*2.1. Recombinant Enzyme Expression*

The enzymes FcnA2, FdlA and FdlB expressed well and the purified enzymes gave the expected band sizes as assessed by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) (Figure 2A). The expression of recombinant Fda1 was high, but the protein remained in the cell debris after sonication (Figure S1). Several culture conditions for enzyme expression (temperature, medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration) were tested to obtain a soluble enzyme, but without success. Fda2 expressed well, but migrated slower in the SDS-PAGE gel than expected (94 kDa). At present, the data do not allow any firm conclusions to be drawn regarding the cause of this retarded migration of the Fda2 protein, but high hydrophobicity and high levels of charged amino acids may cause anomalous SDS-PAGE migration as compared to the soluble, commercial protein standards [31]. For the enzymes FcnA2 and Fda2 more than one band was visible in both the SDS-PAGE gel and in the Western blot (Figure 2), suggesting spontaneous degradation rather than impurities from other proteins. This observation agrees with previously published data for recombinantly expressed FcnA2 reporting "co-elution" with other proteins, which could not be separated by anion exchange or SEC [24]. For the Fda2 enzyme, use of protease inhibitors such as

PMSF (36978) from Thermo Fisher Scientific (Waltham, MA, USA) and a protease inhibitor cocktail (P8849) from Sigma-Aldrich (Steinheim, Germany) during purification did not improve stability, corroborating that the degradation likely occurred during expression in *E. coli* cells or during the subsequent purification.

**Figure 2.** Purified recombinantly expressed fucoidan-modifying enzymes. (**A**) SDS-PAGE, and (**B**) Western blot of purified FcnA2, FdlA, FdlB, and Fda2. (St) is the protein plus molecular weight marker. The expected molecular weights of the recombinant enzymes FcnA2, FdlA, FdlB, and Fda2 were 87, 75, 76, and 94 kDa, respectively. The multiple bands seen for FcnA2 and Fda2, notably in the Western blot, indicate partial degradation of the proteins. Expression of recombinant Fda1 resulted in insoluble enzyme material which is not shown in this figure.

#### *2.2. Substrate Specificity of the Recombinant Fucoidan-Degrading Enzymes*

The six different fucoidan samples were treated with the purified enzymes FcnA2, Fda2, FdlA, and FdlB and the treatments produced different carbohydrate-polyacrylamide gel electrophoresis (C-PAGE) patterns with the six fucoidan samples (the expression of recombinant Fda1 resulted in insoluble enzymes, which is why there are no data for Fda1). The reactions were run for 24 h to ascertain maximal substrate degradation. Preliminary data using higher enzyme dosage or longer reaction time did not show higher extent of degradation except of the *S. mcclurei* fucoidan that gave more visible bands in the C-PAGE after a 48 h reaction (these data are discussed further below). Hence, the data obtained by C-PAGE showed both the selectivity and the maximal extent of fucoidan degradation obtainable for each set of enzyme and substrate. This means that it is presumed that the unreacted higher molecular weight polysaccharides do not contain structural units, i.e., backbone-stretches, linkages, substitutions or branches, attackable by the particular enzyme examined. The positive control standard (St) was the hydrolysate from the enzymatic reaction of the *Formosa algae* FFA2 on *F. evanescens* fucoidan, where the lowest band corresponds to a tetra-saccharide of (1→4)- and (1→3)-linked α-L-fucosyls with each fucosyl residue sulphated at C2 [27] (Figure 3). The data obtained by C-PAGE indicated more extensive degradation of the fucoidan substrates from *Sargassum*

*mcclurei* (1), *Fucus vesiculosus* (2), and *Fucus evanescens* (3) than of substrates predominantly having α(1→3) glycoside bonds in their backbone structures, originating from *Turbinaria ornate* (4), *Saccharina cichorioides* (5), and *Undaria pinnatifida* (6), respectively (Figure 3). In general, the data obtained show that each enzyme produced differently sized sulphated oligomers in the C-PAGE chromatograms, suggesting that the different enzymes target different linkages and/or differently sulphated fucosyl residues. The results also suggest that all the enzymes were endo-acting as the enzymatic action left behind relatively high molecular weight fractions.


**Figure 3.** C-PAGE analysis of fucoidan degradation using purified enzymes: (**A**) substrate control with no enzyme; and (**B**–**E**) enzymatic products from reaction of FcnA2, Fda2, FdlA, and FdlB on different fucoidans, respectively (expression of recombinant Fda1 resulted in insoluble enzymes, which is why there are no data for Fda1): (1) *Sargassum mcclurei*; (2) *Fucus vesiculosus*; (3) *Fucus evanescens*; (4) *Turbinaria ornata*; (5) *Saccharina cichorioides*; and (6) *Undaria pinnatifida*. The extent of degradation is indicated with: (+++) highest, (++) medium, (+) lowest and (+) is positive activity resulting in a high molecular smear, while (−) is no activity. The standard (St) is the product profile of FFA2 treatment of fucoidan from *F. evanescens*. The lowest band (\*\*) of the St is a tetra-saccharide of (1→4)- and (1→3)-linked α-L-fucosyls sulphated at every C2 with an approximate mass of 972 Da [27]. (\*) indicates an enzymatic fucoidan degradation product of either lower mass or higher charge than the lowest St band (\*\*) compound. The reaction time was 24 h.
