*2.2. Expression and Purification of FsAlyPL6*

The gene of FsAlyPL6 was ligated into pET-21a(+) and then the recombinant plasmid was transformed into *E. coli BL21* (DE3) for heterologously expression. The recombinant FsAlyPL6 was then purified by Ni-NTA sepharose affinity chromatography and analyzed by SDS-PAGE (Figure 3). A clear band (about 80 kDa) can be observed in gel, which was consistent with the theoretical molecular mass of 83.09 kDa. Three kinds of substrates (sodium alginate, polyM, and polyG) were employed to determine the substrate specificity of FsAlyPL6. As shown in Table 1, FsAlyPL6 exhibited higher activity towards sodium alginate (483.95 U/mg) and it showed lower activity towards to polyM (221.5 U/mg). However, it showed the lowest activity towards to polyG (19.35 U/mg). Accordingly, FsAlyPL6 is a polyMG-preferred lyase like most of PL6 family alginate lyases with the exceptions of Patl3640 from *Pseudoalteromonas atlantica* T6c and Pedsa0631 from *Pedobacter saltans* [13]. Both of them preferred polyG to polyMG blocks. In addition, TsAly6A from *Thalassomonas* sp. LD5 [14], OalS6 from *Shewanella* sp. Kz7 [15], OalC6 from *Cellulophaga* sp. SY116 [16], and AlyF from *Vibrio* sp. OU02 [17] are all characterized as polyG-preferred alginate lyases. The kinetic parameters of FsAlyPL6 towards sodium alginate, polyM, and polyG were calculated based on the hyper regression analysis. As shown in Table 1, the *Km* values of FsAlyPL6 towards sodium alginate, polyM, and polyG were 0.50 mg/mL, 1.52 mg/mL, and 1.62 mg/mL, respectively. FsAlyPL6 had a lower *Km* value towards sodium alginate than to polyM and polyG. Accordingly, FsAlyPL6 exhibited higher affinity towards MG-block than to M-block and G-block. The *kcat* values of FsAlyPL6 towards sodium alginate, polyM and polyG were 33.98 s<sup>−</sup>1, 17.66 s−1, and 4.98 s−1, respectively. It indicated that FsAlyPL6 had higher catalytic efficiency towards MG-block than to the other two blocks.

**Figure 1.** Multiple amino acid sequences alignment of AlyPL6 and other alginate lyases of PL6 family: AlyGC (BAEM00000000.1) from *Glaciecola chathamensis* S18K6T, polysaccharide lyase (ABD79298.1) from *Saccharophagus degradans* 2–40, and TsAly6A (MF958451) from *Thalassomonas* sp. LD5. Three boxes enclose conserved regions. Residues in FsAlyPL6, which are responsible for the enzymatic activity Ca2<sup>+</sup> binding and catalysis, are marked in triangle, dots, and stars, respectively.

**Figure 2.** Phylogenetic analysis of FsAlyPL6 with other alginate lyases of PL6 family based on amino acid sequence comparisons. The species names are indicated along with accession numbers of corresponding alginate lyase sequences. Bootstrap values of 1000 trials are presented in the branching points. The subfamilies 1, 2, and 3 are marked with stars, dots, and triangle, respectively.

**Figure 3.** Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified FsAlyPL6. Lane M protein: restrained marker (Thermo Scientific, Waltham, MA, USA); lane 1: purified FsAlyPL6.

**Table 1.** Specificity and kinetics of FsAlyPL6.

