Starch debranching enzymes

• US 6,265,197 B1
• Filed: 07/01/1999
• Issued: 07/24/2001
• Est. Priority Date: 07/02/1998
• Status: Expired due to Fees

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S Krogshoejvej
• Inventors: Svendsen, Allan, Bisgård-Frantzen, Henrik
• Primary Examiner(s): Prouty, Rebecca E.
• Assistant Examiner(s): Rao, Manjunath N.
• Application Number:
  US09/346,237
• Time in Patent Office: 754 Days
• Field of Search: 435/96, 435/98, 435/210, 435/253, 435/252.3, 435/202, 435/320.1
• US Class Current: 435/210
• 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: C12N 9/246 Isoamylase (3.2.1.68)
• 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: C12Y 302/01041 Pullulanase (3.2.1.41)
• CPC Class Codes: C12Y 302/01068 Isoamylase (3.2.1.68)

Abstract

The invention relates to a genetically engineered variant of a parent starch debranching enzyme, i.e. a pullulanase or an isamylase, the enzyme variant having an improved thermostability at a pH in the range of 4-6 compared to the parent enzyme and/or an increased activity towards amylopectin and/or glycogen compared to the parent enzyme, to methods for producing such starch debranching enzyme variants with improved thermostability and/or altered substrate specificity, and to a method for converting starch to one or more sugars using at least one such enzyme variant.

Claims

1. A variant of a parent pullulanase, said variant exhibiting improved thermostability at a pH in the range of 4-6 compared to said parent, said parent having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:

1, (ii) SEQ ID NO;

2, and (iii) a sequence that is at least 80% homologous to (i) or (ii) when homology is determined using GAP (version

     8), using a gap creation penalty of 3.0 and a gap extension penalty of 0.1;

said variant comprising at least one amino acid substitution relative to said parent, said at least one amino acid substitution corresponding to a position in SEQ ID NO;

1 selected from the group consisting of A210P, V215P, L249P, K383P, S509P, T811P, G823P;

or corresponding to a position in SEQ ID NO;

2 selected from the group consisting of G306P, V311P, L345P, D605P, T907P, A919P.

2. The variant of claim 1, wherein the variant exhibits an increased thermostability when measured by differential scanning calorimetry (DSC).

3. The variant of claim 1, wherein the variant exhibits an increase of at least about 5% in the half-time (T_½

) of enzymatic activity when measured at a pH of 5.0 and a temperature of 95°

C.

4. The variant of claim 1, wherein the variant exhibits an increase of at least about 5% in residual enzyme activity after incubation for 30 minutes a pH of 5.0 and a temperature of 95°

C.

5. The variant of claim 1, wherein the variant exhibits an increase of at least about 5% in the half-time (T_½

) of enzymatic activity when measured at a pH of 4.5 and a temperature of 70°

C.

6. The variant of claim 1, wherein the variant exhibits an increase of at least about 5% in residual enzyme activity after incubation for 30 minutes at a pH of 4.5 and a temperature of 63°

C.

7. The variant of claim 1, wherein the variant exhibits an increase of at least about 5% in residual enzyme activity after incubation for 30 minutes at a pH of 4.5 and a temperature of 70°

C.

8. A variant of a parent pullulanase, said variant exhibiting an improved thermostability at a pH in the range of 4-6 compared to said parent, said parent having an amino acid sequence selected from the group consisting of (i) SEQ ID NO:

1, (ii) SEQ ID NO;

2, and (iii) a sequence that is at least 80% homologous to (i) or (ii) when homology is determined using GAP (version

     8), using a gap creation penalty of 3.0 and a gap extension penalty of 0.1;

said variant comprising replacing an Asn or Gln residue with another amino acid residue;

wherein said Asn or Gln residue corresponds to (a) a position in SEQ ID NO;

1 selected from the group consisting of N379, N384, N426, Q432, N434, N437, N444, N446, N486, N490, Q502, N512, N515, N521, Q596, N616, N621, Q628, N679, N681, Q684, N720, N722, N731, Q732, and combinations of any of the foregoing;

or (b) a position in SEQ ID NO;

2 selected from the group consisting of N475, N480, N522, N533, N590, N582, N608, N611, N617, Q691, Q698, N72, N717, N764, N775,N815, N817, N820, and combinations of any of the foregoing.