Molecular insights into the oligosaccharide binding, recognition and hydrolysis by a fungal exo-β-(1,3)-glucanase

Carbohydrate hydrolysing enzymes assume special industrial and commercial interest as a source for yielding fermentable glucose especially for the biofuel industry. Among these enzymes, the exo-β-(1,3) glucanases are promising for industrial use as they hydrolyze sugars such as laminarin, a major constituent of the algal cell wall. Exploring the structure and function of these enzymes is of particular interest for the improvement of their functional properties for industrial use. We report the structural and biochemical characterizations of Aspergillus oryzae exo-β-(1,3) glucanase (AoBgl). We have expressed, purified, and performed biochemical characterizations of the recombinant AoBgl. Purified AoBgl is found to hydrolyse β-(1,3)-glycosidic linkages present in the oligosaccharide laminaritriose and the polysaccharide, laminarin effectively while retaining >50% activity at glucose concentrations of around 1.5M. We have determined three high-resolution structures of AoBgl: (a) apo form at 1.75 Å, (b) complexed form with bound disaccharide at 1.73 Å and (c) glucose-bound form at 1.20 Å. Sequence analysis and structural comparison indicate that AoBgl belongs to the GH5 sugar hydrolase family. The sugar-bound structures reveal the mode of substrate binding and interactions at the active site of AoBgl. Further, molecular dynamics simulation and mutational studies indicate that AoBgl can effectively bind trisaccharides and higher oligosaccharides. Our biochemical and structural data provide detailed molecular insights into the active site of this GH5 enzyme and would be helpful in the rational engineering of glycosyl hydrolases belonging to similar families for industrial use.