*2.3. Biochemical Characterization of FsAlyPL6*

The optimal temperature of FsAlyPL6 is 45 ◦C and it retains more than 90% of maximal activity after being incubated at 45 ◦C for 1 h (Figure 4A). Compared with other PL6 family alginate lyases, FsAlyPL6 exhibits preferable thermal characteristics than most PL6 family alginate lyases. For example, AlyF of *Vibrio* OU02 showed the maximal activity at 30 ◦C [17] and AlyGC from *G. chathamensis* S18K6T has an optimal temperature of 30 ◦C [12]. OalC6 of *Cellulophaga* sp. SY116 exhibits highest activity at 40 ◦C and retains about 80% of highest activity after being incubated at 40 ◦C for 1 h [16]. In addition, FsAlyPL6 retains 95% activity after being incubated at 35 ◦C for 60 min and inactivated gradually with temperature increased (Figure 4B). This remarkable characteristic indicated FsAlyPL6 possesses great potential in industrial applications for preparation alginate oligosaccharides. The optimal pH of FsAlyPL6 is 9.0 and it retains about 90% activity incubated at pH 9.0–10.0 for 12 h (Figure 4C,D), which indicated FsAlyPL6 is an alkaline-stable lyase. To the best of our knowledge, few alginate lyases of PL6 family are alkaline-stable lyases, and most of them exhibit the maximal activities around neutral pH values such as OalC6 of *Cellulophaga* sp. SY116 has an optimal pH of 6.6 and it retains only 60% of its maximal activity after being incubated at pH 6.0 for 6 h [16]. The OalS6 from *Shewanella* sp. Kz7 exhibits maximal activity at pH 7.2 and retains 80% after being hatched at pH 6.0–8.0 for 24 h [15]. The influences of metal ions on enzyme activity were also investigated. As shown in Table 2, like TsAly6A from *Thalassomonas* sp. LD5 [14], the activity of FsAlyPL6 can be activated by Ca2<sup>+</sup> and Mg2<sup>+</sup>. FsAlyPL6 is inhibited by various divalent metal ions such as Cu2+, Zn2<sup>+</sup> and Ni2+, which is similar to OalS6 from *Shewanella* sp. Kz7 [15].

**Figure 4.** Biochemical characterization of FsAlyPL6: (**A**) The optimal temperature and thermal stability of FsAlyPL6; (**B**) the thermal-induced denaturation of FsAlyPL6; (**C**) the optimal pH of the FsAlyPL6; (**D**) the pH stability of FsAlyPL6.




**Table 2.** Effects of metal ions on activity of FsAlyPL6.

#### *2.4. Action Pattern and Substrate Docking of FsAlyPL6 Product Analysis*

To elucidate the action mode of FsAlyPL6, the degradation products of three substrates for different times (0–48 h) were analyzed by TLC (Figure 5). As the degrading process continues, three kinds of substrates are degraded into oligosaccharides with lower degrees of polymerization (DPs) (2–5) and monosaccharide, which indicated that FsAlyPL6 can cleave the glycosidic bonds within the substrates in an endolytic manner. The ESI-MS results indicated that degradation products of FsAlyPL6 towards the three different substrates include monosaccharide, and oligosaccharides with different DPs (2–5) can be detected (Figure 6A–C). Most of PL6 family enzymes are endo-type alginate lyases, which produce oligosaccharides with DPs (2–4). However, the Patl3640 from *Pseudoalteromonas atlantica* T6c [13], Pedsa0631 from *Pedobacter saltans* [13], OalS6 from *Shewanella* sp. Kz7 [15], and OalC6 from *Cellulophaga* sp. SY116 degrade the substrates into monosaccharides in an exolytic manner [16].

**Figure 5.** TLC analysis of degrading products of FsAlyPL6 towards alginate (**A**), polyM (**B**), and polyG (**C**). Lane M, the oligosaccharide standard; lanes 0–11, the samples taken by 0 min, 5 min, 10 min, 15 min, 30 min, 60 min, 2 h, 6 h, 12 h, 24 h, and 48 h, respectively.

**Figure 6.** ESI-MS analysis of products of FsAlyPL6 towards alginate (**A**), polyM (**B**), and polyG (**C**).

The three-dimensional model of the FsAlyPL6 was constructed by PHYRE2 and the tetrasaccharide (MMMM) was docked into the FsAlyPL6. Because the sequence similarity between FsAlyPL6 and AlyGC was high (45%), the protein model was successfully constructed with 100% confidence. As shown in Figure 7A, the overall structure of FsAlyPL6 was predicted to fold into a "twin tower-like" structure (Figure 7A), which is similar to the structure of AlyGC (Figure 7B). However, AlyGC is an exo-type alginate lyase and FsAlyPL6 degrade alginate into oligosaccharide in an endolytic manner. The key residues for substrate recognition were identified by the sequence alignment and protein–substrate interactions. As shown in Figure 7C, the residues R239, R263, K218, E213, and Y332 are were highly conserved and involved in the interaction between the protein and substrates in subsites −1, +1, +2 and +3, respectively (Figure 8A,B). Based on the docking and β-elimination mechanism, the residues K218 and R239 acted as the Brønsted base and Brønsted acid, respectively, in the cleavage reaction of FsAlyPL6 on alginate, which is consistent with the residues of AlyGC (Figure 8B).

**Figure 7.** (**A**) Overall structure of FsAlyPL6; (**B**) the structural comparison of FsAlyPL6 (green) and AlyGC (yellow); (**C**) sequence alignments of FsAlyPL6 and AlyGC.

**Figure 8.** (**A**) Stereo view of the alginate tetrasaccharide (MMMM) bound to the tunnel-shaped active site of FsAlyPL6. (**B**) The presentation of catalytic residues responsible for binding and catalyzing the substrate.

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

#### *3.1. Materials and Strains*

Sodium alginate (M/G ratio: 77/23) was purchased from Sigma-Aldrich (St. Louis, MO, USA). PolyG and polyM (purity: about 95%; M/G ratio: 3/97 and 97/3, respectively) were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). *Flammeovirga* sp. NJ-04 was isolated from the South China Sea and conserved in our laboratory. It was cultured at 35 ◦C in 2216E medium (Difoc). *Escherichia coli* DH5α and *E. coli* BL21 (DE3) were used for plasmid construction and as the hosts for

gene expression, respectively. These strains were cultured at 37 ◦C in Luria-Bertani (LB) broth or on LB broth agar plates (LB broth was supplemented with 1.5% agar and contained 100 μg/mL ampicillin).

#### *3.2. Cloning and Sequence Analysis of Alginate Lyase*

As previously reported, a gene cluster for degrading alginate has been identified within the genome of the strain *Flammeovirga* sp. NJ-04 [10]. According to the sequence of the putative alginate lyase gene sequence (WP\_044204792.1), a pair of special primers was designed as described in Supplementary Materials. For gene expression, the alginate lyase gene *FsAlyPL6* was subcloned and then ligated into pET-21a(+) expression vector. The theoretical molecular (Mw) and isoelectric point (*p*I) were calculated using Compute pI/Mw tool (https://web.expasy.org/compute\_pi/). Molecular Evolutionary Genetics Analysis (MEGA) Program version 6.0 (Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Tempe, AZ, USA) was applied to construct a phylogenetic tree through a neighbor-joining method based on alginate lyase protein sequences of PL6 family. The Vector NTI (Invitrogen, Thermo Scientific, Waltham, MA, USA) was used to obtain multiple sequence alignment. The homology modeling and docking was built by Protein Homology/analogY Recognition Engine V 2.0 (Structural Bioinformatics Group, Imperial College, London, Britain).
