*2.3. Vmax (Maximum Reaction Rate) and Km (Michaelis Constant) of AlgM4*

AlgM4 activity was measured when incubated with various concentrations of substrate (SA) in a water bath at 30 ◦C for 10 min, and the resulting values were used to calculate the kinetic constants of AlgM4 activity. The *V*max and *K*cat values of AlgM4 were 2.75 nmol/s and 30.25 S−1, respectively, and the *K*<sup>m</sup> value was 2.72 mg/mL. Two other NaCl-activated enzymes—AlyPM from *Pseudoalteromonas* sp. SM0524 and AlySY08 from *Nitratiruptor* sp. SB155-2—were previously shown to have *K*m values of 74.39 [17] and 0.36 mg/mL [14], respectively. The oligoalginate lyase Alg17C derived from *Saccharophagus degradans* 2-40 had an observed *K*<sup>m</sup> value of 35.2 mg/mL [18]. *Vibrio splendidus* 12B01 expresses three oligoalginate lyases—OalA, OalB, and OalC—which had observed *K*m values of 3.25, 0.76, and 0.53 mg/mL, respectively [29].

#### *2.4. Enzymatic Characteristics of AlgM4*

AlgM4 exhibited high enzymatic activity at 20–40 ◦C, which decreased to 11% of the maximal observed activity when incubated at 70 ◦C. Similar to other NaCl-activated alginate lyases in the PL7 family (such as A1m [15], rA9mT [16], and AlyPM [17]), AlgM4 had highest enzymatic activity at 30 ◦C (Figure 4A).

Many alginate lyases have optimal pH values between pH 7 and 8, in contrast to the optimal high-alkaline pH values of pectate lyases [20]. The optimum pH for AlgM4 activity was assessed, and the results are shown in Figure 4B. The optimum reaction pH was 8.5, indicating that AlgM4 was weakly basophilic. In slightly alkaline environments (pH 7.5–9.0), AlgM4 retained 80% activity, whereas AlgM4 was completely inactive at pH 11.

**Figure 4.** Effect of temperature and pH on AlgM4 activity. (**A**) The optimal temperature for AlgM4 activity. The activity of AlgM4 at 30 ◦C was completely retained; (**B**) The optimal pH for AlgM4 activity. The activity of AlgM4 at pH 8.5 was completely retained.

The activity of AlgM4 remained stable for 30 min at 25 ◦C, regardless of the presence or absence of 1 mol/L NaCl (Figure 5A). After a 30 min incubation at 30 or 35 ◦C in the presence of 1 mol/L NaCl, AlgM4 retained 92% of its initial activity, which decreased rapidly at temperatures exceeding 40 ◦C and decreased by 63% at 45 ◦C. In the absence of NaCl, the enzymatic activity of AlgM4 was reduced by 94% at 45 ◦C. An examination of temperature tolerance showed that NaCl improved the thermal stability of AlgM4.

**Figure 5.** The thermal stability and melting temperature (Tm) of AlgM4. (**A**) The thermal stability of AlgM4. The residual activity of AlgM4 at 25 ◦C was completely retained; (**B**) Circular dichroism signals at 218 nm were used for analysis of the Tm value.

The effects of metal ions and surfactants on AlgM4 activity are shown in Table 2. Ca2+ did not influence enzyme activity, unlike for other alginate lyases [12,17,20]. While Mg2+ promoted the activity of AlgM4, Cu2+, Mn2+, and Zn2+ inhibited AlgM4 activity. The most significant inhibitory effect on AlgM4 was observed by Zn2+, which caused an 82% reduction in AlgM4 activity. Ethylene diamine tetraacetic acid (EDTA) and SDS suppressed AlgM4 activity to varying degrees. The anionic surfactant SDS strongly inhibited the enzymatic activity of AlgM4, causing a 97% reduction in AlgM4 activity, while EDTA reduced AlgM4 activity by 35%.


**Table 2.** Effect of chemical reagents on AlgM4 activity.

The data are expressed as the means ± SD, *n* = 3. The activity of AlgM4 in the absence of chemical reagents was completely retained.

#### *2.5. Effect of NaCl on AlgM4 Activity*

Different concentrations of NaCl were added to 0.1 mg/mL purified AlgM4, and the enzymatic activity of AlgM4 was then examined. AlgM4 was promoted at NaCl concentrations of 0.1–1.4 mol/L and was greatest in the presence of 1.0 mol/L NaCl, exhibiting more than 7 times the activity observed in the absence of NaCl (Table 3). The activity of AlyPM in the presence of 0.5–1.2 mol/L NaCl was 6 times that observed in the absence of NaCl [17]. Under optimum reaction conditions, the activity of A1m in the presence of 0.6–0.8 mol/L NaCl was 20 times that observed in the absence of NaCl [15]. The activity of rA9mT in the presence of 0.4 mol/L NaCl was 24 times that observed in the absence of NaCl [16].


**Table 3.** Effect of NaCl on AlgM4 activity.

The data are expressed as the means ± SD, *n* = 3. The activity of AlgM4 in the absence of NaCl was completely retained.

### *2.6. Determination of the Secondary Structure and Thermal Denaturation Temperature of AlgM4*

The thermal denaturation temperature of AlgM4 was determined by circular dichroism (CD) spectroscopy at 25–75 ◦C. In addition, the secondary structure of AlgM4 was determined by ultraviolet–visible (UV–Vis) spectroscopy and CD spectroscopy. The CD absorption values of AlgM4 protein at 218 nm in different temperatures are shown in Figure 5B, and the CD values were used for analysis of the Tm value. The CD values of AlgM4 relatively remained stable at 25–35 ◦C in the absence of NaCl; subsequently, CD values slowly increased with the increase of temperature and the denaturation of AlgM4, then increased dramatically at 40–45 ◦C and remained stable at temperatures exceeding 45 ◦C. The AlgM4 protein was denatured completely when the temperature was higher than 45 ◦C. However, in the presence of 1 mol/L NaCl, the CD values increased rapidly at 45–50 ◦C and remained stable at temperatures exceeding 50 ◦C. The results showed that NaCl enhanced the ability of AlgM4 to resist thermal denaturation.

As shown in Figure 6A, the UV absorbance of AlgM4 at 220–240 nm was significantly reduced in the presence of 1 mol /L NaCl. CD spectroscopy was used to determine the secondary structure of the purified AlgM4, and the results are shown in Figure 6B. The absorption spectrum of AlgM4 was characterized by positive and negative peaks at approximately 198 and 218 nm, respectively, and the intensity of these peaks was reduced by the addition of 1 mol/L NaCl. The results showed that the α-helix and β-sheet contents in the secondary structure of AlgM4 were decreased after addition of 1 mol/L NaCl. Specifically, the α-helix and β-sheet contents were reduced from 12.4% and 38.2% to 10.8% and 36.5%, respectively. The changes in the contents of secondary structural elements are summarized in Table 4. The secondary structure of AlgM4 was altered in the presence of 1 mol/L NaCl, which may enhance the affinity of the enzyme for its substrates and facilitate enzymolysis. The alginate lyase AlyPM, derived from *Pseudoalteromonas* sp. SM0524, differs from AlgM4 in that the presence of NaCl did not alter the secondary structure of AlyPM. However, NaCl enhanced the affinity of AlyPM for its substrates, thereby promoting enzymolysis [17]. In addition, AlyPM is a cold-adapted enzyme, and its thermal denaturation temperature is relatively low, with a Tm of 37 ◦C [17]. At 1 mol/L, NaCl not only altered the secondary structure of AlgM4 but also enhanced the ability of AlgM4 to resist thermal denaturation. As a result, the midpoint of thermal denaturation (Tm) was increased from 43.3 to 48.2 ◦C (Figure 5B).

**Figure 6.** Determination of the secondary structure of the AlgM4. (**A**) Determination of the secondary structure by UV–Vis absorption spectra; (**B**) Determination of the secondary structure by circular dichroism.


**Table 4.** Secondary structure of AlgM4 as estimated by CD.

*2.7. Analysis of the Products of AlgM4-Mediated Enzymolysis of Alginate by Ultra-Performance Liquid Chromatography (UPLC)–Quadrupole Time-of-Flight (QTOF)–Mass Spectrometry (MS)/MS*

Alginate lyases with endolytic characteristics generally act on glycosidic bonds within the linear polysaccharide chain of alginate, generating unsaturated oligosaccharides that are dominated by disaccharides, trisaccharides, and tetrasaccharides [27]. Exolyases further depolymerize these oligosaccharides into mannuronic acids [18,29,30]. AlgM4 degrades both SA (Figure 7A) and polyG (Figure 7B) to produce oligosaccharides with degree of polymerization DP 2–6 [31,32]. The content of each oligosaccharide can be determined only after quantitative determination. Unlike SA and polyG, degradation of polyM produces oligosaccharides DP7, DP8, and DP9 (Figure 9). Furthermore, AlgM4 may be useful in the preparation of oligosaccharides, especially with high DP 7–9, and the study of their biological functions.

In recent years, the biological activities of oligosaccharides and the application of oligosaccharides in the medical and biotechnology fields has attracted the attention of researchers [33]. Oligosaccharides have higher degrees of polymerization and possibly better bioactivity [26]. An et al. observed that oligosaccharides (DP 6–8) derived from SA stimulated the accumulation of phytoalexin and induced phenylalanine ammonia lyase in soybean cotyledons, resulting in their acquired resistance to *Pseudomonas aeruginosa* [34]. In addition, trisaccharides, tetrasaccharides, pentasaccharides, and hexasaccharides obtained by enzymatic degradation of SA promoted the growth of lettuce seedlings [35].

**Figure 7.** (**A**) Ultra-Performance Liquid Chromatography (UPLC)–Quadrupole Time-of-Flight (QTOF)–MS/MS analysis of hydrolysates of AlgM4 with sodium alginate as the substrate; (**B**) UPLC–QTOF–MS/MS analysis of hydrolysates of AlgM4 with polyG as the substrate. DP indicates the degree of polymerization of oligosaccharides from the alginate lyase hydrolysates.

**Figure 8.** *Cont.*

**Figure 9.** UPLC–QTOF–MS/MS analysis of hydrolysates of AlgM4 with polyM as the substrate. DP indicates the degree of polymerization of oligosaccharides from the alginate lyase hydrolysates. The reaction products of disaccharide, tetrasaccharide and pentasaccharide were detected at 0.8 min (**a**). The major peacks of DP7, DP6, DP9 and DP8 were detected at 1.0 min (**b**), 1.2 min (**c**), 2.2 min (**d**) and 2.4 min (**e**), respectively.

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

#### *3.1. The Bacterium*

The marine bacterium *V. weizhoudaoensis* M0101 was isolated from rotten Sargassum collected from Weizhou Island, Beihai, Guangxi Province, China.

#### *3.2. Cloning and Expression of the algM4 Gene*

The primers were designed according to the nucleic acid sequence of the *algM4* gene. The following primers were used to amplify the *algM4* gene: upstream primer, 5 -GGA ATTCCATATGCTTGCATCTTCTGTG-3 (the *Nde*I restriction site is underlined); downstream primer, 5 -CCGCTCGAGACCTTTATAAGAACCGTG-3 (the *Xho*I restriction site is underlined). The parameters of the polymerase chain reaction (PCR) were as follows: 94 ◦C for 2 min; followed by 30 cycles of 94 ◦C for 30 s, 58 ◦C for 30 s, and 72 ◦C for 1 min 40 s; with a final incubation at 72 ◦C for 10 min. The PCR product was double digested with the *Nde*I and *Xho*I restriction enzymes and then ligated into the pET30a(+) vector to construct the recombinant plasmid pET30a-*algM4*. To induce the expression of AlgM4, the recombinant plasmid was transformed into the *E. coli* strain BL21 (DE3). The positive transformants were picked, inoculated into 10 mL of LB (Luria-Bertani) medium containing 50 μg/mL kanamycin (Kan) and cultured at 37 ◦C with shaking (200 rpm) until the OD600 of the culture reached 0.5. The culture was then inoculated into LB medium containing 50 μg/mL Kan (inoculum volume: 1% (*v*/*v*)) and cultivated until the OD600 value reached 0.5. Subsequently, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added at a final concentration of 0.5 mmol/L. After the addition of IPTG, the bacteria were cultured for another 12 h at 25 ◦C with shaking (200 rpm).

#### *3.3. Purification of AlgM4*

Protein isolation and purification was carried out at 0–4 ◦C. The IPTG-induced bacteria were harvested by centrifugation at 6000 rpm for 10 min. Subsequently, the bacteria were resuspended in buffer (10 mmol/L imidazole, 300 mmol/L NaCl, and 20 mmol/L Tris-HCl; pH 7.0) and lysed by ultrasonication. The lysates were centrifuged at 12,000 rpm for 30 min, and the resulting crude enzyme solution was collected. After washing off the protein impurities with buffer (50 mmol/L imidazole, 300 mmol/L NaCl, and 20 mmol/L Tris-HCl; pH 7.0), AlgM4 bound to the Ni2+ column was eluted with elution buffer containing 150 mmol/L imidazole, 300 mmol/L NaCl, and 20 mmol/L Tris-HCl (pH 7.0). The purified protein was analyzed by SDS-PAGE, and the protein concentration was determined using the Bradford method.

#### *3.4. Determination of the Enzymatic Properties of AlgM4*

Enzymatic properties were determined using 0.1 mg/mL AlgM4. Nine hundred microliters of a 1.0% (*w*/*v*) SA solution was mixed with 100 μL of purified AlgM4 and incubated in a water bath at 30 ◦C for 10 min. The mixture was then boiled in a water bath for 5 min to terminate the reaction. After the reaction system was cooled to room temperature, the absorbance was measured at 235 nm. The unit of enzymatic activity (U) was defined as an increase in absorbance of 0.01 per minute. To determine the optimum reaction temperature, the enzymatic activity of AlgM4 was measured at 10–70 ◦C in 100 mmol/L Tris-HCl (pH 7.0) buffer using 0.2% (*w*/*v*) SA as a substrate. To determine the optimum reaction pH, the activity of AlgM4 was evaluated in 100 mmol/L citric acid–sodium citrate buffer (pH value: 4.5–6.5), 100 mmol/L Tris-HCl buffer (pH value: 7.0–9.0), or 100 mmol/L glycine-sodium hydroxide buffer (pH value: 9.5–12.0) at the optimum reaction temperature. To test the thermal stability of the enzyme, AlgM4 was first incubated at 25–50 ◦C for 30 min, and the residual AlgM4 activity was then measured under the optimum reaction conditions. To examine the effects of metal ions and surfactants on the enzymatic activity of AlgM4, metal ions (Ca2+, K+, Mg2+, Mn2+, Zn2+, and Cu2+), EDTA, or SDS were added to 50 μL of enzyme solution at a final concentration of 1 mmol/L. The activity of AlgM4 was then measured under the optimum reaction conditions using a 0.2% (*w*/*v*) SA solution as a substrate. The effect of NaCl on AlgM4 activity was also examined. NaCl was added to the enzyme solution at final concentrations of 0.1–1.4 mol/L, and the activity of AlgM4 was then compared to that observed under optimum reaction conditions.

#### *3.5. Determination of the Kinetic Constants for AlgM4 Activity*

The enzymatic activity of AlgM4 (0.1 mg/mL) was measured under the optimum reaction conditions using 3,5-dinitrosalicylic acid (DNS) assay. The concentrations of the substrate, SA, assayed were 0.5–10 mg/mL. Lineweaver-Burk (double-reciprocal) plots were generated using 1/[*S*] as the abscissa and 1/*V* as the ordinate. The *V*max and *K*<sup>m</sup> values were calculated using the Michaelis-Menten equation.

#### *3.6. The Effects of NaCl on the Secondary Structure and Thermal Stability of AlgM4*

In one group, NaCl was added to a 300 μL reaction containing 0.28 mg/mL of purified AlgM4 (final concentration of NaCl: 1 mol/L), while another group was not exposed to NaCl. The secondary structure of the purified AlgM4 protein was determined at 25 ◦C using a TU-1901 Dual Beam Ultraviolet Spectrophotometer (PERSEE, Beijing, China) (spectral range: 200–350 nm) and a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd., Surrey, UK) (spectral range: 195–250 nm; optical path length: 10 mm; bandwidth: 0.5 nm). The composition of the secondary structural elements was analyzed using CDpro software (http://sites.bmb.colostate.edu/sreeram/CDPro/CDPro.htm). The thermal denaturation temperature of AlgM4 was measured under the following conditions: spectral range, 200–260 nm; bandwidth, 0.7 nm; 25–75 ◦C. The Tm value was calculated using Global 3 software (Applied Photophysics Ltd., Surrey, UK).
