Cellulase Variants

• US 20080206836A1
• Filed: 07/30/2007
• Published: 08/28/2008
• Est. Priority Date: 09/17/1996
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Andersen, Kim Vilbour, Von der Osten, Claus, Henriksen, Torben, Christiansen, Lars, Damgaard, Bo, Schulein, Martin
• Granted Patent: US 8,017,372 B2
• Time in Patent Office: Days
• US Class Current: 435/209
• CPC Class Codes: C11D 3/38645 containing cellulase
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12Y 302/01004 Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase

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.

Claims

1-78. -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;

Y90F or 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;

L1931 or I193L;

R1961, 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*, E48N1/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, I64D, 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 E106Q;

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 C 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.