Purification and characterization of extracellular alpha-amylase from Aspergillus fumigatus

Extracellular alpha-amylase [(1 → 4)-α-d-glucan glucanohydrolase, EC 3.2. 1.1] from Aspergillus fumigatus (Aspergillus sp. K-27) was purified to homogeneity by anion-exchange (DEAE-cellulose) and affinity (α-cyclodextrin-Sepharose) chromatography. The purified enzyme, a glycoprotein with 15% carbohydrate content, showed an isoelectric point of 3.7, a molecular weight of 65,000 (as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), and an amino acid composition with a high number of neutral hydrophobic residues. Alpha-amylase activity on α-d-glucans in solution was optimal at pH 5.5, and the enzyme was stable at 40°C. It hydrolyzed amylose and amylopectin, with respective Km of 0.42 and 7.7 mg mL−1 and kcat/Km of 3.4 and 2.5 mL, mg−1 min−1. The major end-products of maltohexaose, degradation were glucose and maltose. Maltotriose, maltotetraose, and maltopentaose were formed as intermediate products with an α-anomeric configuration. Despite its ability to slowly degrade some α-(1 → 6) linkages, this purified enzyme should be classified as an alpha-amylase.