Purification,Characterization,and Structural Investigation of a New Moderately Thermophilic and Partially Calcium-Independent Extracellular alpha-Amylase From Bacillus sp.TM1

摘要

A new alphs-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 deg C and 4.5-7.5,respectively,and its activation energy was 1.974 kcal/mol.The Rvalue for the enzyme activity on soluble starch was 4 mg/mL,and the T_m values obtained from the circular dichroism (CD) results of thermal unfolding were 78.7 and 80.2 deg C in the absence and presence of the calcium,respectively.The enzyme was almost completely inhibited by the addition of Fe~(3+),Mn~(2+),and Zn~(2+) and was activated by EDTA,Cr~(3+),and A1~(3+).Moreover,it was partially inhibited by Ca~(2+),Ba~(2+),Ni~(2+),and Co~(2+).Proteolytic digestion of the enzyme using trypsin combined with results from T_m using CD and irreversible thermoinactivation suggests that this enzyme can be considered a moderate thermophile with both mild flexibility and rigidity.