US2018112204A1PendingUtilityA1
AprL-CLADE PROTEASE VARIANTS AND USES THEREOF

Assignee: DANISCO US INCPriority: May 13, 2015Filed: May 13, 2016Published: Apr 26, 2018
Est. expiryMay 13, 2035(~8.8 yrs left)· nominal 20-yr term from priority
Inventors:David A. Estell Frits Goedegebuur Marc Kolkman Rie Mejldal Katherine Augustyn Lilia Maria Babe Richard R. Bott Jian Yao Roopa Santosh Ghirnikar
C11D 3/38681C11D 3/386C12Y 304/21062C12N 9/54
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References
Claims
Abstract

The present disclosure provides AprL-clade protease enzymes, including variant AprL-clade protease enzymes, nucleic acids encoding same, and compositions and methods related to the production and use thereof, including an AprL-clade variant subtilisin enzyme that has improved stability and/or soil removal compared to a parent AprL-clade subtilisin enzyme.
Claims

exact text as granted — not AI-modified
We claim: 
     
         1 . An AprL-clade variant subtilisin enzyme or an active fragment thereof comprising one or more amino acid modification to a parent AprL-clade subtilisin enzyme, wherein the modification is selected from: (i) A001C, A001D, A001E, A001Q, A001S, L010M, L010Q, S100G, S102K, S102R, Y103F, Y103M, S104D, G105M, G105R, S108F, S108H, S108K, S108R, E111Q, T114D, T115D, T115E, T115F, T115K, T115Q, N116E, K012C, K012F, K012H, K012M, K012N, K012Q, K012Y, V120S, M123C, M123I, L125I, G127A, G127D, G127I, G127M, G127S, G127T, G127V, A128E, A128H, A128K, A128P, A128R, A128S, A128T, S129H, S129Q, S129R, S129T, S129V, T132C, T132D, T132E, T132K, T132N, T132P, Q136A, Q136C, Q136K, Q136R, V138C, A143D, A143N, G145S, V147L, V148A, V148C, V148M, V148Q, K015A, K015C, K015E, K015I, K015M, K015S, K015T, K015V, S155D, S155F, S155K, S155N, S155R, S157D, S158D, S158I, S158K, S158R, N160C, N160D, N160I, N160M, N160R, T161C, T161D, T161E, T161K, T161Q, T161R, G165A, G165E, G165Q, G165R, G165S, Q017H, A018D, A018E, S181Q, N182D, S183A, S183G, N184C, N184E, N184Q, A186C, S187D, S187E, S187P, Q019D, Q019E, A193D, A193R, A202D, A202E, A202K, A202Q, A202V, G203A, G203C, G203E, G203N, G203Q, Y205E, T210C, T210E, T210I, T210P, T210V, A214E, T215C, L216C, L216H, L216K, L216M, N217S, L234E, L234F, L234Q, L234S, S235D, S235E, P238T, N239A, N239D, N239H, N239M, N239S, N239T, A024E, A024Q, L240D, S241E, S241N, S241Q, S241V, A242G, A242N, S243E, S243Q, S243V, Q244C, Q244E, V245A, V245C, V245T, N247D, N247W, N025D, N025G, N025Q, S250G, S251E, S258C, S258D, S258E, S258H, S258P, S259E, S259P, S259Q, V026A, V026C, V026E, V026H, V026Q, V026R, F260W, K264A, K264C, K264D, K264H, K264M, K264Q, K264S, K264Y, I267C, N268D, N268E, N268Q, V269C, K027C, K027D, K027E, K027M, K027P, A271E, Q274A, Q274C, Q274D, Q274E, Q274G, A029S, T003E, T003I, T003Q, T003V, V030C, I035A, I035S, I035T, I035V, S038T, N043A, N043C, N043F, N043H, N043Q, V044P, V045A, V045C, V045D, V045E, V045F, V045G, V045M, V045N, V045Q, V045R, V045S, V045T, G046D, A048N, A048W, F050H, V051I, A052F, A052G, A052K, A052N, A052P, A052R, A052V, G053N, G053R, A055C, A055D, A055E, A055G, A055H, A055N, A055S, A055V, N057C, N057E, N057G, N057V, T058C, T058I, T058K, T058W, A068C, A068N, A068S, V071A, L074G, D075E, N076K, T077C, T077D, T077H, T077M, T077N, T077P, T077Q, T077S, T078D, T078I, T078V, S086E, S086H, S086R, V087C, V087G, V087S, V087T, S088E, S088N, S088R, L089N, P009C, P009D, P009E, P009N, P009T, L095A, L095V, N096S, S098K, S098R, and G099Q;
 (ii) A001Q, T003V, K015I, I035A, A068S, V071A, T077N, S086H, V087S, L095A, T115F, M123I, G127T, A128P, A128S, V147L, G165Q, G165S, N184Q, A202V, T210P, N217S, N239S, A242G, S250G, S258P, and Q274A; or   (iii) T003V, A068S, T077N, S086H, T115F, M123I, G127T, A128P, G165Q, N184Q, A202V, T210P, N217S, and S258P; wherein the amino acid positions of the AprL-clade subtilisin variant are numbered by correspondence with the amino acid sequence of  Bacillus  sp. AprL subtilisin set forth in SEQ ID NO:2.   
     
     
         2 . The variant of  claim 1 , wherein said variant has an improved stability when compared to the parent AprL-clade subtilisin enzyme. 
     
     
         3 . The variant of  claim 2 , wherein said improved stability is (i) a stability PI&gt;1.2, or (ii) a stability PI&gt;1.2 and one or more of an HDD cleaning PI&gt;0.8, an HDL pH 6 or pH 8 cleaning PI&gt;0.8, an ADW cleaning PI&gt;0.8, or an expression PI&gt;0.8. 
     
     
         4 . The variant of any preceding claim, further comprising one or more amino acid modification to a parent AprL-clade subtilisin enzyme, wherein the modification is selected from:
 (i) A001C, A048R, A052F, G099S, G099T, G101A, G127F, G127I, G127M, G127Q, G127T, G127V, I121M, L010M, L095A, L095F, L095Q, L095S, L125I, L125V, M123C, M123I, N025F, N025S, N116C, N140R, N184C, P238C, P238D, P238I, P238M, P238N, Q136A, S098F, S098H, S102D, S158Q, S241R, T077C, T077F, T215C, V026I, V026Q, V045R, and V149C; or   (ii) A001C, A024E, A128D, G053C, G099T, G101A, G101N, G165E, G203C, L095Q, L095S, L125S, N061E, N116C, N184C, S098D, S102D, S102E, S104D, S129E, S155D, S158E, and V026T.   
     
     
         5 . The variant of any preceding claim, wherein said variant has improved HDL cleaning at pH 6 and/or pH 8 when compared to the parent AprL-clade subtilisin enzyme. 
     
     
         6 . The variant of any preceding claim, wherein said improved HDL cleaning at pH 6 is (i) an HDL pH 6 cleaning PI&gt;1.1, or (ii) an HDL pH 6 cleaning PI&gt;1.1 and one or more of an HDD Cleaning PI&gt;0.8, an HDL pH 8 cleaning PI&gt;0.8, an ADW cleaning PI&gt;0.8, an expression PI&gt;0.8, or a stability PI&gt;0.8; and wherein said improved HDL cleaning at pH 8 is (i) an HDL pH 8 cleaning PI&gt;1.1, or (ii) an HDL pH 8 cleaning PI&gt;1.1 and one or more of an HDD cleaning PI&gt;0.8, an HDL pH 6 cleaning PI&gt;0.8, an ADW cleaning PI&gt;0.8, an expression PI&gt;0.8, or a stability PI&gt;0.8. 
     
     
         7 . The variant of any preceding claim, further comprising one or more amino acid modification to a parent AprL-clade subtilisin enzyme, wherein the modification is selected from: A029T, A048K, A048R, A052F, A052R, A068G, A113C, D119H, E111Q, G099A, G099S, G127A, G127T, G165A, K012G, K012H, K027C, K027E, K027M, L095F, L125I, L125V, L240Q, N025F, N025G, N025L, N043R, N116R, N140R, N160C, N184C, N239K, P238C, P238I, P238M, Q136F, Q136H, Q136K, Q136R, Q136W, S098F, S098H, S104A, S158Q, S241R, V045R, and Y103M. 
     
     
         8 . The variant of any preceding claim, wherein said variant has improved HDD cleaning when compared to the parent AprL-clade subtilisin enzyme. 
     
     
         9 . The variant of any preceding claim, wherein said improved HDD cleaning is (i) an HDD cleaning PI&gt;1.1, or (ii) an HDD cleaning PI&gt;1.1 and one or more of an HDL pH 6 cleaning PI&gt;0.8, an HDL pH 8 cleaning PI&gt;0.8, an ADW cleaning PI&gt;0.8, an expression PI&gt;0.8, or a stability PI&gt;0.8. 
     
     
         10 . The variant of any preceding claim, further comprising one or more amino acid modification to a parent AprL-clade subtilisin enzyme, wherein the modification is selected from: A001C, A001D, A024E, A024G, A048C, A048D, A048K, A048N, A052F, A052M, A052V, A052Y, A055C, A072G, A091G, A113C, A128P, A128Q, A133G, A133S, A133T, A143E, A143F, A143H, A143N, A143S, A143W, A193D, A202C, A202D, A202E, A202Q, A227S, D119F, D119S, D119Y, G053C, G053E, G053N, G053Q, G060L, G060Y, G099A, G099T, G101A, G117D, G117V, G127A, G127S, G127T, G130D, G130H, G203A, G203C, G203E, G203M, H237I, I035A, I035C, I035M, I035T, I121L, K012N, K015M, K027C, K027L, K027M, K027R, K264A, K264S, L010M, L095F, L125V, L234Q, L240N, L249I, M123C, M123I, M123L, N025F, N025G, N025L, N025S, N057C, N061A, N076G, N096D, N096G, N116A, N116C, N116L, N116Y, N140A, N160C, N182C, N182D, N184C, N211S, N247F, N247H, N247I, N247L, N247Q, N247S, N247V, N247W, N247Y, P009E, P009T, P238A, P238C, P238D, P238F, P238I, P238R, Q017H, Q136A, Q136C, Q136F, Q136H, Q136K, Q136L, R144F, R144L, R248K, S086R, S088N, S088Y, S097G, S097H, S097Y, S098H, S098L, S100L, S102D, S102E, S102G, S102K, S129E, S129H, S155A, S155D, S155E, S155H, S157D, S158I, S158Q, S181A, S181Q, S183G, S241R, S241T, S243I, S243N, S243T, S243V, S243Y, S251E, S258C, S258D, S258P, T058C, T058D, T058F, T058I, T077C, T077F, T077L, T077M, T078Q, T132A, T132Q, T132V, T132Y, T161Q, T210I, T215C, V026Q, V026R, V026T, V028A, V028C, V045I, V071A, V071L, V120A, V120C, V120I, V146A, V146H, V146K, V147L, V149C, Y056H, and Y255C. 
     
     
         11 . The variant of any preceding claim, wherein said variant has improved ADW cleaning when compared to the parent AprL-clade subtilisin enzyme. 
     
     
         12 . The variant of any preceding claim, wherein said improved ADW cleaning is (i) an ADW cleaning PI&gt;1.1, or (ii) an ADW cleaning PI&gt;1.1 and one or more of an HDL pH 6 cleaning PI&gt;0.8, an HDL pH 8 cleaning PI&gt;0.8, an HDD cleaning PI&gt;0.8, an expression PI&gt;0.8, or a stability PI&gt;0.8. 
     
     
         13 . The variant of any preceding claim, wherein said stability PI is measured in accordance with a stability assay of Example 1, HDD Cleaning PI is measured in accordance with an HDD cleaning assay of Example 1, HDL cleaning PI is measured in accordance with an HDL cleaning assay of Example 1, ADW Cleaning PI is measured in accordance with an ADW cleaning assay of Example 1, and/or expression PI is measured in accordance with a protein determination assay of Example 1. 
     
     
         14 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme comprises an amino acid sequence of SEQ ID NO:2, 9, 12, 13, 14, 15, or 16. 
     
     
         15 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme comprises an amino acid sequence of SEQ ID NO:2 or 9. 
     
     
         16 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2. 
     
     
         17 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises:
 (i) a YNT (SEQ ID NO:46) motif between Asp32 and His63;   (ii) a Y56N57T58 (SEQ ID NO:45) motif;   (iii) an F50V51X 1 52G53E54X 3 55Y56N57T58 (SEQ ID NO:48) motif, wherein X 1  is an A or S and X 3  is an A or S;   (iv) an LSX 4 S (SEQ ID NO:55) motif between Lys236 and Arg246, wherein X 4  is A or G;   (v) an L240S241A242S243 (SEQ ID NO:50) motif;   (vi) an L240S241G242S243(SEQ ID NO:56) motif;   (vii) a KXXXLSX 4 SQX 5 R (SEQ ID NO:57) motif, wherein X is any amino acid, X 4  is A or G, and X 5  is an I or V;   (viii) a K236XXXLSASQX 5 R246 (SEQ ID NO: 52) motif, wherein X is any amino acid and X 5  is an I or V;   (ix) K236XXXLSGSQX 5 R246 (SEQ ID NO:60) motif, wherein X is any amino acid and X 5  is an I or V; or   (viii) a motif 1 selected from (i), (ii), and (iii) in combination with a motif 2 selected from (iv), (v), (vi), (vii), (viii), and (ix).   
     
     
         18 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises a loop region between residues 52-60. 
     
     
         19 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises an Arg248 residue. 
     
     
         20 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises (i) an amino acid sequence of SEQ ID NO: 45, 46, 47, or 48; and (ii) an amino acid sequence of SEQ ID NO: 49, 50, 51, 52, 55, 56, 57, 58, 59, or 60. 
     
     
         21 . The variant any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises an amino acid sequence of SEQ ID NO:49 or 58 between Lys236 and Arg246, and an amino acid sequence of SEQ ID NO:46 between Asp32 and His63. 
     
     
         22 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises an Asn96 residue. 
     
     
         23 . The variant of any preceding claim, wherein said parent AprL-clade subtilisin enzyme and/or said variant comprises a hydrogen bond between residues 96 and 98. 
     
     
         24 . The variant of any preceding claim, wherein said variant has at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO:2. 
     
     
         25 . A composition comprising at least one variant of any one of  claims 1 - 24 . 
     
     
         26 . The composition of  claim 25 , wherein said composition is a cleaning composition or a detergent composition. 
     
     
         27 . The composition of  claim 26 , wherein said detergent composition is selected from the group consisting of a laundry detergent, a fabric softening detergent, a dishwashing detergent, and a hard-surface cleaning detergent. 
     
     
         28 . The composition of any one of  claims 25 - 27 , wherein said composition further comprises a surfactant; at least one stabilizer; at least one bleaching agent; at least one adjunct ingredient; one or more additional enzymes or enzyme derivatives selected from the group consisting of acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, xyloglucanases, and xylosidases, additional metallopotease enzymes and combinations thereof. 
     
     
         29 . The composition of any one of  claims 25 - 28 , wherein said cleaning composition is phosphate-free or contains phosphate. 
     
     
         30 . The composition of any one of  claims 25 - 29 , wherein said cleaning composition is boron-free or contains borate. 
     
     
         31 . The composition of any one of  claims 25 - 30 , wherein said composition is a granular, powder, solid, bar, liquid, tablet, gel, unit dose or paste composition. 
     
     
         32 . A method of cleaning a surface or item in need of cleaning, comprising: contacting said surface or item in need of cleaning with a variant of any one of  claims 1 - 24 , or a composition of any one of  claims 25 - 31  to produce a cleaned surface or item. 
     
     
         33 . A textile processing, animal feed, leather processing, feather processing, grain processing, cellulosic ethanol processing, lens cleaning, tissue debridement, or tissue cell culture additive composition comprising the variant of any one of  claims 1 - 24 . 
     
     
         34 . A polynucleotide comprising a nucleic acid sequence encoding the variant of any one of  claims 1 - 24 , wherein said polynucleotide is optionally isolated. 
     
     
         35 . An expression vector comprising the polynucleotide of  claim 34 . 
     
     
         36 . A host cell transformed with the vector of  claim 35 .
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