US 2019/0169587 A1 · VARIANT ENZYMES

Variant enzymes

AssigneeDanisco US Inc.

InventorsMARIA FOUKARAKI, RONALDUS WILHELMUS JOANNES HOMMES, THIJS KAPER, BRADLEY R. KELEMEN, SLAVKO KRALJ, SUZANNE E. LANTZ, IGOR NIKOLAEV, WILHELMUS ANTONIUS HENDRICUS VAN DER KLEIJ, JOHANNES FRANCISCUS THOMAS VAN LIESHOUT, SANDER VAN STIGT THANS

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Abstract

Disclosed are glycosyl hydrolase enzyme variants, particularly variants of certain oxidoreductases of glycosyl hydrolase family 61. Nucleic acids encoding the glycosyl hydrolyase variants, compositions including the glycosyl hydrolase variants, methods of producing the variants, and methods of using the variants are also described.

Claims22

Dependent claims

A variant of a parent glycosyl hydrolase 61 (GH61) enzyme, wherein said variant has cellulase augmenting activity, has at least 80% sequence identity to SEQ ID NO:3, and has at least one improved property over said parent GH61 enzyme selected from:

(a) expression, (b) thermostability and/or Tm, (c) performance in a Whole Hydrolysate Dilute Acid Pretreated Corn Stover (whPCS) hydrolysis assay, (f) performance in a Dilute Ammonia Pretreated Corn Stover (daCS) hydrolysis assay.

The variant of any preceding claim, wherein said variant comprises one or more of:

an amino acid substitution at position T287 selected from the group consisting of:
H, A, F, G, S, and I;
an amino acid substitution at position W13 selected from the group consisting of:
G, L, N, R, T, V, F, Q, Y, A, E, I, K, and M;
an amino acid substitution at position L288 selected from the group consisting of:
E, F, G, I, K, M, N, R, S, T, A, C, D, V, and W;
an amino acid substitution at position P19 selected from the group consisting of:
F, M, E, H, Q, T, K, and W;
an amino acid substitution at position T20 selected from the group consisting of:
K, P, G, M, R, Y, A, and N;
an amino acid substitution at position P29 selected from the group consisting of:
C, E, F, K, T, Y D, G, M, R, and S;
an amino acid substitution at position A37 selected from the group consisting of:
D, S, E, L, W, F, K, and Q;
an amino acid substitution at position L39 selected from the group consisting of:
A, D, E, M, T, Y, F, I, N, P, Q, S, and V;
an amino acid substitution at position D47 selected from the group consisting of:
A, K, T, H, R, P, and W;
an amino acid substitution at position A48 selected from the group consisting of:
C, E, G, K, L, N, T, Y, Q, W, H, I, and S;
an amino acid substitution at position S68 selected from the group consisting of:
E, I, L, M, N, T, V, and Y;
an amino acid substitution at position T107 selected from the group consisting of:
A, D, E, K, M, R, S, and G;
an amino acid substitution at position S121 selected from the group consisting of:
E, F, N, T, C, L, V, D, G, K, M, and Y;
an amino acid substitution at position G122 selected from the group consisting of:
E, N, S, V, A, M, T, F, and L;
an amino acid substitution at position T127 selected from the group consisting of:
A, E, F, I, L, M, V, W, and Y;
an amino acid substitution at position S164 selected from the group consisting of:
A, D, E,G, K, L, M, Q, R, V, and Y;
an amino acid substitution at position G166 selected from the group consisting of:
A, D, E, F, H, K, M, N, Q, and S;
an amino acid substitution at position Q167 selected from the group consisting of:
E, A, D, F, G, K, L, N, R, V, and Y;
an amino acid substitution at position A168 selected from the group consisting of:
E, M, D, F, I, L, N, P, Q, R, T, and Y;
an amino acid substitution at position N216 selected from the group consisting of:
D, Q, T, K, and P;
an amino acid substitution at position 1218 selected from the group consisting of:
T, A, D, E, K, M, N, P, S, and V;
an amino acid substitution at position 1219 selected from the group consisting of:
M, A, D, L, N, P, and Q;
an amino acid substitution at position V224 selected from the group consisting of:
Q, I, A, E, N, P, S, and T;
an amino acid substitution at position T289 selected from the group consisting of:
A, D, K, L, M, R, and S;
an amino acid substitution at position Y291 selected from the group consisting of:
A, F, G, I, K, L, M, N, R, S, V, W, D, and E;
an amino acid substitution at position S297 selected from the group consisting of:
D, E, K, N, Q, R, and T;
an amino acid substitution at position T303 selected from the group consisting of:
K, M, P, R, S, V, and Y;
an amino acid substitution at position R304 selected from the group consisting of:
A, C, D, E, N, Q, T, and V;
an amino acid substitution at position A306 selected from the group consisting of:
E, F, G, I, K, L, M, Q, T, and Y;
an amino acid substitution at position P308 selected from the group consisting of:
A, G, H, I, K, L, Q, R, S, T, V, W, and Y;
an amino acid substitution at position S312 selected from the group consisting of:
Q, F, G, I, K, L, M, N, T, V, D, R, and Y;
an amino acid substitution at position T313 selected from the group consisting of:
S, D, G, L, P, V, E, F, I, K, M, and Y;
an amino acid substitution at position N315 selected from the group consisting of:
S, H, L, Q, R, V, Y, E, K, and M;
an amino acid substitution at position Y317 selected from the group consisting of:
Q, S, V, R, E, G, L, M, P, and T;
a Y318T amino acid substitution;
an amino acid substitution at position N5 selected from the group consisting of:
A, D, G, and S;
an amino acid substitution at position I7 selected from the group consisting of:
L, M, and V;
an amino acid substitution at position V12 selected from the group consisting of:
M and T;
an amino acid substitution at position Q15 selected from the group consisting of:
H, K, and D;
an amino acid substitution at position T21 selected from the group consisting of:
I, K, M, and S;
an amino acid substitution at position F22 selected from the group consisting of:
K, I, L, and R;
an amino acid substitution at position S26 selected from the group consisting of:
M, P, and T;
an amino acid substitution at position P28 selected from the group consisting of:
M, S, and V;
an amino acid substitution at position V32 selected from the group consisting of:
A and P;
an amino acid substitution at position G33 selected from the group consisting of:
Q, A, and S;
an amino acid substitution at position T35 selected from the group consisting of:
A, D, E, K, N, and S;
an amino acid substitution at position A36 selected from the group consisting of:
T, I, N, S, and Y;
an amino acid substitution at position N41 selected from the group consisting of:
D, E, L, and M;
an amino acid substitution at position Q50 selected from the group consisting of:
A, T, Y, D, E, and K;
an amino acid substitution at position N51 selected from the group consisting of:
S, E, H, K, L, and T;
an amino acid substitution at position K58 selected from the group consisting of:
I, R, and V;
an amino acid substitution at position N59 selected from the group consisting of:
G, E, Q, and S;
an amino acid substitution at position V80 selected from the group consisting of:
A, E, L, and M;
an amino acid substitution at position P89 selected from the group consisting of:
T, S, V, and L;
an amino acid substitution at position T108 selected from the group consisting of:
A, Q, R, and S;
an amino acid substitution at position G116 selected from the group consisting of:
H, Q, and S;
an amino acid substitution at position L120 selected from the group consisting of:
V, T, I, and S;
an amino acid substitution at position G123 selected from the group consisting of:
E, Q, and R;
an amino acid substitution at position V132 selected from the group consisting of:
D, E, M, and R;
an amino acid substitution at position S135 selected from the group consisting of:
A, E, G, H, M, and N;
an amino acid substitution at position N147 selected from the group consisting of:
E, M, S, and T;
an amino acid substitution at position A161 selected from the group consisting of:
E, G, L, and S;
an amino acid substitution at position A181 selected from the group consisting of:
K, N, and R;
an amino acid substitution at position S183 selected from the group consisting of:
E, K, N, and T;
an amino acid substitution at position S185 selected from the group consisting of:
D, G, and T;
an amino acid substitution at position Q189 selected from the group consisting of:
E, L, M, R, G, and T;
an amino acid substitution at position G195 selected from the group consisting of:
A, E, and S;
an amino acid substitution at position T196 selected from the group consisting of:
H, Y, and R;
an amino acid substitution at position H200 selected from the group consisting of:
R, A, Q, and S;
an amino acid substitution at position A201 selected from the group consisting of:
P, K, and Q;
an amino acid substitution at position T202 selected from the group consisting of:
S, E, and Q;
an amino acid substitution at position 1208 selected from the group consisting of:
F, L, and V;
an amino acid substitution at position P222 selected from the group consisting of:
D and G;
an amino acid substitution at position T223 selected from the group consisting of:
S, E, and Q;
an amino acid substitution at position S226 selected from the group consisting of:
D, G, K, M, P, and Q;
an amino acid substitution at position S231 selected from the group consisting of:
C and I;
an amino acid substitution at position G235 selected from the group consisting of:
A, F, I, M, N, Q, V;
an amino acid substitution at position L290 selected from the group consisting of:
K, M, and T;
an amino acid substitution at position G295 selected from the group consisting of:
A, F, H, Q, and R;
an amino acid substitution at position G296 selected from the group consisting of:
A and M;
an amino acid substitution at position G298 selected from the group consisting of:
K, A, I, N, and Q;
an amino acid substitution at position Y299 selected from the group consisting of:
N, S, F, and P;
an amino acid substitution at position S300 selected from the group consisting of:
F, L, N, T, and Q;
an amino acid substitution at position G301 selected from the group consisting of:
D, S, and Y;
an amino acid substitution at position P302 selected from the group consisting of:
G, L, T, V, Y, and F;
an amino acid substitution at position P307 selected from the group consisting of:
A, F, H, L, and V;
an amino acid substitution at position A309 selected from the group consisting of:
I, K, R, T, V, and Y;
an amino acid substitution at position T310 selected from the group consisting of:
L, M, Q, S, and W;
an amino acid substitution at position P316 selected from the group consisting of:
D, N, R, and T;
an amino acid substitution at position A319 selected from the group consisting of:
Q, W, D, F, and S;
an amino acid substitution at position L322 selected from the group consisting of:
A, E, S, T, and V;
an amino acid substitution at position 14 selected from the group consisting of:
V and T;
an amino acid substitution at position 19 selected from the group consisting of:
V and A;
an N10D amino acid substitution;
a P23G amino acid substitution;
a Y24T amino acid substitution;
an amino acid substitution at position E25 selected from the group consisting of:
N and Q;
an N27E amino acid substitution;
an amino acid substitution at position D38 selected from the group consisting of:
E and N;
an F43Y amino acid substitution;
a V441 amino acid substitution;
a Y49W amino acid substitution;
an amino acid substitution at position P52 selected from the group consisting of:
A and N;
an amino acid substitution at position 154 selected from the group consisting of:
Q and E;
an amino acid substitution at position T61 selected from the group consisting of:
Q and S;
an N62P amino acid substitution;
an amino acid substitution at position K64 selected from the group consisting of:
A and Q;
a V69Y amino acid substitution;
a K70R amino acid substitution;
an amino acid substitution at position A71 selected from the group consisting of:
Y and P;
an I90V amino acid substitution;
an amino acid substitution at position V91 selected from the group consisting of:
L and I;
an amino acid substitution at position N96 selected from the group consisting of:
A and S;
an N98H amino acid substitution;
an amino acid substitution at position D100 selected from the group consisting of:
P and S;
a T103A amino acid substitution;
an E110K amino acid substitution;
a V117A amino acid substitution;
an amino acid substitution at position L119 selected from the group consisting of:
N and T;
a D124N amino acid substitution;
a P125D amino acid substitution;
an amino acid substitution at position G126 selected from the group consisting of:
M and Q;
a V141T amino acid substitution;
a D146E amino acid substitution;
an A149E amino acid substitution;
an amino acid substitution at position I160 selected from the group consisting of:
F and M;
an H163A amino acid substitution;
an A171T amino acid substitution;
an N173H amino acid substitution;
a P175F amino acid substitution;
an I180L amino acid substitution;
an amino acid substitution at position S187 selected from the group consisting of:
A and D;
an amino acid substitution at position L188 selected from the group consisting of:
A and D;
an S191D amino acid substitution;
an amino acid substitution at position L194 selected from the group consisting of:
D and M;
an amino acid substitution at position D197 selected from the group consisting of:
A and V;
a P204A amino acid substitution;
a V206I amino acid substitution;
an L207F amino acid substitution;
an N209D amino acid substitution;
a Y211A amino acid substitution;
an S213T amino acid substitution;
a P214S amino acid substitution;
a Y217H amino acid substitution;
a T230S amino acid substitution;
an amino acid substitution at position V232 selected from the group consisting of:
F and Y;
comprises an amino acid substitution at position A233 selected from the group consisting of:
F and S;
an amino acid substitution at position G292 selected from the group consisting of:
D and K;
an amino acid substitution at position Q293 selected from the group consisting of:
K and P;
a Q320R amino acid substitution;
and an N323V amino acid substitution.

261 more elements

Dependencies are inferred from the claim text.

Facts

Application

16/185,619Filed 9 Nov 2018

Priority

29 Jul 2013

Published

6 Jun 2019

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

C12N 9/2437
Acting on beta-1,4-glucosidic bonds: cellulases
C12N 9/0083
Acting on paired donors with incorporation of molecular oxygen: miscellaneous
C12N 9/2402
Acting on glycosyl compounds: hydrolysing O- and S- glycosyl compounds
C12N 9/2434
Glucanases: acting on beta-1,4-glucosidic bonds

Family

Application61/859,630Us provisional application · filed 29 Jul 2013

Application61/859,666Us provisional application · filed 29 Jul 2013

Application61/859,680Us provisional application · filed 29 Jul 2013

Application61/859,704Us provisional application · filed 29 Jul 2013

Application61/859,712Us provisional application · filed 29 Jul 2013

Application61/859,721Us provisional application · filed 29 Jul 2013

Application61/859,735Us provisional application · filed 29 Jul 2013

Application14/909,031Continuation · filed 29 Jan 2016

Application16/185,619Continuation

US 10,167,460 B2Related patent

US 2016/0177281 A1Published application

US 2019/0169587 A1This patent

From the US filing history. Foreign family members aren’t listed.