THERMAL AND PRESSURE-TEMPERATURE DENATURATION KINETICS OF BACILLUS-SUBTILIS ALPHA-AMYLASE - A STUDY BASED ON GEL-ELECTROPHORESIS

The commercially available enzyme Bacillus subtilis alpha-amylase was characterized by a molecular weight of about 55 kDa and contained isoz ymes with pH values ranging in a narrow zone (4.6-5.3). Furthermore, t he sample was confirmed to contain no disulfide bonds. Upon thermal de naturation in the presence of sodium dodecyl sulfate, a band at half m olecular weight was noticed, indicating that the native enzyme might b e a dimeric form. Thermal as well as pressure-temperature denaturation kinetics were investigated using gel electrophoresis and both could a ccurately be described by a first order kinetic model. For thermal den aturation an activation energy of 283 kJ/mole was calculated. As far a s pressure-temperature denaturation is concerned, activation energy an d activation volume at a constant pressure of 5500 bar and a constant temperature of 40 degrees C were calculated respectively as 77.6 kJ/mo le and -23.2 cm(3)/mole. These values were compared with those for the rmal and pressure-temperature inactivation of Bacillus subtilis alpha- amylase, as determined from residual enzyme activity measurements. No significant differences between the activation energy and volume chara cterizing denaturation and inactivation of Bacillus subtilis alpha-amy lase were observed.