Lipids and lipolytic enzymes of the microalga Isochrysis galbana

International audienceMarine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme-catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase. Keywords: microalgae / v-3 polyunsaturated fatty acids / phospholipids / lipolytic enzymes Résumé-Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana. Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série v-3 comme l'acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérêt croissant du fait d'un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D'autre part, la lipolyse souvent observée dès l'étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu'I. galbana pourrait contenir des enzymes lipolytiques intéressantes en termes de sélectivité. Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d'intérêt et d'enzymes lipolytiques a été étudié. L'analyse des lipides d'I. galbana a tout d'abord révélé que le DHA était majoritairement greffé sur la position sn-2 des phospholipides. Dans ce contexte, les phospholipides d'I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2-DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. À partir des lipides d'I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants. À partir des ARN totaux d'I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L'expression hétérologue d'une séquence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que laLes microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série ω-3 comme l’acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérèt croissant du fait d’un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D’autre part, la lipolyse souvent observée dès l’étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu’I. galbana pourrait contenir des enzymes lipolytiques intéressantes en terme de sélectivité.Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d’intérêt et d’enzymes lipolytiques a été étudié.L’analyse des lipides d’I. galbana a tout d’abord revélé que le DHA était majoritairement greffé sur la position sn-2 des phopholipides. Dans ce contexte, les phospholipids d’I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2–DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. A partir des lipides d’I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants.A partir des ARN totaux d’I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L’expression hétérologue d’une sequence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la construction plasmidique permettait bien d’obtenir une protéine recombinante avec la masse moléculaire attendue. D’autre part, l’outil d’alignement local de séquences, BLASTP, ainsi que des données biochimiques ont permis de confirmer l’hypothèse que la protéine obtenue était une thioestérase

Challenges for the Development of New Non-Toxic Antifouling Solutions

Marine biofouling is of major economic concern to all marine industries. The shipping trade is particularly alert to the development of new antifouling (AF) strategies, especially green AF paint as international regulations regarding the environmental impact of the compounds actually incorporated into the formulations are becoming more and more strict. It is also recognised that vessels play an extensive role in invasive species propagation as ballast waters transport potentially threatening larvae. It is then crucial to develop new AF solutions combining advances in marine chemistry and topography, in addition to a knowledge of marine biofoulers, with respect to the marine environment. This review presents the recent research progress made in the field of new non-toxic AF solutions (new microtexturing of surfaces, foul-release coatings, and with a special emphasis on marine natural antifoulants) as well as the perspectives for future research directions

Isolation of Cholest-5-en-3-ol formate from the red alga Grateloupia turuturu Yamada and its chemotaxonomic significance

International audienceNo abstrac

Spatial and seasonal variation in density, reproductive status, length and phenolic content of the invasive brown macroalga Sargassum muticum (Yendo) Fensholt along the coast of Western Brittany (France)

International audienceThe introduced macroalga Sargassum muticum (Yendo) Fensholt is currently observed along the coasts of Brittany. From February 2003 till June 2004, variations in density, reproductive status, length and phenolic content were assessed at three sites. Seasonal variability of the density was pronounced at two exposed sites (Fort Bloqué and Pointe du Diable), but absent at the sheltered site (Callot), where the macroalga was observed year-round. Thalli were generally longer in Summer (up to 73.2 ± 2.7 cm at Fort Bloqué), and had reproductive receptacles from May to September at Pointe du Diable, though only from June to September in the other stations (Callot and Fort Bloqué). Phenolic content was maximal during the reproductive period, which could be interpreted as chemical protection of the receptacles. Finally the collected data were used to explain the persistence of S. muticum and its proliferation in Brittany

Anti-microfouling activity of lipidic metabolites from the invasive brown alga Sargassum muticum (Yendo) Fensholt

The purification of the chloroform extract from the brown invasive macroalga Sargassum muticum, through a series of chromatographic separations, yielded 12 fractions that were tested against strains of bacteria, microalgae, and fungi involved in marine biofilm formation. The chemical composition of four (a, c, g, and k) out of the six fractions that exhibited anti-microfouling activity was investigated. Fraction a contained saturated and unsaturated linear hydrocarbons (C 12-C 27). Arachidonic acid was identified as the major metabolite in fraction c whereas fraction g contained mainly palmitic, linolenic, and palmitoleic acids. Fraction k was submitted to further purification yielding the fraction kAcaF1e that was composed of galactoglycerolipids, active against the growth of two of the four bacterial strains (Shewanella putrefaciens and Polaribacter irgensii) and all tested fungi. These promising results, in particular the isolation and the activity of galactoglycerolipids, attest the potential of the huge biomass of S. muticum as a source of new environmentally friendly antifouling compounds. © Springer Science + Business Media, LLC 2009

Glycoglycerolipids From Sargassum vulgare as Potential Antifouling Agents

Unraveling new environmentally friendly antifouling (AF) agents is one of the major quests currently facing marine biotechnology. Marine macroalgae represent a rich source of new compounds with promising biological properties, including AF activity, but most of the macroalgal compounds studied to date are terpenoids or polyphenolics. The aim of this work was to investigate the possible AF role played by a usually neglected class of marine natural products: glycolipids (monogalactosyldiacylglycerols – MGDG, digalactosyldiacylglycerols – DGDG and sulfoquinovosyldiacylglycerols – SQDG) isolated from the Phaeophyceae Sargassum vulgare collected along the coast of south-eastern Brazil. Among the 18 extracts, fractions and sub-fractions tested, 3 sub-fractions demonstrated particularly promising AF activity toward the growth inhibition of marine bacteria and microalgae: F3III117, F4II70a, and F4II70b. The main compounds present in these fractions were identified as MGDG, DGDG, and SQDG, respectively. These results highlight the potential of glycoglycerolipids from S. vulgare as new promising antifouling agents

Antiviral Sulfoquinovosyldiacylglycerols (SQDGs) from the Brazilian Brown Seaweed Sargassum vulgare

Total lipids from the Brazilian brown seaweed Sargassum vulgare were extracted with chloroform/methanol 2:1 and 1:2 (v/v) at room temperature. After performing Folch partition of the crude lipid extract, the lipids recovered from the Folch lower layer were fractionated on a silica gel column eluted with chloroform, acetone and methanol. The fraction eluted with methanol, presented a strong orcinol-positive band characteristic of the presence of sulfatides when examined by TLC. This fraction was then purified by two successive silica gel column chromatography giving rise to fractions F4I86 and F4II90 that exhibited strong activity against herpes simplex virus type 1 and 2. The chemical structures present in both fractions were elucidated by ESI-MS and 1H/13C NMR analysis HSQC fingerprints based on their tandem–MS behavior as Sulfoquinovosyldiacylglycerols  (SQDGs). The main SQDG present in both fractions and responsible for the anti-herpes activity observed was identified as 1,2-di-O-palmitoyl-3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol