid
  • Glut and GSH reduced glutathione
  • GSSG oxidized glutathione
  • TCEP Tris[2-carboxyethyl] phosphine
  • Ts tosyl
  • Bn benzyl
  • Ph phenyl
  • Ms mesyl
  • Et ethyl
  • Me Me
  • Taq Thermus aquaticus DNA polymerase
  • Klenow DNA polymerase I large (Klenow) fragment
  • EGTA ethylene glycol-bis(6-aminoethyl ether) N, N, N', N'-tetraacetic acid
  • EDTA ethylene glycol-bis(6-aminoethyl ether) N, N, N', N'-tetraacetic acid
  • EDTA ethylene glycol-bis(6-aminoethyl ether
  • NA and WE North American (NA) and Western European (WE) heavy duty liquid laundry (HDL) detergents
  • heat inactivation of the enzymes present in commercially-available detergents is performed by placing pre- weighed liquid detergent (in a glass bottle) in a water bath at 95 °C for 2 hours.
  • the incubation time for heat inactivation of NA and WE auto dish washing (ADW) detergents is 8 hours. Both un-heated and heated detergents are assayed within 5 minutes of dissolving the detergent to accurately determine percentage deactivated. Enzyme activity is tested by the AAPF assay.
  • compositions of the detergents used in the assays in BPN' Variant (or Part I) Examples are shown in Table 1-3.
  • BPN' variant protein samples were added to the detergent compositions as described in Part I Example 1 to assay for the various properties tested.
  • liquid laundry detergent compositions suitable for top-loading automatic washing machines (1, 2 & 4) and front loading washing machines (3).
  • Ci6_i7 Branched alkyl sulfate 1.7 1.29 3.09
  • Random graft co-polymer 1 1.46 0.5
  • Amphiphilic alkoxylated grease cleaning polymer 3 1.28 1 0.4 1.93
  • Random graft copolymer is a polyvinyl acetate grafted polyethylene oxide copolymer having a polyethylene oxide backbone and multiple polyvinyl acetate side chains.
  • the molecular weight of the polyethylene oxide backbone is about 6000 and the weight ratio of the polyethylene oxide to polyvinyl acetate is about 40 to 60 and no more than 1 grafting point per 50 ethylene oxide units.
  • modified polynucleotides of the invention that encode protease variants of the invention (such as cold water proteases of the invention), including, but not limited to, e.g., site-saturation mutagenesis, scanning mutagenesis, insertional mutagenesis, deletion mutagenesis, random mutagenesis, site- directed mutagenesis, and directed-evolution, as well as various other recombinatorial approaches.
  • Methods for making modified polynucleotides and proteins include DNA shuffling methodologies (see, e.g., Stemmer WP, Proc. Natl. Acad. Sci.
  • oligonucleotides to insert random and targeted mutations, deletions and/or insertions (Ness et al., Nat. Biotechnol.20:1251-5 [2002]; Coco et al., Nat. Biotechnol. 20:1246-50 [2002]; Zha et al., Chembiochem.4:34-9 [2003]; Glaser et al., J. Immunol.149:3903- 13 [1992]); see also Arkin and Youvan, Biotechnology 10:297-300 (1992); Reidhaar- Olson et al., Methods Enzymol. 208:564- 86 (1991).
  • a full-length parent polynucleotide is ligated into an appropriate expression plasmid, and the following mutagenesis method is used to facilitate the construction of the modified protease of the present invention, although other methods may be used.
  • the method is based on that described by Pisarchik et al. (Pisarchik et al., Prot. Eng. Des. Select. 20:257-265 [2007]).
  • an added advantage is provided in that the restriction enzyme cuts outside its recognition sequence, which allows digestion of practically any nucleotide sequence and precludes formation of a restriction site scar.
  • a naturally-occurring gene encoding a full-length protease is obtained and sequenced and scanned for one or more points at which it is desired to make a mutation (e.g., deletion, insertion, substitution) at one or more amino acids.
  • Mutation of the gene in order to change its sequence to conform to the desired sequence is accomplished by primer extension in accord with generally known methods.
  • Fragments to the left and to the right of the desired point(s) of mutation are amplified by PCR and to include the Eaml 1041 restriction site.
  • the left and right fragments are digested with Earn 11041 to generate a plurality of fragments having complementary three base overhangs, which are then pooled and ligated to generate a library of modified sequences containing one or more mutations.
  • This method avoids the occurrence of frame-shift mutations. This method also simplifies the mutagenesis process because all of the oligonucleotides can be synthesized so as to have the same restriction site, and no synthetic linkers are necessary to create the restriction sites as is required by some other methods.
  • this assay was started using filtered B. subtilis bacterial culture supernatant from microtiter plates grown 3-4 days at 33-37°C with shaking at 230-250 rpm and humidified aeration. A fresh 96-well flat bottom microtiter plate (MTP) was used for the assay. First, 100 ⁇ /well of 0.25 N HC1 was placed in each well. Then, 25 ⁇ of filtered culture broth was added. The light scattering/absorbance at 405 nm (use 5 sec mixing mode in the plate reader) was then determined in order to provide the "blank" reading.
  • TCA trichloroacetic acid
  • a standard curve can be created by calibrating the TCA readings with AAPF assays of clones with known conversion factors.
  • the TCA results are linear with respect to protein concentration from 250 to 2500 micrograms protein per ml (ppm) and can thus be plotted directly against enzyme performance for the purpose of choosing good-performing variants.
  • Tris/HCl, pH 8.6, containing 0.005% TWEENO-80 Tris dilution buffer
  • 100 mM Tris buffer, pH 8.6, containing 10 mM CaCl 2 and 0.005% TWEENO-80 Tris/Ca buffer
  • 160 mM suc- AAPF-pNA in DMSO suc- AAPF-pNA stock solution
  • suc- AAPF-pNA working solution 1 ml suc-AAPF-pNA stock solution was added to 100 ml Tris/Ca buffer and mixed well for at least 10 seconds.
  • Blood milk and ink (BMI) stained microswatches of 5.5 millimeter circular diameter were obtained from CFT. Before cutting the swatches, the fabric (EMPA 116) was washed with water. One microswatch was placed in each well of a 96- well non-binding microtiter plate (Corning 3641).
  • the detergents used for the assays included Detergent Composition 1, Detergent Composition 2, and Detergent Composition 4.
  • the detergents were diluted in Milli-Q (deionized) water to a working strength concentration of 0.788 g/L. These detergents were buffered with 5 mM HEPES pH 8.2 or pH 7.2, which upon addition to detergent, buffers at pH 8 or pH 7, respectively.
  • the master dilution was prepared from the culture supematants at 8 ⁇ g/mL, where the approximate enzyme concentration of the culture supematants and BPN'-v3 or BPN'-v36 parent controls was determined using the AAPF protease activity assay, basing the concentration on a purified BPN'-v3 or BPN'-v36 standard of known concentration.
  • the MTP was sealed with tape and placed in the iEMS incubator/shaker (Thermo/Labsystems) pre-set at 16°C in a refrigerated dairy case or at 32°C on the benchtop for 20 minutes, with agitation at 1400 rpm.
  • the stability of protease variants was determined in the presence of 40% concentrated Detergent Composition 3 diluted in water.
  • the reagents used were Detergent Composition 3 diluted to 50% in Milli-Q water, 10 mM MES 0.01 % TWEENO-80 pH 5.8 master dilution buffer, AAPF reagents: see protocol AAPF assay.
  • F-bottom MTP Coming 9017
  • Biomek FX Beckman Coulter
  • Spectramax Plus 384 MTP Reader Molecular Devices
  • iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation)
  • sealing tape Nunc (236366)
  • circulating reservoir Biller Fx
  • Detergent Composition 3 was initially diluted to 50% in water. This detergent was kept at room temperature and cycled through the circulating reservoir. The iEMS incubators/shakers (Thermo/Labsystems) were pre-set at 43°C. Culture supernatants were diluted into plates containing master dilution buffer to a concentration of ⁇ 20 ppm (master dilution plate). Then, 40 ⁇ of sample from the master dilution plate was added to plates containing 160 ⁇ 50% Detergent Composition 3 to give a final incubation concentration of 4 ppm. The contents were mixed and kept at room temperature and triplicate AAPF assays were performed immediately on these plates and recorded as unstressed reads.
  • the reagents used were dodecyllbenzene sulfonate, sodium salt (DOBS, Sigma No. D-2525), TWEENO-80 (Sigma No. P-8074), di- sodium EDTA (Siegfried Handel No. 164599-02), HEPES (Sigma No.
  • V- or U-bottom MTPs as dilution plates (Greiner 651101 and 650161, respectively), F- bottom MTPs (Corning 9017) for unstressed and LAS/EDTA buffer as well as for suc-AAPF- pNA plates, Biomek FX (Beckman Coulter), Spectramax Plus 384 MTP Reader (Molecular Devices), iEMS Incubator/Shaker (1 mm amplitude) (Thermo Electron Corporation), and Nunc sealing tape (236366).
  • the iEMS incubator/shaker (Thermo/Labsystems) was set at 29°C. Culture supernatants were diluted into plates containing unstressed buffer to a concentration of ⁇ 25 ppm (master dilution plate). Then, 20 ⁇ of sample from the master dilution plate was added to plates containing 180 ⁇ unstressed buffer to give a final incubation concentration of 2.5 ppm. The contents were mixed and kept at room temperature and an AAPF assay was performed on this plate.
  • the performance index provides a comparison of the performance of a variant (actual value) and a standard or reference protease enzyme (theoretical value) at the same protein concentration.
  • the theoretical values can be calculated using the parameters of a performance dose response curve (i.e. using a Langmuir equation to generate the performance curve) of the standard/reference protease.
  • the PI identifies winners (e.g., variants having enhanced proteolytic activity compared to that of the standard/reference protease) as well as variants that may be less desirable for use under certain circumstances (e.g., variants having proteolytic activity lower than the proteolytic activity of the standard/reference protease).
  • protease variants of the invention having performance index values lower than that of a reference or standard protease are nevertheless useful in the applications and methods described herein.
  • protease variants of the invention having performance index values lower than that of a reference or standard protease have proteolytic activity and thus are useful in the compositions of the invention, such as, but not limited to, e.g., cleaning compositions (including, but not limited, to, e.g., detergent cleaning compositions) for cleaning a variety of surfaces and items, including, but not limited to, e.g., laundry, fabrics, and dishware, and in personal care applications and compositions as described elsewhere herein; such protease variants are also useful in fabric and home care products and compositions and in non-fabric and home care products and compositions described elsewhere herein and in methods of the invention, including, but not limited, to, e.g., cleaning methods, methods for personal care, etc., described elsewhere herein.
  • non-deleterious variants have a PI >0.05
  • deleterious variants have a PI less than or equal to 0.05
  • combinable variants are those for which the variant has performance index values greater than or equal to 0.2 for at least one property, and >0.05 for all properties.
  • Combinable variants are those that can be combined to deliver proteins with appropriate performance indices for one or more desired properties.
  • subtilase or protease to be engineered has an amino acid different from that of subtilisin BPN' at one or more particular positions, these data find use in identifying amino acid substitutions that alter the desired properties by identifying the best choices for substitutions, including substitutions of the BPN' wild type amino acid.
  • the BPN'-v3 (BPN' protease containing G097A-G128A-Y217Q substitutions) expression cassette used for combinatorial library construction was generated using the BPN' expression cassette, which comprises the aprE-BPN' hybrid leader sequence (i.e., signal sequence), BPN' pro and BPN' mature sequence from B. amyloliquejaciens.
  • the DNA sequence is shown below as SEQ ID NO: l and encodes the BPN' precursor protein shown below as SEQ ID NO: 168.
  • nucleotide sequence of SEQ ID NO:l the DNA sequence encoding the mature protease is shown in bold, the nucleotide sequence encoding leader sequence (aprE-BPN' hybrid leader sequence) is shown in standard (non-underlined) text, and the nucleotide sequence encoding the pro sequence ( ⁇ ') is underlined.
  • amino acid sequence (aprE-BPN' hybrid leader sequence, BPN' pro sequence, and BPN' mature protein sequence) of the BPN' precursor protein set forth in SEQ ID NO: 168 the bolded portion indicates the mature BPN' subtilisin protease.
  • amino acid sequence of the mature BPN' subtilisin protease is shown as SEQ ID NO: 1
  • the nucleotide sequence of the mature BPN'-v3 gene is that of SEQ ID NO:3 (the signal sequence and propeptide sequence used in the BPN'-v3 expression cassette is the same as that for BPN' shown in SEQ ID NO: 1).
  • the protein sequence of the mature BPN'-v3 protease variant is that of SEQ ID NO:4 (the signal sequence and propeptide sequence used in the BPN'- v3 expression cassette is the same as that for BPN' shown in SEQ ID NO: 168).
  • pHPLT-BPN'-v3 plasmid (see Figure 1) containing the BPN'-v3 expression cassette described above served as template DNA for cloning to provide variants derived from BPN'-v3.
  • the vector pHPLT ( Figure 4 in US Patent No. 6,566,112) contains the B. licheniformis LAT promoter ("Plat”); a sequence encoding the LAT signal peptide ("preLAT”). Additional plasmid elements from plasmid pUBHO disclosed in McKenzie et al., Plasmid 15(2): 93-103 (1986):
  • ori-pUB is the origin of replication from pUB HO;
  • neo is the neomycin/kanamycin resistance gene from pUB HO;
  • Terminator is the transcriptional terminator from B.
  • a combinatorial DNA library was synthesized at DNA 2.0 and delivered as individual ligation reactions.
  • the DNA from the ligation reaction mixtures was amplified by rolling circle amplification (RCA) using the Illustra
  • Templiphi kit (GE Healthcare). The reaction was performed according to the manufacturer's protocol. One microliter of ten-fold diluted amplified DNA was used to transform 50 ⁇ L of competent B. subtilis cells (AaprE, AnprE, amyE::xylRPxylAcomK-phleo). The transformation mixture was shaken at 37°C for 1 hour. Ten micro-liter aliquots of the transformation mixture were plated on skim milk (1.6%) Luria agar plates supplemented with 10 ⁇ g/ml of neomycin
  • the transformants that formed halos on the skim milk plates were picked into microtiter plates containing 150 ⁇ 1 Luria broth (LB) medium supplemented with 10 ⁇ g/ml neomycin. Plates were grown overnight at 37°C with 250-300 rpm shaking and 70-80% humidity using
  • Enzyscreen lids for microtiter plates (Enzyscreen). Using a 96 pin replicating tool, (Enzyscreen) the overnight culture plate was used to inoculate a new microtiter plate containing 180 ⁇ of MBD medium (a MOPS based defined medium) with 2.5 ⁇ g/ml neomycin. MBD medium was prepared essentially as known in the art (see Neidhardt et al., J. Bacterid.
  • micronutrients were made up as a 100X stock solution containing in one liter, 400 mg FeS0 4 -7H 2 0, 100 mg MnS0 4 -H 2 0, 100 mg ZnSCv 7H 2 0, 50 mg CuCl 2 -2H 2 0, 100 mg CoCl 2 -6H 2 0, 100 mg NaMo0 4 -2H 2 0, 100 mg
  • protease variants were tested for cleaning performance using a BMI microswatch assay in Detergent Composition 1 at 16°C and pH 8 and BMI microswatch assay in Detergent Composition 2 at 16°C and pH 8. Protein content was determined using the TCA assay. Assays were performed as described in Example 1 and Performance Indices were calculated relative to BPN'-v3 (with a PI value of 1) as per Test Method 3.
  • BPN' subtilisin protease variant was determined to have a PI value greater than 1.0, at least 1.1, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions G097A-G128A-P210S- Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variant has a PI value of 1.1 relative to BPN'-v3 in this BMI microswatch cleaning assay, and enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) in this assay, the variant having an amino acid sequence comprising amino acid substitutions G097A-G128A-P210S- Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • protease variant having enhanced proteolytic activity compared to SEQ ID NO:2 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising amino acid substitutions G097A- G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value greater than that of BPN' (SEQ ID NO:2) and/or BPN'-v3 in a BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises substitutions X097A-X128A-X210S-X217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2, and optionally wherein the variant comprises at least one substitution selected from the group of G097A, G128A, P210S, and Y217Q.
  • protease variant may be an isolated, recombinant, substantially pure, or non- naturally occurring protease variant.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising such protease variant and methods for cleaning utilizing such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value equal to about 1.0 relative to BPN'-v3 (i.e., having a PI value approximately equivalent to that of BPN'-v3) in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A-E156S-P210S-Y217Q, G097A-G128A-P210S-Y217Q-N218A, G097A-G128A-P210S-Y217Q-N218S, and G097A- Y104F-G128A-E156S-P210I-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO: 2 sequence.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of 1.0 compared to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X097A, X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally at least one substitution selected from the group of G097A, Y104F, G128A, E156A/S, P210I/S, Y217Q, and N218A/S, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • compositions including, but not limited to, e.g., cleaning composition
  • BPN' variants were determined to have a PI value of about 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-E156A-P210S-Y217Q- N218S and G097A-G128A-Y217Q-N218A, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • Such variants have proteolytic activity.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-P210S-Y217Q-N218A and G097A-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1 compared to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X097A, X104F, X128A, X156A/S, X210I/S, X217Q, X218A/S, and optionally at least one substitution selected from the group of G097A, Y104F, G128A, E156A/S, P210I/S, Y217Q, and N218A/
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value equal to about 1.0 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q (i.e., BPN'-v3), G097A-G128A-E156S-P210S-Y217Q, and G097A-G128A-P210S-Y217Q-N218S, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have proteolytic activity and enhanced proteolytic activity compared to BPN' in this assay.
  • the invention includes a protease variant having proteolytic activity, PI value of 1.0 relative to BPN'-v3, and/or enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value of about 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-E156A-P210S-Y217Q- N218S, G097A-G128A-Y217Q-N218A, and G097A-Y104F-G128A-E156S-P210I-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have proteolytic activity.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • GcMlOO SEQ ID NO:23 SEQ ID NO:24 b) Construction of combinatorial libraries CG1-CG5 and CG8 using the synthetic genes GcM90-94 and GcMlOO
  • the parent plasmids (template DNA) containing the synthetic genes GcM90-94, and GcMlOO were methylated were methylated using two micrograms of DNA and methylase (NEB), according to the NEB protocol. Methylated DNA was then purified using DNA Clean and Concentrator kit (Zymo Research). Combinatorial libraries CGI -5 and CG8 were made using a QUIKCHANGE® Multi Site-Directed Mutagenesis kit ("QCMS kit”; Stratagene) following the manufacturer's protocol (see Table 3-1 for respective template and primer combinations), with the exception of libraries CG3 and CG4, which used 86.5ng of each primer in place of the 50ng suggested in the protocol.
  • QCMS kit QUIKCHANGE® Multi Site-Directed Mutagenesis kit
  • BPN'-v3+S78N Additional variants of BPN'-v3+S78N were produced by DNA2.0. The following substitutions were introduced individually into the BPN'-v3+S78N parent molecule: Q59G, N62Q, V68A, S89Y, A92G, I108V, II 15V, M124T, P129L, A138T, V147L, S161P, Y167A, P172V, G211T, L267V, and A273S.
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to the enzyme of SEQ ID NO:4 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N, N061A-S078N-G097A-G128A- Y217Q-S224A, S053G-S078N-G097A-G128A-P129T-Q185
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' and BPN'-v3 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X040E, X053G, X059V, X061A, X062H/Q, X068A, X078N, X087E, X101A, X102A, X108V, X124I, X125A, X126V, X129T, X147Q, X159D, X183T, X185T, X211A, X224A, X244I/N, X252Q, and X27
  • BPN' variants were determined to have a PI value equal to about 1.0 relative to the enzyme of SEQ ID NO:4 (alternatively referred to as BPN'-v3) in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061A-S078N-S087E-G097A-G128A-Y217Q- S224A, Q059V-S078N-G097A-G128A-G211A-Y217Q, Q059V-S078N-G097A-G128A- V147Q-Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G097A-I108V-
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variant was determined to have a PI value of about 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions S078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have proteolytic activity.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising said set of amino acid substitutions above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of Q059V-S078N- G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q- G211A-Y217Q, S078N-G097A-I108V-G128A-V147Q- G211A-Y217Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N- G097A-I108V-G128A-V147Q-Y217Q-N252Q, S078N- G097A-I108V-G128A-V147Q-Y217Q-N252Q, and S078N-S087E-G097
  • the invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S063T-S078N-G097A- S101A-G128A-S183T-Y217Q, S063T-S078N-G097A-S101A-G128A-S183T-Y217Q-T244N, S063T-S078N-G097A-S101A-G128A-Y2
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO: 6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061A-S078N- G097A-G128A-Y217Q-S224A, N061A-S078N-S087E-G097A-G128A-Y217Q-S224A, Q059V-S078N-G097A-G128A-G211A-Y217Q, Q059V-S078N-G097A-G128A-V147Q- Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G097A-G128A-Y217Q, Q059V-S078N-G
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of about 1 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value of about 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S078N-G097A-G128A-V147Q-Y217Q and S078N-G097A-I108V-G128A-V147Q-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have proteolytic activity.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including cleaning compositions, comprising at least one such variant and methods for cleaning an item or surface in need of cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of Q059V-S078N- G097A-I108V-G128A-V147Q-G211A-Y217Q-N252Q, S078N-G097A-I108V-G128A-V147Q- G211A-Y217Q, S078N-G097A-I108V-G128A-V147Q-G211A-Y217Q, S078N-G097A-I108V-
  • the invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including cleaning compositions, comprising at least one such variant and methods for cleaning an item or surface in need of cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were constructed from different parental plasmids using
  • the parental plasmids (Table 4-1) were methylated using a NEB Dam Methylase Kit in a reaction containing 77.5 ⁇ H20 + 10 ⁇ Buffer 10X + 0.25 ⁇ SAM + 2 uL DAM methylase + 10 uL miniprep DNA (-150 ng/ ⁇ ) at 37 °C overnight.
  • the methylated plasmid DNA was purified using a QIAGEN® PCR
  • QUIKCHANGE® Multi Site-Directed Mutagenesis reactions were set up for each of the DNA templates in a reaction mix containing 2.5 ⁇ Buffer 5X + 0.5 ⁇ primer 1 (25 ⁇ ) + 0.5 ⁇ primer 2 (25 ⁇ ) + 1 ⁇ dNTP's + 1 ⁇ enzyme blend + 18 ⁇ H 2 0 + 1.5 ⁇ DNA.
  • the PCR program used was: 95°C for 1 min; (95°C for 1 min, 53°C for 1 min, 65°C for 9:39 min) x 29 cycles; 65°C for 10 min, 4°C hold. Primer sequences are shown in Table 4-2. In all reactions, PCR was performed using a MJ Research PTC-200 Peltier thermal cycler.
  • BPN' variant proteins were produced by growing B. subtilis transformants in 96 well microtiter plates at 37°C for 68 hours in a MOPS based medium containing urea as described in Example 2.
  • LC5 through LC37 BPN' variants are as follows, respectively: BPN'-P52L-V68A-G97A-I111V, BPN-I111V-M124V-Y167A-Y217Q, BPN'- Y104N-G128A-Y217Q, BPN'-M124V-Y167A-Y217Q, BPN'-I111V-M124V-Y217Q, BPN'- P52L-V68A-G97A, BPN'-G97A-I111V-M124V, BPN'-V68A-A92G-G97A, BPN'-G97A- II 11 V-M124V-Y167A-Y217Q, BPN'-P52L-V68A-
  • Transformants were picked into microtiter plates and grown as described in Example 2. The variants were assayed for cleaning performance using a BMI microswatch assay in
  • Detergent Composition 2 at 16°C and pH 8. Protein content was determined using the TCA assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN'-v3 (with a PI value of 1.0).
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 2 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-I111V- M124V-Y217Q, G097A-I111V-Y167A-Y217Q, S024G-N025G-N061P-G097A-S101N-G128S- Y217Q, S024G-N025G-S053G-N061P-G097A-S
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1.0 compared to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X052L, X053G, X055P, X061P, X062Q, X068A, X089Y, X092G, X096T, X097A, X101N, X104N, X111V, X124V, X126A, X128A/S, X167A, X203Y, and X217Q, and optionally at least one substitution selected from the group of S
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128S- Y217Q, G097A-I111V-Y217Q, I111V-G128A-Y217Q, I111V-M124V-Y167A, I111V-M124V- Y217Q, L096T-G097A-Y217Q, N062Q-G097A-I111V, S053G-N061P-G097A-S101N-G128S- V203Y-Y217Q, S089Y-M124V-Y217Q, and V068A-I111V, S053G-N061P-G097A-S101N-G128S- V203Y-Y217Q, S089Y-M124V-Y217Q, and V068A
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to the sequence of SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-I111V-M124V, G097A-L126A- Y217Q, G097A-M124V-Y217Q, I111V-Y167A-Y217Q, M124V-Y167A-Y217Q, P052L- V068A-G097A, S089Y-I111V-M124V, V068A-A092G-G097A, V068A-A092G-I111V, V068A-G097A-I111V, V068A-S089Y-I111V, and Y104N-G128A-Y217Q, where
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-M124V- Y167A-Y217Q, V068A-Y167A-Y217Q, G097A-I111V-M124V-Y167A, I111V-M124V- Y167A-Y217Q, V068A-I111 V-Y167A-Y217Q, G097A-I111 V-M124V-Y167A-Y217Q, P052L-V068A-I111V, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • the invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • Such variants have proteolytic activity.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • Variants based on parent BPN' were made by DNA 2.0.
  • the variants were grown as described in Example 2 and tested for cleaning performance on BMI microswatch assay in Detergent Composition 1 at 16°C and pH 8, BMI microswatch assay in Detergent Composition 4 at 16°C and pH 8, and egg microswatch assay in Detergent Composition 4 at 16°C and pH 8.
  • the protein content was determined using the TCA assay.
  • the assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN'-v3 (with a PI value of 1.0).
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-S101N- G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S101N-G128A-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A-S
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A- P210S-Y217Q, G097A-G128S-P210S-Y217Q, G097A-I111V-M124I-Y217Q, G097A-I111V- M124V-P210S-Y217Q, G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q, N061P- G097A-G128A-P210S-Y217Q, N061P-G097A-G128S-Y217Q, N061P-G097A-G128S-Y217Q,
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, N061P- G097A-M124V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, N061P-N062Q-G097A- G100Q-S101N-Y217Q, N061P-N062Q-G097A-G100Q-Y217Q, N061P-N062Q-G100N- G102A-Y217Q, N061P-N062Q-S078N-G097A-G100N-I111V-Y217Q,
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1.0 compared to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 or 4 at pH 8 and 16°C, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X053G, X061P, X062Q, X078N, X097A, X100D/N/Q, X101N, X102A, X111V, X123A/Q/V, X124I/V, X128A/S, X129S, X210S, X217Q, and optionally at least one substitution selected from the group of
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-N123A- Y217Q, G097A-N123V-Y217Q, N061P-G102A-G128S-Y217Q, N061P-S101N-G102A- G128S-Y217Q, Y217Q, S078N-G097A-I111V-N123Q-Y217Q, and G102A-N123Q-Y217Q, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • the invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-G128S- Y217Q, N061P-G097A-S101N-G128A-P210S-Y217Q, N061P-N062Q-G097A-S101N-I111V- Y217Q, S024G-N025G-N061P-G097A
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, G097A-G128A- P210S-Y217Q, G097A-G128S-P210S-Y217Q, G097A-I111V-M124I-Y217Q, G097A-I111V- M124V-P210S-Y217Q, G097A-N123Q-P210S-Y217Q, G097A-N123Q-Y217Q, N061P- G097A-G128A-P210S-Y217Q, N061P-G097A-I111V-M124V-Y217Q, N061P-G097A-I111V-M124V-Y217Q, N061P-G097A-I111V-M124V-Y217Q, N061P-
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, N061P- G097A-S101N-N123Q-Y217Q, N061P-N062Q-G097A-G100N-Y217Q, N061P-N062Q- G097A-G100Q-P210S-Y217Q, N061P-N062Q-G097A-G100Q-S101N-Y217Q, N061P- N062Q-G100N-G102A-Y217Q, S024G-N025G-S053G-N061P-G097A-
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-N123A- Y217Q, G097A-N123V-Y217Q, N061P-N062Q-G097A-G100D-Y217Q, N061P-S101N- G102A-G128S-Y217Q, Y217Q, N061P-G102A-G128S-Y217Q, S078N-G097A-I111V-N123Q- Y217Q, and G102A-N123Q-Y217Q, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO: 2 sequence.
  • the invention includes a protease variant having proteolytic activity, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value greater than 1.0 to about 5 relative to BPN'-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at 16°C and pH 8: BPN' amino acid sequence (SEQ ID NO:2) comprising the set of amino acid substitutions N061P-G097A-S101N-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO:2.
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions N061P- G097A-S101N-G128A-P210S-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128A-Y217Q, N061P-G102A- P129S-Y217Q, N062Q-G097A-I111V-P210S-Y217Q, S024G-N025G-S053G-N061P-G097A- S101N-G128A-P210S-Y217Q, S024G-N025G-N061P-G097A-S101N-G128A-P210S-Y217Q, N061P-G097A-G128A-P210S-Y217Q, G097A-G128S-Y217Q, G097A-G128S-
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value equal to or greater than 0.5 and equal to or less than 0.9 relative to BPN'-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N061P-G097A-M124I-Y217Q, S053G-N061P-G097A-S101N-N123Q-Y217Q, S053G-N061P- G102A-P129S-P210S-Y217Q, G097A-I111V-M124V-P210S-Y217Q, G097A-N123Q-P210S- Y Y
  • the invention includes a protease variant having proteolytic activity and/or a PI value of equal to or greater than 0.5 and equal to or less than 0.9 relative to BPN'-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1.0 and to about 5 compared to BPN'-v3 in this egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X053G, X061P, X062Q, X078N, X097A, X100D/N/Q, X101N, X102A, X111V, X123A/Q/V, X124I/V, X128A/S, X129S, X210S, and X217Q, and optionally at least one substitution selected from the group
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' combinatorial library based on BPN' parent was made by DNA2.0 and delivered as a ligation reaction.
  • DNA from the ligation reaction mixtures was amplified before transformation and transformants grown as described in Example 2.
  • These variants were tested for cleaning performance using BMI microswatch assay of Test Method 3 in Detergent Composition 1 and Detergent Composition 4 at 16°C and pH 8 as well as egg microswatch assay in Detergent Composition 4 at 16°C and pH 8.
  • Protein content was determined using the TCA assay and protease activity was assayed using the AAPF assay.
  • the assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN'-v3 (with a PI value of 1.0).
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-N025G-S053G- T055P-N061P-G097A-S101N-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, N025G-S038G-S053G-N061P-S078N-G097A
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A- Y217Q, N025G-S078N-G097A-G128A-Y217Q, N025G-T055P-G097A-G128A-Y217Q, S024G-G097A-S101N-G128A-Y217Q, S024G-I035V-T055P-N061P-S078N-G097A-Y217Q, S024G-N025G-N061P-S078N-G097A-S101N-
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN' and/or a PI value of greater than 1.0 compared to BPN'-v3 in this BMI microswatch cleaning assay in Detergent Composition 1 at pH 8 and 16°C, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X024G, X025G, X035V, X038G, X053G, X055P, X061P, X078N, X097A, X101N, X116S, X128A/S, X130G, X216Q, X217Q, and X249N, and optionally at least one substitution selected from the group of S024G, N025G, I035V
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-N025G-N061P- S078N-G097A-S101N-G128A-Y217Q, S024G-N025G-S053G-N061P-S078N-S101N-G128A- Y217Q, S024G-N025G-S053G-T055
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A-Y217Q, N025G-G097A-S101N-G128A-Y217Q, N025G-S038G-S053G- N061P-S078N-G097A-S101N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, N025G-S053G-N061P-S078N-G128A-Y217Q, N025G-S053G-N061P-S078N-S101N-G128A-Y217Q, N
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value of about 0.9 relative to BPN'-v3 in a BMI microswatch cleaning assay of Tets Method 3 in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of T055P-N061P-G097A-A116S-G128A, S024G-N025G-T055P-N061P-S078N-G097A-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • Such variants have proteolytic activity and may have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) in this assay.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of N025G-T055P-N061P- S078N-G097A-S101N-G128A-Y217Q, N061P-S101N-G128A-Y217Q, S024G-N025G-S053G- N061P-S078N-S101N-G128A-Y217Q, S024
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by
  • BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of G097A-G128S-Y217Q, G097A-G128A- Y217Q, S024G-G097A-S101N-G128A-Y217Q, N025G-T055P-N061P-S078N-S101N-G128A- Y217Q, S053G-T055P-N061P-S101N-G128A-Y217Q, S053G-T055P-S078N-G097A-S101N- G128A-Y217Q, N025G-S053G-N061P-S078N-G097A-S101N- G128A-Y217Q, N025G-S053G-N061P-S078N-G097A-S101N- G1
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'-v3 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning comprising utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value of about 0.9 relative to BPN'-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S101N-G128A-Y217Q, S024G-T055P- N061P-G097A-S101N-G128A, S024G-N025G-N061P-S078N-G097A-S101N-G128A, S024G- T055P-N061P-S078N-S101N-G128A-Y217Q, S024G-N025G
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.9 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variant was determined to have a PI value of about 0.8 relative to BPN'-v3 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN' amino acid sequence (SEQ ID NO:2) comprising amino acid substitutions S024G-S053G- S078N-G097A-S101N-G128A-Y217Q, wherein positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • the invention includes a protease variant having proteolytic activity, a PI value of 0.8 relative to BPN'-v3, and/or an enhanced proteolytic activity compared to BPN' in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions S024G-S053G-S078N- G097A-S101N-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN' variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1 to about 12, from greater than 4 to about 12, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v3 in an AAPF proteolytic assay: BPN' amino acid sequence (SEQ ID NO:2) comprising at least one set of amino acid substitutions selected from the group consisting of S024G-G097A-S101N-G128A-Y217Q, S101N-G128A-Y217Q, N025G-T055P-N061P-S078N- S101N-G128A-Y217Q, S053G-T055P-N061P-S101N-G128A-Y217
  • Such variants have enhanced proteolytic activity compared to BPN' protease (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v3 in this AAPF assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • BPN' variant was determined to have a PI value equal to about 1.0 relative to BPN'-v3 in an AAPF proteolytic assay: BPN' amino acid sequence (SEQ ID NO:2) comprising amino acid substitutions G097A-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with the sequence of SEQ ID NO: 2.
  • BPN' amino acid sequence SEQ ID NO:2
  • Such variants have enhanced proteolytic activity compared to BPN' protease (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a PI value of 1.0 relative to BPN'- v3 in this AAPF assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 and comprising amino acid substitutions G097A-G128A-Y217Q, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the amino acid sequence of BPN'-v36 is that of SEQ ID NO:6 and the nucleic acid sequence encoding the BPN'-v36 protease variant is that of SEQ ID NO:5.
  • the amino acid sequence of BPN'-v36 may be represented by reference to the subtilisin BPN' amino acid sequence of SEQ ID NO:2. That is, BPN'-v36 may be represented as the subtilisin BPN' sequence of SEQ ID NO:2 with the six amino acid substitutions S024G-S053G- S078N-S101N-G128A-Y217Q.
  • the BPN'-v36 amino acid sequence may be conveniently designated as BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q or
  • each amino acid position of an amino acid sequence is numbered according to the numbering of a corresponding amino acid position in the amino acid sequence of Bacillus amyloliquefaciens subtilisin BPN' shown in SEQ ID NO:2 as determined by alignment of the variant amino acid sequence with the Bacillus amyloliquefaciens subtilisin BPN' amino acid sequence.
  • SELs Site evaluation libraries
  • each codon to be mutated in the BPN'-v36 protease a pair of partially overlapping, complementary (mutagenic forward and reverse) primers were designed.
  • two PCR reactions were carried out using either a common forward gene-flanking primer (P4974, SEQ ID NO:60) and a mutagenic NNS reverse primer, or the common reverse gene-flanking primer (P4976, SEQ ID NO:61) and a mutagenic NNS forward primer.
  • P4974 common forward gene-flanking primer
  • P4976 SEQ ID NO:61
  • PCR reactions generated two PCR fragments, one encoding the 5' half of the mutant BPN'-v36 gene (5' gene fragment) and the other encoding the 3' half of the mutant BPN'-v36
  • Each PCR amplification reaction contained 30 pmol of each primer and 100 ng of the BPN'-v36 parent template DNA (plasmid pHPLT-BPN'-v36, see Figure 4). Amplifications were carried out using Vent DNA polymerase (NEB). The PCR reaction (20 ⁇ ) was initially heated at 95°C for 2.5 min followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 15 sec. and extension at 72°C for 40 sec.
  • NEB Vent DNA polymerase
  • the 5' and 3' gene fragments were gel-purified by the QIAGEN® gel-band purification kit, mixed (50 ng of each fragment), mixed and amplified by PCR once again using the primers P4973 (SEQ ID NO:62) and P4950 (SEQ ID NO:63) to generate the full-length gene fragment.
  • the PCR conditions were same as described above, except the extension phase, which was carried out at 72°C for 2 min.
  • the full-length DNA fragment was gel-purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HmdIII restriction enzymes and ligated with the pHPLT-BPN' partial opt vector that also was digested with the same restriction enzymes.
  • Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer' s recommendation (GE Healthcare) to generate multimeric DNA for
  • the colonies with halos were inoculated in 150 ⁇ of LB media containing 10 ⁇ g/mL neomycin. The next day, cultures were either frozen with 15% glycerol or grown in MBD medium for biochemical analysis as described in Example 2.
  • Protein variants from BPN'-v36 SEL were tested for cleaning performance using a BMI microswatch assay in Detergent Composition 4 at 16°C and pH 8 and egg microswatch assay in Detergent Composition 4 at 16°C and pH 8. Protein content was determined using the TCA assay. The assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN'-v36 (i.e., BPN'-S24G-S53G-S78N-S101N-G128A-Y217Q).
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q (SEQ ID NO:6) (i.e., BPN'-v36) comprising at least one amino acid substitution selected from the group consisting of A116V, G160S, I111L, II 15V, N109S, N117M, P005G, Q059V, T164S, Y262M, A015Q, A015S, A098
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN'-v3 and BPN'-v36, a PI value greater than that of BPN'-v3, and/or a PI value greater than 1 to about 5 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one amino acid substitution selected from the group consisting of BPN'-v36, A001G, A001Y, A013G, A013V, A015F, A015G, A015K, A015M, A015P, A015T, A015W, A015Y, A029G, A073S, A088C, A088I, A088L, A088T, A088V, A098D, A098K, A098P, A098R, A098W, A116D, A116E, A116R, A1
  • the invention includes BPN'-S024G-S053G-S078N- S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising substitution A128S, e.g., BPN'-S024G-S053G-S078N-S101N-G128S-Y217Q.
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of about 1.0 relative to BPN'-v3, and/or a PI value of 1.0 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined using Test Method 3 to have a PI value equal to about 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one amino acid substitution selected from the group consisting of A001F, A001K, A001L, A001M, A001Q, A001R, A001S, A001T, A001V, A013C, A013S, A015D, A015E, A015L, A015R, A048S, A073N, A073T, A074G, A074S, A085C, A085G, A085S, A085V, A088M, A088S, A092S, A098G, A114G,
  • Such variants may have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and/or a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere here
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in the BMI microswatch cleaning assay of Test Method 3 in Detergent Composition 4 at pH 8 and 16°C: BPN'-S24G-S53G-S78N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting ofAOOlD, A001H, A001N, A015C, A048C, A048E, A085T, A133R, A137R, A142C, A144D, A144R, A152S, A153G, A187P, A187Q, A187T, A187V, A216R, A230S, A272R, A273H, A273T, A274H, D036N, D036S, D181H, D181T, D259N, D259
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this BMI microswatch cleaning assay(Test Method 3), the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at 16°C and pH 8: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one amino acid substitution selected from the group consisting of A216E, L090I, A098R, A098W, A098Y, A116G, A116R, A116S, A133M, I107L, I115V, M124L, N101I, N109H, N109S, N109T,
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN'-v3 and BPN'-v36, a PI value greater than that of BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the S
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one amino acid substitution selected from the group consisting of BPN'-v36, A001D, A001M, A001N, A001R, A001T, A001V, A013C, A013G, A013S, A015D, A015F, A015L, A015M, A015P, A015Q, A074G, A074S, A085S, A085T, A085V, A088C, A088L, A088S, A088T, A088V, A133E, A133G, A133R, A137E
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of about 1.0 relative to BPN'-v3, and/or a PI value of 1.0 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the following BPN'-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one amino acid substitution selected from the group consisting of A001E, A015C, A015E, A048C, A048E, A073T, A085C, A085G, A088I, A088M, A114G, A137R, A187L, A187N, A187P, A187W, A216C, A230S, A273D, A273H,
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), and/or a PI value of 0.9 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in an egg microswatch cleaning assay in
  • Detergent Composition 4 at pH 8 and 16°C BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one amino acid substitution selected from the group consisting of AOOIC, A142C, A187C, A216H, A273Q, A274H, D036Q, D036S, D099S, D197T, E156L, F189A, F189L, G053L, G053R, G157P, G178A, G258P, H039S, H238Y, K012C, K012E, K012L, K012W, K136E, K265Y, L075G, L075V, L082E, L126W, L257D, L257P, M050L, M222A, M222F, M222L, N056S, N062C, N062L, N062
  • the invention includes a protease variant having proteolytic activity and/or a PI value of equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one, two, three, four, five, six or more amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-S24G-S53G-S78N- S101N-G128A-Y217Q BPN'-S24G-S53G-S78N- S101N-G128A-Y217Q
  • DNA 2.0 DNA 2.0.
  • BMI microswatch assay in Detergent Composition 4 at 16°C and pH 7 Egg microswatch assay in Detergent Composition 4 at 16°C and pH 8
  • Protein content was determined using TCA assay and protease activity was assayed using AAPF assay. All assays were performed as described in Example 1 and the Performance Indices were calculated relative to BPN'-v36.
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-L257G, A116T-A128S, N061S- N109G-A128S-N243V-S260P, S009T-N109G-A128S-K141R-N243V,
  • variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A088T, A088T-A116T, A088T-G131H, A088T-K256R, A088T-N109G, A088T- N243V, A088T-Q103H, A088T-S162G, A088T-S248N, A088T-S249A, A088T-T158S, A116T, A116T-G131H, A116T-K256R, A116T-L257G, A116
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this BMI microswatch cleaning assay Test Method 3), the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such
  • BPN'-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay(test Method 3) in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A088T, A001E-A116T, A001E-A128S-G131H-N243V, A001E-G131H-G169A-N243V, A001E-K256R, A001E-N109G, A001E-N243V, A001E- S033T, A001E-S033T-N109G-N218S, A001E-S033T-N109G-N243V, A001E-S162G, A001E-S
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), and/or a PI value of 0.9 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E, A001E-A128S, A001E-G024E, A001E-G131H, A001E-G169A, A001E-L257G, A001E-N218S, A001E-Q103H, A001E- S063G, A001E-S248N, A001E-S249A, G024E-N076D, K043Y-N076D, K043Y-Q206D, N076D
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO: 6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the following BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in a BMI microswatch cleaning assay (test Method 3) in Detergent Composition 4 at pH 7 and 16°C: BPN'-S024G-S053G-S078N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A116T, A088T-N
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in a BMI microswatch cleaning assay(Test Method 3) in Detergent Composition 4 at pH 7 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A001E-A128S, A001E-G131H, A001E-K256R, A001E-N218S, A001E-N243V, A001E-S033T, A001E-S063G, A001E-S162G, A088T, A088T- K256R, A088T-N218S, A088T-Q103H, A088T-S162G, A088T-T158S, A116
  • Such variants have enhanced proteolytic activity compared to BPN' protease (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one
  • BPN'-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 7 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E, A001E-A088T, A001E-A116T, A001E-G169A, A001E- L257G, A001E-N109G, A001E-S033T-N109G-N243V, A001E-T158S, A088T-G169A, A088T-Q206D, A116T-N218S, A128S-G131H, A128S-N243V-S248N-K256R, A128S-
  • the invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay in
  • Detergent Composition 4 at pH 7 and 16°C BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A128S-G131H-N243V, A001E- G024E, A001E-G131H-G169A-N243V, A001E-Q103H, A001E-S033T-N109G-N218S, A001E-S248N, A116T-G169A, A116T-Q206D, G169A-S249A, K043Y-G169A, N109G- G169A, P040E-N109G-A128S-G131H, Q206D-L257G, S033T-A128S-G131H-N243P, S033T- A128S-G131H-N
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-L257G, G024E-K256R, G024E- L257G, N109G-A116T, N109G-L257G, N243V-K256R, S033T-N109G, S03
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A001E, A001E-A116T, A001E-G131H, A001E-G169A, A001E-K256R, A001E- N109G, A001E-S033T-N109G-N243V, A001E-S063G, A001E-S248N, A001E-S249A, A001E-T158S, A088T, A088T-A116T, A088T-G131H, A088T-Q103H, A088
  • Such variants have enhanced proteolytic activity compared to BPN' protease (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this egg microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in an egg microswatch cleaning assay in
  • Detergent Composition 4 at pH 8 and 16°C BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-A088T, A001E-A128S, A001E- A128S-G131H-N243V, A001E-G024E, A001E-G024E-S204E-Q206D, A001E-G131H- G169A-N243V, A001E-K043Y, A001E-N076D, A001E-N076D-N109G-A128S, A001E- N218S, A001E-N243V, A001E-Q103H, A001E-Q206D, A001E-S033T, A001E-S033T- N109G-N218S, A001E-S16
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the following BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of T158S-L257G, K256R, L
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in a grass microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A001E-A088T, A001E-A116T, A088T-A128S, A088T-N243V, A088T-Q103H, A088T-S248N, A088T-S249A, A116T, A116T-G169A, A116T-N218S, A116T-S162G, A116T-S249A, A116T-T158S, A128S-G169A, A128S-K256R,
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO: 6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in a grass microswatch cleaning assay in
  • Detergent Composition 4 at pH 8 and 16°C BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A001E-G169A, A001E-K256R, A001E- N109G, A001E-N218S, A088T-A116T, A088T-G131H, A088T-N109G, A088T-N218S, A116T-A128S, A116T-G131H, A116T-S248N, A128S, A128S-G131H, G024E, G024E- N109G, G131H, G131H-L257G, G169A-K256R, G169A-Q206D, K043Y, K043Y-A088T, K043Y-L257G, K
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this grass microswatch cleaning assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the following BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in an AAPF proteolytic assay: BPN'-S024G- S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of S033T-N076D-A128S- N218S, A001E-S033T-N
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than ⁇ ', BPN'- v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in an AAPF proteolytic assay: BPN'-S024G-S053G-S078N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A001E-G131H, A001E-S063G, A088T-G131H, A088T-T158S, A116T-G131H, A116T-S162G, A116T-T158S, G024E-S063G, G131H-N243V, G131H-N243V-K256R, G131H-Q206D, G131H-S249A, K043Y, K043Y- S063G, K043Y-S248N, K043Y-S249A, K043
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN'-v36 in an AAPF proteolytic assay: BPN'-S024G-S053G-S078N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of G131H-S162G, G131H-S248N, G131H- T158S, K043Y-G131H, K043Y-S162G, S063G-A088T, S063G-G131H, S063G-S248N, T158S-S162G, Q103H-N218S, S033T-Q103H, and
  • the invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having proteolytic activity, enhanced proteolytic activity compared to BPN', or a PI value greater than that of BPN' (SEQ ID NO:2) in a BMI microswatch cleaning assay, the variant comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X001E, X009T, X018T, X021N, X024G, X033T, X040A, X043Y, X061G/P/S, X063G, X076D, X088T, X103H, X109A/G/Q/S, X116T, X128S, X131H, X141R, X158S, X162G, X169A, X204E,
  • a BPN' combinatorial library based on the BPN'-v36 parent molecule was made by DNA 2.0 and delivered as a ligation reaction.
  • DNA from the ligation reaction mixture was amplified before transformation and transformants grown as described in Example 2.
  • the variants were tested for cleaning performance using BMI microswatch assay in Detergent Composition 4 at 16°C and pH 8 and egg microswatch assay in Detergent Composition 4 at 16°C and pH 8. Protein content was determined using the TCA assay. Assays were performed as in Example 1 and Performance Indices were calculated relative to BPN'-v36 (i.e., BPN'-S24G-S53G-S78N-S101N-G128A-Y217Q).
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in a BMI microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S 101N-G128A- Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A116T-N243V-K256R-L257G, A088T-A116T-N243V-L257G, A088T-T158S-N218S-K256R, A088T-T-
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A088T, A088T-A116T-G131H-L257G, A088T- A116T-G131H-N218S-A274T, A088T-A116T-G131H-N218S-K256R, A088T-A116T-G131H- N218S-K256R-L257G, A088T-A116T-G131H-N218S-N243V, A088T-A
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to about 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at H 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A098S-N218S-K256R, A088T-A116T-G131H-K256R, A088T-A116T-G131H-K256R-L257G-L267M, A088T-A116T-G131H-N218S
  • the invention includes a protease variant having proteolytic activity and/or a PI value of 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in a BMI microswatch cleaning assay (Test Method 3) in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N- G128A-Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A015S-A088T-N109G-G131H-T158S- N218S-S248N, A088T-A098S-G131H-S248N-K256R-L257G, A088T-A116T-G131H-N218S- N243V-K256R-L257G, A088T-A116T-G131H-T158S-L257G, A088T-A116T-G131H
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • BPN'-v36 variants were determined to have a PI value greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2, from greater than 1.0 to about 10, from greater than 1.0 to about 8, or from greater than 1.0 to about 5 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-N109G-A116T-T158S-N243V- L257G, A116T-N218S-N243V-L257G-N269S, A088T-A116T-K256R, A088T
  • Such variants have enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, and a greater PI value than BPN', BPN'-v3 and BPN'-v36 in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), enhanced proteolytic activity compared to BPN', BPN'-v3, and BPN'-v36, a PI value of greater than 1.0 to about 5 relative to BPN'-v3, and/or a PI value of greater than 1.0 to about 5 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino
  • BPN'-v36 variants were determined to have a PI value equal to about 1.0 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A-Y217Q amino acid sequence (SEQ ID NO: 6) comprising at least one set of amino acid substitutions selected from the group consisting of BPN'-v36, A015S-A088T-N109G-G131H-T158S-N218S-S248N, A088T, A088T-A116T- G131H-N218S-N243V-S248N, A088T-A116T-G131H-N218S-S248N, A088T-A116T-G131H- N218S-S248N, A088T-A116T-G131H- N218S-S248N
  • Such variants have enhanced proteolytic activity compared to BPN' (SEQ ID NO:2) and a greater PI value than BPN' in this assay.
  • the invention includes a protease variant having enhanced proteolytic activity compared to BPN' (SEQ ID NO:2), a PI value of 1.0 relative to BPN'-v3, and a PI value of 1.0 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of amino acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least
  • BPN'-v36 variants were determined to have a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in an egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C: BPN'-S024G-S053G-S078N-S101N-G128A- Y217Q amino acid sequence (SEQ ID NO:6) comprising at least one set of amino acid substitutions selected from the group consisting of A088T-A098S-G131H-S248N-K256R- L257G, A088T-A116T-G131H-K256R, A088T-A116T-G131H-N218S-S248N-K256R, A088T- A116T-G131H-N243V-S248N-L257G, A088T-A116T-G131H-S248N, A088T-A116T-G131H-S2
  • the invention includes a protease variant having proteolytic activity and/or a PI value equal to or greater than 0.5 and less than 0.9 relative to BPN'-v36 in this assay, the variant comprising an amino acid sequence having at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% or 99% identity to SEQ ID NO:2 or SEQ ID NO:6 and comprising at least one set of acid substitutions selected from said group above, wherein amino acid positions of the variant are numbered by correspondence with amino acid positions of the SEQ ID NO:2 sequence.
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • subtilisin protease variant having enhanced proteolytic activity compared to BPN'-v36 and/or BPN-'v3 and/or a PI value of greater than 1.0 compared to BPN'- v36 in a BMI microswatch or egg microswatch cleaning assay in Detergent Composition 4 at pH 8 and 16°C, the variant comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identity to SEQ ID NO:2, wherein the variant comprises at least one substitution selected from the group of X001R/T, X002R, X003F/P, X004A/L/M/P, X005S, X006H, X014L, X015L/S, X016C, X017T, X018L, X019K, X020A, X021T, X022L,
  • compositions including, but not limited to, e.g., cleaning compositions, comprising at least one such variant and methods for cleaning utilizing at least one such variant as described in greater detail elsewhere herein.
  • the DNA from the site evaluation libraries of the BPN'-v36 was further mutagenized by error-prone PCR. These libraries were amplified with primers P4973 and P4950 (described in Example 7) using Taq DNA polymerase (Promega). Each PCR amplification reaction contained 30 pmol of each primer, 100 ng of the template DNA (SELs of the BPN'-v36) and various amount of MnC .
  • the PCR reaction (20 iL) was initially heated at 95°C for 2.5 min followed by 30 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 15 sec. and extension at 72°C for 2 min.
  • the DNA fragment was gel -purified by the QIAGEN® gel-band purification kit, digested by the BamHI and HzVidlll restriction enzymes and ligated with the pHPLT-BPN' partial opt vector that also was digested with the same restriction enzymes.
  • Ligation mixtures were amplified using rolling circle amplification in an Illustra Templiphi kit according to the manufacturer's recommendation (GE Healthcare) to generate multimeric DNA for transformation into Bacillus subtilis. For this purpose, ⁇ of the ligation mixture was mixed with 5 ⁇ of the sample buffer, heated to 95°C for 3 min and cooled on ice.

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Abstract

La présente invention concerne des produits d'entretien du linge et de la maison comprenant une ou plusieurs protéases présentes dans les eaux froides et des procédés destinés à fabriquer et à utiliser de tels produits. De telles compositions permettent de réaliser un meilleur nettoyage et de la fraîcheur. De telles protéases présentes dans les eaux froides peuvent être dérivées d'enzymes parentes, comprenant la subtilisine BPN' et la subtilisine dérivée de Bacillus lentus, par substitution, insertion et/ou délétion d'un ou de plusieurs acides aminés des enzymes parentes.
PCT/US2010/059669 2009-12-09 2010-12-09 Produits d'entretien du linge et de la maison WO2011072117A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
EP18181021.9A EP3434764A3 (fr) 2009-12-09 2010-12-09 Tissu et produits de soins à domicile
EP22182416.2A EP4159833A3 (fr) 2009-12-09 2010-12-09 Tissu et produits de soins à domicile
CA2782613A CA2782613C (fr) 2009-12-09 2010-12-09 Produits d'entretien du linge et de la maison
CN201080055885.5A CN102652175B (zh) 2009-12-09 2010-12-09 织物和家居护理产品
MX2012006616A MX2012006616A (es) 2009-12-09 2010-12-09 Productos para el cuidado de las telas y el hogar.
EP10796231A EP2510092A1 (fr) 2009-12-09 2010-12-09 Produits d'entretien du linge et de la maison
JP2012543280A JP5882904B2 (ja) 2009-12-09 2010-12-09 布地ケア製品及びホームケア製品

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WO2024137568A1 (fr) 2022-12-23 2024-06-27 The Procter & Gamble Company Procédé de fabrication d'un article en dose unitaire de détergent soluble dans l'eau
WO2024137571A1 (fr) 2022-12-23 2024-06-27 The Procter & Gamble Company Procédé de fabrication d'un article de détergent à lessive
WO2024163584A1 (fr) * 2023-02-01 2024-08-08 Danisco Us Inc. Variants de subtilisine et procédés d'utilisation
WO2024163600A1 (fr) * 2023-02-01 2024-08-08 Danisco Us Inc. Variants de subtilisine et procédés d'utilisation

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CN102652175B (zh) 2016-02-10
US10900000B2 (en) 2021-01-26
US20140193888A1 (en) 2014-07-10
EP3434764A2 (fr) 2019-01-30
US8785171B2 (en) 2014-07-22
JP5882904B2 (ja) 2016-03-09
EP2510092A1 (fr) 2012-10-17
US20110237487A1 (en) 2011-09-29
CA2782613A1 (fr) 2011-06-16
US20210071113A1 (en) 2021-03-11
MX2012006616A (es) 2012-06-21
JP2013513386A (ja) 2013-04-22
EP4159833A2 (fr) 2023-04-05
EP4159833A3 (fr) 2023-07-26
US20180171268A1 (en) 2018-06-21
CA2782613C (fr) 2016-08-23
CN102652175A (zh) 2012-08-29
EP3434764A3 (fr) 2019-04-03

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