*3.3. Sequence Analysis*

In our previous study, genomic sequence analysis of *Vibrio*. sp SY01 showed a putative alginate lyase-encoding gene, *aly08*. The gene was cloned from the genome of strain SY01 and deposited in the Genbank database (accession number MH791447). The open reading frame (ORF) was identified with the program ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/) and the signal peptide was analyzed using the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/). The domain analysis of *aly08*, and its family analysis, is based on a comparison with the CDD (https://www.ncbi.nlm.nih.gov/cdd). The theoretically pI and Mw of *aly08* was determined by pI/Mw Tool (http://web.expasy.org/compute\_ pi/). Afterwards, the BLAST algorithm program on NCBI was used to search for similar sequences of *aly08*. Multiple sequence alignment was constructed using ClustalX 2.1 (National Center for Biotechnology Information, Bethesda, MD, USA), and the phylogenetic tree was created using the bootstrapping neighbor-joining method of MEGA 6.0 (Center for Evolutionary Medicine and Informatics, The Biodesign Institute, Tempe, AZ, USA).

#### *3.4. Heterologous Expression and Purification of Recombinant Aly08*

In order to express Aly08, the primers (PyAly08-F and PyAly08-R) were used to amplify the genomic DNA of *Vibrio* sp. SY01 without a signal sequence or stop codon. The *aly08* gene was then ligated into the expression vector pET-22b(+) with recognition sites *Nde* I and *Xho* I. In addition, the recombinant plasmid pET22b-Aly08 with a C-terminal 6 × His-tag was transformed into the *E. coli* BL21 (DE3) and grown on media (50 μg/mL ampicillin). Single colonies of *E. coli* BL21-pET22b-Aly08 were picked and cultured in LB medium (50 μg/mL ampicillin) at 37 ◦C with shaking at 200 rpm until OD600 reached 0.6–0.8. In order to induce the expression of protein, and the incubation was continued for 20 h at 20 ◦C (containing 0.1 mM isopropyl β-d-thiogalactoside (IPTG)). The cultured supernatant was harvested by high-speed refrigerated centrifuge (Hitachi, Tokyo, Japan) system (12,000 rpm, 5 min, 4 ◦C) and loaded onto a Ni-NTA sepharose column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) using the AKTA150 automatic intelligent protein purification system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) which had been equilibrated with wash buffer (20 mM phosphate buffer (pH 7.6), 500 mM NaCl). The Ni-NTA sepharose column was then eluted with a linear gradient of imidazole (25-500 mM imidazole, 20 mM phosphate buffer, 500 mM NaCl, pH7.6) in order to collect the active fractions. The active fraction was further analyzed by 12% SDS-PAGE, and the PageRuler Prest Protein Ladder (Thermo Scientific, USA) was used as a protein standard marker. Afterwards, the protein concentration of purified Aly08 was determined by a BCA protein assay kit (Beyotime Biotechnology, Shanghai China), bovine serum albumin (BSA) was used as a standard.

#### *3.5. Alginate Lyase Activity Assay*

Absorption at 235 nm (A235) was used to measure the activity of Aly08 as previously described [43–45]. The appropriately diluted enzyme (100 μL) was mixed with 900 μL of 0.3% (*w*/*v*) sodium alginate solution (10 mM glycine-NaOH buffer and 100 mM NaCl, pH 8.35). Then, the reaction system was incubated at 45 ◦C for 10 min and terminated by boiling for 10 min, and its absorbance was measured on a NanoPhotometer Pearl-360 spectrophotometer (IMPLEN, Munich, Germany). Alginate lyase activity was determined by increasing A235 as the production of unsaturated double bonds occurred as the alginate lyase cleaved glycosidic bonds at the non-reducing end of the polymer chain. One unit (U) of enzyme activity was defined as the amount of enzyme required to increase A235 by 0.1 per minute, under the above conditions.

#### *3.6. Biochemical Characterization of the Recombinant Enzyme*

The enzyme and substrate was incubated under 10 mM glycine-NaOH buffer (pH 8.35) at various temperatures (10–70 ◦C) to obtain the optimal temperature for Aly08. The thermal stability of Aly08 was then assayed by measuring its activity after pre-incubation at various temperatures (10–70 ◦C) for 1 h. The influence of pH values on Aly08 was calculated by measuring the residual activities in different buffers. The following buffers were used: 50 mM Na2HPO4-citric acid (pH 4.6–7.0), Tris-HCl (pH 7.6–8.6), glycine-NaOH (pH 8.6–10.6), and Na2HPO4-NaH2PO4 (pH 6.6–7.6). The highest activities represent 100% enzyme activity. The pH stability of Aly08 was determined by measuring the residual activity after incubating the purified enzyme with various pH buffers (pH 4.6–10.6) at 4 ◦C for 12 h. Substrate solution (10 mM glycine-NaOH buffer and 100 mM NaCl, pH 8.35) with three different substrates [0.3% (*w*/*v*) sodium alginate, polyM block and polyG block] were prepared and used for assaying the activities of the purified enzyme Aly08 in order to determine the preferred substrate of Aly08. Afterwards, based on the protein concentration measured by the BCA protein kit, the specific activity of Aly08 with the different substrates was calculated. To measure the effects of chemical compounds and metal ions on enzymatic activity, different metal ions and chemical compounds were added to the reaction system with a final concentration of 1 mM. A reaction mixture containing no metal ion or chemical compounds was used as a control. All reactions were performed in triplicate and the reaction parameters were expressed as the mean ± standard deviation.
