Polypeptides with endoglucanase activity and uses thereof

Abstract

Disclosed herein are cellulase variants, or active fragments thereof, and polynucleotides encoding same, wherein the cellulase variants, or active fragments thereof, hav endoglucanase activity. Also disclosed herein are compositions comprising said cellulase variants, or active fragments thereof; vectors and/or host cells comprising the polynucleotides encoding said cellulase variants, or active fragments thereof; and methods for making and/or using said cellulase variants, or active fragments thereof and/or compositions containing same; wherein said cellulase variants, or active fragments thereof, have endoglucanase activity.

We claim: 
     
         1 . A cellulase variant, or an active fragment thereof, comprising an amino acid sequence comprising a substitution at one or more positions selected from:
 (i) 4, 20, 23, 29, 32, 36, 44, 51, 77, 80, 87, 90, 97, 98, 99, 102, 112, 116, 135, 136, 142, 153, 154, 157, 161, 163, 192, 194, 204, 208, 210, 212, 216, 217, 221, 222, 225, 227, and 232;   (ii) K4X, G/K20X, A/S23X, F29X, S32X, N/Q36X, K44X, S51X, S77X, N80X, G87X, E90X, P97X, V98X, A99X, T102X, G112X, T116X, S135X, P/5136X, A142X, G/H/S153X, Q154X, S157X, A161X, K163X, L192X, A194X, G204X, V208X, T210X, P212X, Q216X, S217X, S221X, S222X, S225X, K227X, and 5232X;   (iii) X4V, X20N, X23L, X29W, X32D/Y, X36T, X44V, X51T, X77K/M, X80S, X87A, X90A, X97S, X98G, X99E/Y, X102K, X112S/T/V, X116V, X135T, X136E/K/S, X142D/E/P/Q, X153D, X154E, X157D, X161E/P, X163V, X192V, X194S, X204S, X208H/K, X210V, X212S, X216D/E/G/P/S/T/V, X217G/M, X221L/M, X222A, X225K, X227R, and X232T; or   (iv) K4V, G/K20L, A/S23L, F29W, S32D/Y, N/Q36T, K44V, S51T, S77K/M, N80S, G87A, E90A, P97S, V98G, A99E/Y, T102K, G112S/Y/V, T116V, S135T, P/S136E/K/S, A142D/E/P/Q, G/H/S153D, Q154E, S157D, A161E/P, K163V, L192V, A194S, G204S, V208H/K, T210V, P212S, Q216D/E/G/P/S/T/V, S217G/M, S221L/M, S222A, S225K, K227R, and S232T, wherein said variant has endoglucanase activity, and wherein the amino acid positions of the variant, or active fragment thereof, are numbered by correspondence with the amino acid sequence of SEQ ID NO:5.   
     
     
         2 . The cellulase variant, or active fragment thereof, of  claim 1 , wherein said one or more positions is selected from: (i) 142 and 216; (ii) A142X and Q216X; (iii) X142D/E/P/Q and X216D/E/G/P/S/T/V; or (iv) A142D/E/P/Q, and Q216D/E/G/P/S/T/V. 
     
     
         3 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said one or more position is (i) 142, (ii) 216, or (iii) 142 and 216. 
     
     
         4 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said variant has at least one improved property selected from improved thermostability, stability in the presence of one or more protease, and stability in the presence of one or more protease and one or more other detergent component when compared to a parent or reference polypeptide. 
     
     
         5 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said variant comprises an amino acid sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to the amino acid sequence of SEQ ID NO:5, 11, 14, 17, or 22. 
     
     
         6 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said variant is derived from a parent or reference polypeptide selected from SEQ ID NOs:5, 11, 14, 17, and 22. 
     
     
         7 . The cellulase variant, or active fragment thereof, of  claim 6 , wherein the parent or reference polypeptide comprises (i) an amino acid sequence having at least 70%, 75%, 80%, 82%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to amino acids 1-215 of SEQ ID NO:5, or (ii) amino acids 1-215 of SEQ ID NO:5. 
     
     
         8 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein the improved property is (i) improved thermostability and wherein said variant, or active fragment thereof, has a thermal PI that is greater than 1 or 1.1; (ii) improved stability in the presence of one or more protease and wherein said variant, or active fragment thereof, has a PI that is greater than 1, 1.1, 1.5, or 2.0 when the stability of said variant, or active fragment thereof is tested in the presence of said protease; and/or (iii) improved stability in the presence of one or more protease and one or more other detergent component, and wherein said variant, or active fragment thereof, has a PI that is greater than 1, 1.1, 1.5, 2.0, or 2.5 when the stability of said variant, or active fragment thereof is tested in the presence of said protease and said other detergent component. 
     
     
         9 . The cellulase variant, or active fragment thereof, of  claim 8 , wherein the PI is measured in accordance with the Cellulase Activity Assay of Example 1. 
     
     
         10 . The cellulase variant, or active fragment thereof, of 8 or 9, wherein the other detergent component is a surfactant. 
     
     
         11 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said variant, or active fragment thereof, comprises an amino acid sequence having:
 a) at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO:5, with the proviso that the substitution is not A142P and/or Q216T and the further proviso that (i) when the substitution is Q216S the cellulase variant, or active fragment thereof, does not comprise CN103343111-0003, CN103343111-0005, AGY80101, US20150299682-0004, or WO2007071818-0012; or (ii) when the substitution is Q216G the cellulase variant, or active fragment thereof, does not comprise WO2014138983-0859;   b) at least 75% amino acid sequence identity to the amino acid sequence of SEQ ID NO:5, with the proviso that when (i) the substitution is A142P the cellulase variant, or active fragment thereof, does not comprise WO2012089024-0002, WO9804663-AAW46618, GB2479462-AZN28533, WO2007071820-0019, CN103343111-0005, CN103343111-0003, US20150299682-0004, AGY80101, KOP50759, or CAD56665; ii) when the substitution is Q216T the cellulase variant, or active fragment thereof, does not comprise WO9743409-0066; or iii) when the substitution is Q216S the cellulase variant, or active fragment thereof, does not comprise CN103343111-0005, CN103343111-0003, US20150299682-0004, or AGY80101; or   c) at least 80% amino acid sequence identity to the amino acid sequence of SEQ ID NO:5, with the proviso that when (i) the substitution is A142P the cellulase variant, or active fragment thereof, does not comprise WO2012089024-0002, WO9804663-AAW46618, GB2479462-AZN28533, WO2007071820-0019, or CN103343111-0005; or ii) when the substitution is Q216S the cellulase variant, or active fragment thereof, does not comprise CN103343111-0005.   
     
     
         12 . The cellulase variant, or active fragment thereof, of any preceding claim, wherein said variant, or active fragment thereof, is a family GH45 cellulase. 
     
     
         13 . A composition comprising the cellulase variant, or active fragment thereof, of any preceding claim. 
     
     
         14 . The composition of  claim 13 , wherein said composition is selected from an enzyme composition, detergent composition, and fabric care composition. 
     
     
         15 . The composition of  claim 13  or  14 , further comprising (i) one or more other enzymes selected from the group consisting of acyl transferases, amylases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinases, arabinosidases, aryl esterases, beta-galactosidases, beta-glucanases, carrageenases, catalases, chondroitinases, cutinases, endo-beta-mannanases, exo-beta-mannanases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipolytic enzymes, lipoxygenases, mannanases, metalloproteases, oxidases, pectate lyases, pectin acetyl esterases, pectinases, pentosanases, perhydrolases, peroxidases, phenoloxidases, phosphatases, phospholipases, phytases, polygalacturonases, proteases, pullulanases, reductases, rhamnogalacturonases, second cellulase, beta-glucanases, tannases, transglutaminases, xylan acetyl-esterases, xylanases, and xylosidases; (ii) one or more surfactants; (iii) one or more ions selected from calcium and zinc; (iv) one or more adjunct ingredients; (v) one or more stabilizers; (vi) from about 0.001% to about 5.0 weight % of said cellulase variant, or active fragment thereof of any one of  claims 1 - 15 ; (vii) one or more bleaching agents; and/or (viii) combinations thereof. 
     
     
         16 . The composition of any one of  claims 13 - 15 , wherein said composition is a laundry detergent. 
     
     
         17 . The composition of any one of  claims 13 - 16 , wherein the composition is in a form selected from a liquid, a powder, a granulated solid, a tablet, a sheet, and a unit dose. 
     
     
         18 . The composition of any one of  claims 13 - 17 , wherein said composition contains phosphate or is phosphate-free and/or contains boron or is boron-free. 
     
     
         19 . A polynucleotide comprising a nucleic acid sequence encoding the cellulase variant, or active fragment thereof of any one of  claims 1 - 12 . 
     
     
         20 . An expression vector comprising the polynucleotide of  claim 19 . 
     
     
         21 . A host cell comprising the expression vector of  claim 20 . 
     
     
         22 . A method for producing the cellulase variant, or active fragment thereof of any one of  claims 1 - 12  comprising: (a) stably transforming the host cell of  claim 21  with the expression vector of  claim 20 ; (b) cultivating said transformed host cell under conditions suitable for said host cell to produce said cellulase variant or active fragment thereof; and (c) recovering said cellulase variant or active fragment thereof. 
     
     
         23 . Use of the cellulase variant, or active fragment thereof, of any one of  claims 1 - 12  in an enzyme composition, detergent composition, fabric care composition, textile finishing process, or paper and pulp process.