Caracterização dos hidratos de carbono de Nannochloropsis oculata e sua utilização em microarrays

Mestrado em Bioquímica - Bioquímica AlimentarMarine 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.As microalgas marinhas são fáceis de cultivar e acumulam compostos, como polissacarídeos (PS) e glicolípidos (GL), aos quais têm sido atribuídas atividades biológicas. O presente trabalho teve como objetivo estudar as características estruturais dos PS e dos hidratos de carbono dos GL da microalga marinha Nannochloropsis oculata. A biomassa foi desengordurada, extraída com água quente (HW) e ultrafiltrada. Isto originou um resíduo rico em (1→4)-Glc, atribuído à presença de celulose na parede celular, um filtrado rico em manitol e um retentato (Mw>10 kDa) que continha os PS solúveis. Os PS solúveis foram fracionados por precipitação em etanol originando três frações, Et50, Et85 e EtSN. Os PS da fração Et85 foram sujeitos a fraccionamento por cromatografia de troca aniónica, obtendo-se as frações #1, #2 e #3. Todas as frações contiveram ácidos urónicos e grupos sulfato, sendo este conteúdo quase negligenciável na fração #1. As análises de açúcares, juntamente com os microarrays usando proteínas de especificidades conhecidas, permitiram verificar que os PS da fração Et50 são constituídos por (β1→4)-Glc, exibindo também domínios de (β1→3, β1→4)-glucanas. Os PS das frações Et85 e EtSN incluíram maioritariamente Rha e Man, contendo também outros açúcares em menores quantidades, Xyl, Fuc e Gal. As frações Et85, #1, #2 e #3, e EtSN possuiram principalmente (1→2)-Rha, com uma ramificação na posição C-3. As frações Et85, #2 e #3 continham também o resíduo substituído 2,3,4-Rha. Na fração Et85 os grupos sulfato encontraram-se na posição C-3, e simultaneamente nas posições C-3 e C-4 de (1→2)-Rha. As frações #2 e #3, além de resíduos de Rha continham Xyl, Fuc e Gal, sugerindo que estes fazem parte de heteropolissacarídeos aniónicos sulfatados. A Man, nas frações Et85 e EtSN, encontrou-se maioritariamente como (1→3)- e (1→4)-Man, fazendo parte de PS não-aniónicos na fração Et85. A análise por microarrays evidenciou que a Man deve encontrar-se em configuração α devido à interação com a ConA. A proteína BT0996 da bactéria Bacteroides thetaiotaomicron, do sistema digestivo humano, usada nos microarrays, mostrou que os PS nas frações Et50, Et85, #1 e #3 podem conter um epítopo comum. Esta interação evidencia que os PS de N. oculata poderão ser metabolizados no sistema digestivo humano. Os GL de N. oculata ocorrem principalmente como mono- e digalactolipídos, sendo a sua presença confirmada pelos microarrays por interação com a proteína RCA120. A análise de açúcares evidenciou também que a fração lipídica deve conter sulfoquinovosil diacilglicerol. Considerando os resultados obtidos, a combinação das análises de açúcares com os microarrays mostraram ser uma abordagem adequada para identificar as características estruturais dos PS e GL da microalga N. oculata

Cyanoflan: A cyanobacterial sulfated carbohydrate polymer with emulsifying properties

Abstract The extracellular polysaccharides produced by cyanobacteria have distinctive characteristics that make them promising for applications ranging from bioremediation to biomedicine. In this study, a sulfated polysaccharide produced by a marine cyanobacterial strain and named cyanoflan was characterized in terms of morphology, chemical composition, and rheological and emulsifying properties. Cyanoflan has a 71% carbohydrate content, with 11% of sulfated residues, while the protein account for 4% of dry weight. The glycosidic-substitution analysis revealed a highly branched complex chemical structure with a large number of sugar residues. The cyanoflan high molecular mass fractions (above 1 MDa) and entangled structure is consistent with its high apparent viscosity in aqueous solutions and high emulsifying activity. It showed to be a typical non-Newtonian fluid with pseudoplastic behavior. Altogether, these results confirm that cyanoflan is a versatile carbohydrate polymer that can be used in different biotechnological applications, such as emulsifying/thickening agent in food or cosmetic industries

Non-enzymatic approaches to depolymerize polysaccharides into oligosaccharides for polysaccharides fingerprinting

Plant polysaccharides are the most abundant biomacromolecules found in nature and frequently used in foods. Despite this, detailed characterization of their structures remains challenging. A method often used to characterize and recognize polysaccharides in full detail relies on the enzymatic digestion of polysaccharides to structure-informative oligosaccharides (enzymatic fingerprinting), prior to full analysis of these oligomers with LC and MS approaches. However, the enzyme-polysaccharide specificity hampers the use of this method as universal polysaccharide depolymerization approach for polysaccharides fingerprinting. In this thesis, various chemical-induced polysaccharide depolymerization approaches were investigated to reach a generic fingerprinting of polysaccharides.A polysaccharide depolymerization method based on TEMPO-oxidation and partial acid-hydrolysis was investigated for the fingerprinting of arabinoxylans (AXs), the main cereal hemicellulose components. TEMPO:NaClO2:NaOCl oxidation of AXs selectively oxidized the arabinose side chains of AXs into arabinuronic acid, resulting in an arabinuronoxylan with a xylan structure having a substitution pattern mostly resembling the parental AX. Subsequently, three structurally different AXs were TEMPO-oxidized, partially acid-hydrolysed and reduced, releasing arabinurono-xylo-oligomer alditols (AUXOS-A). Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) with a stationary phase of porous-graphitized carbon (PGC) allowed the separation and identification of various isomeric AUXOS-A. AX-specific UHPLC-PGC-MS profiles of AUXOS-A were obtained, allowing to distinguish different AXs. Additionally, tandem MS analysis of individual AUXOS-A enabled tentative and conclusive characterization of their structures, which allowed us to substantiate the main structural differences among the AXs investigated.Periodate oxidation of plant polysaccharides with and without subsequent autoclave (AC) thermal treatment was a second approach investigated to reach a generic fingerprinting of plant polysaccharides. After periodate oxidation and AC treatment, all investigated plant polysaccharides, except xyloglucan, and mixes thereof released oligosaccharides. These oligosaccharides had highly complex structures, comprising intact sugar units, and oxidized sugars in the form of dialdehydes, hemialdals, and remnants of oxidized sugars. This high structural complexity resulted in clusters of oxidized oligosaccharides that were polysaccharide structure-dependent, giving unique ESI-Ion trap-MS and MALDI-TOF MS oligosaccharide profiles per polysaccharide. These findings allowed us to distinguish individual plant polysaccharides, within and between polysaccharide classes, and identify the polysaccharide classes present in a polysaccharide mix by their oligosaccharides fingerprint.Thus, partial acid-hydrolysis of TEMPO-oxidized AXs, and thermal treatment of periodate-oxidized plant polysaccharides have potential to more generically recognize AXs and plant polysaccharides, respectively, by oligosaccharides fingerprinting. This can be of high interest for the food industry to study the carbohydrate fraction of a food product

TEMPO/NaClO₂/NaOCl oxidation of arabinoxylans

TEMPO-oxidation of neutral polysaccharides has been used to obtain polyuronides displaying improved functional properties. Although arabinoxylans (AX) from different sources may yield polyuronides with diverse properties due to their variable arabinose (Araf) substitution patterns, information of the TEMPO-oxidation of AX on its structure remains scarce. We oxidized AX using various TEMPO:NaClO2:NaOCl ratios. A TEMPO:NaClO2:NaOCl ratio of 1.0:2.6:0.4 per mol of Ara gave an oxidized-AX with high molecular weight, minimal effect on xylose appearance, and comprising charged side chains. Although NMR analyses unveiled arabinuronic acid (AraAf) as the only oxidation product in the oxidized-AX, accurate AraA quantification is still challenging. Linkage analysis showed that > 75 % of the β-(1→4)-xylan backbone remained single-substituted at position O-3 of Xyl similarly to native AX. TEMPO-oxidation of AX can be considered a promising approach to obtain arabinuronoxylans with a substitution pattern resembling its parental AX.</p