The influence of charged matrix surfaces on the thermostabilizing effect of calcium ions on immobilized fungal alpha-amylase.

The stabilizing effect of calcium ions on fungal alpha-amylase (EC 3.2.1.1) immobilized on a polystyrene anion exchanger (P+ amylase) was investigated and compared to the behaviour of soluble amylase. Moreover, gamma-(1,4-benzoquinone-2-yl)-aminopropyl silica-amylase (Si(n) amylase) as a conjugate with weakly basic amino groups and gamma-succinamidopropyl silica amylase (Si- amylase) as a conjugate with free carboxyl groups were applied for comparison. Depending on the calcium ion concentration the immobilized amylases showed a lower thermal stability than the soluble enzyme. The reduced stability was attributed to matrix effects in the microenvironment of the immobilized amylases and the calcium ion concentration in the carrier phase, which was changed in comparison with the external solution. Contrary to the non-measurable matrix effects in the microenvironment, altered calcium ion concentrations in the carrier phase of the polystyrene anion exchanger (P+) and gamma-succinamidopropyl silica (Si-) could be detected. With increasing calcium ion concentration a greater decrease of activity was observed for the soluble amylase than for the immobilized enzymes. The thermal stability of soluble amylase and P+ amylase was studied in dependence on pH. In the acidic pH-range P+ amylase indicated a higher thermal stability than the soluble enzyme in the presence of Ca2+ as well as in the absence of Ca2+. Contrary to soluble amylase the stabilizing effect of calcium ions on P+ amylase begins already at pH 3.5. Kinetic investigations for thermal inactivation were performed on soluble amylase and P+ amylase in the presence and absence of Ca2+ in the temperature range between 44--60 degrees C. Thermal inactivation proceeded by first order reactions. The inactivation rate constants kin served as a measure of thermal stability for discussing the stabilizing effect by Ca2+ depending on the temperature. The activation energies of inactivation EA were determined from the Arrhenius-plot of the inactivation rate constants.