IP Library › Granted Patent US 8,809,032
Granted Patent B2
US 8,809,032 · App. 12/024,324 · Granted Aug 19, 2014

Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof

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Quick Facts
Patent No.
US 8,809,032
App. No.
12/024,324
Granted
Aug 19, 2014
Kind
B2
Abstract

This invention relates to amylase polypeptides, and nucleic acids encoding the polypeptides 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 exospecifity.

Claims (73)

1. A non-naturally occurring enzyme having at least 95% identity with amino acids 1-429 of SEQ ID NO: 1 and including at least one substitution of the amino acid residue at a position corresponding to a position of SEQ ID NO:1 selected from the group consisting of 4, 33, 34, 70, 71, 87, 99, 108, 113, 121, 134, 141, 157, 158, 171, 178, 179, 188, 198, 199, 223, 290, 307, 315, 334, 343, 399, and 405, wherein the enzyme is an exo-specific non-maltogenic exoamylase.

2. The enzyme of claim 1 , wherein said at least one substitution is selected from the group consisting of G4D, N33Y, D34N, G70D, K71R, G87S, A99V, K108R, V113I, G121D, G134R, A141P, I157L, G158D, Y171S, L178F, A179T, G188A, Y198F Y198L, A199V, G223A, V290I, H307L, 1315V, S334P, D343E, S399P, A405F and A405E.

3. The enzyme of claim 1 wherein said at least one position is selected from the group consisting of 33, 34, 71, 87, 121, 134, 141, 157, 178, 179, 223, 1307, 334 and 343.

4. The enzyme of claim 3 , wherein said at least one substitution is selected from the group consisting of N33Y, D34N, K71R, G878, G121D, G134R, A141P, I157L, L178F, A179T, G223A, H307L, S334P, and D343E.

5. The enzyme of claim 3 , further comprising at least one additional substitution at a position selected from the group consisting of 108, 158, 171 and 188.

6. The enzyme of claim 5 , wherein said at least one additional substitution is selected from KI08R, G158D, Y1718, and G188A.

7. The enzyme of claim 1 , wherein said enzyme comprises one or more substitutions selected from the group consisting of:

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

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, G121D, Y171S and G188A;

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, K71R, L178F and A179T;

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T and G188A;

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

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

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

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

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

G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, G121D and E343D;

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

G87S, G121D, G134R, A141P, I157L, G223A, H307L, S334P, D343E, N33Y, D34N, K71R, L178F, A179T, Y33N, N34D and E343D;

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

G134R, A141P, I157L, G223A, H307L, S334P, 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, 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, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

V113I, G121D, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

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

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, Y198F, G223A, V290I and S399P;

V113I, A141P, Y198W, G223A and V290I;

V113I, A141P, Y198F, G223A and V290I;

Y198F, G223A and V290I;

Y198W, G223A and V290I;

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

V113M;

V113A;

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, A141P, 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;

D34N, V113I, G134R, A141P, I157L, Y198F, G223A, V290I, H307L, S334P and D343E;

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

V113I, G134R, A141P, I157L, 11701, Y198F, G223A, V290I, H307L, S334P and D343E;

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

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 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, V290I, H307L, S334P and D343E;

D34G, V113I, G134R, A141P, 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 enzyme of claim 1 , wherein said enzyme is from a Pseudomonas sp.

9. The enzyme of claim 8 , wherein said Pseudomonas sp. is Pseudomonas saccharophila or Pseudomonas stutzeri.

10. The enzyme of claim 1 , wherein said at least one substitution is at positions 334 and 307.

11. The enzyme of claim 1 , wherein the substitutions include substitutions at positions 33, 34, 121, 134, 141, 157, 178, 179, 223, 307 and 334.

12. A method for preparing a food product comprising: (a) providing a starch medium; (b) adding to the starch medium the exoamylase according to any one of claims 1 to 11 .

13. A process for making a bread product comprising: (a) providing a dough medium; (b) adding to the dough medium the exoamylase of any one of claims 1 to 11 ; and (c) applying heat to the starch medium during or after step (b) to produce a bread product.

Assignments (2)
CHANGE OF NAME Recorded Aug 19, 2013
From: DANISCO A/S
To: DUPONT NUTRITION BIOSCIENCES APS
Reel/Frame 031035/0589 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 21, 2010
From: KRAGH, KARSTEN MATTHIAS; BERG, CASPER TUNE; DERKX, PATRICK M.F.; KELLETT-SMITH, ANJA H.; THOUDAHL, CHARLOTTE REFDAHL; BOJSEN, KIRSTEN; GERRITSE, GIJSBERT; FIORESI, CAROL; LIU, WEI; SHAW, ANDREW
To: DANISCO A/S
Reel/Frame 025171/0469 →