**Table 1.** *Cont*.

\*: Not specified.

### **5. Enzymatic Modification of Native Fucoidans**

Owing to their high molecular weight, therapeutic applications of native fucoidans face many challenges including structure elucidation, solubility, manufacturing, and handling [63,116], in addition to safety as a food supplement [175]. Structure elucidation and quantitation of native fucoidans is highly complicated and requires advanced or hyphenated spectroscopic techniques such asHPLC-MS/MS as it applied in Sea Cucumbers fucoidans [176,177]. Also, these techniques must be applied after a step of enzymatic or acid hydrolysis to transform the fucoidans polymers to oligomers. According to their molecular weight, fucoidans are classified into three classes: LMWF (<10 kDa), medium molecular weight fucoidan (MMWF) (10–10000 kDa), and HMWF (>10000 kDa) [31]. LMWF demonstrated better bioavailability and bioactivities than HMWF [178,179]. As a consequence, several articles reported physical, chemical and enzymatic modification of the native HMWF to ge<sup>t</sup> LMWF of higher biological activity [62]. Specifically, enzymatic modification of macroalgal polysaccharides, including fucoidans by either fucoidanases or sulfatases, is characterized by regioselectivity and

stereospecificity. This new trend is considered crucial and highly promising for current and future applications of polysaccharides [180].

Nevertheless, our publications in 2009 particularly reviewed the specific enzymatic degradation of fucoidans induced by fucoidanases (EC 3.2.1.44) and <sup>α</sup>-L-fucosidases (EC 3.2.1.51), mainly those isolated from marine bacteria [35]. Cumashi, et al. studied the chemical structures of different fucoidans isolated from a number of brown algal species [181]. Their proposed models, which were highly appreciated and recommended by many researchers [60], showed the backbone of fucoidans to be mainly an alternating α-(1-4) and α-(1-3) linked L-fucopyranoside. Regarding the sulfation pattern, C-2 is usually substituted with sulfate ester groups in addition to alternating C-3 or C-4 in L-fucopyranose residue, according to the glycosidic linkages. In addition, branched chain polymers were also found as in *F. serratus*. Other minor sugar units (e.g., mannose, galactose, glucose and xylose) occur as well in fucoidans structure; however, their distribution pattern and positions are still unknown [60,181]. Now, the mechanism of enzymatic degradation can be described in relation to fucoidans chemical structures.

Despite the increasing number of publications investigating fucoidanase activity of different marine species cell extracts, few of these enzymes have been isolated and characterized. Moreover, genome sequences encoding few fucoidanases have been published, including Ffa2 and FFA1 from *Formosa algae* KMM 3553<sup>T</sup> [182,183], FcnA from *Mariniflexili fucanivorans* SW5T [184]. Therefore, specificity of fucoidanases, type of cleaved glycoside bond, structure-activity relationship studies and enzyme stability are still poorly described. It was only observed that identified microbial fucoidanses act only on fucoidans isolated from their respective symbionts [185]. In fact, fucoidanases have not actually been fully utilized ye<sup>t</sup> as a powerful tool either for the structural studies of fucoidans or production of defined and well-characterized bioactive fragments of extracted fucoidans, as shown in Table 2.

Similarly, recent advances in bioinformatics and genome sequencing of microbial species have resulted in a continual increase of novel genome sequences. These genomes demonstrated various potential genes encoding for enzymes with biopolymer-degrading capabilities, such as *Shewanella violacea* DSS12 (NC\_014012.1), *Formosa algae* KMM 3553 (NZ\_LMAK01000014.1) [182], *Formosa haliotis* MA1 (NZ\_BDEL01000001.1) [198], *Wenyingzhuangia fucanilytica* CZ1127 (NZ\_CP014224.1) [199] and *Pseudoalteromonas* sp. strain A601 (MXQF01000000) [200]. Moreover, production of stabilized fucoidanases has been achieved by targeted truncation of the C-terminal of FcnA2, Fda1 and Fda2. This recently developed method may help with enzymatic production of defined degrees of polymerization and more bioactive products from native fucoidan substrates [201].


**Table 2.** Source of fucoidans as a substrate and mode of action of some fucoidanases.

\* n.d.: not determined.

## **6. Conclusion and Future Prospective**

As multifunctional molecules, fucoidans have received special interest based on their proven efficacy in different fields. The current article reviewed many aspects related to fucoidans' production, mainly from brown algae. Biogenic source and downstream processes were shown as major factors determining their application, which is affected by molecular weight and quality grade of fucoidans. Therefore, the alteration of fucoidans' native structure was recommended, especially as performed by fucoidanases. Their production in nanoform or in combination with other polymers can improve or modify their potential uses, allowing their expanded potential as therapeutic agents, e.g., in anti-cancer applications [202].

Production of high-quality purified fucoidans is urgently required to clarify the relationships between chemical structure and the various bioactivities attributed to fucoidans, eliminating any interference from contaminants. However, it was observed in some cases that crude extracts and presence of co-extracted contaminants, especially polyphenolic phlorotannins, have advantageous cosmeceutical effects due to their powerful anti-oxidant activity [203,204].

Novel techniques, either in cultivation or downstream processes, have been established, increasing the global production yields and reducing ecological and economic problems. A new advance toward achieving such goals was established by optimization of water extraction via measurement of kinetic parameters [205]. In addition to this, it is expected that most future trends in marine biotechnology research will focus on the cell wall and extracellular matrix components of brown algae, including fucoidans' biosynthetic genes and production regulators [23,53,63,206–208]. Such trials may enable the scientific community to produce more bioactive molecules of fucoidans with defined characteristics, including degree of polymerization, sulfate content and pattern, in reproducible manners.

**Author Contributions:** R.U. planned the manuscript's topics and is the corresponding author, while A.Z. collected the data and wrote the article. All authors have read and agreed to the published version of the manuscript.

**Acknowledgments:** The research is funded by the "Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)-Project-ID 172116086-SFB 926". The authors would like also to thank Mrs. Aya Abdella and Ms. Gabrielle Phillips for helpful comments and English editing of the manuscript.

**Conflicts of Interest:** The authors declare no conflicts of interests.
