ISOLATION AND CHARACTERIZATION OF ALPHA-AMYLASE DERIVED FROM STARCH-GROWN CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824

An extracellular alpha-amylase was purified to homogeneity from the culture supernatant of Clostridium acetobuylicum 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 alpha-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 alpha-1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5-5.0 and temperature of 55-degrees-C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2-5 mM) increased alpha-amylase activity by ca. 20%, while the addition of 19 mug ml-1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. The V(max) and K(m) of alpha-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.