Starch debranching enzymes
View Patent ↗The invention relates to a genetically engineered variant of a parent starch debranching enzyme, i.e. a pullulanase or an isoamylase, 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.
1. An isolated variant of a parent pullulanase, wherein the variant
(a) has a degree of sequence identity to SEQ ID NO: 2 of at least 95% when sequence identity is determined using GAP (version 8), using a gap creation penalty of 3.0 and a gap extension penalty of 0.1,
(b) comprises a substitution at one or more positions corresponding to positions 515-554 of SEQ ID NO: 2, and
(c) has pullulanase activity.
2. The variant of claim 1 , which comprises a substitution at a position corresponding to position 522 of SEQ ID NO: 2.
3. The variant of claim 1 , which comprises a substitution at a position corresponding to position 533 of SEQ ID NO: 2.
4. The variant of claim 1 , further comprising a substitution at one or more positions corresponding to positions 465-493, 515-554, 580-621, 649-664, 680-711, 714-717, 757-765 and 804-834 of SEQ ID NO: 2.
5. The variant of claim 1 , which has a degree of sequence identity to SEQ ID NO: 2 of at least 97%.
6. The variant of claim 1 , which has a degree of sequence identity to SEQ ID NO: 2 of at least 99%.
7. The variant of claim 1 , wherein the parent pullulanase is SEQ ID NO: 2.
8. A method for converting starch to one or more sugars, comprising
(a) liquefying the starch with at least one variant of claim 1 and at least one alpha-amylase to form dextrin; and
(b) saccharifying the dextrin with at least one glucoamylase to form the one or more sugars.