title: Purification, characterization, and structural investigation of a new moderately thermophilic and partially calcium-independent extracellular alpha-amylase from Bacillus sp. TM1.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modarres University, PO Box 14115-175, Tehran, Iran.
authors.forename: Reza Hassan
authors.initials: RH
authors.lastname: Sajedi
authors.forename: Hossein
authors.initials: H
authors.lastname: Naderi-Manesh
authors.forename: Khosro
authors.initials: K
authors.lastname: Khajeh
authors.forename: Bijan
authors.initials: B
authors.lastname: Ranjbar
authors.forename: Nasser
authors.initials: N
authors.lastname: Ghaemi
authors.forename: Mehdi
authors.initials: M
authors.lastname: Naderi-Manesh
mesh_terms: D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000378:metabolism; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D000516:alpha-Amylases; D002118:Calcium