Optimized cellulase enzymes
View Patent ↗The invention discloses cellulase enzymes with optimized properties for processing of cellulose- and lignocellulose-containing substrates. In particular, cellobiohydrolase enzymes with preferred characteristics are disclosed. The present invention provides fusion, insertion, deletion and/or substitution variants of such enzymes. Enzyme variants have enhanced thermostability, proteolytic stability, specific activity and/or stability at extreme pH. Nucleic acid molecules encoding said enzymes, a composition comprising said enzymes, a method for preparation, and the use for cellulose processing and/or for the production of biofuels are disclosed.
1. A recombinant polypeptide having cellobiohydrolase activity, wherein the recombinant polypeptide comprises at least 90% sequence identity to SEQ ID NO: 5, wherein the recombinant polypeptide is modified at one or more positions selected from the group consisting of Q1, Q2, T7, A8, N10, Q28, E65, S86, D181, E183, D202, P224, S311, N318, T335, D346, Q349, T392, T393, and Y422, and wherein the polypeptide maintains an IT50 for 60 minutes at a temperature of 62° C. or higher.
2. The recombinant polypeptide according to claim 1 , comprising additional substitution(s) deletion(s) or insertion(s) while maintaining at least 90% sequence identity to SEQ ID NO: 5.
3. The recombinant polypeptide according to claim 1 , comprising additional modification at one or more of the following amino acid residues by substitution or deletion at positions G4, P12, T15, A21, G23, S24, T26, T27, N29, G30, A31, V32, N37, W40, V41, G46, Y47, T48, N49, C50, T52, N54, D57, T59, Y60, D64, A68, Q69, A72, V84, S89, S90, K92, S99, Q109, D110, D111, I116, F117, K118, L119, L120, D120, V130, G139, A145, M146, V152, K154, Y155, N157, N158, K159, K163, G167, Q172, F179, I180, E187, G188, Q190, S192, S193, N194, I200, H203, D211, V212, A221, D228, T229, G231, T233, M234, S236, T243, Y244, S245, N246, D247, G251, F260, G266, K275, I276, I277, T280, L290, D293, G294, T295, T297, T299, S301, K304, F306, N310, V313, I314, D320, I321, T325, N327, A340, F341, D343, T344, D345, H350, A354, K355, A358, Q361, Q362, G363, M364, V367, D373, Y374, A375, A376, P386, T387, D390, T392, P394, T400, P402, T403, D404, D410, N417, S418, T421, F427, P429, I430, G431, T433, G434, N435, P436, S437; or additional one or more insertion group(s) after positions G151, or K159, each said insertion group(s) comprising five amino acids, while maintaining at least 90% sequence identity to SEQ ID NO: 5.
4. The recombinant polypeptide according to claim 1 , comprising additional modification at one or more amino acid residues as follows:
Position
Modified to:
G4
C
A6
G, L, or V
P12
Q
T15
S
A21
S, T, or C
G23
A, D, or N
S24
T, C, or N
T26
I, or N
T27
S, or Q
N29
T, or Y
G30
A
A31
S
V32
G
N37
S
W40
R
V41
T
G46
S
Y47
S, or F
T48
A
N49
S
C50
S
T52
D
N54
S
D57
S
T59
M
Y60
H
D64
N
A68
T
Q69
K, or R
A72
V, or C
V84
A
S89
N
S90
T, or F
K92
R
S99
T
Q109
R
D110
G, S, or N
D111
H, or E
I116
V, K, or E
F117
Y
K118
A, T, or Q
L119
L, or I
L120
P, or M
D129
N
V130
I
G139
S
A145
T
M146
C
G151
GCGRSG
V152
A, or E
K154
R
Y155
S, C, H
N157
S
N158
D
K159
E, KCGRNK
K163
C
G167
C
Q172
Q
F179
I
I180
N
E187
K
G188
C
Q190
L, or K
S192
L, I, P, T, or M
S193
L, P, or T
N194
G, L, I, V, S, C, K, R, D, Q, or Y
I200
N or F
H203
R
D211
G
V212
L
A221
V
D228
N
T229
A, S, or M
G231
D
T233
S
M234
L, I, V, T, or K
S236
F, or Y
T243
G, A, L, I, V, P, S, C, M, R, D,
Q, F, Y, or W
Y244
H, F
S245
T
N246
S, K, or D
D247
N
G251
R
F260
C
G266
S
K275
E
I276
V
I277
V
T280
A
L290
H
D293
R, or H
G294
A
T295
S
T297
N
T299
I, or S
S301
C
K304
R
F306
L, Y
N310
D, E
V313
I
I314
F
D320
I, V, E, N
I321
N
T325
A, or I
N327
Y
A340
G, S, or T
F341
C
D343
A
T344
M
D345
E
H350
Y
A354
T
K355
Q
A358
E
Q361
R
Q362
G, R, or H
G363
P
M364
L, or S
V367
A
D373
E
Y374
A, P, S, C, R, H, D
A375
G, L, V, T, C, M, R, D, E, N, Q, or Y
A376
T
P386
L, S
T387
A, S
D390
G, E
P394
C
T400
S
P402
S
T403
K
D404
N
D410
G
N417
Y
S418
P
T421
I
F427
Y
P429
C
I430
L
G431
D
T433
S, or E
G434
S, or GAAATG
N435
Q
P436
S
S437
P
while maintaining at least 90% sequence identity to SEQ ID NO: 5.
5. The recombinant polypeptide according to claim 1 , which is expressed and secreted at a level of more than 100 mg/1 into a supernatant of a yeast culture medium after introduction of a nucleic acid encoding the recombinant polypeptide according to claim 1 into the yeast.
6. The recombinant polypeptide according to claim 1 , wherein the one or more modifications are indicated in the following table:
Position
Modified to:
Q1
L
Q2
P, or S
T7
Q
A8
S
N10
T, or D
Q28
L, K , R, or N
E65
V, M, or K
S86
T
D181
N
E183
V, M, or K
D202
G, I, V, N, F, or Y
P224
L
S311
G, D, or N
N318
I, H, D, or Y
T335
I
D346
G, A, V, or E
Q349
K, or R
T392
S, M, or K
T393
A, I, V, or S
Y422
F.
7. A method of producing a recombinant polypeptide, comprising the steps:
a. obtaining a host cell:
b. cultivating the host cell under conditions which the recombinant polypeptide according to claim 1 is expressed; and
c. recovering the recombinant polypeptide according to claim 1 .
8. A process of enzymatically degrading lignocellulosic biomass comprising, degrading lignocellulosic biomass by the recombinant polypeptide according to claim 1 , or processing textiles by the recombinant polypeptide according to claim 1 or adding the recombinant polypeptide according to claim 1 as ingredient in detergents or adding the recombinant polypeptide according to claim 1 as ingredient in food or feed compositions.