Mannanases
The present invention provides an insertion, deletion and/or substitution mutein of wild-type Trichoderma reesei β-mannanase having enhanced thermostability, proteolytic stability, specific activity and/or stability at low pH, a nucleic acid molecule encoding said mannanase mutein, a composition comprising said mannanase mutein; a method for its preparation, and its use for food and feed processing, for coffee extraction and the processing of coffee waste, as a supplement to food and feed, for enzyme aided bleaching of paper pulps, as bleaching and/or desizing agent in textile industry, for oil and gas well stimulation by hydraulic fracturing, as detergent, for removal of biofilms and in delivery systems, or for the processing of renewable resources intended for the production of biological fuels.
1. A modified β-mannanase having an amino acid sequence that is at least 90% homologous to SEQ ID NO:1, wherein said modified β-mannanase includes a modification by insertion, deletion and/or substitution of the amino acid residue corresponding to position 274 of SEQ ID NO:1.
2. The modified β-mannanase according to claim 1 , wherein said modified β-mannanase further includes an amino acid substitution at one or more of the positions corresponding to SEQ ID NO:1 selected from the group consisting of 4, 7, 11, 13, 31, 41, 66, 70, 74, 97, 101, 113, 132, 146, 148, 170, 173, 181, 182, 185, 186, 187, 189, 201, 207, 215, 216, 220, 252, 255, 259, 280, 281, 282, 313, 316, 317, 325, 331, 341, 345, 351 and 352.
3. The modified β-mannanase according to claim 1 , wherein the total number of amino acid insertions, deletions and substitutions corresponding to SEQ ID NO:1 is from 1 to 30.
4. The modified -mannanase according to claim 1 , wherein said modified β-mannanase includes one or more amino acid substitutions corresponding to SEQ ID NO:1 as shown in the Table below:
variant A
F274L
variant B
L207F; F274L
variant C
T201S; Q280R; F274L
variant F
A215T; F274L; N282D
variant G
Y220F; F274L
variant H
V187M; F274L
variant K
V181A; F274L
variant L
V148I; F274L
variant M
N173H; F274L; N331S;
variant 13
F274M
variant 21
N13D; F274L
variant 22
F274L; Q280L
variant 23
F274L; Q317H
variant 25
L207F; F274M
variant 28
F4Y; I70V; F274L
variant 29
Q101R; F274L; Q281H
variant 30
T201G; L207F; F274L
variant 31
T201S; L207F; F274L
variant 32
V181T; L207W; A215T; F274L
variant 34
K74M; F274L; A313T; V325I
variant 35
N173H; V181H; L207W; F274L
variant 36
T201S; L207Y; F274L; Q280R
variant 37
T201S; L207W; F274L; Q280R
variant 38
T201S; L207R; F274L; Q280R
variant 39
V181T; L207W; F274M; N331S; P352L
variant 40
N173H; L207W; A215T; F274L; Q280R
variant 41
V181H; L207W; A215T; F274L; N282D
variant 42
N173H; V181T; T201S; L207W; F274L
variant 43
N173H; V181H; L207W; A215T; F274L
variant 44
V181H; L207W; A215T; F274L; Q280R;
N282D
variant 45
S66P; N173H; V181H; A215T; F274L;
Q280S; N282D
variant 46
N173H; V181T; L207W; A215T;
F274L; Q280R; N282D.
5. The modified β-mannanase according to claim 1 , wherein said amino acid residue corresponding to position 274 of SEQ ID NO:1 is A, C, G, H, I, L, M, V, W or Y.
6. The modified β-mannanase according to claim 1 , wherein said amino acid residue corresponding to position 274 of SEQ ID NO:1 is L.
7. The modified β-mannanase according to claim 1 , wherein said amino acid corresponding to position 274 of SEQ ID NO:1 is selected from the group consisting of F274L and F274M, and wherein the modified β-mannanase further comprises one or more substitutions corresponding to SEQ ID NO:1 selected from the group consisting of F4Y, I7V, Q11R, Q11H, N13D, F31Y, T41I, S66P, I70V, K74M, Q97R, Q101R, N113Y, T132S, K146Q, V148I, P170L, N173H, N173T, V181A, V181H, V181T, Q182R, T185K, S186N, V187M, V187L, Q189H, T201S, T201A, T201G, L207F, L207W, L207R, L207S, L207Y, A215T, Y216H, Y220F, T252I, N255I, Q259R, Q280R, Q280S, Q280L, Q281H, N282D, A313T, A316T, Q317H, V325I, D341E, G345C, P351L, P352L and N331S.
8. The modified β-mannanase according to claim 4 , wherein said modified β-mannanase has increased thermostability as compared to SEQ ID NO:1.
9. The modified β-mannanase according to claim 1 , wherein said modified β-mannanase includes one or more substitutions corresponding to SEQ ID NO:1 as shown in the Table below:
variant B
L207F, F274L
variant C
T201S, Q280R, F274L
variant 31
T201S, L207F, F274L
variant 42
N173H, V181T, T201S,
L207W, F274L
and wherein the said mannanase has increased stability at acidic pH values and/or resistance to protease degradation as compared to SEQ ID NO:1.
10. A composition comprising the modified β-mannanase according to claim 1 .
11. The modified β-mannanase according to claim 1 , wherein the total number of insertions, deletions and substitutions corresponding to SEQ ID NO:1 is from 1 to 10.
12. The modified β-mannanase according to claim 1 , wherein the total number of insertions, deletions and substitutions corresponding to SEQ ID NO:1 is from 1 to 5.
13. The modified β-mannanase according to claim 1 further comprising one or more amino acid substitutions at a position corresponding to SEQ ID NO:1 as shown in the Table below:
Wild type
residue
Substituted by
F4
A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
I7
A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T,
V, W or Y
Q11
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
N13
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
F31
A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
T41
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y
S66
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T,
V, W or Y
I70
A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T,
V, W or Y
K74
A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T,
V, W or Y
Q97
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
Q101
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
N113
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
T132
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y
K146
A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T,
V, W or Y
V148
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, W or Y
P170
A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,
V, W or Y
N173
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
V181
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, W or Y
Q182
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
T185
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y
S186
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T,
V, W or Y
V187
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, W or Y
Q189
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
T201
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y
L207
A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,
V, W or Y
A215
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
Y216
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V or
Y220
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, V or W
T252
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y
N255
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
Q259
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
Q280
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
Q281
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
N282
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
A313
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
A316
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
Q317
A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T,
V, W or Y
V325
A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
T, W or Y
N331
A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T,
V, W or Y
D341
A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
G345
A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T,
V, W or Y
P351
A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,
V, W or Y
P352
A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T,
V, W or Y.
14. The modified β-mannanase according to claim 1 , further comprising one or more amino acid substitutions corresponding to a residue in SEQ ID NO:1 selected from the group consisting of:
(i) the amino acid N173 is substituted by A, C, D, G, H, S, P, Q, T or V;
(ii) the amino acid V181 is substituted by A, C, F, G, H, I, L, M, W or Y;
(iii) the amino acid V148 is substituted by A, C, F, G, H, I, L, M, W or Y;
(iv) the amino acid V187 is substituted by A, C, F, G, H, I, L, M, W or Y;
(v) the amino acid T201 is substituted by A, C, H, N, P, Q, S, V or Y;
(vi) the amino acid L207 is substituted by A, C, F, G, H, I, M, V, W or Y;
(vii) the amino acid A215 is substituted by C, F, G, H, I, L, M, T, V, W or Y;
(viii) the amino acid Y220 is substituted by A, F, H, I, L, M, N, P, V, or W;
(ix) the amino acid Q280 is substituted by C, D, E, H, K, N, R, S, T or Y;
(x) the amino acid N282 is substituted by C, D, E, H, K, Q, R, S, T or Y; and
(xi) the amino acid N331 is substituted by C, D, E, H, K, Q, R, S, T or Y.
15. The modified β-mannanase according to claim 1 , further comprising one or more amino acid substitutions corresponding to a residue in SEQ ID NO:1 selected from the group consisting of:
(i) the amino acid N173 is substituted by A, C, H, S, Tor V;
(ii) the amino acid V181 is substituted by A, I, L or M;
(iii) the amino acid V148 is substituted by A, I, or L;
(iv) the amino acid V187 is substituted by A, I, L or M;
(v) the amino acid T201 is substituted by A, C, N, Q, S or V;
(vi) the amino acid L207 is substituted by A, F, G, H, I or V;
(vii) the amino acid A215 is substituted by C, F, G, H, I, L or T;
(viii) the amino acid Y220 is substituted by F, H, W or Y;
(ix) the amino acid Q280 is substituted by C, H, N, S, or T;
(x) the amino acid N282 is substituted by C, D, H, Q, S or T; and
(xi) the amino acid N331 is substituted by C, H, Q, S or T.
16. The modified β-mannanase according to claim 1 , further comprising one or more amino acid substitutions corresponding to a residue in SEQ ID NO:1 selected from the group consisting of:
(i) the amino acid N173 is substituted by H;
(ii) the amino acid V181 is substituted by A;
(iii) the amino acid V148 is substituted by I;
(iv) the amino acid V187 is substituted by M;
(v) the amino acid T201 is substituted by S;
(vi) the amino acid L207 is substituted by F;
(vii) the amino acid A215 is substituted by T;
(viii) the amino acid Y220 is substituted by F;
(ix) the amino acid Q280 is substituted by R;
(x) the amino acid N282 is substituted by D; and
(xi) the amino acid N331 is substituted by S.
17. The modified β-mannanase according to claim 1 , wherein the modified mannanase comprises one or more of the combinations of substitutions corresponding to residues in SEQ ID NO:1 selected from the group consisting of F274L/A215T, F274L/N282D, F274L/A215T/N282D, F274L/Y220F, F274L/V187M, F274L/V181A, F274L/V148I, F274L/N173H, F274L/N331S, F274L/N173H/N331S, F274L/L207F, F274L/T201S, F274L/Q280R, N13D/F274L, F274L/Q280L, F274L/Q317H, L207F/F274M, F4Y/I70V/F274L, Q101R/F274L/Q281H, T201G/L207F/F274L, T201S/L207F/F274L, V181T/L207W/A215T/F274L, K74M/F274L/A313T/V325I, N173H/V181H/L207W/F274L, T201S/L207Y/F274L/Q280R, T201S/L207W/F274L/Q280R, T201S/L207R/F274L/Q280R, V181T/L207W/F274M/N331S/P352L, N173H/L207W/A215T/F274L/Q280R, V181H/L207W/A215T/F274L/N282D, N173H/V181T/T201S/L207W/F274L, N173H/V181H/L207W/A215T/F274L, V181H/L207W/A215T/F274L/Q280R/N282D, S66P/N173H/V181H/A215T/F274L/Q280S/N282D, N173H/V181T/L207W/A215T/F274L/Q280R/N282D and F274L/T201S/Q280R.
18. The modified β-mannanase according to claim 1 , wherein the modified mannanase has the sequence shown in SEQ ID NOs: 10 (variant B), 11 (variant C) or 12 (variant 31).
19. The modified β-mannanase of claim 1 , wherein said modified β-mannanase includes one or more amino acid substitutions corresponding to amino acid residue in SEQ ID NO:1 selected from the group consisting of:
(i) the amino acid N173 is substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y;
(ii) the amino acid V181 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
(iii) the amino acid V148 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
(iv) the amino acid V187 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y;
(v) the amino acid T201 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W or Y;
(vi) the amino acid L207 is substituted by A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y;
(vii) the amino acid A215 is substituted by C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y;
(viii) the amino acid Y220 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W;
(ix) the amino acid Q280 is substituted by A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y;
(x) the amino acid N282 is substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; and
(xi) the amino acid N331 is substituted by A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y.