Characterization of a thermostable Bacillus stearothermophilus alpha-amylase.
Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.
Vihinen
M
M
Department of Biochemistry, University of Turku, Finland.
Mäntsälä
P
P
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000494:pharmacology; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000378:metabolism; D012997:Solvents / Q000494:pharmacology; D014162:Transfection; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D002614:Chelating Agents; D008670:Metals; D021382:Protein Sorting Signals; D012997:Solvents; D000516:alpha-Amylases; D010447:Peptide Hydrolases