**3. Materials and Methods**

#### *3.1. Materials*

Sodium alginate derived from brown seaweed was purchased from Sigma (St. Louis, MO, USA). SOURCETM 15Q 4.6/100 PE and Superdex peptide 10/300 gel filtration columns were purchased from GE HealthCare Bio-Sciences (Uppsala, Sweden). DNA polymerase, protein molecular weight markers, and polyacrylamide were purchased from New England Biolabs (Ipswich, MA, USA). Other chemicals and reagents used in this study were of analytical grade.

#### *3.2. Screening and Identification of Strain Alg07*

Sea mud and rotten kelp samples were collected from a seaweed farm in Weihai, China. Five grams of the samples were added to 45 mL of 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. After 48 h of enrichment at 30 ◦C, the culture was serially diluted with deionized water and spread on modified marine broth 2216 agar containing 10 g/L sodium alginate. The plates were incubated at 30 ◦C for two days until colonies appeared. Single colonies were inoculated into marine broth 2216 medium containing 10 g/L sodium alginate and incubated for 48 h at 30 ◦C. Then, the activities of alginate lyase in the supernatants were determined. The alginate lyase from one of the isolates, strain Alg07, showed the highest activity among all lyases.

To identify the Alg07 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 [42] through neighbor-joining method [43].

#### *3.3. Production and Purification of AlgA*

Strain Alg07 was cultured for 24 h at 30 ◦C and 180 rpm in the optimized liquid medium, which contained 1g/L peptone, 3 g/L yeast extract, 9 g/L sodium alginate, 5 g/L NaCl, 1 g/L MgSO4·7H2O, 5 g/L KCl, and 4 g/L CaCl2 (pH 6.5). The supernatant was collected after 30 min of centrifugation at 10,000 rpm and 4 ◦C and then concentrated and desalted using a tangential flow filtration system (Vivaflow 50, Sartorius, Goettingen, Germany). The concentrated solution was subjected to AKTA FPLC (GE Healthcare Life Science, Marlborough, MA, USA) equipped with a SOURCETM (Barrie, ON, Canada) 15Q 4.6/100 PE column that had been equilibrated with 20 mM Tris–HCl buffer (pH 7.0). Adsorbed proteins were eluted with a linear gradient of 0–0.5 M NaCl in equilibrating buffer under a flow rate of 1 mL/min. Fractions possessing the highest specific activity among all fractions were pooled and dialyzed against 20 mM Tris-HCl buffer (pH 7.0) for further enzyme characterization. All purification procedures were performed at 4 ◦C. Protein concentration was determined with a protein quantitative analysis kit (Solarbio, Beijing, China) using bovine serum albumin as the calibration standard. The purity of the isolated alginate lyase was analyzed through 12.5% SDS-PAGE in accordance with the method of Laemmli (1970) [44].

#### *3.4. Enzyme Activity Assay*

To determine the activity of alginate lyase, 100 μL of appropriately diluted enzyme was added to 1900 μL of substrate solution containing 10 g/L sodium alginate, 20 mM Tris-HCl, and 200 mM NaCl (pH 7.5). The reaction was allowed to proceed for 20 min at 40 ◦C and terminated by the addition of 20 μL of 10 M NaOH. Absorbance at 235 nm was recorded. One unit (U) was defined as the amount of enzyme required to increase the absorbance at 235 nm by 0.1 per min. For kinetic parameter analysis, the 3,5-dinitrosalicylic acid method was performed to determine alginate lyase activity based on the release of reducing sugars from substrates [45]. One unit (U) was defined as the amount of enzyme required to release 1 μmol of reducing sugar per min. All enzyme reactions were performed in triplicate, and reaction parameters were expressed as mean ± standard deviation.

#### *3.5. Characterization of AlgA*

Enzyme reactions were carried out at different temperatures (25 ◦C–60 ◦C) to determine the effect of temperature on AlgA. To evaluate the thermal stability of AlgA, the enzyme was incubated for different intervals at 40 ◦C, 45 ◦C, and 50 ◦C. Then, the residual activities of the enzyme were tested. The activity of the enzyme stored at 4 ◦C was used to represent 100% enzyme activity. The effect of different pH values on AlgA was determined by calculating the residual activities of AlgA after 20 h of incubation at 4 ◦C in 20 mM sodium acetate (pH 3.0–6.0), Tris-HCl (pH 6.0–9.0), or glycine-NaOH (pH 9.0–11.0) buffers. The initial activities in different pH buffers represented 100% enzyme activity.

Enzyme reactions were performed in the presence of different concentrations of NaCl (0–500 mM) to evaluate the effect of NaCl on AlgA. To determine the effects of metal ions and EDTA on the activity of AlgA, the highest enzyme activity was considered as 100% enzyme activity. AlgA was subjected to an activity assay after 12 h of incubation at 4 ◦C in the presence of 2 mM of different metal ions and EDTA.

#### *3.6. Analysis of the N-Terminal Amino Acid Sequence of AlgA*

After SDS-PAGE, the protein band of AlgA was electro-transferred onto a polyvinylidene difluoride membrane (Imobulon; Millipore, Darmstadt, Germany). Amino acid sequences were determined with a PPSQ-31A protein sequencer (Shimadzu Corporation; Kyoto, Japan).

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

A standard enzymatic assay was performed to test the substrate preference of AlgA by using pectin, hyaluronan, chitin, agar, sodium alginate, polyM, and polyG as substrates. PolyM and polyG were prepared in accordance with the method of Haug et al. [46].

The kinetic parameters of AlgA toward alginate and polyM were determined by measuring the initial velocities of enzyme activity under various substrate concentrations and were calculated on the basis of the nonlinear regression fitting of the Michaelis–Menten equation using Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA).

#### *3.8. FPLC and ESI-MS Analysis of the Degradation Products of AlgA*

To elucidate the mode of action of AlgA toward alginate, alginate degradation was performed at 40 ◦C with 10 g/L sodium alginate as a substrate. The reaction was initiated by the addition of 2 μg of purified AlgA in a 10 mL reaction volume. Reaction solutions were withdrawn at appropriate time intervals, and AlgA was inactivated by 5 min of boiling. The samples were analyzed through FPLC with a Superdex peptide 10/300 gel filtration column (GE Health, Marlborough, MA, USA) with 0.2 M ammonium bicarbonate as the mobile phase at a flow rate of 0.4 mL/min [34]. The reaction was monitored at 235 nm.

To determine the oligosaccharide compositions of the final digests, an excess of AlgA was used to completely degrade 10 g/L sodium alginate, polyM, or polyG. The reaction was carried out in a 10 mL reaction volume at 40 ◦C for 24 h and then terminated by boiling for 5 min. Oligosaccharides were separated through gel filtration as described above. Peak fractions containing unsaturated oligosaccharide products were collected and repeatedly freeze-dried to remove NH4HCO3 for ESI-MS analysis. The molecular weight of each oligosaccharide fraction was determined using the ESI-MS method on microTOF-Q II equipment (Bruker, Billerica, MA, USA) with the following conditions: capillary voltage of 4 kV, dry temperature of 180 ◦C, gas flow rate of 4.0 L/min, and scan range of 50–1000 *m*/*z*.

#### **4. Conclusions**

An alginate lyase–producing marine bacterium was isolated and identified as *Bacillus* sp. Alg07. AlgA, the alginate lyase derived from *Bacillus* sp. Alg07, was purified through the two simple steps of

tangential flow filtration concentration and anion-exchange chromatography. The results of SDS-PAGE indicated that the molecular mass of AlgA is approximately 60 kDa. The optimal temperature and pH for the activity of AlgA is 40 ◦C and 7.5, respectively. The activity of AlgA is dependent on NaCl and is promoted by the addition of Mg2+ and Ca2+. Under optimal conditions, the specific activity of AlgA reaches up to 8306.7 U/mg, which is the highest activity recorded for all reported alginate lyases. Moreover, AlgA is stable over a broad pH range (5.0–10.0) and under its optimal temperature (40 ◦C). AlgA is an endolytic polyM-specific alginate lyase and mainly produces disaccharides, trisaccharides, and tetrasaccharides from alginate and disaccharides and trisaccharides from polyM. The highly efficient and thermostable AlgA can have potential applications in the production of mannuronic oligosaccharides and polyG blocks from alginate.

**Supplementary Materials:** The following are available online at www.mdpi.com/1660-3397/16/3/86/s1, Figure S1: Purification of alginate lyase by anion exchange chromatograph (Source 15Q); Figure S2. The N-terminal amino acid sequence of the purified AlgA; Figure S3. Effects of substrate concentration on the activity of AlgA.

**Acknowledgments:** This work was supported by the National key R&D Program of China (2017YFD200900), the Science Technology Planning Project of Tianjin (16YFXTNC00160), and Youth Innovation Promotion Association, Chinese Academy of Sciences (to Yueming Zhu).

**Author Contributions:** Peng Chen and Yueming Zhu conceived and designed the experiments; Peng Chen performed the experiments; Peng Chen and Yan Zeng analyzed the data; Yan Men contributed reagents/materials/analysis tools; Peng Chen wrote the paper. Yuanxia Sun edited the paper.

**Conflicts of Interest:** The authors declare no conflict of interest.
