Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris–HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides.</p

Détermination de la composition et de la distribution des carraghénanes par hydrolyse enzymatique

Carrageenans are linear sulphated polysaccharides occuring as cell wall constituents in red algae and are used texturing agents in the food industry. The backbone of the polymer is composes of alternating α(1→3) et β (1→4)-Linked D-galactopyranose units. The structure of the repeating units differs in the degree and position of sulphate esterification. 3,6-anhydro-α-D-galactopyranoses units can also replace the α(1→3)-D-galactopyranose units can also replace the α(1→3)-D-galactopyranose units. Strucural studies of carrageenans have been conducted using carrageenases which are glycoside hydrolases producted by marine bacteria. They degrade specifically carrageenans by cleaving their β(1→4) bounds. The oligo-carrageenans obtained have been analysed by liquid chromatography and characterised by mass spectrometry and NMR. The structure and the proportions of hydrolysis products are representative of the polymer structure. These studies were performed with k- and i-carrageenases on carrageenans containing their biosynthetic precursors, the κ-/μ- an ι-/ν-carrageenans, as well as other hybrid carrageenans. Characterisation of hybrid oligosaccharides within the hydrolytic products has confirmed the heterogeneity of these strucures. Different organisation models (block, random) of the repetitive units within these heterogeneous chains have been proposed.Les carraghénanes sont des polysaccharides linéaires sulfatés qui composent la paroi cellulaire des algues rouges et qui sont utilisés comme agents texturants dans l’industrie alimentaire. Leur squelette est composé de D-galactopyranoses reliés alternativement par des liaisons α(1→3) et β(1→4). La structure des motifs disaccharidiques de répétition des carraghénanes varie par la position et le nombre des groupements sulfates. Des unités 3,6-anhydro-α-D-galactopyranoses peuvent aussi remplacer les unités α(1→3)-D-galactopyranoses. L’étude structurale des carraghénanes a été entreprise en utilisant des carraghénases, glycosides hydrolases produites par des bactéries marines, qui dégradent spécifiquement les carraghénanes en coupant leurs liaisons β(1→4). Les oligo-carraghénanes formés ont été analysés par chromatographie liquide et caractérisés par spectrométrie de masse et RMN. La nature et les proportions des produits de l’hydrolyse sont représentatives de la structure du polymère. Ces études ont été menées à l’aide des κ- et ι-carraghénases, sur des carraghénanes riches en précurseurs biosynthétiques, les κ-/μ- et ι-/ν-carraghénanes, ainsi que d’autres carraghénanes hybrides. La caractérisation d’oligosaccharides hybrides parmi les produits d’hydrolyse a confirmé l’hétérogénéité de ces structures et des modèles d’organisation (bloc, aléatoire) des motifs de répétition de ces chaînes hétérogènes ont été proposés

Détermination de la composition et de la distribution des carraghénanes par hydrolyse enzymatique

Les carraghénanes sont des polysaccharides linéaires sulfatés qui composent la paroi cellulaire des algues rouges et qui sont utilisés comme agents texturants dans l industrie alimentaire. Leur squelette est composé de D-galactopyranoses reliés alternativement par des liaisons a(1->3) et b(1->4). La structure des motifs disaccharidiques de répétition des carraghénanes varie par la position et le nombre des groupements sulfates. Des unités 3,6-anhydro-a-D-galactopyranoses peuvent aussi remplacer les unités a(1->3)-D-galactopyranoses. L étude structurale des carraghénanes a été entreprise en utilisant des carraghénases, glycosides hydrolases produites par des bactéries marines, qui dégradent spécifiquement les carraghénanes en coupant leurs liaisons b(1->4). Les oligo-carraghénanes formés ont été analysés par chromatographie liquide et caractérisés par spectrométrie de masse et RMN. La nature et les proportions des produits de l hydrolyse sont représentatives de la structure du polymère. Ces études ont été menées à l aide des - et -carraghénases, sur des carraghénanes riches en précurseurs biosynthétiques, les -/ - et -/ -carraghénanes, ainsi que d autres carraghénanes hybrides. La caractérisation d oligosaccharides hybrides parmi les produits d hydrolyse a confirmé l hétérogénéité de ces structures et des modèles d organisation (bloc, aléatoire) des motifs de répétition de ces chaînes hétérogènes ont été proposés.PARIS-BIUSJ-Physique recherche (751052113) / SudocROSCOFF-Observ.Océanol. (292393008) / SudocSudocFranceF

Carrageenan biosynthesis in red algae: A review

In this review, we summarize the current state of knowledge on the biosynthesis of carrageenan by exploring both the enzyme activities and their localizations. Genomic data, with the sequencing of the genome of Chondrus crispus and the first transcriptomic study into the life cycle stages of this organism, as well as fine carbohydrate structural determination of matrix glycans, provide leads in the study of carrageenan anabolism. Comparison to related carbohydrate-active enzymes, detailed phylogenies alongside classic histochemical studies and radioactivity assays, help predict the localization of the carrageenan-related enzyme biochemistries. Using these insights, we provide an updated model of carrageenan biosynthesis which contributes to understanding the ancestral pathway of sulfated polysaccharide biosynthesis in eukaryotes

Matrix-assisted laser desorption/ionization mass spectrometric analysis of polysulfated-derived oligosaccharides using pyrenemethylguanidine.

International audienc

Regulation of alginate catabolism involves a GntR family repressor in the marine flavobacterium Zobellia galactanivorans DsijT

International audienceMarine flavobacteria possess dedicated Polysaccharide Utilization Loci (PULs) enabling efficient degradation of a variety of algal polysaccharides. The expression of these PULs is tightly controlled by the presence of the substrate, yet details on the regulatory mechanisms are still lacking. The marine flavobacterium Zobellia galactanivorans DsijT digests many algal polysaccharides, including alginate from brown algae. Its complex Alginate Utilization System (AUS) comprises a PUL and several other loci. Here, we showed that the expression of the AUS is strongly and rapidly (<30 min) induced upon addition of alginate, leading to biphasic substrate utilization. Polymeric alginate is first degraded into smaller oligosaccharides that accumulate in the extracellular medium before being assimilated. We found that AusR, a GntR family protein encoded within the PUL, regulates alginate catabolism by repressing the transcription of most AUS genes. Based on our genetic, genomic, transcriptomic and biochemical results, we propose the first model of regulation for a PUL in marine bacteria. AusR binds to promoters of AUS genes via single, double or triple copies of operator. Upon addition of alginate, secreted enzymes expressed at a basal level catalyze the initial breakdown of the polymer. Metabolic intermediates produced during degradation act as effectors of AusR and inhibit the formation of AusR/DNA complexes, thus lifting transcriptional repression

Kinetics of Fibril Formation of Bovine κ-Casein Indicate a Conformational Rearrangement as a Critical Step in the Process

International audienceS-carboxymethylated (SCM) κ-casein forms in vitro fibrils that display several characteristics of amyloid fibrils, although the protein is unrelated to amyloid diseases. In order to get insight into the processes that prevent the formation of amyloid fibrils made of κ-caseins in milk, we have characterized in detail the reaction and the roles of its possible effectors: glycosylation and other caseins. Given that native κ-casein occurs as a heterogeneous mixture of carbohydrate-free and carbohydrate-containing chains, kinetics of fibril formation were performed on purified glycosylated and unglycosylated SCM κ-caseins using the fluorescent dye thioflavin T in conjunction with transmission electron microscopy and Fourier transform infrared spectroscopy for morphological and structural analyses. Both unglycosylated and glycosylated SCM κ-caseins have the ability to fibrillate. Kinetic data indicate that the fibril formation rate increases with SCM κ-casein concentration but reaches a plateau at high concentrations, for both the unglycosylated and glycosylated forms. Therefore, a conformational rearrangement is the rate-limiting step in fibril growth of SCM κ-casein. Transmission electron microscopy images indicate the presence of 10- to 12-nm spherical particles prior to the appearance of amyloid structure. Fourier transform infrared spectroscopy spectra reveal a conformational change within these micellar aggregates during the fibrillation. Fibrils are helical ribbons with a pitch of about 120–130 nm and a width of 10–12 nm. Taken together, these findings suggest a model of aggregation during which the SCM κ-casein monomer is in rapid equilibrium with a micellar aggregate that subsequently undergoes a conformational rearrangement into a more organized species. These micelles assemble and this leads to the growing of amyloid fibrils. Addition of αs1-and β-caseins decreases the growth rate of fibrils. Their main effect was on the elongation rate, which became close to that of the limiting conformation change, leading to the appearance of a lag phase at the beginning of the kinetics

Internal Water Dynamics Control the Transglycosylation/Hydrolysis Balance in the Agarase (AgaD) of Zobellia galactanivorans

International audienc

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris–HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides