IP Library › Granted Patent US 7,776,576
Granted Patent B2
US 7,776,576 · App. 10/886,905 · Granted Aug 17, 2010

Thermostable amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

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Quick Facts
Patent No.
US 7,776,576
App. No.
10/886,905
Granted
Aug 17, 2010
Kind
B2
Abstract

This invention relates to amylase polypeptides, and nucleic acids encoding the polpypeptides and uses thereof. The amylases of the present invention have been engineered to have more beneficial qualities. Specifically, the amylases of the current invention show an altered thermostability.

Claims (89)

1. An isolated thermostable non-maltogenic exoamylase comprising an amino acid sequence having at least 95% identity with SEQ ID NO: 1 and having at least one substitution of the amino acid residue at a position selected from the group consisting of 4, 13, 33, 34, 42, 70, 71, 87, 99, 100, 108, 113, 121, 131, 134, 135, 141, 153, 157, 158, 160, 161, 166, 170, 171, 178, 179, 184, 188, 198, 199, 221, 223, 238, 270, 277, 290, 307, 315, 334, 335, 342, 343, 372, 392, 398, 399, 405, 415, and 425.

2. The exoamylase of claim 1 , wherein said at least one substitution is selected from G4D, N33Y, D34N, G70D, K71 R, G87S, A99V, K108R, V113I, G121D, G134R, A141P, I157L, G158D, S161A, Y171S, 178F, 179T, G188A, Y198F, Y198L, A199V, G223A, V290I, H307L, I315V, S334P, D343E, S399P, A405F and A405E.

3. The exoamylase of claim 1 wherein said at least one position is selected from 33, 34, 70, 71, 87, 121, 134, 141, 157, 161, 178, 179, 223, 307, 334, and 343.

4. The exoamylase of claim 3 , wherein said at least one substitution is selected from N33Y, D34N, G70D, K71 R, G87S, G121D, G134R, A141P, I157L, S161A, L178F, A179T, G223A, H307L, S334P, and D343E.

5. The exoamylase of claim 4 , further comprising at least one additional substitution at a position selected from 108, 158, 171, and 188.

6. The exoamylase of claim 5 , wherein said at least one additional substitution is selected from K108R, G158D, Y171 S, and G188A.

7. The exoamylase of claim 1 , wherein said at least one substitution comprises a combination selected from the following:

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T and G121D;

G87S, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T and G121D;

G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F, A179T and G121D;

G134R, A141P, I157L, G223A, H307L, S334P and D343E;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, G158D, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, D343E and G158D;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N and G158D;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y and G121D;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y and G121D;

G134R, A141P, I157L, G223A, H307L, S334P, D343E and N33Y;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, G87S, G121 D, S214N and T375A;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, G121 D, Y171 S, G188A and N138D;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T and G121D;

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71 R, L178F and A179T;

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71 R, L178F, A179T and G188A;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, K71 R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T, G87S and G121 D;

G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, 178F, A179T, G87S and G121D;

G134R, A141P, I157L, G223A, H307L, S334P, L178F, A179T and G121D;

G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, K71R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, N33Y, D34N, K71R, L178F, A179T and G121 D;

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71 R, L178F and A179T;

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, K71 R, L178F and A179T;

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T and G121D;

G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E; 113F, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

A99V, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

A199V, D343E, V113I, A141P, I157L, Y198F, G223A, V290I, H307L and S334P;

V113I, A141P, I157L, Y198F, G223A, V290I, S334P and D343E;

V113I, A141P, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, A141P, Y198W, G223A and V290I;

V113I, A141P, Y198F, G223A, V290I and H307L;

V113I, A141P, Y198F, G223A, V290I, S334P and D343E;

V113I, A141P, Y198F, G223A, A268P, V290I and S399P;

V113I, A141P, Y198W, G223A and V290I;

V113I, A141P, Y198F, G223A and V290I;

V113I, A141P, Y198F, G223A and V290I;

Y198F, G223A and V290I;

Y198W, G223A and V290I;

V113I, A141P, I157L, Y198F, G223A and V290I;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, 1315V, S334P and D343E;

D34N, V113I, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, G188S, Y198F, G223A, V290I, H307L, S334P and D343E;

K71R, V113I, G134R, A141P, I157L, L178L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, A179V, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, G313G, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E, G400G and A405S;

G87S, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E and A405E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E and A405F;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P, D343E and A405V;

A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198L, G223A, V290I, H307L, S334P and D343E;

V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

K71R, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

K108R, V113I, G134R, A141P, I157L, Y198F, G223A, V290 I, H307L, S334P and D343E;

D34G, V113I, G134R, A141 P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

G4D, V113I, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E; and

A141P, G134R, G223A, H307L, I157L, V113I, V290I, Y198F and G188A.

8. The exoamylase of claim 4 , wherein said at least one substitution comprises a combination selected from the following:

N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;

N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P; and

N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P.

9. The exoamylase of claim 1 , wherein said exoamylase is derived from a Pseudomonas sp.

10. The exoamylase of claim 9 , wherein said Pseudomonas sp. is selected from Pseudomonas saccharophilia and Pseudomonas stutzeri.

11. The exoamylase of claim 4 , wherein said at least one substitution comprises a combination selected from the following:

G134R, A141 P, I157L, G223A, H307L and S334P;

G121D, G134R, A141 P, I157L, G223A, H307L and S334;

G87S, G121D, G134R, A141P, I157L, G223A, H307L and S334P;

G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;

G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;

G121D, G134R, A141 P, I157L, L178F, A179T, G223A, H307L and S334P;

N33Y, D34N, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P;

N33Y, D34N, G121D, G134R, A141 P, I157L, L178F, A179T, G223A, H307L and S334P; and

N33Y, D34N, G87S, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L and S334P.

Assignments (2)
CHANGE OF NAME Recorded Aug 19, 2013
From: DANISCO A/S
To: DUPONT NUTRITION BIOSCIENCES APS
Reel/Frame 031035/0463 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 25, 2009
From: KRAGH, KARSTEN MATTHIAS; BERG, CASPER TUNE; DERKX, PATRICK M.F.; KELLETT-SMITH, ANJA H.; THOUDAHL, CHARLOTTE REFDAHL; VELTMAN, OENE R.; GERRITSE, GIJSBERT; FIORESI, CAROL; LIU, WEI; SHAW, ANDREW
To: DANISCO A/S
Reel/Frame 022450/0294 →