*4.4. Bioinformatics Analyses*

The amino acid sequence of Ps-Mn-SOD was translated using ExPASy translation tool (http: //web.expasy.org/translate/). The signal peptide, secondary structure, motif sequences, and 3D homology model were predicted by SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/), Scratch Protein Predictor (http://scratch.proteomics.ics.uci.edu/), InterPro Scan (http://www.ebi.ac. uk/InterProScan/), and Swiss model server (http://swissmodel.expasy.org/) [40], respectively. The physicochemical properties of Ps-Mn-SOD were predicted using ExPASy ProtParam tool (http://web. expasy.org/protparam/). The possible cleavage sites of trypsin and chymotrypsin on Ps-Mn-SOD were predicted using the peptide cutter software (http://web.expasy.org/peptide\_cutter/). Multiple alignments of Ps-Mn-SOD were processed using DNAMAN 7.0.2 software. Homology analysis was constructed by pairwise alignment tool (https://www.ebi.ac.uk/Tools/psa/emboss\_needle/). The neighbor-joining phylogenic tree was generated in MEGA 7.0 with bootstrap values 1000.

### *4.5. Enzyme Assays*

SOD activity was determined via spectrophotometric method using the SOD assay kit from Nanjing Jiancheng Institute of Biology and Engineering (Code No. A001-1-1, Nanjing, China). Each measurement point contained three replicates, and the results are shown as mean (n = 3) ± standard deviation (SD). The 1 × TBS was used as the blank control. One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of chromogen production at 550 nm.

The purified Ps-Mn-SOD was quantified, and residual activities were determined after incubation under different variables, including temperature, pH, chemicals, digestive enzymes, and high hydrostatic pressure. Considering temperature, proteins were treated from 0 ◦C to 80 ◦C for 15 min with an interval of 10 ◦C [33,34]. Proteins were treated at pH 2.2–13 for 1 h at 25 ◦C [4,34]. The enzymatic activity at optimum temperature and pH was set as 100%. With regard to chemicals, the proteins were mixed with an equal treatment solution at different final concentrations for 40 min at 25 ◦C [34,41]. The incubation time of urea, guanidine hydrochloride, hydrogen peroxide, and sodium azide was expanded to 1 h. The enzyme activity without chemicals was set as 100%. For proteolytic susceptibility assay, the mass ratio of recombinant Ps-Mn-SOD and trypsin/chymotrypsin complex was 1:100, and the group incubated for 0 h was considered with 100% enzyme activity [34,42]. For high hydrostatic pressure, proteins were treated at 0.1, 30, and 100 MPa for 2 h at 5 ◦C. The enzyme activity at 0.1 MPa was set as 100%, and bovine erythrocyte SOD was selected for comparison from atmospheric organism. Kinetics of Ps-Mn-SOD were measured as previously described by Li et al. [35].

#### *4.6. Statistical Analysis*

Independent sample *T*-test was used for statistical analysis for each of the two groups using SPSS 21.0 (IBM Company, Armonk, NY, USA); *p* < 0.05 was considered statistically significant.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/2/84/s1, Figure S1: The predicted 3D model of Ps-Mn-SOD. Red spheres represent manganese ions. (**A**) homodimer. (**B**) close-up of the manganese ion binding site. Figure S2: Analysis of SDS-PAGE. M: protein marker, Lane 1: total proteins before induction, Lane 2: total proteins after induction, Lane 3: inclusion body after ultrasonication, Lane 4: supernatant after ultrasonication, Lane 5: Western blot of recombinant protein, Lane 6: purified protein. Figure S3: The curve of kinetic parameters of Ps-Mn-SOD. Figure S4: SOD type assay. The result is expressed using specific activity. Table S1. Pairwise alignment analysis between Ps-Mn-SOD and other species. Table S2. The prediction of cleavage site of Ps-Mn-SOD.

**Author Contributions:** H.Z. collected the sample, Y.L. designed and performed the experiments, Y.L. and H.Z. prepared the manuscript, and X.K. gave advice during the experiments.

**Funding:** We thank J.C. for total RNA extraction. We also thank for language modification given by L.Y. in South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences. This work was We changed "supported by The National Key Research and Development Program of China (2017YFC0306600, 2018YFC0309804), Hundred Talents Program of CAS (SIDSSE–BR–201401).

**Conflicts of Interest:** The authors declare no conflict of interest.
