*3.3. Hereologous Expression and Purification of the Recombinant Enzyme*

The recombinant plasmid pET-21a(+)–*FsAlyPL6* was transformed into *E. coli* BL21 (DE3). It was then cultured in an LB medium (containing100 μg/mL of ampicillin) at 37 ◦C by shaking at 200 rpm for 5 h, followed by being induced with 0.1 mM IPTG at 25 ◦C for 36 h when OD600 reached 0.6. The purification of FsAlyPL6 was performed as follows. The cells were harvested by centrifugation and then sonicated in lysis buffer (50 mM Tris-HCl with 300 mM NaCl, pH 8.0). The cell homogenate that contained recombinant protein were purified by using a His-trap column (GE Healthcare, Uppsala, Sweden). SDS on 12% (*w*/*v*) resolving gel was applied to detect the purity of the recombinant protein.

#### *3.4. Substrate Specificity and Enzymatic Kinetics*

The reaction was performed using 20 μL FsAlyPL6 (4 μg) mixed with 180 μL 0.8% alginate, polyM, and polyG respectively. The enzyme activity was measured using the ultraviolet absorption method [11]. One unit was defined as the amounts of enzyme required to increase absorbance at 235 nm (extinction coefficient: 6150 M−1·cm<sup>−</sup>1) by 0.1 per min. The kinetic parameters of the FsAlyPL6 towards alginate, polyM, and polyG were investigated by measuring the enzyme activity with these substrates at different concentrations (0.4–10 mg/mL). Velocity (V), *Km*, and *Vmax* values were calculated as previously reported [10]. The radio of *Vmax* versus enzyme concentration ([*E*]) was used to calculate the turnover number (*k*cat) of the enzyme.

#### *3.5. Biochemical Characterization of the Recombinant Enzyme FsAlyPL6*

The effects of temperature on the enzyme activity were determined by testing the activity at different temperatures (35 ◦C to 60 ◦C). The thermal stability was characterized by measuring the residual activity after the purified FsAlyPL6 was incubated at 35–60 ◦C for 1 h. Furthermore, the thermally induced denaturation was also determined by measuring the residual activity after incubating the enzyme at 35–50 ◦C for 0–60 min. To investigated the optimal pH of the FsAlyPL6, 1% alginate mixed with different buffers at 45 ◦C (50 mM phosphate–citrate (pH 4.0–5.0), 50 mM NaH2PO4–Na2HPO4 (pH 6.0–8.0), 50 mM Tris–HCl (pH 7.0–9.0), and glycine–NaOH (pH 9.0–12.0)) were used as the substrates and the purified enzyme incubated with these substrates under standard conditions. Moreover, the pH stability was evaluated based on the residual activity after being incubated with indifferent buffers (pH 4.0–12.0) for 20 h. The effects of metal ions on the enzymatic activity were performed by incubating the FsAlyPL6 with substrates that contained various metal compounds with a final concentration of 1 mM. The reaction performed under standard tested conditions and the substrates blend without any metal ion was taken as the control.

#### *3.6. Action Pattern and Degradation Product Analysis*

In order to elucidate the action pattern of the FsAlyPL6, the thin-layer chromatography (TLC) was applied to analyze the degrading products of FsAlyPL6 towards sodium alginate, polyM and polyG. The reaction and treatment of the samples were performed as previously reported [10]. In order to investigate the composition of the degrading products, ESI-MS was employed as follows: The supernatant (2 μL) was loop-injected to an LTQ XL linear ion trap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) after centrifugation. The oligosaccharides were detected in a negative-ion mode using the following settings: ion source voltage, 4.5 kV; capillary temperature, 275–300 ◦C; tube lens, 250 V; sheath gas, 30 arbitrary units (AU); and scanning the mass range, 150–2000 *m*/*z*.

#### *3.7. Molecular Modeling and Docking Analysis*

Protein Homology/analogY Recognition Engine V 2.0 was applied to construct the three-dimensional structure of FsAlyPL6 according to the known structure of alginate lyase AlyGC from *Glaciecola chathamensis* S18K6T (PDB: 5GKD) with a sequence identity of 45%. The molecular docking of the FsAlyPL6 and MMMM was performed using Molecular Operating Environment (MOE, Chemical Computing Group Inc., Montreal, QC, Canada). The ligand-binding sites were defined using the bound ligand in the homology models. PyMOL (http://www.pymol.org) was used to visualize and analyze the modeled structure and to construct graphical presentations and illustrative figures.

#### **4. Conclusions**

In this study, we reported a new PL family alginate lyase FsAlyPL6 from the marine *Flammeovirga* sp. NJ-04. It preferred to degrade the polyMG block and showed highest activity at 45 ◦C and could retain 50% of activity after being incubated at 45 ◦C for 1 h. The FsAlyPL6 endolytically degraded alginate polysaccharide and released oligosaccharides with DPs of 1–5. In addition, it could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3 to release oligosaccharides. The research provides extended insights into the degradation pattern of PL6 alginate lyases and further expands the application of alginate lyases.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/17/6/323/s1, Table S1: The primers for cloning the gene of FsAlyPL6.

**Author Contributions:** Q.L. and F.H. conceived and designed the experiments; B.Z., Q.L., and F.H. performed the experiments; Y.S., Y.S., and Z.Y. analyzed the data; B.Z. wrote the paper. All authors reviewed the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant number: 31601410 and 21776137).

**Acknowledgments:** The work was supported by the National Natural Science Foundation of China (grant numbers: 31601410 and 21776137).

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

#### **References**


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