Background: Starch processing requires a combination of enzymes with other chemical
and physical processes, which increases cost and time. Enzymes used in these processes have a
characteristic (α/β)8 barrel domain architecture, although, show variable activity. Pullulanase type
1 and isoamylase act on α-1-6 linkage, amylase on α-1-4 linkage whereas pullulanase type 2 acts
on both α-1-6, and α-1-4 linkages of starch.
Objective: This article focusses on elucidating the importance of sequence and structural-based
differences in pullulanase, that may lead to its dual catalytic nature.
Methods: Initially, sequences and structures of pullulanase type 1, pullulanase type 2, amylase and
isoamylase were retrieved from the database (NCBI and PDB). Homology modelling using
SWISS-MODEL and PHYRE2 was carried out for predicting the structure of the enzymes with
unavailable structures. Further, the modelled structures were validated using ANOLEA, Verify 3D
and PROCHECK, structures with high confidence value were selected and used for analysis.
Finally, the selected structures were compared by using PDBefold, and their domain alignment and
analysis was performed manually using Pymol.
Results: Modelled structures of pullulanase and isoamylase were validated and selected based on
the confidence score. Comparative analysis of complete structures low similarity between the
enzymes, although, domain analysis showed good similarity. Moreover, alignment of catalytic site
residues showed high similarities with the change in orientation of critical site residues (HIS 242,
ASP 347 and GLN 375).
Conclusion: The change in orientation of active site residues along with the absence or presence of
few residues might play a crucial role in imparting dual functionality.