Deletion analysis of the starch-binding domain of Aspergillus glucoamylase

The large form of glucoamylase (GAI) from Aspergillus awamori (EC 3.2.1.3) binds strongly to native granular starch, whereas a truncated form (GAII) which lacks 103 C-terminal residues, does not, This C-terminal region, conserved among fungal glucoamylases and other starch-degrading enzymes, is part of an independent starch-binding domain (SBD), To investigate the SBD boundaries and the function of conserved residues in two putative substrate-binding sites, five gluco-amylase mutants were constructed with extensive deletions in this region for expression in Saccharomyces cerevisiae, Progressive loss of both starch-binding and starch-hydrolytic activity occurred upon removal of eight and 25 C-terminal amino acid residues, or 21 and 52 residues close to the N-terminus, confirming the requirement for the entire region in formation of a functional SBD, C-terminal deletions strongly impaired SBD function, suggesting a more important role for one of the putative binding sites, A GAII phenocopy showed a nearly complete loss of starch-binding and starch-hydrolytic activity, The deletions did not affect enzyme activity on soluble starch or thermo-stability of the enzyme, confirming the independence of the catalytic domain from the SBD.