*2.5. FdlA and FdlB Action*

The FdlA and FdlB enzymes originating from *Flavobacterium* sp. SA-0081 (previously referred to as "*Fucobacter marina*") (Table 1) have been claimed to be specific for certain sulphated fuco-glucuronomannan (SFGM) structural fragments containing uronic acid and D-mannosyl α-linkages in fucoidan molecules [25]. The enzymes were purified from the SA-0082 strain and were shown to catalyse cleavage of SFGM fractions from the brown algae *Kjellmaniella crassifolia* (now *S. sculpera*) via a lyase mechanism cleaving the α-linkage between D-mannosyl and D-glucuronate in the SFGM fractions [33].


nd, not determined in this study. <sup>a</sup> Wild type signal peptide had been removed for codon-optimised synthesised construct; <sup>b</sup> Includes his-tags; <sup>c</sup> groES-groEL chaperone expressed from the pGro7 plasmid.

In this study, FdlA and FdlB both exerted activity on the fucoidans from *S. mcclurei*, *F. vesiculosus*, and *F. evanescens.* Only weak action was observed on fucoidans from *T. ornata* and essentially no activity on *S. cichorioides* and *U. pinnatifida* was found (Figure 3D,E). Fucoidan preparations from *S. mcclurei*, *T. ornata*, *F. evanescens* and *F. vesiculosus* may contain low amounts of uronic acid and sometimes traces of mannose [12,14,33–35] but until now no data show that D-mannosyl and D-glucuronate are present in the backbone of these fucoidans. Moreover, no lyase activity was detected by monitoring absorbance at 232 nm, indicating that the degradation products did not include unsaturated uronic oligosaccharides. FdlA and FdlB most likely cleave α(1→4) fucosyl bonds in the backbone of these fucoidans, since lack of activity on fucoidan from *S. cichorioides* and from *U. pinnatifida* (and weak action on *T. ornata* fucoidan) indicate that FdlA and FdlB do not cleave the α(1→3) bonds in fucoidan.

The similar weak degradation of the fucoidan substrates from *F. vesiculosus* and *F. evanescens* by both enzymes, i.e., producing almost similar oligomer profiles in the C-PAGE, suggests a preference for rare or complex fucosyl-sulphatation (e.g., C2 and C4) in the *Fucus* fucoidan substrates. Such substrates may occur more abundantly in *S. mcclurei* fucoidan (Figure 2), and the enzyme most likely prefers to attack only α(1→4) fucosyl-bonds. Enzyme FdlB appeared to exert a more profound action on

the *F. evanescens* substrate than FdlA. Interestingly, the action of the two enzymes on *S. mcclurei* galacto-fucan substrate produced a band that travelled further in the gel than the sulphated tetra-saccharide of the control, suggesting that both FdlA and FdlB are able to catalyse disintegration of sulphated fucoidan oligomers. Due to the high degree of depolymerisation, down to oligosaccharides of less than DP4 (Figure 3), and due to the high abundance of galactosyl residues in *S. mcclurei* fucoidan, we cannot rule out that FdlA and FdlB may cleave galactosyl-α(1→4) bonds (Figure 3D,E), and further analysis could confirm this conclusion.

#### *2.6. Further Assessment of Sargassum mcclurei Fucoidan Degradation*

C-PAGE and SEC of oligosaccharides released by the enzymes FcnA2, Fda2, FdlB and FdlA after extended reaction for 48 h, showed that each enzyme catalysed profound degradation of *S. mcclurei* fucoidan (Figure 4).

**Figure 4.** Degradation of *Sargassum mcclurei* fucoidan (S.m) by fucoidanase enzymes. (**A**) C-PAGE; and (**B**) size exclusion chromatography (SEC) of the products produced by: (1) FcnA2; (2) Fda2; (3) FdlA; and (4) FdlB on *S. mcclurei* fucoidan and molecular weight standards. 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 approximately 972 Da [27]. Reaction time was 48 h. (\*) indicates an enzymatic fucoidan degradation product of either lower mass or higher charge than the lowest St band (\*\*) compound.

In all cases, the smallest oligosaccharide ran further than the lowest of the standard, suggesting that the released oligosaccharides are either smaller or more charged, i.e., more sulphated, than the tetra-saccharide in the standard. The SEC profiles of the FcnA2 and Fda2 were similar, but the product profile differed from those of FdlA and FdlB which contained a smaller peak at around 22.5 min (corresponding to a molecular weight SEC standard of around 1.3 kDa), indicating that they acted slower if at all on certain fucoidan fragments <1.3 kD. Taken together with the C-PAGE results (Figure 4A), these data suggest that FdlA and FdlB exerted similar substrate attack preferences and left behind some oligomers around 1.3 kDa, whereas FcnA2 and Fda2 appeared to degrade the lower molar weight oligomers to a more significant extent.

#### *2.7. New Construct of FcnA2*

Western blot analysis of FcnA2 (Figure 2B) indicated that the spontaneous degradation of FcnA2 occurred from the C-terminal end, since the N-terminal His-tag was still present, making the protein visible in the Western blot. To avoid this degradation, a further truncation was made by removing an additional 80 amino acids from C-terminal end of the FcnA2 protein. This truncated enzyme was thus 229 amino acids shorter than the original FcnA enzyme and was called FcnAΔ229 (Table 1). FcnAΔ229 could be expressed very well and was purified with no apparent protein degradation, as illustrated by SDS-PAGE and Western blot analysis, giving the expected band size of 80 kDa (Figure 5A,B). FcnAΔ229 showed activity on the same substrates as FcnA2 (Figure 5C).

**Figure 5.** Purification and activity of enzyme FcnAΔ229. (**A**) SDS-PAGE indicating the expected molecular weight of 80 kDa and purity; (**B**) Western blot of purified FcnAΔ229. (St) is the protein plus molecular weight marker; and (**C**) enzyme activity by C-PAGE of (a) FcnA2 and (b) FcnAΔ229 on fucoidans from *S. mcclurei*, *F. vesiculosus* and *F. evanescens*. FcnA2 and FcnAΔ229 have similar profiles on *F. vesiculosus* and *F. evanescens* fucoidans. The reaction time was 24 h. 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 approximate 972 Da [27]. (\*) An oligosaccharide of lower molecular weight or higher charge than the lowest band in the standard (\*\*).

However, an oligosaccharide was released after 24 h that was running further than what was observed for FcnA2 (Figure 5C). This result indicated that the change in stability conferred by deletion of the 80 amino acids in FcnA2 in turn apparently enhanced substrate degradation, but the truncation did not confer any other apparent changes in the *S. mcclurei* degradation profile.

#### *2.8. Stabilisation through C-Terminal Truncation of Fda1 and Fda2*

The expression of Fda1 was high but the protein remained in the cell debris after sonication (Figure S1). By sequence analyses of Fda1 and Fda2, it was found that both enzymes contained two predicted Laminin G domains (IPR001791) (LamG domains) towards the C-terminal of each protein (Figure S2). Western blot analysis of Fda2 (Figure 2B) also indicated that enzyme destabilisation occurred via degradation from the C-terminal end as was observed for FcnA2. Hence, a strategy to stabilise the enzymes by deletion of the two predicted LamG domains in Fda1 and in Fda2 was

developed and new constructs of Fda1, called Fda1Δ145 (one LamG domain deleted) and Fda1Δ395 (both LamG domains deleted), were prepared (Table 1). An additional his-tag was included with these new constructs to ensure better binding to the Ni2+ Sepharose column. Notably for Fda2, in addition to being highly unstable, substantial amounts of protein were lost during purification, presumably due to lack of binding to the column (data not shown). This new construct was called Fda2-His (Table 1). In addition, as for Fda1, new constructs devoid of either one or both of the two predicted LamG domains of Fda2 were also constructed. These Fda2 C-terminal deletion mutants were named Fda2Δ146 and Fda2Δ390 (Table S1 and Figure S2).

SDS-PAGE and Western blot analysis showed that all modified enzyme constructs expressed well. Some protein degradation was evident, but notably the double LamG deletion constructs, Fda1Δ395 and Fda2Δ390, appeared more stable than full length enzymes (Figure 6A,B). All the truncated enzymes exerted activity on *S. mcclurei* fucoidan, verifying the enzyme stabilisation strategy by LamG deletion (Figure 6C). Further study verified that Fda1Δ395 was stable but that degradation of the other truncated enzymes (Fda1Δ145, Fda2-C-His, Fda2Δ146, and Fda2Δ390) occurred already inside the *E. coli* cells, presumably via action of proteases in *E. coli*, recognising sites in the C-terminal of the enzymes, since degradation was evident in the *E. coli* cells before sonication (Figure S3).

**Figure 6.** Purification and activity of Fda1 and Fda2 deletion mutants. (**A**) SDS-PAGE; and (**B**) Western blot of purified: (1) Fda1Δ145; (2) Fda1Δ395; (3) Fda2-C-His; (4) Fda2Δ146; and (5) Fda2Δ390. (St) is the protein plus molecular weight marker. The expected sizes of the proteins were 90, 50, 125, 110, 70 kDa respectively. (**C**) Enzymatic *S. mcclurei* fucoidan (S.m) degradation by C-PAGE: (1) Fda1Δ145; (2) Fda1Δ395; (3) Fda2-His; (4) Fda2Δ146; (5) Fda2Δ390; (6) Fda2; and the standard (St) resulting from FFA2 treatment of fucoidan from *F. evanescens*. 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]. (\*) indicates an enzymatic fucoidan degradation product of either lower mass or higher charge than the lowest St band (\*\*) compound. The reaction time was 48 h.
