Isolation and culture of a marine bacterium degrading the sulfated fucans from marine brown algae

Abstract

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an alpha-L-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of alpha-1-->3-L-fucopyranose-2-sulfate-alpha-1-->4-L-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.

Fig. 1

Phylogenetic relationships of strain SW5 to some marine representatives of the family Flavobacteriaceae. Square brackets indicate a generically misnamed taxon and quotation marks indicate a name not yet validated. Accession numbers of 16S rDNA sequences are given in brackets. The topology shown is the tree obtained using the neighbor-joining method (Jukes and Cantor distance correction). Numbers at the nodes refer to the bootstrap values (100 replicates) as obtained in distance, maximum-likelihood, and maximum parsimony analyses, respectively, while dashes instead of numbers indicate that the node was not observed in the corresponding analysis. The scale bar represents the expected number of changes per sequence position.

Fig. 2

Growth of the SW5 strain (A), as seen from the culture optical density measured at 600 nm. SW5 was grown in a 5-L fermenter in the presence of ZoBell medium supplemented with fucoidan from Pelvetia canaliculata. (B) Fucoidanolytic activity in the culture supernatant was monitored using C-PAGE analysis. Culture aliquots (2 ml) were centrifuged, supernatant aliquots (20 μl) were incubated for 3 h with 100 μl of 0.2% (wt/vol) fucoidan from P. canaliculata, and the hydrolysates (5 μl aliquots) were analyzed by C-PAGE. Numbers above the lanes refer to the culture duration (in days), with T corresponding to unhydrolyzed fucoidan.

Fig. 3

(A) C-PAGE electrophoresis of the hydrolysates by SW5 ammonium sulfate fucoidanase fraction of various brown algal sulfated fucans (0.5%): the FS28 fraction of Pelvetia canaliculata fucoidan (Pc1), a CPC-purified fraction from the same species (Pc2), and fucoidans purified using CPC from Fucus spiralis (Fs) and Ascophyllum nodosum (An). The control (T) consisted of the FS28 fraction incubated with boiled enzyme. Bands numbers were assigned relative to their elution order on gel filtration (see Figure 4B) and by comparison with C. (B) C-PAGE analysis of the hydrolysis kinetics of P. canaliculata fucoidan (FS28) by SW5 ammonium sulfate fucoidanase fraction. The fucoidan (400 μl, 0.2%) was incubated with the ammonium sulfate enzyme fraction (40 μl) and aliquots (20 μl) of this mix were boiled and electrophoresed as described previously. Band numbers were assigned by comparison to A and C. Numbers at the top of the gel refer to the duration of hydrolysis (in minutes and hours). (C) C-PAGE analysis of purified low molecular weight fucoidans. P. canaliculata fucoidan (FS28) was hydrolyzed with SW5 fucanase and the products were recovered by ultrafiltration through a 500-Da membrane (OF fraction). This fraction was further purified by chromatography on DEAE Sepharose CL6B and then on Biogel P6, and carbohydrate-containing peaks 1, 2, 3, and 4 (see Figure 4B) were submitted to C-PAGE (lanes 1 to 4). Arrows indicate the corresponding oligosaccharide bands in the OF fraction.

Fig. 4

Purification of the end-products of the hydrolysis of Pelvetia canaliculata fucoidan (FS28) by the SW5 ammonium sulfate fucanase fraction. (A) Fractionation of the hydrolysate (OF fraction) from P. canaliculata fucan on DEAE Sepharose. The OF fraction (250 mg) was chromatographed on DEAE Sepharose CL6B with a 0 to 2 M NaCl gradient, eluted fractions were assayed for total sugars, and carbohydrate-containing fractions (fractions 85 to 100) were collected. (B) Fractions 85 to 100 were pooled and then chromatographed on Biogel P6 using 50 mM NaNO3 as eluent. The four peaks resolved as fractions 1 to 4 were collected. (C) HPAEC elution profiles of peaks 1 to 4 from fractions 85 to 100 on Biogel P6 chromatography. Fractions were desalted on Sephadex G10 and injected at 250 μg/ml on an AS11A anion-exchange column, using a NaOH linear gradient and conductivity detection.

Fig. 5

Expansion of the double ¹H-¹H ROESY spectrum of oligosaccharide 4, at 400 MHz, 25°C, in D₂O. Cross-peaks between the various sugar residues are indicated as, for example: d1/c4 for H1 of unit d with H4 of unit c. 1024 experiments of 2000 data points and eight transients each with a recycling time of 1.16 s were recorded and transformed on 2 × 2 K data points with unshifted sine bell multiplication in both dimensions. The ROESY spinlock pulse duration was 700 ms.

Fig. 6

Expansion of the double ¹H-¹³C HMBC spectrum of oligosaccharide 3, at 400 MHz, 25°C, in D₂O. Cross-peaks between the various sugar residues are indicated using the same nomenclature as in Figure 5. 512 experiments of 2000 data points and 64 transients, each with a recycling time of 1.2 s, were recorded and transformed on 1 × 2 K data points on the F1 and F2 dimensions, respectively after exponential multiplication in F1 (line broadening of 0.1 Hz) and unshifted sine bell multiplication in F2. The evolution time for long range interactions was set at 75 ms.