*2.3. FcnA2 Catalyses Cleavage of α(1*→*4) Fucosyl Bonds in Sulphated Fucoidan Backbones*

The recombinantly expressed FcnA2 enzyme exerted highest activity on the fucoidan from *F. evanescens*, and the degradation of this substrate was much more profound than on *F. vesiculosus*, even though both substrates have similar alternating α(1→3) and α(1→4) glycoside bonds in the backbone. The degradation of fucoidan from *F. evanescens* was in agreement with previous data showing that FcnA2 is able to degrade fucoidan from *Pelvetia canaliculata* [24]. The fucoidans from *F. evanescens* and *P. canaliculata* presumably have less if any C2, C4 disulphates in the "−1" position of the α(1→4)-L-fucosyl linkage compared to the fucoidan substrate from *F. vesiculosus*, which likely contains more fucosyl residues with C2/C4 and even some with C2/C3 disulphatation than the *F. evanescens* fucoidan. The lesser degree of C2/C4 and C2/C3 disulphatation might be the reason for the *F. evanescens* fucoidan being more degraded than the *F. vesiculosus* fucoidan (Figure 3). Hence, FcnA2 most likely catalyses cleavage of (1→4)-α-glycosidic bonds between the −1 fucosyl residues having the sulphate group at C2, but not at both C2, C4. However, detailed structural elucidation of the fucoidan products and modelling of the substrate accommodation in the enzyme's active site are warranted to substantiate this hypothesis. The differences in the degradation of fucoidan from *F. evanescens* and *F. vesiculosus* thus indicate that differences in the sulphatation pattern or in other types of substitutions on the substrate backbones may influence the action of FcnA2 on these two *Fucus* sp. derived fucoidans. The data suggest that the presumed presence in *F. vesiculosus* of fucosyl residues with disulphate at C2, C4 (on either the −1 or +1 position of the α(1→4) glycoside bond) may retard the enzymatic action of FcnA2.

The smallest oligomers released from *F. evanescens* by FcnA2 also differed from those released by the FFA2 treatment of fucoidan from *F. evanescens* in the standard (st) (Figure 3). FFA2 catalyses the cleavage of (1→4)-α-glycosidic bonds in the *F. evanescens* fucoidan within the structural fragment [→3)-α-L-Fucp2S-(1→4)-α-L-Fucp2S-(1→]n but not in the fragment [→3)-α-L-Fucp2S,4S-(1→4)-α-L-Fucp2S-(1→]n. The difference in the oligomers released suggests that the sulphatation preferences of the FFA2 and FcnA2 may differ, which invites to further elucidation of the enzyme structures and detailed analyses and modelling of enzyme-substrate interactions. FcnA2 also catalysed degradation of the sulphated galacto-fucan fucoidan from *S. mcclurei* resulting in production of several low molecular weight bands in the C-PAGE (Figure 3B). The partial degradation is in agreement with the enzyme attacking α(1→4) linked (sulphated) L-fucosyl residues. Nevertheless, this enzymatic degradation of *S. mcclurei* fucoidan is a novel finding, as enzymatic modification of the *S. mcclurei* fucoidan has not previously been reported. The apparent lack of action of FcnA2 on the fucoidan from *T. ornata*, *S. cichorioides*, and *U. pinnatifida* suggests that FcnA2 does not catalyse cleavage of α(1→3) bonds between fucosyl residues, whereas the activity on the other three substrates supports the hypothesis that the enzyme attacks α(1→4) bonds between L-fucosyl residues as previously shown [24].
