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

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Abstract
An extracellular α-amylase was purified to homogeneity from the culture supernatant ofClostridium 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. TheV max andK 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.