*2.3. Expression, Purification, and Validation of Ps-Mn-SOD*

The Ps-Mn-SOD gene was expressed with a His-tag in *E. coli*. Supplementary Figure S2 shows the SDS-PAGE analysis results. Recombinant Ps-Mn-SOD was expressed under 0.1 mM IPTG at 15 ◦C for 24 h and produced a distinct band at approximately 30 kDa, consistent with the previously estimated molecular weight (Supplementary Figure S2, lanes 1 and 2). The protein was purified under native conditions due to its highly soluble expression in the supernatant (Supplementary Figure S2, lanes 3 and 4). The maximum protein yield approximated 4.39 mg/L culture. Western blot analysis was performed to verify its successful expression (Supplementary Figure S2, lanes 5 and 6).

#### *2.4. Characterizations of Ps-Mn-SOD*

#### 2.4.1. Effects of Temperature on Ps-Mn-SOD

The activity of Ps-Mn-SOD was determined from 0 ◦C to 80 ◦C, with the optimum temperature observed at 0 ◦C. A stable activity was observed at low temperatures, with > 70% activity highlighted from 0 ◦C to 60 ◦C. The activity was maintained at 2.53% at 70 ◦C and lost at 80 ◦C (Figure 4A).

**Figure 4.** Effects of temperature (**A**), pH (**B**), urea and guanidine hydrochloride (**C**), and high hydrostatic pressure (**D**) on Ps-Mn-SOD. Ps-SOD and Be-SOD represent SOD from *Paelopatides* sp. and bovine erythrocytes, respectively.
