*2.3. Expression and Purification of the rPsLeuDH*

The gene coding for the PsLeuDH was cloned into the pET-28a (+) vector and expressed in *E. coli* BL21 (DE3) under IPTG induction (Figure 3, Lane 3). rPsLeuDH was purified in a single step using His-tag affinity chromatography. A major band was observed on SDS-PAGE with about the molecular weight 44.4 kDa (Figure 3, Lane 4, 5). It is noteworthy that the last purified rPsLeuDH exhibited the highest specific activity of 275.13 U/mg.

**Figure 3.** Expression and purification analysis of PsLeuDH. Lane 1: molecular weight standard marker; Lane 2: crude extract from the BL21/pET-28a (+); Lane 3: crude extract from the BL21/pET-28a (+)-PsLeuDH with IPTG induction; Lane 4: rPsLeuDH eluted with 50 mM imidazole; Lane 5: rPsLeuDH eluted with 100 mM imidazole.

#### *2.4. Effects of Temperature and pH on Activity and Stability of rPsLeuDH*

The temperature characteristic of rPsLeuDH was shown in Figure 4a. It exhibited the highest activity at 30 ◦C, and that of a cold-adapted LeuDH was 30 ◦C [12], whereas thermophilic LeuDH was approximately 40–65 ◦C [6,22], or (60–75 ◦C) [5]. It is worth pointing out that rPsLeuDH retained 40% of the highest activity at 0 ◦C, suggested that the enzyme is a cold-adapted enzyme [23]. Furthermore, the thermostability of rPsLeuDH was assessed in Figure 4b. It was stable and retained 85% of its initial activity after incubating at 30 ◦C after 120 min. While, after incubating at 50 ◦C for 20 min, it was only 30% of its activity lower than other cold-adapted LeuDHs from *Alcanivorax dieselolei* [12] and *Sporosarcina psychrophila* [7]. However, thermostable LeuDH could retain full activity after incubation at 65 ◦C for 10 min [24]. The above results indicated that rPsLeuDH had thermal instability, which was another significant feature of cold-adapted enzyme [25]. The effect of pH on rPsLeuDH activity was shown in Figure 4c. The activity of rPsLeuDH was higher under alkaline conditions (pH 7.0–10.0), with the highest activity at pH 9.0. Similar results were described in other LeuDHs such as *Sporosarcina psychrophile* (pH 8.5–11.0) [7], *Laceyella sacchari* (pH 9.5–11) [6] and *Citrobacter freundii* (pH 9.0 to 11.0) [5]. After 30 min of exposure to pH 6.0–10.0, the stability of rPsLeuDH showed a similar pattern with that of the activity response to pH (Figure 4d). This broad range of pH dependence for the activity and stability made the rPsLeuDH probably useful for medical industrial applications.

**Figure 4.** Effects of temperature and pH on the activity and stability of rPsLeuDH. (**a**) Effect of temperature on the activity of rPsLeuDH. (**b**) Effect of temperature on the stability of rPsLeuDH. (-) 30 ◦C, () 40 ◦C, () 50 ◦C. (**c**) Effect of pH on the activity of rPsLeuDH. (**d**) Effect of pH on the stability of rPsLeuDH. Data are presented as mean ± SD (*n* = 3).

#### *2.5. Effects of NaCl Concentration and Different Reagents on the Activity of PsLeuDH*

The effect of NaCl concentration on the rPsLeuDH activity was shown in Figure 5. It could be seen that rPsLeuDH was stable at 0–3.0 M NaCl, with the highest activity at 2.0 M NaCl, which may be related to high salinity in the Antarctic sea ice environment. The similar result was also found in LeuDH from *Bacillus licheniformis* [3] and *Thermoactinomyces intermedius* [24] after high salt concentration treatment. The effect of various reagents on the rPsLeuDH activity was listed in Table 2. rPsLeuDH was completely inhibited by 1 mM Pb(NO3)2 and BaCl2. Inhibitions by 1 mM CrCl2 and CdCl2 were 86.7% and 92.4%, respectively, while only partially inhibited by other metals salt in some extent. In addition, rPsLeuDH was sensitive to Thiourea and ethanol, but Triton X-100 kept the enzyme activity.

**Figure 5.** Effect of salt concentration on the activity of rPsLeuDH.


**Table 2.** Effects of different reagents on the activity of rPsLeuDH.
