Abstract
The granular starch hydrolyzing enzyme, glucoamylase from Rhizopus oryzae, is a commonly used glycoside hydrolase in industry. It comprises a C-terminal catalytic domain and an N-terminal starch-binding domain which belongs to carbohydrate-binding module (CBM) family 21 rather than family 20. Extensive structural and biochemical investigations of family 20 CBMs have been reported; however, those of family 21 CBMs are quite limited. In the present study, the functional SBD from R. oryzae was expressed and purified in Escherichia coli. Its optimal pH range for binding was between 5 and 6. The starch-binding isotherm indicated that the starch-binding affinity and capacity were greater than Aspergillus SBDs. In addtion, a molecular model of the family 21 CBM from R. oryzae glucoamylase (RoGACBM21) was constructed according to progressive secondary structure correlation (PSSC) modified structure-based sequence alignment, and used site-directed mutagenesis to identify and characterize potential ligand binding sites. Our model suggests that RoGACBM21 contains two ligand binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83, and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former is not only involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharide; whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. We have first demonstrated that the key ligand binding residues of RoGACBM21 can be identified and characterized employing combination of novel bioinformatics methodology in the absence of resolved three-dimensional structural information.