Effect of additives on kinetic thermal stability of polygalacturonase II from Aspergillus carbonarius:: Mechanism of stabilization by sucrose

The thermal inactivation of polygalacturonase (PG II; M-w = 42 kDa) from Aspergillus carbonarius at optimum pH of the enzyme activity followed first-order kinetics both in the presence and in the absence of additives. The enzyme was stable below its optimum pH. Sodium chloride enhanced the thermal stability, the midpoint of thermal stability (T-m), shifted by 6 degrees C, and the half-life at T-m increased by 68-fold. Except for ethylene glycol, the other polyhydric alcohols enhanced thermal stability in a concentration-dependent manner; the stabilizing effect by polyhydric alcohols increased with increase in hydroxyl content of the polyol. The activation enthalpy increased in the presence of 1 M sucrose and 1 M NaCl from 77 to 116 kcal mol(-1); activation entropy increased from 169 to 290 cal deg(-1) mol(-1). The net free energy change of stabilization in the presence of stabilizers was 3-4 kcal mol(-1). The thermal inactivation of the enzyme involved conformational changes and unfolding of the enzyme molecule; the major secondary structural element, beta structure, decreased and the tryptophan residues were exposed to solvent. Stabilization of enzyme by sucrose was by preventing unfolding of the enzyme.