Pullulanase, microorganisms which produce it, processes for the preparation of this pullulanese and the uses thereof

• US 6,074,854 A
• Filed: 12/23/1997
• Issued: 06/13/2000
• Est. Priority Date: 12/28/1992
• Status: Expired due to Term

• Current Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Genencor International Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Deweer, Philippe, Amory, Antoine
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Stole, Einar
• Application Number:
  US08/996,733
• Time in Patent Office: 903 Days
• Field of Search: 435/99, 435/100, 435/210
• US Class Current: 435/99
• CPC Class Codes: C12N 1/205 Bacterial isolates
• CPC Class Codes: C12N 9/2451 acting on alpha-1,6-glucosidic bonds
• CPC Class Codes: C12N 9/2457 Pullulanase (3.2.1.41)
• 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
• CPC Class Codes: C12R 2001/07 Bacillus
• CPC Class Codes: C12Y 302/01041 Pullulanase (3.2.1.41)

Abstract

The invention relates to a heat-stable pullulanase having the property of hydrolysing glucosidic bonds of the α-1,6 type in amylopectin and having an enzymatic activity in an acid medium and at a temperature of about 60° C.

The invention also relates to strains of microorganisms which produce this pullulanase and processes for the preparation of this pullulanase.

The invention also relates to the uses thereof and compositions comprising the product.

The invention also relates to a DNA molecule. The invention relates to an expression vector containing this DNA molecule and to a chromosomal integration vector containing this DNA molecule.

Claims

1. An enzymatic composition comprising a pullulanase produced by Bacillus deramificans, or by a derivative or a mutant strain thereof that retains all identifying characteristics of said species.

2. The composition of claim 1 wherein the B. deramificans has the following

N-terminal sequence:

Asp Gly Asn Thr Thr Thr Ile Ile Val His Tyr Phe Cys Pro Ala Gly Asp Tyr Gln Pro.

3. The composition of claim 1 wherein the pullulanase comprises the amino acid sequence illustrated in FIG. 4 (SEQ ID NO:

11) from amino acid 1 to 928 or a modified sequence derived therefrom, wherein said modified sequence catalyzes hydrolysis of α

-1,6-glucosidic bonds.

4. The composition of claim 3 wherein the pullulanase is synthesized in the form of a precursor containing an additional sequence of 29 amino acids identified in SEQ ID NO:

12.

5. The composition of claim 1 wherein the pullulanase is encoded by nucleic acid having the sequence as shown in SEQ ID NO:

10.

6. The composition of claim 1 further comprising glucoamylase.

7. The composition of claim 6 wherein the glucoamylase is derived from an Aspergillus strain.

8. The composition of claim 7 wherein the Aspergillus strain is selected from the group consisting of Aspergillus niger, Aspergillus awamori and Aspergillus foetidus.

9. The composition of claim 6 comprising at least 60% pullulanase.

10. The composition of claim 1 wherein said composition is in solid form.

11. The composition of claim 1 wherein said composition is in liquid form.

12. A process for the saccharification of starch, wherein said process allows for reduced concentrations of saccharification by-products, comprising the step of contacting aqueous liquefied starch with an enzyme composition comprising pullulanase produced by B. deramificans or by a derivative or a mutant strain thereof that retains all the identifying characteristics of said species or a B. deramificans pullulanase derivative or mutant thereof, wherein said pullulanase derivative or mutant is capable of hydrolyzing α

-1,6-glucosidic bonds.

13. The process of claim 12 further comprising the steps of heating said liquefied starch, and recovering products.

14. The process of claim 12 wherein said enzyme composition further comprises glucoamylase.

15. The process of claim 14 wherein said enzyme composition further comprises at least 60% pullulanase.

16. The process of claim 12 wherein said starch comprises pullulane and amylopectin.

17. The process of claim 12 wherein said contacting is at a pH of less than or equal to 7 and greater than or equal to 3.

18. The process of claim 17 wherein said pH is in a range of about 3.8 to about 4.9.

19. The process of claim 17 wherein said pH is about 4.3.

20. The process of claim 13 wherein said heating is at a temperature range of between 55 and 65 degrees C.

21. The process of claim 20, wherein said temperature is about 60 degrees C.