Abstract
Fungal glucoamylase (GA; 1,4-□-D-glucan glucohydrolase, EC. 3.2.1.3) from Rhizopus oryzae consists of 579 amino acids with starch binding domain (SBD) and a catalytic domain connected by a highly glycosylated linker region. The function and structure of the catalytic domain have been thoroughly investigated; here this study focuses on the characterization and application of linker and ligand binding domain. Many GAs have linker regions separating the SBD from the catalytic domain. The extent of interaction between the catalytic modules in GAs with carbohydrate binding module family 21 (CBM21) remains an open question. Our data provide direct evidence that although the length, composition, and glycosylation of the interdomain linker vary in structural organization and in flexibility of the functional domains in GAs, presence of the linker indeed promotes the hydrolytic action towards the natural substrates. Insoluble polysaccharides can be degraded by hydrolytic enzymes appended to one or more non-catalytic CBMs. The CBM21 of RoGA (RoSBD) containing 106 amino acid residues is situated N-terminally and retained functionality in its isolated form. The properties and applications of recombinant RoSBD are described here and compared with other SBD-containing CBM families, including 20, 25, 26, 34, 41, 45, 48 and 53. Understanding of the CBMs properties and mechanisms in ligand binding may provide the basis for new strategies in functional predictions and industry applications.