*2.9. C-Terminally Truncated Fda1 Attacks α(1*→*3)-Linkages*

The truncated Fda1 proteins Fda1Δ145 and Fda1Δ395 both catalysed degradation of most of the fucoidan substrates, although compared to the degradation achieved on the *S. mcclurei* fucoidan, the extent of degradation appeared to be lower (Figure 7). Both truncated enzymes produced comparable degradation patterns, releasing fucoidan oligo-saccharides that migrated to the same extent within the C-PAGE gels. Interestingly, Fda1 mutants were able to catalyse the degradation of fucoidans rich in α(1→3) fucosyl linkages from *T. ornata*, *S. cichorioides* and *U. pinnatifida* (Figure 7), indicating that the C-terminally truncated Fda1 enzymes attack α(1→3)-linkages as previously described [25]. Removal of up to 47% of the Fda1 sequence from the C-terminal thus resulted in a more stable enzyme that retain activity.

**Figure 7.** Enzyme activity of truncated Fda1 mutants by C-PAGE. Enzyme activity of (c) Fda1Δ145 and (d) Fda1Δ395 on fucoidans from *F. vesiculosus* (F.ve), *F. evanescens* (F.ev), *T. ornata* (T.o), *S. cichorioides* (S.c) and *U. pinnatifida* (U.p), and standard (st). Both enzymes show activity on all the tested substrates to a comparable degree. The lowest band (\*\*) of the standard (St), resulting from FFA2 treatment of fucoidan from *F. evanescens*, corresponds to a tetra-saccharide of (1→4)- and (1→3)-linked α-L-fucosyls with each fucosyl residue sulphated at C2; total mass has been calculated to be 972 Da [27].

#### **3. Discussion**

This work showed that different microbially derived fucoidan-degrading enzymes exert activity on an array of different fucoidan substrates from brown macroalgae, even the very complex *S. mcclurei* fucoidan. FcnA2, Fda2, FdlA, and FdlB were found to degrade *S. mcclurei* fucoidan, with Fda2, FdlA and FdlB having particularly high activity on this fucoidan, which is known to contain sulphated galacto-fucan structural units and both α(1→4) and α(1→3) L-fucosyl linkages (Figure 1). FcnA2 and FcnA2Δ229 were more active than all the other enzymes on fucoidan from *F. evanescens* and they were also more active on fucoidan from *F. evanescens* than on fucoidan from *F. vesiculosus* suggesting an effect

of the substrate sulphatation pattern or of other structural features of the substrate. Fda2 was the only enzyme that degraded fucoidans rich in α(1→3) L-fucosyl linkages, but FdlA and FdlB were also able to at least partially degrade the fucoidan from *T. ornata*. FdlA and FdlB were previously claimed to be lyases acting on manno-glucurono-linkages in fucoidan from *K. crassifolia* (i.e., *S. sculpera*). In the present work these enzymes were specifically found to act as endo-fucoidanases on fucoidans devoid of these types of bonds and did not produce any unsaturated 4–5 oligosaccharide uronides.

Enzyme stabilization was successfully achieved by targeted truncation of the C-terminal ends of FcnA2, Fda1 and Fda2. Interestingly, for FcnA2, the stabilisation by C-terminal truncation, to the enzyme variant FcnAΔ229, resulted in an enzyme which appeared able to foster more profound degradation of the *S. mcclurei* fucoidan than the parent enzyme. For Fda1 and Fda2, successful expression and stabilisation were attained by LamG domain deletion, in turn this stabilisation allowed us to show the ability of the otherwise unstable Fda1 to catalyse degradation of the *S. mcclurei* fucoidan. The data obtained have implications for use of these enzymes, including the stabilised versions, in fucoidan processing.

Enzymatically produced short sulphated fuco-oligosaccharides, with degree of polymerisation of 4–10, derived from *Sargassum horneri*, obtained via treatment with a recombinant GH family 107 endo-fucoidanase, FFA1 (originating from the marine bacterium *Formosa algae*), were recently reported unable to suppress growth of DLD-1 human colon cancer cells in vitro, whilst this ability, i.e., potential anti-cancerogenic activity, is significant for native fucoidan from *S. horneri* [28]. In contrast, enzymatically produced sulphated fucoidan products from *F. evanescens* have been reported to have a better effect than the corresponding native, higher molecular weight fucoidan, on the functional activity of innate immunity cells in vitro [36]. Partially depolymerised fucoidan fractions from *Saccharina cichorioides* exert strong inhibition of colony formation of colorectal carcinoma cells HT-29 in vitro [37]. It is not yet known whether specific structural units of fucoidan backbones or if particular sidechains or substitutions on fucoidans confer specific bioactivity functions. The results of the present work enable targeted production of defined fucoidan oligomer products. The availability of such homogenous fucoidan oligomers will permit rigorous research studies on the putative pharmaceutical functions of fucoidans of different structural configurations.

#### **4. Materials and Methods**
