*2.1. Screening and Identification of Strain Alg07*

Twenty-one strains with alginate lyase activity were isolated using alginate as the sole carbon source. Alg07 secreted the alginate lyase with the highest activity in the fermentation culture.

The 16S rRNA gene of Alg07 was cloned, sequenced, and submitted to GeneBank (accession number KM040772) for strain identification. The alignment of 16S rRNA gene sequences from different *Bacillus* species showed that strain Alg07 is closely related to *Bacillus litoralis* S20409 (97%) and *Bacillus simplex* J2S3 (97%). However, the low similarity shared by the 16s rRNA gene sequence of Alg07 with that of other known *Bacillus* species indicated that Alg07 may be a novel *Bacillus* species. In accordance with the neighbor-joining phylogenetic tree, the strain was assigned to the genus *Bacillus* and designated as *Bacillus* sp. Alg07 (Figure 1).

**Figure 1.** Neighbor-joining phylogenetic tree generated on the basis of the 16S rRNA gene sequences of strain Alg07 and other known *Bacillus* species.

#### *2.2. Purification of Alginate Lyase from Bacillus sp. Alg07*

*Bacillus* sp. Alg07 was cultured in optimized liquid medium for 24 h until its alginate lyase reached the highest activity. The supernatant containing 510 U/mL of alginate lyase was subjected to further purification through the two simple steps of tangential flow filtration concentration and anion exchange chromatography with Source 15Q (Figure S1). After purification, the alginate lyase was purified 8.34-fold with a yield of 62.4% (Table 1). The final specific activity of the purified alginate lyase was 8306.7 U/mg. The purified alginate lyase from *Bacillus* sp. Alg07 was designated as AlgA.


**Table 1.** Summary of the purification of AlgA.

The activities of AlgA and those of other well-studied strains are shown in Table 2, which shows that AlgA has the highest activity among all reported alginate lyases. The simple purification, high recovery, and high specific enzyme activity of AlgA indicated that it may be produced industrially on a large scale.

**Table 2.** Comparison of the properties of AlgA with those of alginate lyases from different microorganisms.


Figure 2 shows that the purified AlgA exhibited a clear and unique band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This result suggested that the two-step purification process is successful. The molecular weight of AlgA was approximately 60 kDa, which is similar to molecular weights of alginate lyases from *Vibrio* sp.YKW-34 [30] and *Saccharophagus degradans* [26]. AlgA belongs to the class of large alginate lyases on the basis of its molecular weight [31].

The N-terminal amino acid sequence of the purified AlgA was analyzed. The sequence was identified as Glutamic acid–Glutamic acid–Glutamic acid–Glutamic acid–Aspartic acid–Valine–Threonine–Tyrosine (Figure S2). The results from homology search using BLASTp and CLUSTAL X indicated that the N-terminal amino acid sequence of AlgA is absent from the sequences of the previously reported alginate lyases. Furthermore, a protein with four Glu residues at its N-terminal is unusual. Therefore, AlgA may be a novel alginate lyase.

**Figure 2.** Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) result for AlgA. Lane M, protein ladder; Lane 1, purified AlgA.

#### *2.3. Biochemical Characterization of AlgA*

The optimal temperature of AlgA is 40 ◦C, which is similar to alginate lyases derived from *Vibrio* sp.YKW-34 [30], *Flavobacterium* sp. LXA [11], *Pseudomonas aeruginosa* PA1167 [32], and *Stenotrophomnas maltophilia* KJ-2 [33]. The activity of AlgA significantly decreased under temperatures exceeding 50 ◦C (Figure 3A). Thermostability analysis indicated that the activity of AlgA remained relatively unchanged after 5 h of incubation at 40 ◦C and did not decrease even after 24 h of incubation at 40 ◦C (data not shown). However, the enzyme was less stable under high temperatures, and the half-life of AlgA is approximately 3 h under 45 ◦C and 0.75 h under 50 ◦C (Figure 3B). This result indicated that at its optimal temperature, AlgA might be the most stable enzyme among all reported alginate lyases. Alginate lyases from *Favobacterium* sp. LXA [11], *Vibrio* sp. W13 [34], and *Zobellia galactanivorans* [35] are stable only under temperatures less than 40 ◦C. The activities of many alginate lyases considerably decrease after incubation at 40 ◦C. AlyL2 from *Agarivorans* sp. L11 has a half-life of 125 min at 40 ◦C [36], and OalS17 from *Shewanella* sp. Kz7 retains 88% of its activity after 1 h of incubation at 40 ◦C [25]. Furthermore, alginate lyases from *Flammeovirga* sp. MY04 [37] and *Vibrio* sp. SY08 [27] retain approximately 80 and 75%, respectively, of their activities after 2 h of incubation at 40 ◦C. Thermostable enzymes are more advantageous than thermolabile counterparts due totheir long half-lives and low production costs. Thus, AlgA has potential industrial applications given its excellent thermostability.

The optimal pH of AlgA was 7.5 in 20 mM Tris-HCl buffer. AlgA exhibited more than 90% of its maximal activity in pH 8.0 buffer. These results suggested that AlgA is basophilic (Figure 3C). Similarly, the optimal activities of most alginate lyases from marine bacteria are observed at pH 7.5–8.0 (Table 2). The results of the pH stability assay showed that AlgA presented the highest stability for an extended period over a pH range of 5.0–10.0 (Figure 3D). The good stability of AlgA over a wide pH range indicated its suitability for industrial application. Furthermore, the use of AlgA may decrease production costs given that pH adjustment will be unnecessary even when various alginates from different sources are employed as substrates.

**Figure 3.** Effects of temperature and pH on the relative activity of AlgA. (**A**) Optimal temperature of AlgA. (**B**) Thermostability of AlgA at 40 ◦C (filled square), 45 ◦C (filled circle), and 50 ◦C (filled triangle). (**C**) Optimal pH for the relative activity of AlgA was determined in 20 mM CH3COOH-CH3COONa buffer (filled square), 20 mM Tris-HCl buffer (filled circle), or 20 mM Glycine-NaOH buffer (filled triangle). (**D**) pH stability of AlgA in 20 mM CH3COOH-CH3COONa buffer (filled square), 20 mM Tris-HCl (filled circle), and 20 mM Glycine-NaOH (filled triangle).

To determine the effect of NaCl on AlgA, the activity of AlgA in the presence of various NaCl concentrations was measured. As shown in Figure 4A, the optimal NaCl concentration for AlgA activity was 200 mM, and no activity was detected in the absence of NaCl. These results indicated that the activity of AlgA is dependent on NaCl. Thus, NaCl concentration is crucial for the activity of AlgA, which is a salt-activated alginate lyase. However, high NaCl concentrations decreased the activity of AlgA. Similarly, the activities of marine bacterial alginate lyases, such as AlyV5 from *Vibrio* sp. QY105 [28] and AlyYKW-34 from *Vibrio* sp. YKW-34 [30], are dependent on NaCl.

**Figure 4.** Effect of NaCl (**A**) and metal ions (**B**) on the activity of AlgA.

The effects of metal ions and EDTA on the activity of AlgA were determined in the presence of 200 mM NaCl. Mg2+ or Ca2+ significantly enhanced enzymatic activity by 300% or 215%, and Co2+ and Mn2+ slightly increased enzymatic activity (Figure 4B). By contrast, Hg2+, Fe3+, Fe2+, and Cu2+ completely inhibited lyase activity. Ba2+ and EDTA partially inhibited lyase activity. Ca2+ and Mg2+ increase the activity of many alginate lyases, such as aly-SJ02 from *Pseudoalteromonas* sp. SM0524 [38] and AlyV5 from *Vibrio* sp. QY105 [28]. However, Mg2+ and Ca2+ decrease the activity of Alg7D from *S. degradans* [26].

#### *2.4. Substrate Specificity and Kinetic Parameters of AlgA*

AlgA exhibited activity toward alginate but not toward pectin, hyaluronan, chitin, or agar. This behavior suggested that AlgA is indeed an alginate lyase. As shown in Figure 5, the relative activities of AlgA toward alginate, polyM, and polyG blocks are 100 ± 4.3, 87.2 ± 6.9, and 10.5 ± 1.7%, respectively. The slight activity toward polyG block might result from the presence of a few M residues in polyG substrates. These results indicated that the polyM block of substrates is the preferred substrate of AlgA. Thus, AlgA is a mannuronate lyase. Alginate lyases from *Flavobacterium* sp. UMI-01 [16], *Vibrio* sp. JAM-A9m [39], and *Pseudomonas* sp. QD03 [40] also belong to the mannuronate lyase class of enzymes.

**Figure 5.** Relative activities of AlgA toward alginate, polyM, and polyG.

The kinetic parameters of AlgA were determined through nonlinear regression analysis (Figure S3) and are shown in Table 3. AlgA has a lower *Km* value for polyM than for sodium alginate. This result suggested that AlgA has high affinity for polyM blocks and further confirmed that AlgA is a mannuronate lyase. However, the *kcat* values of AlgA for sodium alginate were higher than those for polyM. Therefore, AlgA has equivalent catalytic efficiency for sodium alginate and polyM.

**Table 3.** Kinetic parameters of the activity of AlgA toward sodium alginate and polyM blocks.

