Thermal and pressure‐temperature denaturation kinetics of bacillus subtilis α‐amylase: A study based on gel electrophoresis

Abstract

The commercially available enzyme Bacillus subtilis α‐amylase was characterized by a molecular weight of about 55 kDa and contained isozymes with pI values ranging in a narrow zone (4.6–5.3). Furthermore, the sample was confirmed to contain no disulfide bonds. Upon thermal denaturation in the presence of sodium dodecyl sulfate, a band at half molecular weight was noticed, indicating that the native enzyme might be a dimeric form. Thermal as well as pressure‐temperature denaturation kinetics were investigated using gel electrophoresis and both could accurately be described by a first order kinetic model. For thermal denaturation an activation energy of 283 kJ/mole was calculated. As far as pressure‐temperature denaturation is concerned, activation energy and activation volume at a constant pressure of 5500 bar and a constant temperature of 40°C were calculated respectively as 77.6 kJ/mole and ‐23.2 cm³/mole. These values were compared with those for thermal and pressure‐temperature inactivation of Bacillus subtilis α‐amylase, as determined from residual enzyme activity measurements. No significant differences between the activation energy and volume characterizing denaturation and inactivation of Bacillus subtilis ct‐amylase were observed.