RESUMEN
The best amylolytic activity production by Aspergillus clavatus UEM 04 occurred in submersed culture, with starch, for 72 h, at 25 °C, and 100 rpm. Exclusion chromatography partially purified two enzymes, which ran as unique bands in SDS-PAGE with approximately 84 kDa. LC-MS/MS identified a glucoamylase (GH15) and an α-amylase (GH13_1) as the predominant proteins and other co-purified proteins. Zn2+, Cu2+, and Mn2+ activated the glucoamylase, and SDS, Zn2+, Fe3+, and Cu2+ inhibited the α-amylase. The α-amylase optimum pH was 6.5. The optimal temperatures for the glucoamylase and α-amylase were 50 °C and 40 °C, and the Tm was 53.1 °C and 56.3 °C, respectively. Both enzymes remained almost fully active for 28-32 h at 40 °C, but the α-amylase thermal stability was calcium-dependent. Furthermore, the glucoamylase and α-amylase KM for starch were 2.95 and 1.0 mg/mL, respectively. Still, the Vmax was 0.28 µmol/min of released glucose for glucoamylase and 0.1 mg/min of consumed starch for α-amylase. Moreover, the glucoamylase showed greater affinity for amylopectin and α-amylase for maltodextrin. Additionally, both enzymes efficiently degraded raw starch. At last, glucose was the main product of glucoamylase, and α-amylase produced mainly maltose from gelatinized soluble starch hydrolysis.
