Cellulase variants

Abstract

The present invention relates to a method for improving the properties of a cellulolytic enzyme by amino acid substitution, deletion or insertion, the method comprising the steps of: a. constructing a multiple alignment of at least two amino acid sequences known to have three-dimensional structures similar to endoglucanase V (EGV) from Humicola insolens known from Protein Data Bank entry 4ENG; b. constructing a homology-built three-dimensional structure of the cellulolytic enzyme based on the structure of the EGV; c. identifying amino acid residue positions present in a distance from the substrate binding cleft of not more than 5 Å; d. identifying surface-exposed amino acid residues of the enzyme; e. identifying all charged or potentially charged amino acid residue positions of the enzyme; f. choosing one or more positions wherein the amino acid residue is to be substituted, deleted or where an insertion is to be provided; and g. carrying out the substitution, deletion or insertion by using conventional protein engineering techniques. Also described are cellulase variants obtained by this method.

1 - 78 . (canceled) 
     
     
         79 . A method of producing an enzyme variant, comprising introduction a mutation in the amino acid sequence of a parent endoglucanase at one or more positions selected from the group consisting of:
 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21a, 22, 24, 25, 26, 28, 29, 32, 33, 34, 35, 37, 40, 42, 42a, 43, 44, 45, 47, 48, 49, 49a, 49b, 50, 53, 54, 55, 58, 59, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 79, 80, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 95, 95d, 95h, 95j, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 110, 111, 112, 113, 114, 115, 116, 117, 119, 121, 123, 127, 128, 129, 130, 131, 132, 132a, 133, 134, 136, 137, 138, 139, 140, 140a, 141, 143a, 145, 146, 147, 148, 149, 150b, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160c, 160e, 160k, 161, 162, 163, 164, 165, 166, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 183, 184, 185, 186, 188, 191, 192, 193, 195, 196, 197, 200, and 201,   wherein each mutation is independently a substitution, insertion or deletion, the enzyme variant has endoglucanase activity and each position is numbered according to the amino acid sequence of the cellulase of SEQ ID NO: 1.   
     
     
         80 . The method of  claim 79 , wherein the parent endoglucanase comprises the amino acid sequence of SEQ ID NO: 1. 
     
     
         81 . The method of  claim 79 , wherein the mutation is one or more of the following:
 K13L or L13K;   P14A or A14P;   S15H or H15S;   K20E, K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E, A20E, G20A, or A20G;   K21N or N21K;   A22G, A22P, G22A, P22A, G22P, or P22G;   V24*, V24L, *24V, L24V, *24L, or L24*;   V28A, V28L, A28V, L28V, A28L, or L28A;   N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K, S32D, K32D, S32K, or K32S;   N34D or D34N;   I38L, I38F, I38Q, L38I, F38I, Q38I, L38F, L38Q, F38L, Q38L, F38Q, or Q38F;   S45N or N45S;   G46S or S46G;   E48D, E48N, D48E, N48E, D48N, or N48D;   G50N or N50G;   A53S, A53G, A53K, S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, or K53G;   Y54F or F54Y;   W62F or F62W;   A63D or D63A;   V64I, V64D, I64V, D64V, I64D, or D64I;   N65S, N65D, N65E, S65N, D65N, E65N, S65D, S65E, D65S, E65S, D65E, or E65D;   D66N, D66P, D66T, N66D, P66D, T66D, N66P, N66T, P66N, T66N, P66T, or T66P;   F68V, F68L, F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T, V68P, L68V, T68V, P68V, L68T, L68P, T68L, P68L, T68P, or P68T;   A69S, A69T, S69A, T69A, S69T, or T69S;   L70Y or Y70L;   G71A or A71G;   F72W, F72Y, W72F, Y72F, W72Y, or Y72W;   A73G or G73A;   A74F or F74A;   T75V, T75A, T75G, V75T, A75T, G75T, V75A, V75G, A75V, G75V, A75G, or G75A;   G79T or T79G;   W85T or T85W;   A88Q, A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R, or R88G;   Y90For F90Y;   L92A or A92L;   T93Q, T93E, Q93T, E93T, Q93E, or E93Q;   T95E or E95T;   S96T or T96S;   G97T, G97A, T97G, A97G, T97A, or A97T;   P98A or A98P;   V99L or L99V;   M104L or L104M;   V106F or F106V;   S110N or N110S;   T111I, T111V, I111T, V111T, I111V, or V111I;   G113Y or Y113G;   L115V or V115L;   G116S, G116Q, S116G, Q116G, S116Q, or Q116S;   N118T, N118G, N118Q, T118N, G118N, Q118N, T118G, T118Q, G118T, Q118T, G118Q, or Q118G;   H119Q, H119N, Q119H, or N119H;   V129L or L129V;   I131L, I131A, L131I, A131I, L131A, or A131L;   G134A or A134G;   Q138E or E138Q;   G140N or N140G;   R146Q or Q146R;   S152D or D152S;   R153K, R153L, R153A, K153R, L153R, A153R, K153L, K153A, L153K, A153K, L153A, or A153L;   L163V, L163W, V163L, W163L, V163W, or W163V;   G166S or S166G;   W169F or F169W;   R170F or F170R;   F171Y, F171A, Y171F, A171F, Y171A, or A171Y;   D172E, D172S, E172D, S172D, E172S, or S172E;   W173E or E173W;   F174M, F174W, M174F, W174F, M174W, or W174M;   A177N or N177A;   D178P or P178D;   N179V or V179N;   P180L or L180P;   L193I or I193L;   R196I, R196K, I196R, K196R, I196K, or K196I; and/or   T197S or S197T.   
     
     
         82 . The method of  claim 79 , wherein the mutation is one or more of the following:
 in position 4 holds R, H, K, Q, V, Y, or M;   in position 5 holds S, T, or A;   in position 13 holds K, or L;   in position 14 holds P, or A;   in position 15 holds H, or S;   in position 16 holds C, or A;   in position 19 holds A, D, S, P, T, or E;   in position 20 holds A, E, G, or K;   in position 21 holds K, or N;   in position 21a holds V or *;   in position 22 holds A, G, or P;   in position 24 holds L, V, or *;   in position 28 holds A, L, or V;   in position 32 holds D, K, N, or S;   in position 34 holds D or N;   in position 38 holds F, I, L, or Q;   in position 42 holds D, G, T, N, S, K, or *;   in position 44 holds K, V, R, Q, G, or P;   in position 45 holds N, or S;   in position 46 holds G, or S;   in position 47 holds C, or Q;   in position 48 holds D, E, N, or S;   in position 49 holds P, S, A, G, or *;   in position 49a holds C, or *;   in position 49b holds N, or *;   in position 50 holds G, or N;   in position 53 holds A, G, K, or S;   in position 54 holds F, or Y;   in position 62 holds F, or W;   in position 63 holds A, or D;   in position 64 holds D, I, or V;   in position 65 holds D, E, N, or S;   in position 68 holds D, N, P, or T;   in position 69 holds A, S, or T;   in position 70 holds L, or Y;   in position 71 holds A, or G;   in position 72 holds F, W, or Y;   in position 73 holds A, or G;   in position 74 holds A, or F;   in position 75 holds A, G, T, or V;   in position 79 holds G, or T;   in position 82 holds E, or *;   in position 88 holds A, G, Q, or R;   in position 90 holds F, or Y;   in position 92 holds A, or L;   in position 93 holds E, Q, or T;   in position 95 holds E, or T;   in position 95j holds P, or *;   in position 96 holds S, or T;   in position 97 holds A, G, or T;   in position 98 holds A, or P;   in position 99 holds L, or V;   in position 104 holds L, or M;   in position 106 holds F, or V;   in position 110 holds N, or S;   in position 111 holds I, T, or V;   in position 113 holds G, or Y;   in position 115 holds L, or V;   in position 116 holds G, Q, or S;   in position 118 holds G, N, Q, or T;   in position 119 holds H, N, or Q;   in position 129 holds L, or V;   in position 131 holds A, I, or L;   in position 132 holds A, P, or T;   in position 133 holds D, K, N, or Q;   in position 134 holds A, or G;   in position 138 holds E, or Q;   in position 145 holds A, D, N, or Q;   in position 146 holds Q, or R;   in position 150b holds A, or *;   in position 152 holds D, or S;   in position 153 holds A, K, L, or R;   in position 163 holds L, V, or W;   in position 166 holds G, or S;   in position 169 holds F, or W;   in position 170 holds F, or R;   in position 171 holds A, F, or Y;   in position 172 holds D, E, or S;   in position 173 holds E, or W;   in position 174 holds F, M, or W;   in position 177 holds A, or N;   in position 178 holds D, or P;   in position 179 holds N, or V;   in position 180 holds L, or P;   in position 193 holds I, or L;   in position 196 holds I, K, or R; and/or   in position 197 holds S, or T.   
     
     
         83 . The method of  claim 79 , wherein the mutation is one or more of the following: C16M+C86G, D42W, D42Y, E48D/P49*, E48N/P49* and/or L70Y. 
     
     
         84 . The method of  claim 79 , wherein the mutation is one or more of the following:
 K13L or L13K;   P14A or A14P;   S15H or H15S;   K20E, K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E, A20E, G20A, or A20G;   K21N or N21K;   A22G, A22P, G22A, P22A, G22P, or P22G;   V24*, V24L, *24V, L24V, *24L, or L24*;   N32D, N32S, N32K, D32N, S32N, K32N, D32S, D32K, S32D, K32D, S32K, or K32S;   N34D or D34N;   G50N or N50G;   A53S, A53G, A53K, S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, or K53G;   Y54F or F54Y;   V64I, V64D, I64V, D64V, 164D, or D64I;   F68V, F68L, F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T, V68P, L68V, T68V, P68V, L68T, L68P, T68L, P68L, T68P, or P68T;   A69S, A69T, S69A, T69A, S69T, or T69S;   L70Y or Y70L;   G71A or A71G;   G79T or T79G;   W85T or T85W;   A88Q, A88G, A88R, Q88A, G88A, R88A, Q88G, Q88R, G88Q, R88Q, G88R, or R88G;   L92A or A92L;   T93Q, T93E, Q93T, E93T, Q93E, or E93Q;   V106F or F106V;   Q138E or E138Q;   G140N or N140G;   S152D or D152S;   R153K, R153L, R153A, K153R, L153R, A153R, K153L, K153A, L153K, A153K, L153A, or A153L;   G166S or S166G;   W169F or F169W;   R170F or F170R;   F171Y, F171A, Y171F, A171F, Y171A, or A171Y;   D172E, D172S, E172D, S172D, E172S, or S172E;   W173E or E173W;   F174M, F174W, M174F, W174F, M174W, or W174M;   L193I or I193L; and/or   T197S or S197T.   
     
     
         85 . The method of  claim 79 , wherein the mutation is one or more of the following:
 the amino acid residue at position 22 is A, G, or P;   the amino acid residue at position 24 is L, V, or *;   the amino acid residue at position 32 is D, K, N, or S;   the amino acid residue at position 34 is D or N;   the amino acid residue at position 42 is D, G, T, N, S, K, or *;   the amino acid residue at position 49 is P, S, A, G, or *;   the amino acid residue at position 49a is C or *;   the amino acid residue at position 49b is N or *;   the amino acid residue at position 50 is G or N;   the amino acid residue at position 53 is A, G, K, or S;   the amino acid residue at position 54 is F or Y;   the amino acid residue at position 64 is D, I, or V;   the amino acid residue at position 68 is D, N, P, or T;   the amino acid residue at position 69 is A, S, or T;   the amino acid residue at position 70 is L or Y;   the amino acid residue at position 71 is A or G;   the amino acid residue at position 79 is G or T;   the amino acid residue at position 88 is A, G, Q, or R;   the amino acid residue at position 92 is A or L;   the amino acid residue at position 93 is E, Q, or T;   the amino acid residue at position 95j is P or *;   the amino acid residue at position 106 is F or V;   the amino acid residue at position 138 is E or Q;   the amino acid residue at position 150b is A or *;   the amino acid residue at position 152 is D or S;   the amino acid residue at position 153 is A, K, L, or R;   the amino acid residue at position 166 is G or S;   the amino acid residue at position 169 is F or W;   the amino acid residue at position 170 is F or R;   the amino acid residue at position 171 is A, F, or Y;   the amino acid residue at position 172 is D, E, or S;   the amino acid residue at position 173 is E or W;   the amino acid residue at position 174 is F, M, or W;   the amino acid residue at position 193 is I or L; and/or   the amino acid residue at position 197 is S or T.