Purification, characterization, and structural investigation of a new moderately thermophilic and partially calcium-independent extracellular alpha-amylase from Bacillus sp. TM1.

A new alpha-amylase was extracted from a recently found strain of Bacillus sp. and purified by ion-exchange chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed a single band for the purified enzyme with an apparent molecular weight of 59 kDa. The optimum temperature and pH range of the enzyme were 40-60 degrees C and 4.5-7.5, respectively, and its activation energy was 1.974 kcal/mol. The Km value for the enzyme activity on soluble starch was 4 mg/mL, and the Tm values obtained from the circular dichroism (CD) results of thermal unfolding were 78.7 and 80.2 degrees C in the absence and presence of the calcium, respectively. The enzyme was almost completely inhibited by the addition of Fe3+, Mn2+, and Zn2+ and was activated by EDTA, Cr3+, and Al3+. Moreover, it was partially inhibited by Ca2+, Ba2+, Ni2+, and Co2+. Proteolytic digestion of the enzyme using trypsin combined with results from Tm using CD and irreversible thermoinactivation suggests that this enzyme can be considered a moderate thermophile with both mild flexibility and rigidity.