A lipoxygenase with dual positional specificity is expressed in olives (Olea europaea L.) during ripening.

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenases catalysing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological roles remain to be clarified. With the aim to clarify the occurrence of LOXs in olives and their contribution to the elaboration of the olive oil aroma, we cloned and characterized the first cDNA of the LOX isoform which is expressed during olive development. The open reading frame encodes a polypeptide of 864 amino acids. This olive LOX is a type-1 LOX which shows a high degree of identity at the peptide level towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme shows a dual positional specificity, as it forms both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio, and would be defined as 9/13-LOX. Although a LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest that there are at least two Lox genes expressed in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity and its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

Caractérisation et rôle des enzymes impliquées dans les qualités organoleptiques de l'huile d'olive

La "voie de la lipoxygénase" est activée dans la plante en réponse à un stress environnemental (blessures, attaque de pathogènes, sécheresse,...) ou durant certaines étapes du développement de la plante (floraison, sénescence,...). Elle doit son nom à la première enzyme intervenant dans la cascade réactionnelle, la lipoxygénase, qui catalyse la formation d'hydroperoxydes à partir d'acides gras libres: l'acide linoléique et l'acide linolénique dans la plante. Ces hydroperoxydes peuvent ensuite être transformés par différentes enzymes pour former des composés impliqués dans des voies de signalisation et de défense. Nous avons étudié plus précisément la voie enzymatique de formation des composés volatils à six atomes de carbone dans l'olive car ces composés participent également à la qualité organoleptique de l'huile d'olive. D'une part, nous avons mis en évidence l'influence de la variété et du stade de maturation des olives sur l'activité enzymatique de la lipoxygénase et de l'hydroperoxyde lyase. De plus, la quantité de composés volatils à six atomes de carbone formés dans le fruit varie en fonction de l'activité enzymatique de la lipoxygénase. D'autre part, nous avons entrepris la purification de la lipoxygénase de pulpe d'olive. Le protocole mis au point au laboratoire se déroule en trois étapes au bout desquelles 4,7 mg d'enzyme purifiée sont obtenus avec un rendement de 18% et un facteur de purification de 65 fois. La masse moléculaire de la lipoxygénase d'olive a été estimée à 98kDa. La lipoxygénase de pulpe d'olive semble avoir une préférence pour l'acide linoléique (Km=82,44 M) par rapport à l'acide linolénique (Km=306,26 M). L'analyse par RMN des produits obtenus après incubation de la lipoxygénase d'olive avec l'acide linoléique met en évidence la prédominance de l'isomère 13 de l'hydroperoxyde d'acide gras. L'enzyme semble relativement stable d'un point de vue thermique et peut être inhibée de façon compétitive (propyl gallate) et non compétitive (NDGA).The "lipoxygenase" pathway is a mechanism of response to environnemental stress (wounding, pathogen attack, dryness,...) or is activated in some step of development of plant (flowering, pollen formation, senescence,...). The lipoxygenase is the first enzyme of the pathway and catalyse the formation of hydroperoxides from free fatty acids, especially linoleic and linolenic acid in plant kingdom. Hydroperoxides can then be transformed by several enzymes into signalisation (jasmonic acid and methylic ester of jasmonic acid) or defense ((E)-2-hexenal) compounds. We choose to study the enzymatic pahway of C6 volatil compounds formation in olive because they are involved in olive oil aroma. Indeed, aldehydes, alcohols and esters from the lipoxygenase pathhway are associated to "fiuity or green notes" which is an important aspect of organoleptic quality of olive oil. In first hand, we show that lipoxygenase and hydroperoxide lyase activities are influenced by variety and maturation stage of olive fruits. The amount of C6 volatil compounds in fruits is related to lipoxygenase enzymatic activity. In second hand, we purify to homogeneity lipoxygenase from olive pulp. The purification is made in 3 steps and lead to 4,7 mg of purified enzyme with a yield of 18 % and a purification factor of 65-fold. The molecular mass of olive lipoxygenase is estimated to 98 kDa. The linoleic acid (Km=82,44 M) seems to be the preferential substrat of olive lipoxygenase rather than linolenic acid (Km=306,26 M). Olive lipoxygenase seems to be a 13-LOX. Indeed, only 13-hydroperoxide isomer is detected alter NMR analysis of products obtained from incubation of olive lipoxygenase with linoleic acid. Olive LOX is thermically stable and can be competitively (propyl gallate) or non competitively (NDGA) inhibited.CORTE-BU (200962101) / SudocSudocFranceF

Primary Mode of Action of Cistus ladaniferus L. Essential Oil Active Fractions on Staphylococcus aureus Strain

International audienceThe purpose of this study was to investigate the primary mode of action of Cistus ladaniferus essential oil active fractions on Staphylococcus aureus strain ATCC6538P (CIP 53.156). The mode of inhibition of the active fractions was assessed by determining the minimum inhibitory concentration (MIC). The effects of time on cell integrity were determined by time-kill, bacteriolysis and loss of 260 and 280-nm-absorbing material assays. Measurement of intra-and extracellular ATP was used to evaluate the energy remaining in the cells after treatment. A bacteriostatic and a bac-tericidal mode of inhibition were established respectively for acetate and alcohol fractions at their MIC. No intracellular material leakage and no lysis occurred after treatments with these fractions. In both cases, we observed a decrease of the ATP level within S. aureus cells whilst there was no proportional increase outside the cells. However, the effects induced by alcohols are more pronounced than those provoked by acetates. Indeed, marked structural changes were observed by transmission electron microscopy (TEM). The septal material of cells undergoing division became thicker and stained more lightly. The proportion of septa is also markedly increased and defective with respect to placement. These observations suggest a blocking in cell division, probably caused by the inhibition of ATPase or a disturbance in proton motrice force by the hydrophobic molecules viridiflorol and ledol, mainly present in alcohol fraction

A novel lipoxygenase in olives (Olea europaea L.), with dual positional specificity, expressed late during fruit development

International audiencePlant lipoxygenases (LOXs) are a class of widespread dioxygenase catalyzing the hydroperoxidation of polyunsaturated fatty acids. Although multiple isoforms of LOX have been detected in a wide range of plants, their physiological role is still unclear. With the aim to clarify the occurrence of LOXs and their contribution to the elaboration of the olive oil aroma, we carried out the biochemical and molecular characterization of the LOX isoform expressed during olive development. A full-length cDNA was isolated by Reverse Transcription PCR and Rapid amplification of cDNA end carried out on total RNA from mature olives. The 2852 bp sequence displays an open reading frame of 2592 bp encoding a putative polypeptide of 864 amino acids with a calculated molecular mass of 98.4 kDa and a pI of 5.95. The olive LOX is a type-1 LOX and shows a high degree of identity towards hazelnut (77.3%), tobacco (76.3%) and almond (75.5%) LOXs. The recombinant enzyme produced in E. coli shows a dual positional specificity, as it releases both 9- and 13-hydroperoxide of linoleic acid in a 2:1 ratio. Although LOX activity was detected throughout the olive development, the 9/13-LOX is mainly expressed at late developmental stages. Our data suggest the presence of at least two LOX isoforms in black olives, and that the 9/13-LOX is associated with the ripening and senescence processes. However, due to its dual positional specificity ant its expression pattern, its contribution to the elaboration of the olive oil aroma might be considered

Geraniol Restores Antibiotic Activities against Multidrug-Resistant Isolates from Gram-Negative Species▿ †

The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine β-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of β-lactams, quinolones, and chloramphenicol

Anti-Quorum Sensing Activity of 12 Essential Oils on chromobacterium violaceum and Specific Action of cis-cis-p-Menthenolide from Corsican Mentha suaveolens ssp. Insularis

Quorum sensing (QS) is a bacterial communication mechanism used to express various survival or virulence traits leading to enhanced resistance. Chromobacterium violaceum is a commonly used strain that highlights anti-QS action of bioactive substances. Here, we wanted to see if 12 selected essential oils (EO) could exert anti-QS activity. We measured the sublethal minimal QS inhibitory concentration (MQSIC) by assessing violacein production of C. violaceum along with bacterial growth. To confirm the QS disruption, we also proceed to surface bacterial observations using scanning electron microscopy (SEM). We showed that cis-cis-p-menthenolide extracted and isolated from a plant endemic to occidental Mediterranean Sea islands, Mentha suaveolens ssp. insularis, acts as an inhibitor of violacein production and biofilm formation. Measured MQSIC was much lower than the minimal inhibitory concentration (MIC): 0.10 mg·mL−1 vs. 3.00 mg·mL−1. Moreover, disturbance of QS-related traits was confirmed by the degradation of C. violaceum biofilm matrix. There is a clear structure–activity relationship between cis-cis-p-menthenolide and anti-QS activity. Indeed, its isomer molecule (mintlactone) exerts a poor anti-QS action. These results indicate that inhibition of violacein production and biofilm formation by cis-cis-p-menthenolide might be related to a disruption in the QS mechanism

Key role of hydrogen peroxide in antimicrobial activity of spring, Honeydew maquis and chestnut grove Corsican honeys on Pseudomonas aeruginosa DNA

International audienceIn honeys, several molecules have been known for their antibacterial or wound healing properties. Corsican honeys just began to be tested for their antimicrobial activity with promising results on Pseudomonas aeruginosa. So, identification of active molecules and their mode of action was determined. Hydrogen peroxide concentrations were evaluated and, in parallel, the minimal inhibitory concentrations (MIC) values were performed with and without catalase. More, the quantity of phenolic compounds and ORAC assay were measured. Observation of antibacterial action was done using scanning electron microscopy (SEM) followed by plasmidic DNA extraction. MIC values of chestnut grove and honeydew maquis honeys vary between 7 and 8%, showing a strong antimicrobial capacity, associated with a plasmidic DNA degradation. When catalase is added, MIC values significatively increase (25%) without damaging DNA, proving the importance of H2O2. This hypothesis is confirmed by SEM micrographies which did not show any morphological damages but a depletion in bacterial population. Although, such low concentrations of H2O2 (between 23 μmol l−1 and 54 μmol l−1) cannot explain antimicrobial activity and might be correlated with phenolic compounds concentration. Thus, Corsican honeys seem to induce DNA damage when H2O2 and phenolic compounds act in synergy by a putative pro‐oxidant effect