Characterization of carbohydrates from Nannochloropsis oculata and their use in microarrays

• Autor(a) principal: Pandeirada, Carolina Oliveira
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/22402
• topic: Bioquímica alimentar; Polissacarídeos; Hidratos de carbono; Proteínas imobilizadas

Marine microalgae are of easy culturing and able to accumulate compounds, such as polysaccharides (PS) and glycolipids (GL), which have been attributed biological activities. The present work aimed to study the structural features of the PS and the carbohydrates of the GL of the marine microalga Nannochloropsis oculata using carbohydrate analyses and microarrays. Biomass was defatted, extracted with hot-water (HW), and ultrafiltered. This yielded a residue rich in (1→4)-Glc that was assigned to the presence of a cellulose cell-wall, one filtrate rich in mannitol, and one rentante (Mw>10 kDa) that comprised the soluble PS. The soluble PS were fractionated through ethanol precipitation giving three fractions, Et50, Et85 and EtSN. The PS in Et85 were further fractionated through an anion-exchange chromatography, yielding fractions #1, #2, and #3. All PS fractions comprised uronic acids and sulfate groups, being this content almost negligible in fraction #1. Carbohydrate analyses together with microarrays experiments using proteins with known carbohydrate-binding specificities allowed to verify that the PS in Et50 are dominated by (β1→4)-Glc and exhibited also domains of (β1→3, β1→4)-glucans. PS in Et85 and EtSN fractions were composed by Rha and Man, comprising other sugars in lower content, Xyl, Fuc, and Gal. Fractions Et85, #1, #2, #3, and EtSN comprised mainly (1→2)-Rha with a branching point occurring at position C-3. Fractions Et85, #2, and #3 included also the substituted residue 2,3,4-Rha. In fraction Et85, the sulfate group occurred at position C-3, and simultaneously at positions C-3 and C-4 of (1→2)-Rha. Fractions #2 and #3, besides Rha residues, contained Xyl, Fuc, and Gal, suggesting that these are part of anionic sulfated heteropolysaccharides. The Man, in Et85 and EtSN, occurred mainly as (1→3)- and (1→4)-Man residues, being these part of non-anionic PS in fraction Et85. Carbohydrate microarrays highlighted that Man may be present in α-configuration due to the interaction with ConA. The protein BT0996 of the Bacteroides thetaiotaomicron, a human gut bacteria, used in microarrays, displayed that the PS in fractions Et50, Et85, #1, and #3 may share a common epitope. This interaction is remarkable as it shows that these PS are probably metabolized in the human gut. The GL occur mainly as mono- and digalactolipids, being the presence of galactolipids confirmed through microarrays by interaction with RCA120. Carbohydrate analyses highlighted that the lipid fraction may comprise sulfoquinovosyl diacylglycerol. Considering the results obtained, the joint use of carbohydrate analyses with microarrays showed to be a suitable approach to identify the structural features of the PS and the GL of the marine microalga N. oculata.