*2.2. E*ff*ect of Ultrasonication on PPO Activity*

The PPO activities of the ultrasonicated protein solutions are presented in Figure 2A,B, respectively. As can be seen from Figure 2A, the PPO activity was very high. Ultrasonic processing at lower duration stimulated the activation of PPO. With constant power of 20 W/mL at different times (5, 10, 15, 20 and 30 min) in ultrasonic processing, the relative enzyme activity changed. The PPO activity increased with the increase of time up to 15 min and decreased with a further increase in time. As seen in Figure 2B, with constant ultrasonication for 20 min under different powers, the relative enzyme activity changed. With increasing ultrasonic power, a peak of the relative enzyme activity was observed at 20 W/mL. At above 20 W/mL, the relative enzyme activity began to reduce. Under an ultrasonic power of 40 W/mL, the enzyme activity was restrained; in particular, PPO activity was reduced by 48% under ultrasonic processing at 50 W/mL for 20 min.

These results indicate that low-intensity (20 W/mL or lower) ultrasonic processing stimulates the PPO activity, but high-intensity ultrasonic processing can inhibit the PPO activity. Previous studies demonstrated the accordant effect of ultrasonic processing on PPO activity [7,10,16]. The effect of ultrasonication on the PPO activity may be attributed to the ability of ultrasonic processing to break down the molecular aggregates. Ultrasonic processing at low intensity may dissociate protein monomers which results in the exposure of the active site to the substrate, causing the activation of the PPO enzyme [16]. However, the catalytic center was destroyed under strong ultrasonic processing and more treatment time, which might cause higher levels of denaturation and inactivation of PPO protein.

**Figure 2.** Relative activities of ultrasonic-processed purified PPO at 20 W/mL for 5, 10, 15, 20 and 30 min (**a**); processed for 20 min at 10, 20, 30, 40 and 50 W/mL (**b**).
