Ultrasound-Assisted Optimization of the Activation and Inactivation of Thermostable α-Amylase

Ultrasound is a non-thermal technology increasingly applied in food processing to modulate enzyme activity. This study investigated the effects of ultrasonic irradiation on the activity of a commercial thermostable α-amylase. Enzyme activity was determined by quantifying reducing sugars released from starch using the 3,5-dinitrosalicylic acid method, and protein concentration was measured by the Bradford assay. A one-factor-at-a-time approach was used to evaluate the effects of ultrasonic amplitude, treatment time, enzyme concentration, incubation temperature, and calcium ion concentration. Subsequently, a Box-Behnken design was applied to analyze the combined influence of amplitude, treatment duration, temperature, and calcium concentration on residual activity. The enzyme exhibited an initial activity of 46.27 ± 3.63 U/mL under standard assay conditions. Moderate ultrasonic amplitudes enhanced activity, whereas prolonged exposure and elevated temperatures promoted inactivation. Statistical analysis showed that the incubation temperature and calcium concentration significantly influenced residual activity, and the quadratic model provided a good fit (R² = 0.94). Optimal inactivation conditions were identified at 60% amplitude, 9 min treatment, 85 °C, and 9 ppm calcium, resulting in 66.3% enzyme inactivation. These findings support the use of ultrasound-assisted processing as a controllable strategy to regulate thermostable α-amylase activity in industrial enzyme applications.