Thermal and Molecular Characterization of Aspergillus awamori Glucoamylase Catalytic and Starch-Binding Domains
Aspergillus awamori glucoamylase catalytic domain, linker, and starch-binding domain, the first and third expressed from yeast, have molecular masses of 56.2, 12.6 and 12.9 kDa, respectively, as determined by MALDI-TOF mass spectroscopy, and have 10.2, 73.2 and 7.0 % (w/w) carbohydrate, respectively, showing overglycosylation by yeast. Unfolding of the starch-binding domain monitored by circular dichroism is reversible at pH 6.0-8.0, with the unfolding T m and βH increasing from 49.7 to 58.5 Β°C and from 284 to 351 kJ/mol, respectively, as pH decreases from 8.0 to 6.0. The catalytic domain unfolds irreversibly at pH 7.5, producing a single asymmetric endotherm by differential scanning calorimetry, with T m and βH at a 1 Β°C/min heating rate being 60.9 Β°C and 1720 kJ/mol, but with both increasing as the heating rate increases. This suggests that unfolding is partially under kinetic control, while various tests show that it does not follow a simple two-state irreversible model. Values of βH from calculated solvent-accessible surface areas of unglycosylated catalytic and starch-binding domains are about 100 kJ/mol lower than experimentally determined βH values of the corresponding glycosylated domains, showing the effect of glycosylation on unfolding enthalpies.