Cellulase variants

• US 20050009166A1
• Filed: 08/16/2004
• Published: 01/13/2005
• Est. Priority Date: 09/17/1996
• Status: Abandoned Application

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Andersen, Kim Vilbour, Von der Osten, Claus, Christiansen, Lars, Damgaard, Bo, Schulein, Martin, Dela, Hanne
• Application Number:
  US10/919,195
• Publication Number:
  US 20050009166A1
• 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

1-36. -36. (Cancelled).

37. A modified cellulase having endoglucanase activity, comprising a mutation in an amino acid sequence of a cellulase, wherein the mutation comprises a substitution at one or more positions selected from the group consisting of:

4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 21a, 22, 24, 25, 26, 29, 32, 33, 34, 35, 40, 42, 42a, 43, 44, 45, 47, 48, 49, 49a, 49b, 50, 53, 54, 64, 68, 69, 70, 71, 74, 76, 79, 80, 82, 84, 85, 86, 87, 88, 89, 91, 92, 93, 95d, 95h, 95j, 106, 110, 111, 112, 113, 114, 115, 116, 121, 123, 127, 128, 129, 130, 131, 132, 132a, 133, 137, 138, 139, 140, 140a, 141, 145, 146, 147, 148, 149, 150b, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 159, 160c, 160e, 160k, 161, 164, 165, 166, 168, 169, 170, 171, 172, 173, 174, 178, 179, 188, 191, 192, 193, 195, 197, 200 and 201,

wherein each mutation is independently a substitution, insertion or deletion and each position is numbered according to the amino acid sequence of the cellulase of SEQ ID NO;

1.

38. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 22.

39. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 24.

40. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 32.

41. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 34.

42. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 42.

43. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 49.

44. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 50.

45. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 53.

46. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 54.

47. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 64.

48. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 68.

49. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 69.

50. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 70.

51. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 71.

52. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 79.

53. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 85.

54. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 86.

55. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 88.

56. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 92.

57. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 93.

58. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 95j.

59. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 106.

60. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 138.

61. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 140.

62. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 150b.

63. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 152.

64. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 153.

65. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 166.

66. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 169.

67. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 170.

68. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 171.

69. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 172.

70. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 173.

71. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 174.

72. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 193.

73. A modified cellulase of claim 37, wherein the mutation comprises a substitution at position 197.

74. The cellulase variant of claim 37, wherein the cellulase has an amino acid sequence of SEQ ID NO:

1.

75. The cellulase variant of claim 74, wherein the mutation comprises C16M+C86G, D42W, D42Y, E48D/P49*, E48N/P49* and/or L70Y.

76. A cellulase variant of claim 37, wherein the mutation comprises:

K13L or L13K;

P14A or A14P;

S15H or H15S;

K20E, K20G, K20A, E20K, G20K, A20K, E20G, E20A, G20E, A20E, G20A, or A20G;

K21 N or N21 K;

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;

V641, V64D, 164V, D64V, 164D, or D641;

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;

L1931 or 1193L; and

/or

T197S or S197T.

77. A cellulase variant of claim 37, wherein as a result of the mutation, 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.

78. A detergent composition comprising a cellulase variant of claim 37 and a surfactant.