**1. Introduction**

Fucoidan polysaccharides are a family of sulphated, fucose-rich polysaccharides uniquely produced by brown marine macroalgae (seaweeds) and certain marine invertebrates, such as sea

cucumbers [1,2]. In general, fucoidans, also known as fucose-containing sulphated polysaccharides (FCSPs), consist of a backbone of α-L fucosyl residues linked together by (1→3) and/or (1→4)-glycoside bonds. The bonds are organised in stretches of α(1→3) or of alternating α(1→3)- and α(1→4)-glycoside linkages, depending on the macroalgal origin of the fucoidan, i.e., the species, age, geographical origin, and collection time (season) [3]. The L-fucosyl residues may be sulphated (−SO3 −) at position C2 and/or C4 (rarely at C3). Some fucoidans have fucose, galactose, glucuronic acid or other mono- and oligosaccharides as short branches [1,4,5]. Galactofucans are the most structurally diverse group of fucoidans that have been characterised from brown algae to date. The galactofucans have galactose residues in their backbone or in their branches; the position and number of these galactose residues depend on the type of algae [6,7].

The structural diversity of fucoidans or FCSPs is very high as both the sulphatation pattern and the backbone bond pattern of α(1→3) and α(1→4)-glycosidic bonds vary significantly depending on the fucoidan source. The fucoidan from *Fucus vesiculosus*, which is available commercially, is known to be made up of a backbone of repeating disaccharide units of α(1→3)- and α(1→4)-linked sulphated L-fucosyl residues (C2, C2/C3, C2/C4, C4 sulphatation) [8–10] (Figure 1). Fucoidan from *Fucus evanescens* has a similar L-fucosyl backbone of alternating α(1→4) and α(1→3) L-fucosyls with sulphate substitution at C2. An additional sulphate may occupy position 4 in some of the α(1→3)-linked fucosyls, and the remaining hydroxyl groups may be randomly acetylated [1] (Figure 1). In contrast, the bonds in the backbone of the fucoidan from *Undaria pinnatifida* and *Saccharina cichorioides* are exclusively α(1→3). The backbone *U. pinnatifida* fucoidan is moreover assumed to be rich in 2,4-disulphate substituted fucosyl residues and to contain some β(1→4)-linked galactosyl residues as branches [11] (Figure 1). Some fucoidans have even more complex backbone structures as is the case, e.g., for fucoidan from the brown macroalgae *Sargassum mcclurei* and *Turbinaria ornata* commonly found along the Pacific Ocean coastline of Vietnam. The *S. mcclurei* fucoidan is essentially a sulphated galactofucan polysaccharide having both α(1→3) and α(1→4) linked fucosyl residues, as well as galactosyl-β(1→3) links to fucosyl, and α(1→6) linkages from fucosyl to galactosyl in the reducing end of the backbone (Figure 1). The fucosyl residues in *S. mcclurei* fucoidan are moreover differentially sulphated at C2 and/or at C4 and some of the galactosyl moieties are sulphated at C6 [12] (Figure 1). Fucoidan extracted from *T. ornata* collected at Nha-Trang bay, Vietnam, also seems to be a galactofucan. The backbone of *T. ornata* fucoidan has thus been proposed to consist of α(1→3)-linked L-fucosyls with galactosyl branches (Fuc:Gal ≈ 3:1) and has been found to have a high sulphate content of about 25% with sulphate attached mostly at C2, and to a lesser extent at C4, of both the fucosyl and the galactosyl residues [13,14] (Figure 1). The biological function of fucoidans in brown macroalgae is uncertain, but fucoidans have long been known to exert beneficial biological activities including anti-tumorigenic, immune-modulatory, anti-inflammatory, anti-coagulant and anti-thrombotic effects, as demonstrated in vitro and in vivo [14–16]. Fucoidan from *S. mcclurei*, including the unique galactofucan structural moieties with sulphated α(1→3) L-fucosyl and α(1→4) linked galactosyl residues, have for example been shown to inhibit colony formation of DLD-1 human colon cancer cells in vitro [12], and crude, sulphated fucoidan products extracted from *F. vesiculosus* and *Sargassum* spp. are known to cause growth inhibition and apoptosis of melanoma B16 cells in vitro and to enhance the activity of natural killer cells in vivo in mice resulting in the specific lysis of YAC-1 cells (a murine T-lymphoma cell line sensitive to natural killer cells) [15]. However, the high molecular weight, irregular structure, and viscosity of fucoidans are an obstacle for providing homogeneous preparations for soluble and concentrate pharmaceutical use. One approach to solve this problem is to use enzymes that can depolymerise the fucoidans providing a preparation that is easier to handle and also with potentially bioactive properties.

**Figure 1.** Representative fucoidan structures of brown macroalgae *Fucus evanescens*, *Fucus vesiculosus*, *Sargassum mcclurei*, *Turbinaria ornata*, *Saccharina cichorioides*, and *Undaria pinnatifida*: (**A**) main chain of *S. cichorioides* composed of α(1→3)-L-fucosyls; (**B**) main chain of *U. pinnatifida* fucoidan also composed of α(1→3)-L-fucosyls; (**B'**) branches of *U. pinnatifida* fucoidan [11]; (**C**) main chain of *F. evanescens* [1] and *F. vesiculosus* fucoidan [8–10], both composed of α(1→3)- and α(1→4)-linked L-fucosyls; (**D**) main chain of *T. ornata* fucoidan composed of α(1→3)-L-fucosyls [13,14]; (**D'**) branches of *T. ornata* of α(1→3)-L-fucosyls or of β(1→4)galactosyls and mixed fucosyl-galactosyls; (**E**) main chain of *S. mcclurei* fucoidan made up of mainly α(1→3)-L-fucosyls [12]; and (**E'**) branches or inserts in the main chain of *S. mcclurei* fucoidan. In all fucoidan structures: R1: −H or −SO3 <sup>−</sup>; R2: −H, −SO3 <sup>−</sup> or H3COC−; R3: SO3 <sup>−</sup>, H3COC− or branches; and R4: SO3 − or branches.

About 20 microorganisms, mainly marine bacteria, have been described that produce fucoidanases [17–21]. In addition, a few fucoidanases have been found in marine molluscs [22,23]. In 2006, the gene encoding a fucoidanase from the marine bacterium *Mariniflexile fucanivorans* SW5T was cloned and the recombinant enzyme named FcnA. A C-terminal truncated version of FcnA named FcnA2 was previously reported to exert endo α(1→4) action on fucoidan from *Pelvetia canaliculata* (a type of fucoidan encompassing both α(1→4) and α(1→3) fucosyl-linkages in the backbone) [24]. In 2002, the genes encoding for two endo-fucoidanases referred to as Fda1 and Fda2, from the marine bacterium *Alteromonas* sp. SN-1009 were sequenced and their use for degradation of sulphated fucoidan originating from the brown seaweed *Kjellmaniella crassifolia* (now called *Saccharina sculpera*) were patented [25]. In the patent, these enzymes were reported to catalyse cleavage of α(1→3)-glycosidic bonds in the *K. crassifolia* (*S. sculpera*) fucoidan [25]. FcnA, Fda1, and Fda2 all belong to the new glycoside hydrolase family GH107 in CAZy [26]. In 2017, two endo-fucoidanases, FFA1 and FFA2, from the marine bacterium *Formosa algae* (KMM 3553T) were characterised and also suggested to belong to GH family 107 [27,28]. The FFA2 enzyme was proposed to be a poly[(1→4)-α-L-fucoside-2-sulphate] glycano hydrolase [27]. Already in 2003 Sakai et al. reported the finding of a new type of extracellular endo-fucoidan-lyase activity from "*Fucobacter marina*" SA-0082, or more correctly *Flavobacterium* sp. SA-0082, which acted on sulphated fucoglucurono-mannan from *K. crassifolia* (*S. sculpera*) [29,30]. By sequence analyses, it was found that this lyase activity was apparently encoded by two separate coding regions. Recombinant expression of these two putative fucoidan degrading enzymes, referred to as FdlA and FdlB, respectively, showed that the two enzymes had about 56% amino acid sequence identity and both were claimed to act as (glucurono-) fucoidan lyases on *K. crassifolia* (*S. sculpera*) fucoidan [25].

The objective of this work was to compare the catalytic properties, notably the substrate degradation patterns, on different fucoidans of the three GH107 endo-fucoidanases (EC 3.2.1.-) referred to as FcnA2, Fda1, and Fda2, and the two enzymes previously reported to be endofucoglucuronomannan-lyases, referred to as FdlA and FdlB. The action of the enzymes on different fucoidan substrate structures was compared by assessing oligomer product profiles resulting after treatment with recombinantly produced enzymes on fucoidans originating from six different types of brown seaweeds: *Sargassum mcclurei*, *Turbinaria ornata*, *Fucus evanescens*, *Fucus vesiculosus*, *Saccharina cichorioides*, and *Undaria pinnatifida*. We also report stabilisation of the recombinantly produced enzymes by targeted gene truncation resulting in deletion of large parts of the C-terminal end of several of the enzymes.

#### **2. Results**
