Artem S. Silchenko

Hydrolysis of Fucoidan by Fucoidanase Isolated from the Marine Bacterium, Formosa algae

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.

Expression and biochemical characterization and substrate specificity of the fucoidanase from Formosa algae

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.

Polysaccharides from brown algae Sargassum duplicatum: the structure and anticancer activity in vitro

The laminaran SdL and fucoidan SdF were isolated from brown algae Sargassum duplicatum. SdL was 1,3;1,6-β-d-glucan (1,3:1,6=6:1) with a main chain, represented by 1,3-linked glucose residues, due to NMR spectroscopy data. Single glucose residues could form branches at C6. Unusual structure of fucoidan SdF was studied by chemical and enzymatic methods, NMR spectroscopy of desulfated and deacetylated polysaccharide and mass spectrometry of fucoidan fragments labeled with 18O. Fucoidan was sulfated (31.7%) and acetylated galactofucan (Fuc:Gal∼1:1) with a main chain of 1,4-linked alternating α-l-fucose and β-d-galactose residues. Side chains were represented by extensive (DP≥5) 1,3-linked 2,4-disulfated α-l-fucose residues with branching points at C2. Fucose residues in the main chain were sulfated at C2 and less at C3, while galactose residues were sulfated at C2, C3, and less at C4, C6. The fucoidan SdF was effective against colony formation of colon cancer cells in vitro.

Structure, enzymatic transformation, anticancer activity of fucoidan and sulphated fucooligosaccharides from Sargassum horneri.

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.

Brown Alga Metabolites – Inhibitors of Marine Organism Fucoidan Hydrolases

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.

Alginate Lyases: Substrates, Structure, Properties, and Prospects of Application

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.

Modification of native fucoidan from Fucus evanescens by recombinant fucoidanase from marine bacteria Formosa algae

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.

Fucoidan Sulfatases from Marine Bacterium Wenyingzhuangia fucanilytica CZ1127T

Fucoidans belong to a structurally heterogeneous class of sulfated polysaccharides isolated from brown algae. They have a wide spectrum of biological activities. The complex structures of these polysaccharides hinder structure-activity relationships determination. Fucoidan sulfatases can make useful tools for the determination of the fine chemical structure of fucoidans. In this study, identification and preparation of two recombinant sulfatases able to catalyze the cleavage of sulfate groups from fragments of fucoidan molecules is described for the first time. Two genes of sulfatases swf1 and swf4 of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T were cloned and the proteins were produced in Escherichia coli cells. Sulfatases SWF1 and SWF4 are assigned to S1_17 and S1_25 subfamilies of formylglycine-dependent enzymes of S1 family (SulfAtlas). Some molecular and biochemical characteristics of recombinant fucoidan sulfatases have been studied. Detailed specificity and catalytic features of sulfatases were determined using various sulfated fucooligosaccharides. Structures of products produced by SWF1 and SWF4 were established by nuclear magnetic resonance (NMR) spectroscopy. Based on the obtained data, the enzymes are classified as fucoidan exo-2O-sulfatase (SWF1) and fucoidan exo-3O-sulfatase (SWF4). In addition, we demonstrated the sequential action of sulfatases on 2,3-di-O-sulfated fucooligosacchrides, which indicates an exolitic degradation pathway of fucoidan by a marine bacterium W. fucanilytica CZ1127T.