Hitomi Kimura

A marine strain of flavobacteriaceae utilizes brown seaweed fucoidan.

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from Kjellmaniella crassifolia (class Phaeophyceae, order Laminariales, family Laminariaceae) with a yield of about 3.8% dry weight. To isolate enzymes that degrade fucoidan, we first screened marine bacteria for their ability to utilize fucoidan, and isolated one strain of Flavobacteriaceae from seawater that could do this. Phylogenetic analysis of the 16S ribosomal DNA sequence suggested that this strain appeared to belong to a new genus, and was tentatively named Fucobacter marina. The strain utilized L-fucose (17%), D-mannose (91%), D-galactose (46%), and D-glucuronic acid (66%) in the fucoidan from K. crassifolia. The strain partially utilized fucoidan from 2 other seaweeds that belong to the order Laminariales, Undaria pinnatifida (10%) and Lessonia nigrescens (48%).

Marine bacterial sulfated fucoglucuronomannan (SFGM) lyase digests brown algal SFGM into trisaccharides

Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as Δ4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp, Δ4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp(6-O-sulfate), and Δ4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)α1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase, the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and its sugar sequence was determined as (-4-D-GlcpUAβ1-2D-Manpα1-)n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAβ1-2(L-Fucp(3-O-sulfate)α1-3)D-Manpα1-)n. Consequently, the glycosidase was deduced to be an endo-α-D-mannosidase that eliminatively cleaves the α-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively.

Purification of Sulfated Fucoglucuronomannan Lyase from Bacterial Strain of Fucobacter marina and Study of Appropriate Conditions for Its Enzyme Digestion

A marine bacterial strain, Fucobacter marina, produced extracellular sulfated fucoglucuronomannan (SFGM) lyase when cultivated in the presence of crude SFGM obtained from fucoidan of Kjellmaniella crassifolia (brown algae) by cetyl pyridinium chloride fractionation. For the SFGM lyase assay, SFGM fraction separated from K. crassifolia fucoidan by anion exchange column chromatography was used as the substrate. The extracellular SFGM lyase was purified to homogeneity on an electrophoresis gel with 4240-fold purity at 13.8% yield. The enzyme proved to be a monomer, since gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis gave the same relative molecular mass of 67,000. The enzyme specifically digested SFGM but did not digest any other uronic-acid-containing polysaccharides tested. The optimum conditions for the enzyme reaction were around pH 7.5, 43°C, and 0.4 M NaCl concentration. The enzyme was strongly inhibited by CuCl2 and ZnCl2, and also by some sulfhydryl reagents.