Expression system for novel pullulanase

• US 5,736,375 A
• Filed: 06/07/1995
• Issued: 04/07/1998
• 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: Amory, Antoine, Deweer, Philippe
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): HOBBS, LISA JOE
• Application Number:
  US08/474,545
• Time in Patent Office: 1,035 Days
• Field of Search: 435/69.1, 435/252.31, 435/320.1, 435/210, 536/23.2
• US Class Current: 435/210
• 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. Process for the preparation of isolated Bacillus deramifcans pullulanase having N-terminal SEQ ID NO:

1 or a modified sequence derived therefrom that catalyzes hydrolysis of α

-1,6-glucosidic bonds, comprising isolation of a DNA fragment which codes for the pullulanase, insertion of this DNA fragment into a suitable vector, introduction of this vector into a suitable host or introduction of this DNA fragment into the chromosome of a suitable host, culture of this host, expression of the pullulanase and harvesting of the pullulanase.

2. Isolated DNA molecule comprising the nucleotide sequence (SEQ ID NO:

10) which codes for the pullulanase of Bacillus deramificans or a modified sequence derived therefrom, wherein said modified sequence catalyses hydrolysis of α

-1,6-glucosidic bonds.

3. Isolated DNA molecule according to claim 2, characterized in that it comprises the entire gene (SEQ ID NO:

8) of the pullulanase of Bacillus deramificans T 89.117D.

4. Expression vector or chromosomal integration vector containing the DNA molecule according to claim 2.

7. Transformed strain of Bacillus licheniformis comprising the DNA molecule according to claim 2.

8. Transformed strain of Bacillus lichenformis comprising the expression vector or the chromosomal integration vector containing the DNA molecule according to claim 4.

5. Expression vector pUBDEBRA1.

6. Chromosomal integration vector pUBCDEBRA11DNSI.

9. Transformed strain of Bacillus lichenformis comprising the expression vector pUBDEBRA1 or the chromosomal integration vector pUBCDEBRA11DNSI.

10. A biologically pure culture of Bacillus licheniformis SE2 having accession number LMG P-14034.

11. A process for the production of Bacillus deramificans pullulanase having N-terminal SEQ ID NO:

1 or a modified sequence derived therefrom that catalyzes hydrolysis of α

1,6-glucosidic bonds, comprising the steps of;

isolating a DNA molecule encoding said pullulanase,

inserting said DNA molecule into a suitable host,

culturing the host in a suitable nutrient medium containing sources of carbon and nitrogen and mineral salts under aerobic conditions, and

harvesting the pullulanase obtained.

12. The process according to claim 11, wherein said inserting comprises inserting said DNA molecule into a suitable vector and introducing said vector into a suitable host.

13. The process according to claim 12, wherein said vector is expression vector pUBDEBRA1.

14. The process according to claim 11, wherein said inserting comprises introducing sad DNA molecule into a chromosome of said suitable host.

15. The process according to claim 14, wherein said DNA molecule is chromosomal integration vector pUBCDEBRA11DNSI.

16. The process according to claim 11, wherein said host is a microorganism of the genus Bacillus.

17. The process according to claim 16, wherein said Bacillus is selected from the group consisting of B. subtilis, B. licheniformis, B. alcalophilus, B. pumilus, B. lentus, B. amyloliquefaciens and B. deramificans T 89.117D (LMG) P-13056.

18. The process according to claim 16, wherein said Bacillus is a strain of Bacillus licheniformis.

19. The process according to claim 18, wherein said Bacillus is Bacillus lichentformis SE-2 (LMG P-14034).

20. The process according to claim 11, wherein said aerobic conditions comprise a temperature between 25 and 40°

C., a pH of between 3.5 and 6, and with stirring.

21. The process according to claim 11, wherein said harvesting comprises centrifugation, ultrafiltration, evaporation, precipitation, filtration and microfiltration.

22. The process according to claim 11, comprising an optional step of purifying said obtained pullulanase.