US2005009166A1PendingUtilityA1
Cellulase variants
Est. expirySep 17, 2016(expired)· nominal 20-yr term from priority
C12Y 302/01004C11D 3/38645C12N 9/2437
64
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Claims
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
exact text as granted — not AI-modified1 - 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.Cited by (0)
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