*2.4. Fluorescence Spectroscopic Analysis*

The fluorescence property of the native and ultrasonicated purified enzyme was studied by fluorescence spectroscopy. It is a valuable technique to investigate the transition in the tertiary structure of proteins because the fluorescence from the tryptophan of amino acid is sensitive to the polarity of its local environment [16,22]. As shown in Figure 4, the λmax of the native protein was 334 nm with an intensity of 39.74, signifying that Trp residues in enzymes were located in the nonpolar hydrophobic environment. Figure 4a shows the effect of the ultrasonic duration (5, 10, 15, 20 and 30 min at 20 W/mL) on the intrinsic fluorescence spectrum of protein. The ultrasonically treated enzyme showed a red-shift of 0.6–1.2 nm in λmax, with a gradual decrease in fluorescence intensity following the increasing treatment duration. This finding indicated the exposure of Trp residues to the polar environment, thereby disrupting the tertiary structure. More exposure of fluorophores to the polar environment might cause the release and transfer of energy, which consequently leads to the quenching of fluorescence intensity [23,24].

Figure 4B illustrates the effect of increasing ultrasonic intensities (10 W/mL to 50 W/mL) on the fluorescence spectra of purified protein. The purified protein showed red-shifts of 0.2–2.0 nm in λmax when treated with low ultrasonic intensities of 10, 20 and 30 W/mL. In the meantime, the fluorescence intensity gradually decreased with increasing ultrasonic intensity. The fluorophores were exposed to a more polar environment due to the polydispersity of protein aggregate after ultrasonic processing as described above, which caused the red-shift and consequently led to the quenching of fluorescence intensity. However, the extreme ultrasonic processing at 50 W/mL induced aggregation and may bury the exposed fluorophores inside the molecules, thereby decreasing the λmax. PPO structural change is responsible for the activity change. The changes in fluorescence intensity and λmax indicated a possible change in PPO's tertiary structure, which ultimately led to the activity reduction of PPO [25]. The result was similar to the observation that the fluorescence intensity of mushroom tyrosinase decreased in an aqueous system following mild thermal and supercritical CO2 treatments [24].

**Figure 4.** Fluorescence spectra of native and ultrasonic-processed 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**).

#### *2.5. Circular Dichroism Spectroscopy Analysis*

The secondary structures of the native and ultrasonicated enzyme were analyzed through CD spectroscopy. The CD spectra of protein are shown in Figure 5a,b. The native protein showed a positive peak at 193 nm with two double-negative slots (208 and 222 nm), which were considered as typical α-helix conformation in the secondary structures [16,26,27]. As shown in Figure 5a,b, the negative peak (208 nm) increased with the negative peak decrease at 222 nm after ultrasonic processing. As the ultrasonic time and intensity increased, the change grew and the two double-negative slots gradually disappeared. These changes show that the ultrasonic processing triggered α-helix conformation of secondary structure loss [3,16,26–28]. The contents based on the CONTIN algorithms of the protein secondary structure of the native and ultrasonicated protein [24] are shown in Table 1. At the high ultrasonic intensity of 40 W/mL, the α-helix conformation remarkably decreased, while β-turn contents were increased. A similar study was reported by Liu et al. [10], where ultrasonic processing at high intensity caused a loss of α-helix conformation in protein structure. The decrease in α-helix was found to be correlated with the enzymatic activity of PPO molecules [29]. Ultrasonic processing induced the changes in molecular interaction, thus leading to changes in secondary structure and eventually causing the loss of PPO activity.

**Figure 5.** Circular Dichroism (CD) spectra of native and ultrasonic-processed PPO at 20 W/mL for 15, 20 and 30 min (**a**); processed for 20 min at 10, 20 and 40 W/mL (**b**).


**Table 1.** Secondary structure contents of native and ultrasonic-processed PPO.
