*2.7. The Thermodynamic Parameters of rPsLeuDH*

Thermodynamic parameters such as Δ*H*, Δ*S* and Δ*G* at different temperature (0–30 ◦C) were calculated and listed in Table 4. At 0, 10, 20, and 30 ◦C, the *k*cat value of rPsLeuDH were 12.25, 14.96, 20.20 and 30.13/s, respectively, indicating that the *k*cat value increased with increasing temperature, which was similar to the *k*cat change trend of cold-adapted β-D-galactosidase at different temperatures [26]. rPsLeuDH also exhibited lower Δ*H*, Δ*S* and Δ*G* and higher *k*cat at low temperature, as compared to mesophilic enzyme, which may be mainly related to the conformation of cold adapted protein [27]. On the other hand, it may also be related to increasing the efficiency of binding of the substrate to the catalytic site [28].

**Table 4.** Thermodynamic parameter of the rPsLeuDH.


### **3. Materials and Methods**

#### *3.1. Microorganisms and Growth Conditions*

The strain *Pseudoalteromonas* sp. ANT178, isolated from sea ice in Antarctica (68◦30 E, 65◦00 S), was used as a source of *psleudh* gene. The strain ANT178 was cultivated in the 2216E sea water medium (initial pH 7.5, 5 g/L peptone, and 1 g/L yeast extract) for 96 h at 12 ◦C. *E. coli* BL21 (DE3) was used as the plasmid host.

#### *3.2. Sequence Analysis of LeuDH Gene*

The open reading frame and amino acid sequences of *psleudh* gene were computed (https://www. ncbi.nlm.nih.gov/orffinder/). The theoretical molecular weight and p*I* were also analyzed using the ExPASy Compute p*I*/Mw tool (http://web.expasy.org/computepi). Multiple sequence alignment of the amino acids of PsLeuDH was performed using Bioedit 7.2 and ESPript 3.0 [29].

#### *3.3. Protein Homology Modeling*

A homology model of LeuDH was built with SWISS-MODEL. LeuDH from mesophilic bacteria *Bacillus sphaericus* ATCC4525 (PDB ID:1LEH) [16] was selected as the template. The structure figures were created with PyMOL software (DeLano Scientific LLC, San Carlos, CA, USA). Salt bridges were carried out using VMD 1.9.3. (University of lllinois Urbana-Champaign, Champaign, IL, USA). For the hydrogen bonds, a cut-off distance of 3.3 Å was set. Cation-pi interactions, aromatic interactions, ionic interactions, and hydrogen bonds were predicted by the Protein Interactions Calculator program (http://pic.mbu.iisc.ernet.in).

#### *3.4. Molecular Cloning, Expression and Purification of rPsLeuDH*

The genome of *Pseudoalteromonas* sp. ANT178 was sequenced and annotated using high-throughput technologies (data not shown). The full-length gene of *psleudh* was amplified by PCR using the primers 5 -GATGGATCCATGGAATTT TTATGTG-3 (*Bam*HI site underlined) and 5 -CAGAAGCTTGAAGACCGTTTT TAAG-3 (*Hin*dIII site underlined) according to its genome sequence. PCR was performed with Taq DNA polymerase (TaKaRa Bio, Dalian, China). The product was then directly cloned into the corresponding sites of the pET-28a (+) vector and transformed into *E. coli* BL21. The transformants with the *psleudh* gene were grown in Luria-Bertani (LB) medium supplemented with 100 mg/L kanamycin and cultured by shaking at 37 ◦C until the OD600 reached 0.6–0.8. Then, 1.0 mM sopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction. The bacterial cells were cultured at 37 ◦C for 2–3 h, and then the culture temperature was shifted to 28 ◦C to induce the protein expression for 6 h. The induced cells centrifuged at 4 ◦C and 7500× *g* for 15 min and subjected to ultrasonic disruption with 150 W (JY96-IIN, Shanghai, China). The insoluble debris was removed by centrifuged at 4 ◦C and 7500× *g* for 15 min, and the supernatant was harvested as crude protein (21.99 mg). Purification of rPsLeuDH with the His-tagged was purified using Ni-NTA affinity chromatography. The purified protein (1.11 mg) was eluted with 10, 50, 100 and 250 mM imidazole buffer (20 mM Tris-HCl, 500 mM NaCl, pH 8.0) at a flow rate of 1.0 mL/min. The purity and the molecular mass of the rPsLeuDH were determined by SDS-PAGE, using 12.0% polyacrylamide gels.

#### *3.5. Assay of rPsLeuDH Activity*

The standard enzyme assay were based on traditional method and modified on basis [1,30]. The oxidation reaction activity assay was determined by 200 μL reaction system. It contained 0.1 M Glycine-NaOH (pH 10.4) buffer, 10 mM L-leucine and 10 μL purified enzyme (0.62 μg), which incubated at 30 ◦C for 2 min. After adding 1 mM NAD+, the changes of absorbance at 340 nm within 1 min were detected. Futhermore, the reductive amination reaction system containing (200 μL) 0.2 M NH4Cl-NH4OH buffer (pH 9.0), 5 mM TMP and 10 μL purified enzyme at 30 ◦C for 2 min, after

adding 0.2 mM NADH, changes in absorbance at 340 nm within 1 min were measured. One unit of LeuDH activity was defined as the amount of enzyme catalyzed the formation or reduction of 1 μmoL NADH/min at 30 ◦C.
