2.4.2. Effects of pH on Ps-Mn-SOD

The activity of recombinant Ps-Mn-SOD was measured under pH 2.2–13.0, with an optimum pH observed at 10.5 (Figure 4B). Ps-Mn-SOD could resist extreme pH values (> 20% at pH 3.0–13.0) and showed optimal activity (> 70%) at pH 5.0–12.0.

#### 2.4.3. Effects of Chemicals on Ps-Mn-SOD

The effects of metal ions on Ps-Mn-SOD activity were determined at 0.1 or 1 mM final concentration (Table 1). Ps-Mn-SOD activity was inhibited by Mn2+, Co2+, Ni2+, Zn2+, and 1 mM Cu2+ and Ba2+. In particular, Co2+ showed more significant inhibition effect on Ps-Mn-SOD activity. Mg2+ and Ca2+ showed minimal effects.

Table 2 provides the effects of inhibitors, detergents, and denaturants on Ps-Mn-SOD activity. Ps-Mn-SOD activity was strongly inhibited by ethylene diamine tetraacetic acid (EDTA) and SDS and especially sensitive to SDS. Reductant dithiothreitol (DTT) and β-mercaptoethanol (β-ME) minimally affected enzyme activity. Detergents of Tween 20, Triton X-100, and Chaps slightly enhanced enzyme activity at 0.1% concentration.


**Table 1.** Effects of metal ions on Ps-Mn-SOD. \*\* *p* < 0.01.

**Table 2.** Effects of inhibitors, reductant, and detergents. \* *p* < 0.05; \*\* *p* < 0.01.


The enzyme could resist the strong denaturation of urea and guanidine hydrochloride (Figure 4C) and maintain an almost full activity after 1 h treatment in 5 M urea or 4 M guanidine hydrochloride.

Hydrogen peroxide and sodium azide were used to determine the SOD type (Figure 5 and Supplementary Figure S4). After treatment of the recombinant Ps-Mn-SOD using 10 mM hydrogen peroxide and sodium azide at 25 ◦C for 1 h, the relative activities were 7.73% and 90.39%, respectively. This showed that the SOD from *Paelopatides* sp. belongs to Fe/Mn-SOD family, in accordance with previous phylogenetic analysis and 3D structure prediction.

**Figure 5.** SOD type assay.

#### 2.4.4. Effects of Digestive Enzymes on Ps-Mn-SOD

Digestion experiment was performed to test the stability of recombinant Ps-Mn-SOD in digestive fluid. Residual enzyme activity was measured after different incubation times for 0–4 h at 37 ◦C and pH 7.4. As shown in Table 3 and Supplementary Table S2, although the Ps-Mn-SOD sequence putatively contains 30 chymotrypsin and 23 trypsin cleavage sites, the enzyme could still maintain intact activity after 4 h treatment at an enzyme/substrate (*w*/*w*) ratio of 1/100.

**Table 3.** Cleavage effect of digestive enzyme on Ps-Mn-SOD at different time periods. Results are shown as mean (*n* = 3) ± SD. \*\* *p* < 0.01.


#### 2.4.5. Effects of High Hydrostatic Pressure on Ps-Mn-SOD

As shown in Figure 4D, the recombinant Ps-Mn-SOD could maintain full activity with increasing hydrostatic pressure until 100 MPa. By contrast, the SOD from bovine erythrocytes exhibited reduced activity of 84.57% when the pressure reached 100 MPa.

#### 2.4.6. Kinetic Parameters

The kinetic parameters of recombinant Ps-Mn-SOD were determined using a series of xanthine (0.006–0.6 mM) concentrations at 37 ◦C and pH 8.2 (Supplementary Figure S3) based on the Michaelis–Menten equation. The Km and Vmax values of Ps-Mn-SOD were 0.0329 ± 0.0040 mM and <sup>9112</sup> ± 248 U/mg, respectively. The R2 value of the curve fitting was 0.9815.

#### **3. Discussion**

Mn-SODs are predominantly found in mitochondria, as the first line of antioxidant defense, which are involved in cellular physiology, such as cell impairment and immune-responsive [8]. The important biological functions of Mn-SODs have attracted increasing attention among researchers. Novel Mn-SODs with remarkable characteristics will have great applications in food, cosmetic, and pharmaceutical industries. In the present study, a novel and kinetically stable Mn-SOD derived from hadal sea cucumber was cloned, expressed, and characterized.

Based on preliminary data, the Ps-Mn-SOD is frigostabile, consistent with the fact that the protein was derived from hadal area, which maintained > 90% activity below 20 ◦C with the optimum

temperature observed at 0 ◦C. In contrast, Mn-SOD from ark shell, *Scapharca broughtonii*, showed <40% activity below 20 ◦C [4]. Mn-SOD from seahorse, *Hippocampus abdominalis*, showed <80% and continuously reduced activity below 20 ◦C [8].

Mn-SODs in several sources have been found to function at wide pH values. For example, a hyperthermostable Mn-SOD from *Thermus thermophilus* HB27 maintained >70% activity at pH 4.0–8.0 [29]; Mn-SOD from deep-sea thermophile *Geobacillus* sp. EPT3 maintained >70% activity at pH 7.0–9.0 [30]; and Mn-SOD from *Thermoascus aurantiacus* var. *levisporus* only maintained >40% activity at pH 6.0–9.0 [31]. In contrast, the present Ps-Mn-SOD could maintain >70% activity at pH 5.0–12.0, showing remarkably wide pH values adaptation. Furthermore, after 1 h treatment in extremely acidic (pH 2.2) or alkaline (pH 13.0) conditions, Ps-Mn-SOD still maintained ~20% activity, showing remarkable stability to extreme pH values. The pH assays also showed that Ps-Mn-SOD is more stable under alkaline (pH 8.5–12.0) than acidic (pH 2.2–5.0) conditions. Metal ligands may undergo protonation at low pH but exhibit stability in alkaline conditions [32]. Similar studies on seahorse and bay scallop SODs were also reported [8,33].

Ps-Mn-SOD is relatively stable in chemicals, such as urea, guanidine hydrochloride, β-ME, DTT, etc. It maintained almost 100% activity after 1 h treatment of 5 M urea or 4 M guanidine hydrochloride at 25 ◦C, showing excellent resistance to strong protein denaturants. By comparison, the Mn-SOD from deep-sea thermophile *Geobacillus* sp. EPT3 maintained > 70% residual activity in 2.5 M urea or guanidine hydrochloride after 30 min treatment [30]. Fe-SOD from Antarctic yeast *Rhodotorula mucilaginosa* showed relatively low tolerance to urea [34]. However, based on our obtained data (unpublished and [35]), SODs from hadal sea cucumbers constantly exhibited excellent resistance to perturbation of denaturants. In addition, Ps-Mn-SOD maintained 97.00% and 99.22% residual activity after 1 h treatment of 10 mM DTT and 1% Triton X-100, respectively. While Mn-SOD from deep-sea thermophile *Geobacillus* sp. EPT3 only maintained 84.10% and 70.30% activity after 30 min treatment of corresponding chemicals [30].

As expected, Ps-Mn-SOD could also resist the perturbation by high hydrostatic pressure compared to the homolog from atmospheric pressure organism, because it was derived from a hadal field. Given the limitations of our equipment, the experiment was not performed at pressure more than 100 MPa. In fact, Ps-Mn-SOD might resist >100 MPa hydrostatic pressure. Similar results have been reported in other deep-sea enzymes, such as RNA polymerase from *Shewanella violacea* [36], N-acetylneuraminate lyase from *Mycoplasma* sp. [37], and lactate dehydrogenase b from *Corphaenoides armatus* [38]. Nonetheless, the sensitivity of enzymes to high hydrostatic pressure is not always related to the depth where the organisms lived. For example, two polygalacturonases from the hadal yeast *Cryptococcus liquefaciens* strain N6 exhibited an almost constant activity from 0.1 to 100 MPa. While, at the same pressure, polygalacturonase from *Aspergillus japonicus,* which lives under atmospheric pressure, increased by approximately 50% [39]. However, limited studies reported in detail the pressure assays of SODs, proving the difficulty in the interpretation of their pressure tolerance mechanism.

Altogether, these features render Ps-Mn-SOD a potential candidate in the biopharmaceutical and nutraceutical fields.

#### **4. Materials and Methods**

#### *4.1. Material and Reagents*

Hadal sea cucumber was collected at the depth of 6500 m in the Mariana Trench (10◦ 57.1693 N 141◦ 56.1719 E). Total RNA was extracted using RNeasy Plus Universal Kits from Qiagen, Hilden, Germany, and reverse-transcribed to cDNA. The transcriptome was obtained by sequencing assembly and annotation by Novogene Company (Tianjin, China). The following reagents were purchased from Takara, Tokyo, Japan: PrimeScriptTM II 1st strand cDNA Synthesis Kit, PrimeSTAR® GXL DNA Polymerase, *E. coli* DH5α, and pG-KJE8/BL21 competent cells, pCold II vector, restriction enzymes *BamH I,* and *Pst I*, T4-DNA ligase, and DNA and protein markers. The 1 mL Ni-NTA affinity column, BCA protein assay kit, primers, and trypsin/chymotrypsin complex (2400:400) were obtained from Sangon Biotech Company, Shanghai, China. Polyvinylidene difluoride (PVDF) membrane was obtained from Millipore Company, USA. The primary (ab18184) and secondary antibodies (ab6789) were obtained from Abcam, Cambridge, UK. Pierce™ ECL Plus Western blot analysis substrate was obtained from ThermoFisher, Waltham, MA, USA.

#### *4.2. Cloning and Recombinant*

For the manganese SOD (Ps-Mn-SOD) gene, the Mn-SOD sequences of Holothuroidea in GenBank were submitted to the transcriptome database of *Paelopatides* sp. to run a local blast using Bioedit 7.0 software. The open reading frame (ORF) of Ps-Mn-SOD (deleted signal peptide) was amplified by primers Ps-Mn-SOD-S: CGGGATCCAAGGCTCCGTATGAAGGCCTGGAGA and Ps-Mn-SOD-A: AACTGCAGTCACAATTCTTCATGTTTAGATGGC using the cDNA as template (the underlined restriction enzyme sites). The sequence was submitted to GenBank database with accession numbers MK182093. The purified and digested PCR product was ligated with pCold II vector. The recombinant plasmids, that is, pCold II-Ps-Mn-SOD, were transformed into *E. coli* DH5α, and positive clones were verified by sequencing.

#### *4.3. Protein Overproduction, Purification, and Confirmation*

The recombinant plasmids were transformed into *E. coli* chaperone competent cells pG-KJE8/BL21, which were inoculated in liquid Luria-Bertani medium (containing 100 μg/mL ampicillin, 20 μg/mL chloramphenicol, 0.5 mg/mL L-arabinose, and 2 ng/mL tetracycline), proliferated at 37 ◦C until the OD600 reached 0.4–0.6, cooled on an ice–water mixture for 40 min, added isopropyl β-D-1-thiogalactopyranoside (IPTG) with a final concentration of 0.1 mM, and then incubated for 24 h at 15 ◦C to produce the recombinant protein. Cells were harvested, washed with **1** *×* phosphate-buffered saline, resuspended in binding buffer (50 mM Na3PO4, 300 mM NaCl, and 20 mM imidazole, pH 7.4), and then sonicated on ice. The supernatant harboring the recombinant protein was separated from cell debris by centrifugation at 12000 g and 4 ◦C for 20 min and then applied to 1 mL Ni-NTA column for purification of the target protein based on its 6× His-tag, according to the manufacturer's instructions. The harvested target protein was dialyzed with 1 × tris buffered saline (TBS) at 4 ◦C for 24 h against three changes of 1 × TBS and finally stored at −80 ◦C for further experiments. The expression condition was analyzed on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed using Western blot analysis. The recombinant protein on 12% SDS-PAGE gel was transferred to a PVDF membrane, which was successively incubated with primary (diluted 1:5000) and secondary antibodies (diluted 1:10000), dyed with Pierce™ ECL Plus Western blot analysis substrate, and detected under chemiluminescent imaging system. Additional details were as described by Li et al. [35].
