Alpha(1,6)-linked glucose oligosaccharide hydrolyzing enzyme polynucleotides and polypeptides and methods of use thereof

• US 7,268,221 B2
• Filed: 08/25/2003
• Issued: 09/11/2007
• Est. Priority Date: 08/23/2002
• Status: Expired due to Fees

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Zhang, Yuying, Caimi, Perry G., Wang, Siqun, Chen, Mario W., Tomb, Jean-Francois, Nigarajan, Vasantha Nagarajan
• Primary Examiner(s): Prouty; Rebecca S.
• Assistant Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US10/648,152
• Publication Number:
  US 20040171023A1
• Time in Patent Office: 1,478 Days
• Field of Search: 435/6, 435/210, 435/320.1, 435/69.1, 536/23.2
• US Class Current: 536/23.2
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 9/2451 acting on alpha-1,6-glucosidic bonds
• CPC Class Codes: C12N 9/246 Isoamylase (3.2.1.68)
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: C12P 7/20 Glycerol
• CPC Class Codes: C12Y 302/01068 Isoamylase (3.2.1.68)
• CPC Class Codes: C12Y 302/01133 Glucan 1,4-alpha-maltohydrolase (3.2.1.133), i.e. maltogenic alpha-amylase

Abstract

This invention relates to a method for utilizing less purified starch in fermentation processes. One example is a recombinant E. coli containing a exogenous extracellular isoamylase activity that is capable of utilizing small oligomers containing (1,6) linkages (including but not limited to isomaltose and panose) in fermentations to produce useful products. The invention is useful in large-scale industrial biofermentations by reducing the cost of the substrate carbohydrate.

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO;

6;

(b) a nucleic acid molecule that hybridizes with (a) under following the hybridization conditions;

0.1×

SSC, 0.1% SDS, 65°

C. and washed with 2×

SSC, 0.1% SDS followed by 0.1×

SSC, 0.1% SDS, wherein said nucleic acid molecule encodes an amino acid sequence having α

(1,6)-linked glucose oligosaccharide hydrolyzing activity; and

(c) a nucleic acid molecule that is fully complementary to (a) or (b).

2. The isolated nucleic acid molecule of claim 1 which is the nucleic acid molecule of SEQ ID NO:

5.

3. An isolated nucleic acid molecule selected from the group consisting of:

(a) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide operably linked to an α

(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide having the amino acid sequence set forth in SEQ ID NO;

6;

(b) a nucleic acid molecule that hybridizes with (a) under the following hybridization conditions;

0.1×

SSC, 0.1% SDS, 65°

C. and washed with 2×

SSC, 0.1% SDS followed by 0.1×

SSC, 0.1% SDS, wherein said nucleic acid molecule encodes an amino acid sequence having α

(1,6)-linked glucose oligosaccharide hydrolyzing activity; and

(c) a nucleic acid molecule that is fully complementary to (a) or (b).

4. The isolated nucleic acid molecule of claim 3, wherein the signal peptide is SEQ ID NO:

24 or SEQ ID NO;

25.

5. The isolated nucleic acid molecule of claim 3 encoding the α

(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide, the isolated nucleic acid molecule having the sequence as set forth in SEQ ID NO;

5.