Abstract
Glucoamylase (GA) is an exo-acting carbohydrase which cleaves glucose units from the non-reducing ends of starch molecules. The Rhizopus GA gene encodes 604 amino acids and the mature GA consists of 579 amino acids. The molecular weight of the mature GA is calculated to be 62,197 daltons. GA is composed of three domains: a catalytic domain (CD), an O-glycosylation linker region and a starch binding domain (SBD). To find out the smallest functional part of Rhizopus GA, four deletion mutants, GAD26-131, GAD26-145, GAD26-160 and GAD26-167, were constructed and expressed in Saccharomyces cerevisiae. GAD26-131, GAD26-145, and GAD26-160 showed clear zones around the colonies, and the catalytic activity was about 100 %, 50 %, and 50%, respectively of that of wild type GA. On the other hand, GAD26-167 yeast colonies showed no clear zone and the deletion also caused loss of catalytic activity. This result indicated that the deletion of the SBD does not affect the starch hydrolytic ability of the catalytic domain. Besides, five residues in the catalytic domain were separately substituted by site-directed mutagenesis. They all showed no clear zone, indicating that the five residues were required and critical for catalytic function of GA.