Ultrasonic inactivation of Bacillus α-amylase. I. effect of gas content and emitting face of probe

Sonoinactivation of α-amylase from Bacillus amyloliquefacience was studied at a constant frequency of 30 kHz. The effect of sonotrode emitting face and gas content of medium on the efficiency of enzyme inactivation were investigated at different time–temperature combinations and generation of OH free radicals was also monitored. The results showed that ultrasound effectively inactivated α-amylase with a minimum overall inactivation rate at 50 °C. The tip diameter of the sonotrode and the gas content of the medium both significantly affected the rate of enzyme inactivation. The increase of tip diameter increased the effect of ultrasound on the enzyme, while the removal of dissolved gas adversely influenced the cavitational events and reduced the rate of enzyme inactivation. Calculation of the kinetic and activation parameters revealed that ultrasound decreased the activation energy, Ea, activation enthalpy, ΔH#, and the activation Gibbs free energy, ΔG#, and strongly reduced the activation entropy, ΔS#, down to negative values. This huge reduction in activation entropy was attributed to the different mechanisms of inactivation induced by heat and ultrasound. It is proved in this study that ultrasonically generated OH free radicals and shear forces, which arise from pulsation- or collapse of bubbles, both can destabilize the enzyme, although their contribution to the overall inactivation varies depending on the temperature and the tip diameter of the sonotrode.