*2.3. Substrate Specifity and Enzymatic Kinetics of the Enzyme*

Seven kinds of polysaccharide substrates were used to investigate the substrate specificity of the enzyme (Table 1). The alginate lyase showed higher activity towards sodium alginate and polyM, but no activity towards polyG. Additionally, the AlgNJ-07 displayed no activity towards pullulan, pectin, xylan, and heparin. Therefore, the AlgNJ-07 is a novel polyM-specific alginate lyase. Until now, hundreds of alginate lyases have been identified and characterized. However, only a few enzymes exhibited the polyM-specific activity, such as AlgA from *Pseudomonas* sp. E03 [34], ALYII from *Pseudomonas* sp. OS-ALG-9 [32], the AlyA from *Azotobacter chroococcum* 4A1M [31], AlgL from *Pseudomonas aeruginosa* [30], AlyA from *Pseudomonas aeruginosa* [36], AlyA from *Pseudomonas* sp. strain KS-408, and AlyM from unknown marine bacterium [33,35]. They all displayed preference to polyM substrate and very low activity toward polyG substrate. However, compared with these

characterized enzymes, AlgNJ-07 showed no activity toward polyG, indicating it is a novel alginate lyase with strict polyM-specific substrate specificity.


**Table 1.** The substrate specificity of AlgNJ-07 towards various substrates.

\* No activity detected.

The kinetics of AlgNJ-07 towards sodium alginate and polyM were calculated according to the hyperbolic regression analysis. As shown in Table 2, the *Km* values of AlgNJ-07 with sodium alginate and polyM as substrates were 0.53 mM and 0.27 mM. The results showed that AlgNJ-07 had a much lower *Km* values towards polyM than sodium alginate, indicating that it showed higher affinity towards polyM than that to sodium alginate. The *kcat*/*Km* values of AlgNJ-07 towards polyM (115 mM−1·s−1) was higher than alginate (64 mM−1·s−1), which indicates that the enzyme possesses higher catalytic efficiency towards M block than to MG block. The polyM-specific alginate lyase AlgL from *Pseudomonas aeruginosa* showed different *Km* and *kcat* values towards polyM substrates with various Dps and it exhibited different affinity and catalytic efficiency towards those substrates. The variation in *kcat*/*Km* with substrate length suggests that AlgL operates in a processive manner [30].

**Table 2.** The kinetics parameters of AlgNJ-07.


#### *2.4. Biochemical Characterization of AlgNJ-07*

The enzyme showed maximum activity at 40 ◦C (Figure 3A) and was stable below 40 ◦C (Figure 3B). It possessed approximately 50% activity after incubation at 40 ◦C for 30 min and was gradually inactivated as the temperature increased. The thermal degeneration curve of AlgNJ-07 was shown in Figure 4. The enzyme could retain more than 70% of its total activity after being incubated at 40 ◦C for 60 min, which indicates it possesses better thermal stability. The optimal temperature for polyM-specific alginate lyase from *Pseudomonas* sp. strain KS-408 was 37 ◦C [33]. The AlgA from *Pseudomonas* sp. E03 and ALYII from *Pseudomonas* sp. OS-ALG-9 both exhibited their maximal activity at 30 ◦C [33,34]. While the AlyA from *Azotobacter chroococcum* 4A1M showed the highest activity at 60 ◦C, which shows potential in industrial applications [31].

The optimal pH for the enzyme activity was 9.0 (Figure 3C) and retained more than 80% activity at a broad pH range from pH 8.0 to 10.0 (Figure 3D) after incubation for 24 h. However, this enzyme was mostly stable at pH 9.0 and retained more than 80% activity at a broad pH range from 7.0 to 10.0. Interestingly, it could retain about 40% of its activity at pH 11.0. Thus, AlgNJ-07 was an alkaline-stable lyase and it could retain stability in a broader pH range. While most of the other characterized polyM-specific alginate lyases exhibited their maximal activity around neutral pH. For instance, the AlgA from *Pseudomonas* sp. E03 possessed its optimal pH of 8.0 [34], the AlyA from

*Pseudomonas* sp. strain KS-408 displayed its maximal activity at pH of 9.0 [34]. While the AlyA from *Azotobacter chroococcum* 4A1M had a lower optimal pH of 6.0 [31].

**Figure 3.** The biochemical characteristics of AlgNJ-07. (**A**) The optimal temperature of AlgNJ-07. (**B**) The thermal stability of AlgNJ-07. (**C**) The optimal pH of AlgNJ-07. (**D**) The pH stability of AlgNJ-07. Each value represents the mean of three replicates ± standard deviation.

**Figure 4.** The thermal degeneration curve of AlgNJ-07. The maximal activity of the treated enzyme was regarded as 100% and the other relative activity was determined.

The effects of metal ions on the activity of AlgNJ-07 are shown in Table 3. It was observed that Na<sup>+</sup> could enhance the activity of the enzyme, while some divalent ions such as Zn2+, Cu2+, Mn2+, and Co2+ inhibited the activity. Interestingly, the reported activators such as Mg2+ and Ca2+ displayed

slight inhibitory effects on activity of AlgNJ-07. While Ca2+ can activate the activities of the AlyA from *Pseudomonas* sp. strain KS-408 [33], the AlyA from *Pseudomonas* sp. E03 [34], the AlyA from *Azotobacter chroococcum* 4A1M [31], and ALYII from *Pseudomonas* sp. OS-ALG-9 [32] could enhance the substrate-binding ability of the enzyme.


**Table 3.** The effect of metal ions on activity of AlgNJ-07.

To determine the number of substrate binding subsites in the active tunnel of AlgNJ-07, we compared the degrading capability of AlgNJ-07 to oligosaccharide substrates with different Dps. As shown in Figure 5, purified disaccharide cannot be further degraded by the enzyme even under more focused conditions (high enzyme concentration and prolonged incubation time). The trisaccharide was the shortest chain that can be recognized and cleaved by AlgNJ-07, producing monosaccharide and disaccharide. The result indicated that trisaccharide was the shortest substrate for AlgNJ-07.

**Figure 5.** TLC analysis of hydrolysis products of oligosaccharides with Dps (2–8) for determination of substrate binding sites of AlgNJ-07 (−Enz: enzyme free; +Enz: AlgNJ-07 added).

The degradation products of sodium alginate and polyM by AlgNJ-07 were analyzed by TLC plate (Figure 6). As the proceeding of hydrolysis, oligosaccharides with high Dp (6–8) appeared. After incubation for 48 h, dimers, trimers, and tetramers turned out to be the main hydrolysis products for sodium alginate and polyM. Interestingly, the enzyme could release monosaccharide with processing of the hydrolysis. The distributions of the degradation products for the above two kinds of substrates were similar, and the results indicate that AlgNJ-07 can hydrolyze the substrates in a unique endolytic manner.

**Figure 6.** TLC analysis of the AlgNJ-07 hydrolysis products for different times. Lane 1–15, the samples taken by 0 min, 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, 60 min, 2 h, 4 h, 12 h, 24 h, 36 h, and 48 h. Lane M, the oligosaccharide standards of tetramer and pentamer.

In order to further determine the composition of the degradation products, the hydrolysates (1 mL) were then loaded onto a carbograph column to remove salts after removing other proteins, followed by being concentrated, dried, and re-dissolved in 1 mL methanol with the final concentration of 1 mg/mL. The degradation products were then analyzed by ESI-MS. As shown in Figure 7, monosaccharides, disaccharides, and trisaccharides account for a major fraction of the hydrolysates of two kinds of substrates. This result indicate that AlgNJ-07 may be a potential tool for the enzymatic hydrolysis of sodium alginate to produce oligosaccharides with lower Dps. The distribution of degradation products of other polyM-specific enzymes is similar, such as AlgA from *Pseudomonas* sp. E03 [34] and AlyA from *Pseudomonas* sp. strain KS-408 [33], which mainly produced oligosaccharides with Dp of 2–5 in an endolytic manner. However, the AlgL from *Pseudomonas aeruginosa* generated dimeric and trimeric products, and the rapid-mixing chemical quench studies indicate that AlgL can operate as an exopolysaccharide lyase [30]. None of those enzymes could produce monosaccharide during the hydrolytic procedure, which indicates that the AlgNJ-07 possesses a unique manner for releasing products.

**Figure 7.** ESI-MS analysis of the degradation products of AlgNJ-07 with (**A**) alginate and (**B**) the polyM as substrate. The data highlighted in red represent the relative abundance of peaks.

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

#### *3.1. Materials*

Sodium alginate derived from brown seaweed was purchased from Sigma (St. Louis, MO, USA). PolyM (purity: about 99%) and polyG (purity: about 99%) were purchased from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China). The SOURCETM 15Q 4.6/100 PE column was purchased from GE HealthCare Bio-Sciences (Uppsala, Sweden). Other chemicals and reagents used in this study were of analytical grade.

#### *3.2. Screening and Identification of Strain NJ-07*

The samples were collected from the coast of the Yellow Sea, washed by sterilized sea water and then spread on sodium alginate-agar plates. The plates were incubated at 30 ◦C for 36 h and the positive colonies showing clear zones were picked out from the selection plates. The re-screening process was conducted as follows. Strains with clear hydrolytic zones were selected and incubated aerobically in a fermentation medium (modified marine broth 2216 medium containing 5 g/L (NH4)2SO4, 19.45 g/L NaCl, 12.6 g/L MgCl2·6H2O, 6.64 g/L MgSO4·7H2O, 0.55 g/L KCl, 0.16 g/L NaHCO3, 1 g/L ferric citrate, and 10 g/L sodium alginate) at 30 ◦C and 200 rpm. Furthermore, the activity of alginate lyase was determined by 3,5-dinitrosalicylic acid (DNS) colorimetry [37]. Among the isolates, the most active strain NJ-07 was selected for further studies. To identify the NJ-07 strain, the 16S rRNA gene of the strain was amplified through PCR by using universal primers. The purified PCR fragment was sequenced and compared with reported 16S rRNA sequences in GenBank by using BLAST. A phylogenetic tree was constructed using CLUSTAL X and MEGA 6.0 through neighbor-joining method [38].

#### *3.3. Production and Purification of the Alginate Lyase*

The strain NJ-07 was propagated in a fermentation medium with shaking for 40 h at 30 ◦C. The culture medium was centrifuged (10,000× *g*, 60 min) to completely remove the sludge and the cell-free supernatant was fractionated at 30% and 80% ammonium sulfate saturation. The precipitated protein with 30% ammonium sulfate saturation was discarded, and the precipitated protein with 80% ammonium sulfate saturation was suspended in distilled water and dialyzed in a dialysis bag (MWCO: 8000–14,000 Da) against the distilled water and freeze-dried successively. Protein contents were determined by the Bradford method [39]. The obtained enzyme powder was dissolved in 5 mL Tris-HCl buffer (pH 9.0) with 4% as the final concentration, then the enzyme solution was applied to a SOURCETM 15Q 4.6/100 PE column equilibrated with a linear gradient of 0–0.5 M NaCl in an equilibrating buffer under a flow rate of 1 mL/min. The eluents were monitored continuously at 280 nm for protein and fractions were assayed for activity against sodium alginate. Fractions were collected and monitored for the presence of alginate lyase. The purity of the fractions was assessed by SDS-PAGE. Pure fractions with activity were stored at −80 ◦C.

#### *3.4. Enzyme Activity Assay*

The purified enzyme (0.1 mL) was mixed with 0.9 mL Tris-HCl (20 mM, pH 8.0, 1% sodium alginate) and incubated at 40 ◦C for 10 min. The reaction was stopped by heating in boiling water for 10 min. The enzyme activity was then assayed by measuring the increased absorbance at 235 nm due to the formation of double bonds between C4 and C5 at the nonreducing terminus by β-elimination. One unit was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.01 per min [40].

#### *3.5. Substrate Specificity and Kinetic Measurement of Alginate Lyase*

The purified enzyme was reacted with 1% of sodium alginate, polyM, polyG, pectin, xylan, and heparin. The assays of enzyme activity for sodium alginate, polyM, and polyG were defined as described previously, whereas the assays for pectin, xylan, and heparin were determined by using the DNS method. The kinetic parameters of the purified enzyme toward sodium alginate and polyM were determined by measuring the enzyme activity with substrates at different concentrations (0.1–8.0 mg/mL). As sodium alginate is a polymer consisting of random combinations of mannuronic acid and guluronic acid residues. Since they both have the same molecular weight (MW), substrate molarity was calculated using the MW of 176 g/mol for each monomer of uronic acid in the polymer. The concentrations of the product were determined by monitoring the increase in absorbance at 235 nm using the extinction coefficient of 6150 M−<sup>1</sup> cm−1. Velocity (V) at the tested substrate concentration was calculated as follows: V (mol/s) = (milliAU/min × min/60 s × AU/1000 milliAU × 1 cm)/(6150 M−<sup>1</sup> cm−1) × (2 × <sup>10</sup>−<sup>4</sup> L). The *Km* and *Vmax* values were calculated by hyperbolic regression analysis as described previously [41]. Additionally, the turnover number (*kcat*) of the enzyme was calculated by the ration of *Vmax* versus enzyme concentration ([E]).

#### *3.6. Biochemical Characterization of AlgNJ-07*

The effects of pH on the enzyme activity were evaluated by incubating the purified enzyme in buffers with different pHs (4.0–12.0) at 40 ◦C under the assay conditions described previously. The pH stability depended on the residual activity after the enzyme was incubated in buffers with different pH (4.0–12.0) for 24 h and then residual activity was determined at 40 ◦C under the assay conditions. Meanwhile, the effects of temperatures (20–60 ◦C) on the purified enzyme were investigated at pH 9.0. The thermal stability of the enzyme was determined at pH 9.0 under the assay conditions described previously after incubating the purified enzyme at 30–50 ◦C for 30 min. The buffers with different pHs used were phosphate-citrate (pH 4.0–5.0), NaH2PO4-Na2HPO4 (pH 6.0–8.0), Tris–HCl (pH 7.0–9.0), glycine-NaOH (pH 9.0–10.0), and Na2HPO4–NaOH (pH 11.0–12.0). In addition, the thermally-induced denaturation was also investigated by incubating the enzyme at 30–50 ◦C for 0–60 min.

The influence of metal ions on the activity of the enzyme was performed by incubating the purified enzyme at 4 ◦C for 24 h in the presence of various metal compounds at a concentration of 1 mM. Then, the activity was measured under standard test conditions. The reaction mixture without any metal ions was used as a control.

#### *3.7. Substrate Binding Subsites of AlgNJ-07*

To determine the smallest substrate and the number of substrate binding subsites in its catalytic tunnel of AlgNJ-07, hydrolysis reactions were carried out using oligosaccharides with different Dps (Dp 2–8) at a concentration of 10 mg/mL in 10 μL reaction mixture (pH 9.0). The reaction mixtures were incubated at 40 ◦C with AlgNJ-07 for 24 h. The hydrolysates were loaded onto a carbograph column (Alltech, Grace Davison Discovery Sciences, Carnforth, UK) to remove salts after removing proteins, and then concentrated, dried, and re-dissolved in 1 mL methanol. The degradation products were analyzed by TLC with the solvent system (1-butanol/formic acid/water 4:6:1) and visualized by heating TLC plate at 130 ◦C for 5 min after spraying with 10% (*v*/*v*) sulfuric acid in ethanol.
