Polypeptide

• US 20070141693A1
• Filed: 07/07/2006
• Published: 06/21/2007
• Est. Priority Date: 07/07/2005
• Status: Abandoned Application

• Current Assignee: Danisco A/S (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco A/S (International Flavors & Fragrances Incorporated)
• Inventors: Fioresi, Carol, Liu, Wei, Shaw, Andrew, Gerritse, Gijsbert, Kragh, Karsten, Sorensen, Bo, Thoudahl, Charlotte, Berg, Casper, Derkx, Patrick Maria, Kellet-Smith, Anja
• Application Number:
  US11/483,220
• Publication Number:
  US 20070141693A1
• Time in Patent Office: Days
• US Class Current: 435/204
• CPC Class Codes: A21D 8/042 with enzymes
• CPC Class Codes: A23L 33/18 Peptides; Protein hydrolysates
• CPC Class Codes: C12N 9/2411 Amylases
• CPC Class Codes: C12N 9/2414 Alpha-amylase (3.2.1.1.)
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12N 9/2425 Beta-amylase (3.2.1.2)
• CPC Class Codes: C12N 9/2428 Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase

Abstract

The invention provides for a PS4 variant polypeptide derivable from a parent polypeptide having amylase activity which may be selected from the group consisting of: (a) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334; (b) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 145, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334; (c) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334; and (d) a polypeptide comprising an amino acid mutation at each of positions 3, 33, 34, 70, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334; with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO: 1, uses of such a polypeptide as a food or feed additive, and nucleic acids encoding such.

Claims

1. A PS4 variant polypeptide derivable from a parent polypeptide having amylase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 161, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

(b) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 145, 146, 157, 178, 179, 223, 229, 272, 303, 307 and 334;

(c) a polypeptide comprising an amino acid mutation at each of positions 33, 34, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

(d) a polypeptide comprising an amino acid mutation at each of positions 3, 33, 34, 70, 121, 134, 141, 146, 157, 178, 179, 223, 229, 272, 303, 307, 309 and 334;

with reference to the position numbering of a Pseudomonas saccharophilia exoamylase sequence shown as SEQ ID NO;

1.

2. A PS4 variant polypeptide according to claim 1, in which each of the amino acid mutations in polypeptide (a) are independently selected from the group consisting of:

33Y, 34N, 121F, 134R, 141P, 146G, 157L, 161A, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P, preferably N33Y, D34N, G121F, G134R, A141P, Y146G, I157L, S161A, L178F, A179T, G223E, S229P, H272Q, G303E, H307L, A309P and S334P.

3. A PS4 variant polypeptide according to claim 2, which comprises the sequence pSac-pMD229 (SEQ ID NO:

13).

4. A PS4 variant polypeptide according to claim 1, in which each of the amino acid mutations in polypeptide (b) are independently selected from the group consisting of:

33Y, 34N, 121F, 134R, 141P, 145D, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L and 334P, preferably N33Y, D34N, G121F, G134R, A141P, N145D, Y146G, I157L, L178F, A179T, G223E, S229P, H272Q, G303E, H307L and S334P.

5. A PS4 variant polypeptide according to claim 4, which comprises the sequence pSac-pMD248 (SEQ ID NO:

15).

6. A PS4 variant polypeptide according to claim 1, in which each of the amino acid mutations in polypeptide (c) are independently selected from the group consisting of:

33Y, 34N, 121D, 134R, 141P, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P, preferably N33Y, D34N, G121D, G134R, A141P, Y146G, I157L, L178F, A179T, G223E, S229P, H272Q, G303E, H307L, A309P and S334P.

7. A PS4 variant polypeptide according to claim 6 which comprises the sequence pSac-pMD253 (SEQ ID NO:

17).

8. A PS4 variant polypeptide according to claim 1, in which each of the amino acid mutations in polypeptide (d) are independently selected from the group consisting of:

3S, 33Y, 34N, 70D, 121D, 134R, 141P, 146G, 157L, 178F, 179T, 223E, 229P, 272Q, 303E, 307L, 309P and 334P, preferably A3S, N33Y, D34N, G70D, G121D, G134R, A141P, Y146G, I157L, L178F, A179T, G223E, S229P, H272Q, G303E, H307L, A309P and S334P.

9. A PS4 variant polypeptide according to claim 8 which comprises the sequence pSac-pMD271 (SEQ ID NO:

19).

10. A PS4 variant polypeptide according to claim 1, in which the parent polypeptide comprises exoamylase activity, preferably a non-maltogenic exoamylase, more preferably a glucan 1,4-alpha-maltotetrahydrolase (EC 3.2.1.60).

11. A PS4 variant polypeptide according to claim 1, in which the parent polypeptide is or is derivable from Pseudomonas species, preferably Pseudomonas saccharophilia or Pseudomonas stutzeri.

12. A PS4 variant polypeptide according to claim 1, in which the parent polypeptide is a non-maltogenic exoamylase from Pseudomonas saccharophilia exoamylase having a sequence shown as SEQ ID NO:

1 or SEQ ID NO;

5.

13. A PS4 variant polypeptide according to claim 12 having an amino acid sequence which at least 75% identical to SEQ ID NO:

1 or SEQ ID NO;

5.

14. A PS4 variant polypeptide according to claim 1, in which the parent polypeptide is a non-maltogenic exoamylase from Pseudomonas stutzeri having a sequence shown as SEQ ID NO:

7 or SEQ ID NO;

11.

15. A PS4 variant polypeptide according to according to claim 14 having an amino acid sequence which at least 75% identical to SEQ ID NO:

7 or SEQ ID NO;

11.

16. A PS4 variant polypeptide according to claim 1, in which the PS4 variant polypeptide has a higher thermostability compared to the parent polypeptide or a wild type polypeptide when tested under the same conditions.

17. A PS4 variant polypeptide according to claim 16, in which the half life (t½

), preferably at 60 degrees C., is increased by 15% or more, preferably 50% or more, most preferably 100% or more, relative to the parent polypeptide or the wild type polypeptide.

18. A PS4 variant polypeptide according to claim 1, in which the PS4 variant polypeptide has a higher exo-specificity compared to the parent polypeptide or a wild type polypeptide when tested under the same conditions.

19. A PS4 variant polypeptide according to claim 18, in which the PS4 variant polypeptide has 10% or more, preferably 20% or more, preferably 50% or more, exo-specificity compared to the parent polypeptide or the wild type polypeptide.

20. A PS4 variant polypeptide according to claim 1, in which a food product treated with a the PS4 variant polypeptide has any one or more, preferably all of the following properties:

(a) lower firmness;

(b) higher resilience; and

(c) higher cohesiveness compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

21. A PS4 variant polypeptide according to claim 20, in which the resilience or cohesiveness of the food product is independently increased by 15% or more, preferably 50% or more, most preferably 100% or more, relative to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

22. A PS4 variant polypeptide according to claim 21, in which each of resilience and cohesiveness of a food product treated with a the PS4 variant polypeptide is increased compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

23. A PS4 variant polypeptide according to claim 20, in which the firmness of the food product is independently decreased by 15% or more, preferably 50% or more, most preferably 100% or more, relative to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

24. A PS4 variant polypeptide according to claim 23, in which the firmness of a food product treated with a the PS4 variant polypeptide is increased compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

25. A polypeptide derivable from a PS4 variant polypeptide according to claim 1 by mutation at one or more residues of the PS4 variant polypeptide sequence, in which the polypeptide has a higher thermostability or a higher exo-specificity, or both, compared to the parent polypeptide of the PS4 variant polypeptide or a wild type polypeptide, or in which a food product treated with a the PS4 variant polypeptide has any one or more, preferably all of the following properties:

(a) lower firmness;

(b) higher resilience;

or (c) higher cohesiveness as compared to a food product which has been treated with a parent polypeptide or a wild type polypeptide.

26. A polypeptide comprising a fragment of at least 20 residues of a PS4 variant polypeptide according to claim 1, in which the polypeptide has non-maltogenic exoamylase activity.

27. A polypeptide derivable from a polypeptide according to claim 1 by mutation at one or more residues of the PS4 variant polypeptide sequence, in which the polypeptide has a higher thermostability or a higher exo-specificity, or both, compared to the parent polypeptide of the PS4 variant polypeptide or a wild type polypeptide.