Glucoamylase (GA) from Aspergillus (EC 3.2.1.3) exists in two forms: GAI (amino acids 1-616) and GAII (amino acids 1-512). GAII is identical to GAI but lacks the C-terminal region (amino acids 513-616). While both forms of glucoamylase are able to digest soluble starch, only GAI has the ability to bind to and therefore hydrolyze native starch granules. To further examine the location of the starch binding domain at the C-terminus of GAI and to determine how many amino acid residues of GAI are required for starch binding, six [beta]-galactosidase fusion proteins were constructed. Affinity purified fusion proteins were tested for native starch binding. Fusion proteins containing 119 and 133 amino acids from the C-terminus of GAI, respectively, had much higher affinity to starch granules than [beta]-galactosidase and a size-control fusion protein. The results confirmed the hypothesis that the starch binding domain of Aspergillus GAI resides in the C-terminus of the enzyme and indicated that the C-terminal 119 amino acids of GAI is near the optimum functional size when fused to [beta]-galactosidase and expressed in E. coli. The binding specificity of the fusion proteins native starch granules was also tested. The results showed that the fusion proteins could specifically as well as strongly bind to native starch granules in the presence of crude soluble cell extracts and could be eluted from starch granules with similar purity to that achieved by affinity chromatography, suggesting the potential application of native starch as an adsorbent to facilitate the purification or immobilization of fusion proteins containing the starch binding region;In order to test the effect of deletions in the starch binding domain on glucoamylase itself, five deletion mutants of glucoamylase were constructed and expressed in Saccharomyces cerevisiae. Progressive loss of starch binding and starch hydrolytic activity was observed upon removal of 8 to 52 amino acid residues from the C-terminus of GAI. Removal of the C-terminal 103 amino acids resulted in nearly complete loss of starch binding and hydrolytic activity of GAI. These deletions in the starch binding domain did not affect enzyme activity on soluble starch or thermostability of the enzyme, confirming the independence of the catalytic domain from the starch binding domain. Study of the starch binding domain will facilitate understanding of the interaction between glucoamylase and starch and also the future utilization of the domain.