Recently, ultrasound was demonstrated to increase enzyme activity in food industry. In most studies, an enzyme-substrate mixture was ultrasonicated. Very little is reported on the ultrasonication of enzyme preparation; the effects of ultrasonication variables on the enzyme activity and properties remained unclear. In this study, an -amylase preparation was ultrasonicated under different conditions. At the ultrasonic frequency, power, temperature, and time of 20 kHz, 25 W/mL, 30 degrees C, and 75 s, respectively, the secondary structure of the -amylase was changed and that improved the catalytic activity by 47% over the control. The optimal temperature and pH of the -amylase preparation did not change under the ultrasonic treatment. In addition, ultrasonic treatment increased kinetic parameters of the -amylase including maximal velocity V-max, Michaelis constant K-m, turnover number K-cat, and catalytic specificity constant K-cat/K-m. Nevertheless, the ultrasonicated -amylase had lower thermal inactivation rate constant and half-life value than the nonultrasonicated enzyme. Ultrasonic treatment can be considered as a novel solution for improvement in enzyme activity. (c) 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:702-710, 2018