*4.1. Fucoidan Substrates*

Crude fucoidans from *Sargassum mcclurei*, *Fucus evanescens*, *Undaria pinnatifida*, and *Saccharina cichorioides* were extracted as described by Zvyagintseva et al. (1999) [38]. Fucoidan from *S. mcclurei* was purified further by ion-exchange chromatography [12]. *Turbinaria ornata* fucoidan was extracted as described by Thanh et al. (2013) [13]. Fucoidans from *F. evanescens*, *U. pinnatifida*, and *S. cichorioides* were purified as described by Kusaykin et al. (2006) [39]. Pure fucoidan from *Fucus vesiculosus* (F8190) was from Sigma-Aldrich (Steinheim, Germany).

#### *4.2. Enzymes and Gene Constructs*

Amino acid sequences for the five enzymes FcnA2, the C-terminal truncated version of FcnA (CAI47003.1) from *Mariniflexile fucanivorans* SW5 [24]; Fda1 (AAO00508.1) and Fda2 (AAO00509.1) from *Alteromonas* sp. SN-1009 (); FdlA (AAO00510.1) and FdlB (AAO00511.1) from *Flavobacterium* sp. SA-0082, were retrieved from GenBank (Table 1). The construct containing the gene encoding FcnA2 was designed to harbour an N-terminal 6xhistidine tag, while the gene constructs Fda1, Fda2, FdlA, and FdlB encoding the Fda1, Fda2, FdlA, FdlB proteins, respectively, were designed to harbour an N-terminal 10× histidine tag. The synthetic codon-optimised genes (optimised for *E. coli* expression), all devoid of their original signal peptide, were synthesised by GenScript (Piscataway, NJ, USA) and delivered as inserted either into the pET-45b(+) vector between the KpnI and PacI restriction

sites (FcnA2) or into the pET-19b(+) plasmid vector between the NcoI and XhoI restriction sites (all other enzymes).

For FcnA2 C-terminal deletion of 80 amino acids of the enzyme equivalent to deletion of 229 amino acids of FcnA (GenBank No. CAI47003.1) was constructed, and the truncated protein was named FcnAΔ229 (Table 1).

Both Fda1 and Fda2 contain two predicted laminin G (LamG) domains in the sequence. Deletion mutants devoid of one or both predicted LamG domains were constructed by PCR amplification of the codon-optimised genes, each with an additional C-terminal 10× histidine tag, using CloneAmp HiFi polymerase premix (Takara Bio USA Inc., Mountain View, CA, USA) (primer sequences are listed in Table S1). For Fda1, the truncated proteins were named Fda1Δ145 and Fda1Δ395, as 145 and 395 amino acids had been removed from the C-terminal end, respectively. Analogously, for Fda2, the truncated versions were named Fda2Δ146 and Fda2Δ390, indicating that 146 and 390 amino acids, respectively, had been removed from the C-terminal. The construct of Fda2-His was done by adding 10× histidine tag at the C-terminal end. After PCR amplification, products were digested with BsaI and XhoI and ligated into the pET19b (+) vector between the NcoI and XhoI sites. Positive clones were confirmed by DNA sequencing.

The *Escherichia coli* strain DH5α (Invitrogen® Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), was used as plasmid propagation host. BL21 (DE3) and C41 (DE3) (also from Invitrogen® Life Technologies) were used as expression hosts for the fucoidan-degrading enzymes (Table 1). Protein expression was done as described below.

### *4.3. Production of Recombinant Enzymes*

Expression of FcnA2 and FcnAΔ229 was performed in *E. coli* (BL21 (DE3) harbouring the Pch2 (pGro7) plasmid. Overnight cultures grown at 37 ◦C with agitation (180 rpm) in lysogeny broth (LB) medium containing 100 μg mL−<sup>1</sup> ampicillin and 34 μg mL−<sup>1</sup> chloramphenicol were used to inoculate 500 mL LB containing 100 μg mL−<sup>1</sup> ampicillin, 34 μg mL−<sup>1</sup> chloramphenicol and 0.05% arabinose. The inoculated LB was incubated at 37 ◦C with 180 rpm shaking until cultures reached 0.6-0.8 OD600. Enzyme expression was induced with 1mM IPTG for 20 h at 20 ◦C and 180 rpm.

Expression of Fda1, Fda1Δ145, Fda1Δ395, Fda2, Fda2-His, Fda2Δ146, and Fda2Δ390 was also performed in *E. coli* (BL21 (DE3) with Pch2 (pGro7)). Overnight cultures grown at 37 ◦C and 180 rpm in LB medium containing 100 μg mL−<sup>1</sup> ampicillin and 34 μg mL−<sup>1</sup> chloramphenicol were used to inoculate 500 mL auto-induction media containing 0.6% Na2HPO4, 0.3% KH2PO4, 2%, tryptone, 5% yeast extract, 5% NaCl, 0.6% glycerol, 0.05% glucose, 0.2% lactose, 0.05% arabinose, 100 μg mL−<sup>1</sup> ampicillin and 34 μg mL−<sup>1</sup> chloramphenicol. Cells were grown at 20 ◦C, 180 rpm for 20 h. Expression of FdlA, FdlB was performed in *E. coli* (C41 (DE3)). Overnight cultures grown at 37 ◦C and 180 rpm in LB medium containing 100 μg mL−<sup>1</sup> ampicillin were used to inoculate 500 mL LB containing 100 μg mL−<sup>1</sup> ampicillin and were grown at 37 ◦C and 180 rpm until cultures reached 0.6-0.8 OD600. The expression of the recombinant fucoidanases was induced with 1 mM IPTG during cell growth for 20 h at 20 ◦C and 180 rpm.

Cells were harvested by centrifugation at 5000× *g* for 20 min and 4 ◦C and the pellet was re-suspended in binding buffer (20 mM Tris-HCl buffer, 250 mM NaCl, 20 mM imidazole, pH 7.5) before being disrupted by UP400S Ultrasonic processor (Hielscher, Teltow, Germany)with 0.5 cycle and 100% amplitude. Cell debris was pelleted by centrifugation (20,000× *g*, 20 min at 4 ◦C). The supernatant obtained by centrifugation was then filtered through a 0.45 μm filter and applied to a 5 mL Ni2+ Sepharose HisTrap HP column (GE Healthcare, Uppsala, Sweden) which was equilibrated with binding buffer using an Äkta purifier (GE Healthcare, Uppsala, Sweden). The resin was washed 3 times with 20 mM Tris-HCl buffer, 250 mM NaCl, and 20 mM imidazole at pH 7.5 and proteins were eluted by a linear gradient of elution buffer (20 mM Tris-HCl buffer, 250 mM NaCl, and 20–500 mM imidazole, pH 7.5). The eluted fractions were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting as described below to assess the purity and

homogenous fractions were pooled. Protein content was determined by the Bradford assay [40] with bovine serum albumin as standard.

#### *4.4. SDS-PAGE*

The homogeneity and molecular weight of the recombinantly expressed proteins were estimated by (SDS–PAGE) electrophoresis according to the Laemmli protocol [41]. Electrophoresis was performed in 12% acrylamide gels with the addition of 4× Laemmli loading-buffer, to 40 μg of crude protein and 5 μg purified protein and 5 mM DTT. The analysis of total intracellular proteins was conducted by using the biomass from 300 μL culture with 100 μL of 4× Laemmli loading-buffer, 10 μL of samples were loaded on the 12% acrylamide gels. The Protein Plus molecular weight marker (Bio-Rad Laboratories, Hercules, CA, USA) with molecular weights of 10–250 kDa was used as standard.

### *4.5. Western Blot Analysis of Proteins*

Total intracellular protein, crude enzymes (40 μg) and pure enzymes (5 μg) were separated using 12% acrylamide gels with the addition of 4× Laemmli loading-buffer. Separated proteins were transferred onto a PVDF blotting membrane (GE Healthcare No. 1060022) and blotted in Tris-glycine pH 8.3 running buffer at 100 V for 45 min, after activation of the membrane in 96% ethanol for around 10 s. The membrane was blocked with 2% skim milk in 0.01 M TBS (Tris-based sodium chloride pH 7.6) buffer containing 0.1% Tween 20 (TBS\_T buffer) for 60 min. The membrane was then incubated in TBS\_T buffer with monoclonal anti-polyhistidine peroxidase conjugated antibody (Sigma-Aldrich, Steinheim, Germany) at 1:10.000 dilutions in a total volume of 30 mL for 1 h. The membrane was washed in TBS\_T buffer 3 × 10 min and TBS with 0.1% Tween20 for 20 min. The bound antibodies were detected by horse radish peroxidase using the AEC Kit (Sigma-Aldrich, Steinheim, Germany) according to manufacturer's protocol.

#### *4.6. Carbohydrate–Polyacrylamide Gel Electrophoresis (C-PAGE)*

Reaction mixtures containing 0.5 μg/μL enzyme solution in 20 mM Tris–HCl buffer pH 7.4, 250 mM NaCl and 10 mM CaCl2 (buffer A) and 1% weight/volume fucoidan in buffer A were incubated at 35 ◦C for 24–48 h. Each reaction mixture (10 μL) was mixed with 5 μL loading buffer (20% glycerol and 0.02% phenol red). Samples (5 μL) were electrophoresed at 400 V through a 20% (*w*/*v*) 1 mm thick resolving polyacrylamide gel with 100 mM Tris-borate buffer pH 8.3 for 1 h. Gel staining was performed in two steps, first with a solution containing 0.05% alcian blue 8GX (Panreac, Barcelona, Spain) in 2% acetic acid for 45 min and then with 0.01% O-toluidine blue (Sigma-Aldrich, Steinheim, Germany) in 50% aqueous ethanol and 1% acid acetic. The hydrolysate standard was obtained after enzymatic reaction of FFA2 on *Fucus evanescens* fucoidan [27].

#### *4.7. SEC Analysis*

High Performance Size Exclusion Chromatography was performed using an Ultimate iso-3100 SD pump with a WPS-3000 sampler (Dionex, Sunnyvale, CA, USA) connected to an RI-101 refractive index detector (Shodex, Showa Denko K.K., Tokyo, Japan). One hundred microliters of three times diluted reaction mixtures were loaded on a Shodex SB-806 HQ GPC column (300 × 8 mm) equipped with a Shodex SB-G guard column (50 mm × 6 mm) (Showa Denko K.K., Tokyo, Japan). Elution was performed with 100 mM sodium acetate pH 6 at a flow rate of 0.5 mL/min at room temperature. Pullunan standards were used as references.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/16/11/422/ s1, Figure S1: Recombinant expression of Fda1 in *E. coli*. (A) SDS-PAGE; and (B) Western blot of: (1) Autoinduced cells; (2) the cell debris (after sonication and protein extraction); and (3) crude extract after sonication and centrifugation. (St) is the protein plus molecular weight marker; Figure S2: Predicted protein domain structures of Fda1 and Fda2. Domains were predicted using NCBI conserved domain database (cdd) search tool and both proteins were found to contain two predicted LamG (Laminin G) superfamily domains. In Fda1, the domains

*Mar. Drugs* **2018**, *16*, 422

span from position 429 to 574 aa and from 670 to 809 aa. For Fda2, the domains span from 496 to 641 aa and from 737 to 876 aa. Arrows indicate the points of truncation. Deletion mutants were named according to deletion from the C-terminal end, i.e., Fda1Δ145, Fda1Δ395, Fda2Δ146, and Fda2Δ390; Figure S3. (A) SDS-PAGE; and (B) Western blot of induced cells of: (1) Fda1; (2) Fda1Δ145; (3) Fda1Δ395; (4) Fda2-His; (5) Fda2; (6) Fda2Δ146; and (7) Fda2Δ390. St is the protein plus molecular weight marker (Bio-Rad Laboratories, Hercules, CA, USA). Table S1: Primers for constructing C-terminal deletion mutants.

**Author Contributions:** H.T.T.C., M.D.M., M.J.L., and A.S.M. designed the research, analysed and interpreted the data, and prepared the manuscript. A.S.S. and M.I.K. prepared the fucoidan from *F. evanescens*, *S. cichorioides* and *U. pinnatifida* and enzyme activity standard for C-PAGE. L.M.B., V.T.T.T., T.D.P. and B.H.T. prepared the *S. mcclurei* and *T. ornata* fucoidans, and J.H. contributed the SEC analyses. All authors have read and approved the final manuscript.

**Acknowledgments:** This work was supported by grants from Vietnam Academy of Science and Technology (VAST.ĐA47.12/16-19, VAST.HTQT.NGA.06/16-17), the Fucosan Interreg Germany–Denmark project, and the Seaweed Biorefinery Research Project in Ghana (SeaBioGha) supported by Denmark's development cooperation (Grant DANIDA-14-01DTU), The Ministry of Foreign Affairs of Denmark.

**Conflicts of Interest:** All authors declare no conflicts of financial or non-financial interest.
