*2.1. Sequence Analysis of Aly08*

The marine bacterium *Vibrio* sp. SY01 was isolated from Yellow sea sediment, China. It grew rapidly in the alginate sole-carbon medium and efficiently degraded brown seaweed with a high alginate lyase activity (more than 50 U/mL). The genome sequence analysis of *Vibrio* sp. SY01 showed that it contained the putative alginate lyase-encoding gene, *aly08*, consisting of 897 bp of an open reading frame (ORF). The identified alginate lyase, Aly08, contained 299 amino acid residues. Signal peptide analysis showed that Aly08 predicted a putative signal peptide (Met<sup>1</sup> to Phe22) in its N-terminal. Furthermore, the theoretically isoelectric point (pI) and theoretical molecular weight (Mw) of mature Aly08 were 4.57 and 32.89 kDa, respectively. According to a search of Conserved Domain Database (CDD) of NCBI, Aly08 is a new alginate lyase with a single-domain belonging to the alginate lyase superfamily 2.

Based on the sequences of Aly08 and other reported PL family 7 alginate lyases, phylogenetic trees were created. Pectate lyase (Genbank number CAD56882) from *Bacillus licheniformis* 14A was included as a control (Figure 1). Among all of the reported alginate lyase, Aly08 had the highest identity sequence of amino acids (78%) with a PL family 7 alginate lyase, AlyL2 (Genbank number MH791447), from *Agarivorans* sp. L11 [19].

A deeply branched cluster was formed in the phylogenetic tree among the enzymes Aly08 (Genbank number MH791447), AlyL2 (Genbank number AJO61885), AlgMsp (Genbank number BAJ62034), and Alg7D (Genbank number ABD81807). According to the multiple sequence alignment (Figure 2), the enzyme contains three conserved regions "RTELREMLR", "QIH", and "MYFKAG" which are related to substrate binding and catalytic activity in PL family 7 [20]. These results identified Aly08 as a new member of the PL family 7. Thus far, several alginate lyases have been identified from various bacteria, such as *Pseudoalteromona, Flavobacterium*, *Nitratiruptor*, *Agarivorans*, and *Vibrio* [7,17,21–23]. After determining their various properties, most of the alginate lyases showed a preference towards polyM blocks. In this study, the purified alginate lyase Aly08 is a polyG-preferred alginate lyase containing the "QIH" conserved region, according to their sequence analysis (Figure 2). The conserved region, "QIH" or "QVH", plays a key role in the substrate preferences of alginate lyases. Aly08, along with AlgMsp, A1-II and Alg2A containing the "QIH" conserved region showed preferences for polyG [24–26]. In addition, other alginate lyases derived from PL family 7, such as AlxM from *Photobacterium* sp. ATCC 43367, and A9mT from *Vibrio* sp. A9m, possess "QVH" regions show preference for degrading polyM blocks (Table 1) [27,28]. Through further sequence screening, it was found that "QIH" or "QVH" may be an indicator for substrate-preferred analysis. Furthermore,

Aly08 can be used for the next part of combining a polyM-preferred alginate lyase for synergetic degradation alginate or brown seaweeds.

**Figure 1.** Phylogenetic analysis of Aly08 with other reported alginate lyases. The reliability of the phylogenetic reconstructions was determained by boot-strapping values (1000 replicates). Branch-related numbers are bootstrap values (confidence limits) representing the substitution frequency of each amino acid residue. A pectate lyase (CAD56882) from *Bacillus licheniformis* 14A was taken as control.

**Figure 2.** Sequence comparison of Aly08 with related alginate lyases from PL family 7: Alg7D (ABD81807) from *Saccharophagus degradans* 2–40, AlgMsp (BAJ62034) from *Microbulbifer* sp. 6532A, AlyV4 (AGL7859) from *Vibrio* sp. QY104, and AlyL2 (AJO61885) from *Agarivorans* sp. L11. The conserved regions and identical residues are marked with bands and black star, respectively.


**Table 1.** Comparison of the properties of Aly08 with other alginate lyases.

#### *2.2. Expression, Purification, and Characterization of Aly08*

The expression strain *E. coli* BL21-pET22b-Aly08 was grown in LB broth and Aly08 was purified by a Ni-NTA affinity column. The specific activity of purified Aly08 was 841.1 U/mg with high viscosity sodium alginate as substrate. Moreover, the purified enzyme Aly08 was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and observed as a single band on the gel with an approximate Mw of 35 kDa (Figure 3), which was corresponding to the theoretical molecular mass of 32.89 kDa.

**Figure 3.** SDS-PAGE analysis of the recombinant enzyme Aly08. Lane M, protein marker; Lane 1, the purified Aly08.

Then, the characterization of purified Aly08 was analyzed as follows. The enzyme Aly08 showed maximum activity at 45 ◦C, and maintained activities of 82.8% and 48.7% when the enzyme was incubated at a low temperature for 1 h, 10 ◦C and 20 ◦C, respectively (Figure 4A,B). The optimal pH for Aly08 was found to be 8.35 (Figure 4C). In addition, Aly08 holds more than 60% of activity in a wide pH range from 7.0–11.0 after incubation in different buffers at 4 ◦C for 12 h, and was particularly stable under alkaline conditions. (Figure 4D). As previous study, most of the alginate lyases showed optimal pH and stability close to a neutral environment, such as AlyH1 from *Vibrio furnissii* H1 shows high activity at the optimal pH 7.5 and it was stable at pH 6.5–8.5. In addition, AlyH1 only retains about 20% of residual activity when incubated at pH 9.5 for 12 h [35]. AlgNJU-03 from *Vibrio* sp. NJU-03 possessed a neutral optimal pH at 7.0 and its pH-stability range from 6.0 to 8.0 [20]. Those alginate lyases prefer neutral pH and they only show high activities within a narrow pH range after incubating for several hours and always exhibit instability under alkaline conditions. Another reported high-alkaline alginate lyase A1m from marine bacteria *Agarivorans* sp. JAM-A1m, exhibited high activity at pH 9.0 under glycine-NaOH buffer with 0.2 M NaCl added to the reaction mixture. However, A1m was only stable and maintain more than 60% of residual activity in a short period of 1 h over a narrow pH range of 7.0–9.0 [33]. Comparing with A1m, Aly08 was stable over 12 h with its 80% residual activity even at pH 9.0–11.0, while another enzyme derived from *Vibrio* sp. NJ-04 maintains good stability at pH 4.0–10.0, but the enzyme only maintains its maximum activity under neutral conditions (pH 7.0) [36]. Thus, Aly08 is an alkaline-stable lyase with industrial application potential as it has been proven to conduct catalysis reactions and maintain activity in a broader pH range.

In particular, the substrate preferred by Aly08 was determined by experimenting with three polymeric substrates (sodium alginate, polyM block and polyG block). Aly08 was found to prefer polyG blocks (1078.2 U/mg) rather than polyM blocks (297.1 U/mg) and native alginate (841.1 U/mg).

**Figure 4.** The biochemical characteristics of Aly08. (**A**) The optimal temperature of Aly08. (**B**) The thermal-stability of Aly08. (**C**) Optimal pH for the relative activity of Aly08 was determined in 20 mM Tris-HCl buffer (solid circle), 20 mM phosphate buffer (solid triangle), 20 mM citic-Na2HPO4 (solid square), or 20 mM glycine-NaOH buffer (hollow circle). (**D**) pH stability of Aly08 in 20 mM Tris-HCl buffer (solid circle), 20 mM phosphate buffer (solid triangle), 20 mM citic-Na2HPO4 (solid square), or 20 mM glycine-NaOH buffer (hollow circle).

Moreover, activities of Aly08 were enhanced by NaCl (different concentrations from 10 mM to 3 M), and the activity reached its maximum at 300 mM NaCl, at which point the activity was about eight times higher than the activity in the absence of NaCl (Table 2). Similarly, the activity of AlgM4 from *V. weizhoudaoensis* M0101 was increased about seven times at 1 M NaCl and activated by a concentration range of NaCl at 0–1 M [37]. For these alginates from marine bacteria, a certain level of NaCl concentration is essential for strain survival and enzyme activation. The effects of other metal ions on the activity of Aly08 were also shown in Table 2. Aly08 showed no obvious activated effect in the presence of NH4 <sup>+</sup>, Li<sup>+</sup>, Zn2<sup>+</sup>, Ba2<sup>+</sup>, and Co2<sup>+</sup>, while Al3<sup>+</sup> and Ni2<sup>+</sup> showed no obvious inhibiting effects on relative enzymatic activity. Enzyme activity was activated by divalent ions, such as Ca2<sup>+</sup> and Mn2<sup>+</sup>. Different concentrations of KCl had little effect on the activities of Aly08 (Table S2). Interestingly, the enzyme activity of the reaction system containing divalent ions of Ca2<sup>+</sup> was about twice as high as that of the reaction system without any ions. However, other reagents such as SDS and EDTA showed significant inactivation effects wherein relative activity was reduced to 50% and 55.9%, respectively.


**Table 2.** Effects of metal ions, EDTA and SDS on the activity of Aly08. Notes: Activity without addition of chemicals was defined as 100%. Data are shown as means ± SD (*n* = 3).
