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The review presents materials describing the seaweed-derived sulfated polysaccharides (SPS) as potential means for prevention and treatment of viral diseases of the respiratory tract, mainly influenza and COVID-19. The literature materials on the pathogenetic targets of influenza viruses and SARS-CoV-2, on the antiviral potential of SPS derived from red, brown and green algae, as well as on the mechanisms of antiviral action of these unique compounds are summarized. Seaweed SPS are characterized by high antiviral activity, good solubility, and almost complete absence of toxicity. Pathogens of respiratory infections do not form resistance under the SPS influence. The abovementioned facts allow us to consider these compounds as promising candidates for the creation of medicines, dietary supplements, and functional food products with antiviral and, above all, anti-influenza and anti-coronavirus activity on their basis in the future.

Genomic analysis of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T revealed the presence of four fucoidanase genes fwf1, fwf2, fwf3, fwf4 that belonged to the glycoside hydrolase family 107 (GH107, CAZy), which is located in one gene cluster putatively involved in fucoidan catabolism. Genes encoding two fucoidanases fwf1 and fwf2 were cloned, and the proteins FWf1 and FWf2 were produced in Escherichia coli cells. The recombinant fucoidanases were purified and the biochemical properties of these enzymes were studied. The amino acid sequences of FWf1 and FWf2 showed 41 and 51% identity respectively with a fucoidanase FcnA from the marine bacterium Mariniflexile fucanivorans, with the established 3D structure. Structures of the oligosaccharides produced during enzymatic hydrolysis of fucoidan by FWf1 and FWf2 have been determined by NMR spectroscopy. Detailed substrate specificities of FWf1 and FWf2 were studied using fucoidans and sulfated fucooligosaccharides with different structures. Both fucoidanases catalyzed hydrolysis of 1→4-glycosidic bonds between sulfated α-l-fucose residues but had different specificities regarding sulfation patterns of the fucose residues in fucoidan molecules. Specific cleavage sites recognizable by the fucoidanases in fucoidan molecules were determined. The obtained results provide new knowledge about differences between specificities of the fucoidanases belonging to the GH107 family.

The review considers the current views on pathogenesis and therapeutic targets in infectious diarrhea that develops as a result of exposure of enterocytes to viruses or bacterial toxins. The main methods of infectious diarrhea treatment and disadvantages of standard therapy are described. Modern data on the biological activity (bactericidal/bacteriostatic, antiviral, anti-biofilm, antiinflammatory, immunomodulatory, and antioxidant properties) of marine algae polysaccharides are presented. The possibilities of using seaweed polysaccharides as the basis of medicines, dietary supplements and functional food products for the prevention and treatment of infectious diarrhea are evaluated.

The substances contained in the aqueous–ethanol and aqueous extracts from freshly collected and destroyed brown algae Saccharina cichorioides in their second vegetation season and from excrement of the sea urchins Strongylocentrotus intermedius that had been fed these algae specimens were studied for effects on the digestive enzyme 1,3-β-D-glucanase, sperm, egg cells, and developing embryos of the sea urchin. The yield, composition, and structure of substances from fresh and destroyed S. cichorioides and from sea urchin excrement were analyzed. Sea urchins assimilated mannitol, proteins, and almost completely laminarans from destroyed thalli of S. cichorioides. The detection of activators of sea urchin 1,3-β-D-glucanase in the aqueous–ethanol extracts of destroyed algae and inhibitors of fertilization processes in the extracts of fresh algae is consistent with field observations. The biochemical study has shown that the digestion and reproduction processes in S. intermedius are controlled by metabolites of S. cichorioides to a significant extent.

The disease-preventive and medicinal properties of plant polyphenolic compounds have long been known. As active ingredients, they are used to prevent and treat many noncommunicable diseases. In recent decades, marine macroalgae have attracted the attention of biotechnologists and pharmacologists as a promising and almost inexhaustible source of polyphenols. This heterogeneous group of compounds contains many biopolymers with unique structure and biological properties that exhibit high anti-infective activity. In the present review, the authors focus on the antiviral potential of polyphenolic compounds (phlorotannins) from marine algae and consider the mechanisms of their action as well as other biological properties of these compounds that have effects on the progress and outcome of viral infections. Effective nutraceuticals, to be potentially developed on the basis of algal polyphenols, can also be used in the complex therapy of viral diseases. It is necessary to extend in vivo studies on laboratory animals, which subsequently will allow proceeding to clinical tests. Polyphenolic compounds have a great potential as active ingredients to be used for the creation of new antiviral pharmaceutical substances.

Enzymatic depolymerization of fucoidans attracts many researchers due to the opportunity of obtaining standardized fucoidan fragments. Fucoidanase catalyzes the cleavage of fucoidan from Fucus evanescens (FeF) to form low molecular weight products (LMP) and a polymeric fraction (HMP) with 50.8 kDa molecular weight and more than 50% yield. NMR spectroscopy shows that the HMP fraction has regular structure and consists of a repeating fragment [→3)-α- l -Fucp2,4OSO3−-(1 → 4)-α- l -Fucp2,4OSO3−-(1 → 4)-α- l -Fucp2OSO3−-(1→]n. The anticancer effects of FeF fucoidan and its derivative (HMP) were studied in vitro on colon cancer cells HCT-116, HT-29, and DLD-1. The anticancer activity of the HMP fraction was found to be slightly lower than that of the FeF fucoidan. Research and practical applications of the enzyme include modification of native fucoidans for purposes of regular and easier characterized derivatives acquisition.

This review is devoted to the analysis of publications on studies of phlorotannins, which are polyphenolic metabolites of brown algae. Their properties, the pathway of biosynthesis, and the structural diversity and classification of phlorotannins, as well as the methods for their isolation and determination of their metabolic profiles, are examined. The antioxidant, antitumor, and antimicrobial activities of phlorotannins and their role in the regulation of enzyme activity are discussed. These properties of phlorotannins determine the prospects of using phlorotannins in pharmacology.

Alginate lyases catalyze degradation of alginic acids and their salts, alginates, which are one of the main components of brown algae cell walls and comprise up to 40% algae’s dry weight. Alginates are interesting due to their high biological activity, particularly the ability of charged groups to bind tightly to oppositecharged protein amino acid residues, and chelating and jelling properties in presence of bivalent metal cations. Alginate lyases can digest substrates by β-elimination. They can be classified by the type of cleaved bonds. For today, more than 50000 amino acid sequences are referred to alginate lyases, 47000 of them belonging to bacterial genomes. Alginate lyases are one of the most common tools for degrading biofilms. Alginate digestion products display antitumor, anti-inflammatory, and antioxidant properties.

Fucoidans are a class of sulfated fucose-containing bioactive polysaccharides produced by brown algae. The biological effects exhibited by fucoidans are thought to be related to their sulfation. However, the lack of methods for sulfation control does not allow for a reliable conclusion about the influence of the position of certain sulfate groups on the observed biological effects. We identified the gene encoding the endo-acting fucoidan sulfatase swf5 in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. This is the first report on the sequence of fucoidan endo-sulfatase. Sulfatase SWF5 belongs to the subfamily S1_22 of the family S1. SWF5 was shown to remove 4O-sulfation in fucoidans composed from the alternating α-(1→3)- and α-(1→4)-linked residues of sulfated L-fucose but not from fucoidans with the α-(1→3)-linked backbone. The endo-sulfatase was used to selectively prepare 4O-desulfated fucoidan derivatives. It was shown that the 4O-desulfated fucoidans inhibit colony formation of DLD-1 and MCF-7 cells less effectively than unmodified fucoidans. Presumably, 4O-sulfation makes a significant contribution to the anticancer activity of fucoidans.

A search for enzymes involved in the degradation of polyanionic polysaccharides (fucoidans and alginic acid) was conducted among bacterial epiphytes of the brown alga Sargassum polycystum that grows in the territorial waters of the Socialist Republic of Vietnam. Two resistant bacterial strains, F10 and F14, have been isolated from the algal microflora that degrade the thallus of the alga under laboratory conditions. These bacterial strains differed in the morphological, physiological, and biochemical characteristics and in the composition of enzymes. The strains were studied for the ability to synthesize intracellular oligo-and polysaccharide hydrolases and alginate lyases. The optimal conditions for the growth of bacterial strain F14 and the biosynthesis of fucoidanase and polymannuronate-specific alginate lyase were determined. The partially purified alginate lyase was stable at a temperature up to 40°C and had an optimal pH 6.0 and an optimal temperature 35°C.

Fucoidans are a group of homo-and hetero-polysaccharides, which necessarily contains residues of sulfated α-L-fucose. Fucoidans are found only in brown algae. These polysaccharides exhibit a wide spectrum of biological activity and have a great therapeutic potential. Enzymes capable of catalyzing the degradation of fucoidans are absent in the mammalian enzyme system. The question arises: is the transformation of fucoidan in mammals, particularly in human possible? Studies in vivo (in situ) and in vitro have demonstrated that high molecular weight fucoidans are absorbed across rat intestinal epithelial cells, accumulated by liver macrophages, and characterized by low levels in blood and urine. Using the example of the Okinawa Prefecture (Japan) residents, it was shown that Cladosiphon okamuranus alga is digested and the fucoidan contained in this alga is absorbed in the human body.

Fucoidan hydrolase inhibitors were discovered for the first time. Metabolites in the aqueous EtOH extract of Fucus evanescens inhibited specifically the activity of enzymes involved in catabolism of brown-alga polysaccharides, i.e., fucoidanases (recombinant FFA from the marine bacterium Formosa algae KMM 3553T and PPF from the marine mollusk Patinopecten yessoensis) and glycosidases (recombinant α-L-fucosidase FucFa from F. algae and β-D-glucosidase G-II from the marine mollusk Littorina sitkana). The purified fucophlorethol fraction isolated from this extract inhibited recombinant fucoidanase FFA in the concentration range 10–50 μg/mL.

Brown seaweeds have drawn worldwide attention due to their involvement in many industrial applications. They produce neutral low molecular weight polysaccharides (laminarans) with beneficial biological activities. However, the anticancer activity and molecular mechanism of the laminarans and their sulfated derivatives have not been investigated in detail. Herein, the laminarans from brown seaweeds Saccharina cichorioides, Saccharina japonica, and Fucus evanescens were isolated. The laminarans from S. cichorioides and S. japonica were confirmed to contain a main chain of β-(1→3)-D-glucopyranose with single branches at C6. The laminaran from F. evanescens consisted of not only β-(1→3)-linked D- glucopyranose but also includes single β-(1→6)-linked D-glucose residues. The branches at C6 are presented as glucose or as gentiobiose. The sulfated laminarans with different degrees of sulfation were obtained by the chlorosulfonic acid-pyridine assay. In modified polysaccharides, the positions of sulfates are directly predetermined by the structure of native laminarans. The laminarans and their sulfated derivatives inhibited proliferation, colony formation, and migration of human colorectal adenocarcinoma, melanoma, and breast adenocarcinoma cells in different manners. The sulfated laminaran from F. evanescens possessed the highest anticancer activity in vitro and effectively prevented migration of breast adenocarcinoma cells by inhibiting of the Matrix Metalloproteinases 2 and 9 activity.

This review presents an analysis of works devoted to the anti-human immunodeficiency virus (HIV) activity of algae metabolites—sulfated polysaccharides (fucoidans, carrageenans), lectins, laminarans, and polyphenols. Despite the presence of a significant number of antiretroviral drugs, the development of new therapeutic and prophylactic agents against this infection remains very urgent problem. This is due to the variability of HIV, the absence of an animal model (except monkeys) and natural immunity to this virus and the toxicity of therapeutic agents and their high cost. In this regard, the need for new therapeutic approaches and broad-spectrum drugs, which in addition to antiviral effects can have anti-inflammatory, antioxidant, and immunomodulatory effects, and to which the minimum resistance of HIV strains would be formed. These requirements meet the biologically active substances of marine algae. The results of experimental and clinical studies conducted in vitro and in vivo are presented, and the issues of the anti-HIV activity of these compounds are considered depending on their structural features. On the whole, the presented data prove the high efficiency of seaweed metabolites and justify the possibility of their use as a potential basis for the development of new drugs with a wide spectrum of activity.

The laminarans are neutral water-soluble β-D-glucans of brown algae possessing potent immunomodulating, radioprotective, and anticancer activities. The aim of the present study was to investigate in vitro anticancer, radioprotective, and radiosensitizing activities of laminaran from brown alga Dictyota dichotoma and its sulfated derivative. The native and sulfated laminarans by themselves at non-toxic doses possessed significant anticancer activity against melanoma cells. Both polysaccharides protected normal epidermal cells, while only sulfated laminaran was able to sensitize melanoma cells to X-rays irradiation resulting in significant inhibition of cell proliferation, colony formation, and migration of cancer cells. The molecular mechanism of this action was related to the inhibition of MMP-2 and MMP-9 proteinases activity as well as down-regulation of kinases' phosphorylation of ERK1/2 signaling cascade. Taken together, the combination of sulfated derivative of laminaran from D. dichotoma with X-ray may serve as a potential treatment strategy for human melanoma.

// Olesia S. Vishchuk 1, 2, * , Huimin Sun 3, * , Zhe Wang 4 , Svetlana P. Ermakova 2 , JuanJuan Xiao 1 , Tao Lu 1 , PeiPei Xue 1 , Tatyana N. Zvyagintseva 2 , Hua Xiong 1 , Chen Shao 3, 5 , Wei Yan 4 , Qiuhong Duan 1 , Feng Zhu 1 1 Department of Biochemistry and Molecular Biology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China 2 G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of Russian Academy of Sciences, Laboratory of Enzyme Chemistry, 690022 Vladivostok, Russian Federation 3 Department of Urology, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China 4 Department of Pathology, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China 5 State Key Laboratory of Cancer Biology & Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi’an, Shaanxi 710032, PR China * These authors have contributed equally to this work Correspondence to: Wei Yan, e-mail: yanwei@fmmu.edu.cn Qiuhong Duan, e-mail: duanqhwz@hust.edu.cn Feng Zhu, e-mail: fengzhu@hust.edu.cn Keywords: fucoidan, Fucus evanescens , TOPK, cell transformation, colon carcinoma Received: November 01, 2015 Accepted: January 29, 2016 Published: February 25, 2016 ABSTRACT The fucoidan with high anticancer activity was isolated from brown alga Fucus evanescens . The compound effectively prevented EGF-induced neoplastic cell transformation through inhibition of TOPK/ERK1/2/MSK 1 signaling axis. In vitro studies showed that the fucoidan attenuated mitogen-activated protein kinases downstream signaling in a colon cancer cells with different expression level of TOPK, resulting in growth inhibition. The fucoidan exerts its effects by directly interacting with TOPK kinase in vitro and ex vivo and inhibits its kinase activity. In xenograft animal model, oral administration of the fucoidan suppressed HCT 116 colon tumor growth. The phosphorylation of TOPK downstream signaling molecules in tumor tissues was also inhibited by the fucoidan. Taken together, our findings support the cancer preventive efficacy of the fucoidan through its targeting of TOPK for the prevention of neoplastic cell transformation and progression of colon carcinomas in vitro and ex vivo .

The review summarizes results of studies of effects of sulfated polysaccharides from seaweed on herpesviruses and the course of herpesvirus infections. Importance of this problem is determined by the prevalence of herpesviruses that can persist in the human body and demonstrate a high degree of immune mimicry and resistance to antiviral agents. A wide range of physiological action of sulfated polysaccharides, receptor agonists of innate and adaptive immune cells, which possess potent antiviral, antioxidant and anti-inflammatory activities, open the possibility of their use for creation of new generation pharmacological substances and agents with associated activity for the treatment of herpesvirus infections.Obobshcheny rezul'taty issledovaniĭ deĭstviia sul'fatirovannykh polisakharidov iz morskikh vodorosleĭ na virusy gerpesa i techenie gerpesvirusnykh infektsiĭ. Aktual'nost' étoĭ problemy opredeliaetsia shirokim rasprostraneniem virusov gerpesa, sposobnykh pozhiznenno persistirovat' v organizme cheloveka, obladaiushchikh vysokoĭ stepen'iu immunnoĭ mimikrii i rezistentnost'iu k antivirusnym preparatam. Shirokiĭ spektr fiziologicheskogo deĭstviia sul'fatirovannykh polisakharidov – agonistov retseptorov kletok vrozhdennogo i adaptivnogo immuniteta, obladaiushchikh vyrazhennoĭ protivovirusnoĭ, antioksidantnoĭ i protivovospalitel'noĭ aktivnost'iu, otkryvaiut vozmozhnost' ikh ispol'zovaniia dlia sozdaniia farmakologicheskikh substantsiĭ i lekarstvennykh sredstv novogo pokoleniia s assotsiirovannoĭ aktivnost'iu dlia lecheniia gerpesvirusnykh infektsiĭ.

The toxic effects of a water–ethanol extract of the brown alga Saccharina (= Laminaria) cichorioides and the isolated I1 and I2 proteins on egg fertilization and the development of embryos of the sea urchin Strongylocentrotus intermedius were revealed and studied. The protective effects of fucoidans and 1,3;1,6-β-D-glucans (laminarans, translam, and glucan II) that were isolated from the brown algae S. cichorioides and Fucus evanescens against the toxic impacts of the I1 and I2 proteins were examined. It was found that the lifespan of sea urchin embryos increased from 1.5 to 3 times in the presence of 1,3;1,6-β-D-glucans and fucoidans, whereas the I1 and I2 proteins decreased embryo viability by 2 times. Preadministration of polysaccharides completely neutralized the inhibitory effects of the proteins on developing embryos. It was also shown that a water–ethanol extract of S. cichorioides, derived I1 and I2 proteins, and fucoidans has a contraceptive effect.

Structure and anticancer activity of fucoidan from Sargassum horneri and from products of its enzymatic transformation were investigated. A gene that encodes fucoidanase ffa1 in the marine bacteria F. algae was identified, cloned and the protein (FFA1) was produced in Escherichia coli. The mass of the gene product FFA1 is 111kDa. Sequence analysis has revealed that fucoidanase FFA1 belongs to the GH107 (CAZy) family. Recombinant fucoidanase FFA1 was used to produce fucooligosaccharides. Structure of 5 sulphated oligosaccharides with polymerization degree 4-10 was established by NMR-spectroscopy. The fucoidan extracted from S. horneri is almost pure fucan. The main chain of the fucoidan is established to consist mostly of the repeating →3-α-l-Fucp(2SO3-)-1→4-α-l-Fucp(2,3SO3-)-1→ fragment, with insertions of →3-α-l-Fucp(2,4SO3-)-1→ fragment. Unsulphated side chains with the α-l-Fucp-1→2-α-l-Fucp-1→ structure connect to the main one at the C4 of monosaccharide residue.

The fucoidanase was isolated and purified from the digestive glands of the marine mollusk Lambis sp. The molecular mass of homogeneous enzyme as estimated by SDS electrophoresis was about 50 kDa. Optima of pH and temperature were at 4.9 and 37 °C, respectively. Its half-inactivation time was 20 min at 54 °C. The fucoidanase had a Km value of 1.3 mg ml−1 for the hydrolysis of fucoidan from Fucus evanescens. The fucoidanase catalysed the hydrolysis of 1→3;1→4-α- l -fucans from Fucus evanescens and Fucus vesiculosus, but not 1→3-α- l -fucan from Saccharina cichorioides. Native fucoidan from F. evanescens was hydrolysed weakly in contrast to desulphated fucoidan. Based on the analysis of substrate specificity and the structure of the reaction products the fucoidanase from Lambis sp. catalyzed hydrolysis of 1→4-bonds in a fucoidan molecule as endo-enzyme.

Based on our own and current literature data, we analyzed the prebiotic properties of polysaccharides and extracts of seaweeds. The role of prebiotics, particularly polysaccharides, in the normalization of intestinal microflora is discussed; consideration is also given to the possibility of seaweed polysaccharide fermentation by colonic microflora and selective stimulation of the growth of colonic bifidobacteria. This review also analyzes other useful properties of seaweed polysaccharides and discusses the prospects for inclusion of seaweed polysaccharides in the composition of functional nutrition products for the purposes of correcting intestinal bacterial disorders and inflammatory processes and normalizing the immune and metabolic status.

Many marine-derived polysaccharides and their analogues have been reported as showing anticancer and cancer preventive properties. These compounds demonstrate interesting activities and special modes of action, differing from each other in both structure and toxicity profile. Herein, literature data concerning anticancer and cancer preventive marine polysaccharides are reviewed. The structural diversity, the biological activities, and the molecular mechanisms of their action are discussed.

The effects of six 1,3;1,6-β-D-glucooligo- and polysaccharides with different structures (ranging from 1 to 10 kDa in molecular mass and containing 10-25% of β-1,6-linked glucose residues) from brown algae, Saccharina cichorioides, on development of the chum salmon, Oncorhynchus keta (Walbaum), were evaluated. Exposure of chum salmon eggs to 1,3;1,6-β-D-glucans with a molecular mass of more than 2 kDa increased the survival of embryos and juveniles and their resistance to Saprolegnia infection by up to 2.5-fold, leading to a weight gain in juveniles of 40-55% compared with The control chum salmons. The 1,3;1,6-β-D-glucans with molecular mass of 6-8 kDa and used at a at concentration of 0.5 mg mL(-1) rendered the best stimulative effect.

The antiviral activity of different structure fucoidans (α-l-fucans and galactofucans) was studied using two model viral systems based on a lentiviral vectors and a replication competent Moloney murine leukemia virus (Mo-MuLV). It was found that investigated fucoidans have no cytotoxic effects on Jurkat and SC-1cell at the concentration range of 0.001–100 µg/mL. Fucoidans with different efficiency suppressed transduction of Jurkat cell line by pseudo-HIV-1 particles carrying the envelope protein of HIV-1 and infection of SC-1 cells by Mo-MuLV. According to our data, all natural fucoidans can be considered as potential anti-HIV agents regardless of their carbohydrate backbone and degree of sulfating, since their activity is shown at low concentrations (0.001–0.05 µg/mL). High molecular weight fucoidans isolated from Saccharina cichorioides (1.3-α-l-fucan), and S. japonica (galactofucan) were the most effective inhibitors.

Intracellular fucoidanase was isolated from the marine bacterium, Formosa algae strain KMM 3553. The first appearance of fucoidan enzymatic hydrolysis products in a cell-free extract was detected after 4 h of bacterial growth, and maximal fucoidanase activity was observed after 12 h of growth. The fucoidanase displayed maximal activity in a wide range of pH values, from 6.5 to 9.1. The presence of Mg2+, Ca2+ and Ba2+ cations strongly activated the enzyme; however, Cu2+ and Zn2+ cations had inhibitory effects on the enzymatic activity. The enzymatic activity of fucoidanase was considerably reduced after prolonged (about 60 min) incubation of the enzyme solution at 45 °C. The fucoidanase catalyzed the hydrolysis of fucoidans from Fucus evanescens and Fucus vesiculosus, but not from Saccharina cichorioides. The fucoidanase also did not hydrolyze carrageenan. Desulfated fucoidan from F. evanescens was hydrolysed very weakly in contrast to deacetylated fucoidan, which was hydrolysed more actively compared to the native fucoidan from F. evanescens. Analysis of the structure of the enzymatic products showed that the marine bacteria, F. algae, synthesized an α-l-fucanase with an endo-type action that is specific for 1→4-bonds in a polysaccharide molecule built up of alternating three- and four-linked α-l-fucopyranose residues sulfated mainly at position 2.

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.

Homogeneous alginate lyase with rare specificity exhibiting maximum activity at pH 5.0 and temperature range 40–55°C that catalyzed cleavage of 1→4 glycoside bonds between α-L-guluronic acid residues was isolated from liver-pancreas of the marine gastropod mollusk Lambis sp. The alginate lyase was classified as a poly-1→4-α-L-guluronate lyase (EC 4.2.2.11).

A specific endo-1,3-β-d-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-β-d-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-β-d-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it’s substrate specificity and classify this enzyme as glucan endo-1,3-β-d-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45–85% to β-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45 °C, pH 5.5. Half-life period at 45 °С is 20 min, complete inactivation happens at 55 °C within 10 min. Km for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-β-d-xylopyranoside, glycerol and methyl-α-d-glucopyranoside. The enzyme can be used in structure determination of β-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-β-d-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.

Water-soluble polysaccharides were isolated from the brown alga Eisenia bicyclis , which was collected near the coast of the Republic of Korea. The structures of laminaran and fucoidan were investigated. Laminaran from E. bicyclis was determined to be a glucan with β-(1 → 6) side chains linked to a β-(1 → 3) backbone with relatively few branch points. Based on nuclear magnetic resonance (NMR) data, the ratio of the β-(1 → 3) and β-(1 → 6) linkages was estimated as 2.6:1. Fucoidan from E. bicyclis was found to contain 1,3-linked fucose residues, some 1,6-, 1,2,6-, 1,4,6-linked galactose residues and traces of mannose and xylose. In addition, the amount of sulfate in fucoidan was 13.2%. Those polysaccharides were non-cytotoxic to human melanoma SK-MEL-28 and colon cancer DLD-1 cells. Laminaran and fucoidan from E. bicyclis inhibited the colony formation of those cells. Therefore, they may have potential as antitumor agents.

Accumulating data clearly indicate that the induction of apoptosis by nontoxic natural compounds is a potent defense against the development and progression of many malignancies, including colon cancer. Resveratrol and the fucoidans have been shown to possess potent anti-tumor activity in vitro and in vivo. The aim of the present study was to examine whether the combination of a fucoidan from the brown alga Saccharina cichorioides Miyabe and resveratrol would be an effective preventive and/or therapeutic strategy against colon cancer. Based on NMR spectroscopy and MALDI-TOF analysis, the fucoidan isolated and purified from Saccharina cichorioides Miyabe was (1→3)-α-l-fucan with sulfate groups at C2 and C4 of the α-l-fucopyranose residues. The fucoidan enhanced the antiproliferative activity of resveratrol at nontoxic doses and facilitated resveratrol-induced apoptosis in the HCT 116 human colon cancer cell line. Apoptosis was realized by the activation of initiator caspase-9 and effector caspase-7 and -3, followed by the cleavage of PARP. Furthermore, significant inhibition of HCT 116 colony formation was associated with the sensitization of cells to resveratrol by the fucoidan. Taken together, these results demonstrate that the combination of the algal fucoidan with resveratrol may provide a potential therapy against human colon cancer.

The effect of various pretreatment conditions of brown algae Fucus evanescens, Saccharina japonica, and Sargassum oligocystum on the yield and structural characteristics of fucoidans was studied. The conditions included extraction by aqueous EtOH (70%) and supercritical CO2 with 5% EtOH as solvent and without it. It was shown that highly pure fucoidans from each alga species were similar in yield and structural characteristics for various pretreatment conditions. The exception was 5% EtOH as co-solvent for pretreatment with supercritical CO2, which enabled only highly sulfated fucoidan fractions with homogeneous monosaccharide compositions to be selectively obtained.

A gene that encodes fucoidanase ffa2 in the marine bacterium Formosa algae strain KMM 3553T was cloned, and the protein (FFA2) was produced in Escherichia coli. Recombinant fucoidanase FFA2 was purified, and the biochemical properties of this enzyme were studied. The amino acid sequence of FFA2 showed 57% identity with known fucoidanase FcnA from Mariniflexile fucanivorans. The mass of the gene product FFA2 is 101.2 kDa (918 amino acid residues). Sequence analysis has revealed that fucoidanase FFA2 belongs to the GH107 (CAZy) family. Detailed substrate specificity was studied by using fucoidans from brown seaweeds as well as synthetic fucooligosaccharide with distinct structures. Fucoidanase FFA2 catalyzes the cleavage of (1→4)-α-glycosidic bonds in the fucoidan from Fucus evanescens within a structural fragment (→3)-α-l-Fucp2S-(1→4)-α-l-Fucp2S-(1→)n but not in a fragment (→3)-α-l-Fucp2S,4S-(1→4)-α-l-Fucp2S-(1→)n. Using synthetic di-, tetra- and octasaccharides built up of the alternative (1→4)- and (1→3)-linked α-l-Fucp2S units, the difference in substrate specificity and in the rate of enzymatic selectivity was investigated. Nonsulfated and persulfated synthetic oligosaccharides were not transformed by the enzyme. Therefore, FFA2 was specified as poly[(1→4)-α-l-fucoside-2-sulfate] glycanohydrolase. This enzyme could be used for the modification of natural fucoidans to obtain more regular and easier characterized derivatives useful for research and practical applications.

Glycosidase activities of 177 strains of the phylum Bacteroidetes, belonging to 18 genera and isolated from the algae Chondrus sp., Polysiphonia sp., Neosiphonia japonica, Saccharina crassifolia, Saccharina japonica, Chorda filum, Acrosiphonia sonderi, and Ulva fenestrata collected in the littoral zones of the Sea of Okhotsk and the Sea of Japan, Pacific Ocean, were studied. According to the data obtained, glycosidases catalyzing hydrolysis of the β-glycoside bond were present in over 70% epiphytes of marine algae. It should be noted that α-galactosidases and the extremely rare enzymes, α-N-acetylgalactosaminidases, were more frequent in the Bacteroidetes than in the proteobacteria analyzed previously. It was found that the overwhelming majority of the bacteria of the dominant genera Zobellia and Maribacter contained the complete set of the tested glycosidases involved in degradation of algal polysaccharides. Apparently, the presence of the wide range of glycosidases in bacterial strains of these genera makes it possible for them to occupy diverse ecological niches under extreme conditions of the tidal zone. However, such important enzymes of the microbial lytic complex as α-galactosidases, β-galactosidases, or β-xylosidases, were not detected in the numerically important genus Winogradskyella. The noted difference in the metabolic profiles of the strains of these genera suggests the assumption that Winogradskyella strains play an unique role in the microbial communities, unrelated to the hydrolysis of such polysaccharides as agar and carrageenan. Significant differences in production of glycosidases among the different taxonomic groups were revealed, which is of importance for directed search of promising enzymes for biotechnology.

Extracellular beta-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70 degrees C. Glucosidase catalyzed the hydrolysis of beta-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbon-containing compounds.

Occurrence of α-N-acetylgalactosaminidases among 177 strains of marine bacteria of the phylum Bacteroidetes, epiphytes of marine algae growing on the littoral of the Seas of Okhotsk and Japan, was studied. About 36% of the isolates studied contained α-N-acetylgalactosaminidase. All of the bacteria of the genus Arenibacter (species A. latericius, A. certesii, and A. palladensis), irrespective of the source of isolation, synthesized this enzyme. The greatest number of α-N-acetylgalactosaminidase producers was found among the isolates from the algae Neosiphonia japonica, Acrosiphonia sonderi, and Ulva fenestrata sampled in the Cove of Trinity, Posyet Bay, the Sea of Japan. These were mainly bacteria of the genera Zobellia (50%) and Maribacter (58%). Among the epibionts studied, the bacteria Arenibacter latericius KMM 3523, an epiphyte of the brown alga Chorda filum from the Sea of Okhotsk, and Cellulophaga sp. KMM 6488, an epiphyte of the green alga Acrosiphonia sonderi from the Sea of Japan, were marked as the most promising sources of the enzyme. The results of this study showed that aerobic nonpathogenic marine Bacteroidetes, algal associants not requiring special cultivation conditions, are the promising, economical, and ecologically pure sources of unique and biotechnologically significant α-N-acetylgalactosaminidases.

The effect of fucoidan, extractive substances from Fucus evanescens and the Laminaria cichorioides protein (an inhibitor of endo-1→3-β-D-glucanase) on the degradation of F. evanescens thallus and on the growth of bacteria involved in this process was studied. The complex of O-glycosyl hydrolases and the level of their enzymatic activity in bacteria cultivated under various conditions depended significantly on the composition of the growth medium. The highest taxonomic diversity was observed for bacteria isolated from the thallus degraded in the control medium (sea water). These bacteria were characterized by very low levels of activity of the enzymes degrading polysaccharides (fucoidan, laminaran, and pustulan). In the presence of 1→3-β-D-glucanase inhibitors, the taxonomic diversity of the microorganisms degrading F. evanescens thallus was decreased, and the activity of O-glycosyl hydrolases (particularly, of fucoidan hydrolases) was increased.

The cDNA encoding the glucanase GI from Littorina sitkana (formerly named Littorina kurila ) was cloned and sequenced, and the enzyme was assigned to glycoside hydrolases family 16 (GHF16) on the basis of amino acid sequence similarity. Structural features of the products of transglycosylation reaction using 6-O-methyl-β- d -glucuronic acid as an acceptor were established. Using colisionally induced dissociation (CID) tandem electrospray ionization mass spectrometry (ESIMS/MS) it was shown that GI transfers the residues of glyconic parts of the substrate mainly at C3 and C4 positions of 6-O-methyl-β- d -glucuronic acid and strictly at C3 position of glucose residue. The semi-quantitative characteristics of simultaneously passing hydrolysis and transglycosylation reactions catalyzed by retaining endo-(1→3)-β- d -glucanases GI from L. sitkana and LIV from Pseudocardium sachalinensis (formerly named Spisula sachalinensis ) have been obtained by ESIMS. Laminaran was used as a donor, while glycerol was employed as an acceptor. The significant distinctions of catalytic properties of LIV and GI were revealed.

The interaction of sulfated polysaccharides - fucoidans from brown seaweeds Laminaria japonica, Laminaria cichorioides and Fucus evanescens with Toll-like receptors (TLRs) expressed on membranes of embryonic human kidney epithelial cells (HEK293-null, HEK293-TLR2/CD14, HEK293-hTLR4/CD14-MD2 and HEK293-hTLR2/6) was investigated. In vitro fucoidans specifically interacted with ТLR-2, ТLR-4, and the heterodimer ТLR-2/6 resultated in activation of transcription nuclear factor NF-κB. Analysis of composition the hydrolyzed fucoidan from F. evanescens was carried out by gas-liquid chromatography and chromatography-mass spectrometry. Results indicated the absence of 3-3-hydroxytetradecanoic acid (3-OHC14), the basic component of lipopolysaccharides in the preparation. Thus, the obtained results suggested that fucoidans from brown seaweeds possessing immunotropic activity are independent ligands for TLRs, and are able to induce genetically determined biochemical processes of protection organisms against pathogenic microorganisms.

The variation of the polysaccharide composition of the brown alga Padina pavonica from the Mediterranean Sea was studied as a function of the collection season (April–July). It was shown that the principal polysaccharide (8.0-13.3% of dry alga weight) was alginic acid. Its content did not undergo significant changes from April to July. The content of water-soluble polysaccharides (laminarans and fucoidans) was low (

The polysaccharide compositions of the brown algae Dictyopteris polypodioides and Sargassum sp. from the Mediterranean Sea were determined. The principal polysaccharide of the studied algae (about 12% of the dry alga weight) was alginic acid. The content of water-soluble polysaccharides was low. The amount of fucoidan was less than 1% of the dry alga weight; of neutral polysaccharides, less than 0.25%. The monosaccharide compositions of fucoidans and neutral polysaccharides were investigated. Experiments on soft agar-agar models showed that fucoidans from D. polypodioides and Sargassum sp. exhibited antitumor activity against RPMI-7951 human melanoma cells.