Polypeptides with starch debranching activity

• US 8,021,863 B2
• Filed: 02/15/2008
• Issued: 09/20/2011
• Est. Priority Date: 02/19/2007
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes, Novozymes A/S (Novozymes)
• Inventors: Behr, Regine Kopp, Andersen, Carsten, Fukuyama, Shiro, Viksoe-Nielsen, Anders
• Primary Examiner(s): Saidha; Tekchand
• Assistant Examiner(s): MEAH, MOHAMMAD Y
• Application Number:
  US12/032,376
• Publication Number:
  US 20080241887A1
• Time in Patent Office: 1,313 Days
• Field of Search: None
• US Class Current: 435/102
• CPC Class Codes: C07K 2319/20 containing a tag with affinity for a non-protein ligand
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/2417 from microbiological source
• CPC Class Codes: C12P 19/16 produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin non-biological hydrolysis of starch C08B30/00

Abstract

Polypeptides comprising a catalytic starch debranching domain and a starch binding domain are disclosed. The polypeptides comprising the starch binding domain have an improved functionality in raw starch degradation compared with same catalytic unit in same amount but without the starch binding domain.

Claims

1. An isolated polypeptide having pullulanase activity, comprising a catalytic domain and a starch binding domain, wherein the catalytic domain comprises the sequence of the amino acids numbered 1-829 of SEQ ID NO:

14.

2. The polypeptide of claim 1, wherein the starch binding domain belongs to a family selected from the group consisting of CBM-20, CBM-21, CBM-25, CBM-26, CBM-34, CBM-41 and CBM-45.

3. The polypeptide of claim 2, wherein the starch binding domain belongs to CBM-20.

4. The polypeptide of claim 3, wherein the starch binding domain is the starch binding domain of an Anoxybacillus contaminans alpha-amylase or the starch binding domain of a Thermoanaerobacter sp. CGTase.

5. The polypeptide of claim 1, wherein the starch binding domain comprises the sequence of the amino acids numbered 838-936 of SEQ ID NO:

14.

6. The polypeptide of claim 1, further comprising a linker connecting the catalytic domain and the starch binding domain.

7. The polypeptide of claim 6, wherein the linker consists of less than 100 amino acids.

8. The polypeptide of claim 7, wherein the linker is proline rich.

9. The polypeptide of claim 8, where the linker is PEPTPEPN.

10. A method for raw starch degradation comprising incubating non-gelatinized starch and water with the polypeptide of claim 1.

11. The method of claim 10 comprising:

(a) providing a mixture comprising raw starch and water;

(b) adding the polypeptide together with an endo-acting amylase or an exo-acting amylase; and

(c) incubating the mixture at a temperature and for a time which are effective to degrade the starch.

12. The method of claim 11, wherein the exo-acting amylase is a glucoamylase.

13. An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO:

13.

14. A method for raw starch degradation comprising incubating non-gelatinized starch and water with the polypeptide of claim 13.

15. The method of claim 14 comprising:

(a) providing a mixture comprising raw starch and water;

(b) adding the polypeptide together with an endo-acting amylase or an exo-acting amylase; and

(c) incubating the mixture at a temperature and for a time which are effective to degrade the starch.

16. The method of claim 15, wherein the exo-acting amylase is a glucoamylase.