Isolation and characterization of α-amylase derived from starchgrown Clostridium acetobutylicum ATCC 824

An extracellular α-amylase was purified to homogeneity from the culture supernatant of Clostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified α-amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the α-1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5-5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2-5 mM) increased α-amylase activity by ca. 20%, while the addition of 19 μg ml^-1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. The V_max and K_m of α-amylase were 2.17 mg min^-1 and 3.28 mg ml^-1 on amylose, and 1.67 mg min^-1 and 1.73 mg ml^-1 on soluble starch, respectively.