0000000000459617
AUTHOR
Selim Kermasha
Fusarium proliferatum: induction and intracellular location of a lipoxygenase
The fungus Fusarium proliferatum was grown in a soya oil- and glucose-supplemented medium. Induction of lipoxygenase activity (13-fold) in an extract of Fusarium biomass has been observed at day four on soya oil culture medium. A band at 232 kDa was detected using a specific lipoxygenase stain combined with native PAGE. The method of fungal homogenate obtained has been checked via subcellular marker enzymes activities: succinate dehydrogenase (SDH) for mitochondrial fraction and NADH-cytochrome C reductase (NADH-cyt. C) for the microsomal fraction. Protoplasts production and disintegration followed by subcellular fractionation using differential centrifugation was the best method to recover…
Stabilization of an enzymatic extract from Penicillium camemberti containing lipoxygenase and hydroperoxide lyase activities
International audience; The stabilization of an enzymatic extract, obtained from Penicillium camemberti containing lipoxygenase (LOX) and hydroperoxide lyase (HPL) activities, was investigated using selected additives. Although the addition of KCl (86%, w/w) to the enzymatic extract decreased slightly (7%) the LOX activity, it increased HPL activity by 2.25 fold; however, the addition of dextran resulted in the inactivation of both enzymatic activities. The stability of the solid lyophilized enzymatic extract was greater in the presence of KCl than that without it, with ∼100% residual activity after 8 and 4 weeks of storage at −80 °C, for LOX and HPL, respectively. The rate constants of ina…
Optimization of the Hydrolysis of Safflower Oil for the Production of Linoleic Acid, Used as Flavor Precursor
Commercial lipases, from porcine pancreas (PPL),Candida rugosa(CRL), andThermomyces lanuginosus(Lipozyme TL IM), were investigated in terms of their efficiency for the hydrolysis of safflower oil (SO) for the liberation of free linoleic acid (LA), used as a flavor precursor. Although PPL, under the optimized conditions, showed a high degree of hydrolysis (91.6%), its low tolerance towards higher substrate concentrations could limit its use for SO hydrolysis. In comparison to the other investigated lipases, Lipozyme TL IM required higher amount of enzyme and an additional 3 h of reaction time to achieve its maximum degree of SO hydrolysis (90.2%). On the basis of the experimental findings, C…
Predicted secondary structure of hydroperoxide lyase from green bell pepper cloned in the yeast Yarrowia lipolytica
International audience; Fatty acid hydroperoxide lyase (HPL) is a member of the cytochrome P450 family acting on fatty acid hydroperoxides in many organisms. The active green bell pepper HPL, cloned and expressed in the yeast Yarrowia lipolytica, was purified by immobilized metal-ion affinity chromatography (IMAC) in the presence of 2% of Triton X-100R. The secondary structure prediction by bioinformatics servers of HPL was realized by ANTHEPROT software, using the GOR, DPM and Predator methods. The theoretical results which are average values obtained from three different calculation methods showed 33% α-helix, 18% β-sheet, 7% turn and 42% coil. On the other hand, the secondary structure a…
Recombinant hydroperoxide lyase for the production of aroma compounds: Effect of substrate on the yeast Yarrowia lipolytica
International audience; The aim of this study was to investigate the action mechanism of linoleic acid hydroperoxides (HPOD), which are the major substrates of hydroperoxide lyase for the production of flavour compounds, on the yeast Yarrowia lipolytica by evaluating their effect on the oxidative state of the cells. The total antioxidant capacity (TAC) and the activity of the main antioxidant enzymes, such as glutathione reductase, glutathione peroxidase and superoxide dismutase, of cells treated with HPOD were studied. The potential role of intracellular glutathione, including reduced glutathione (GSH) and oxidized glutathione (GSSG), in conferring HPOD resistance was also been examined. T…
Secondary structure conformation of hydroperoxide lyase from green bell pepper, cloned in Yarrowia lipolytica, and its activity in selected media
International audience; Circular dichroism (CD) spectroscopy of secondary structure conformation of the purified green bell pepper hydroperoxide lyase (HPL), cloned in the yeast Yarrowia lipolytica, was investigated. The CD spectra of HPL in iso-octane, obtained at 60 °C, in the presence of the reducing agent dithiothreitol showed dramatic increase in α-helix content. The enzyme conformation remained unchanged over a range of pH values of 5.0–7.0. Using 13-hydroperoxide of linoleic acid (13-HPOD) as substrate, the biocatalysis of HPL in organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene, was investigated. The results indicated an increase in…
Biogeneration of 1-octen-3-ol by lipoxygenase and hydroperoxide lyase activities of Agaricus bisporus
Abstract The biogeneration of 1-octen-3-ol by lipoxygenase and associated hydroperoxide lyase activities of a homogenate of the cultivated mushroom Agaricus bisporus was investigated using linoleic acid as a substrate. The optimal substrate and protein concentrations were 1.5 mM and 1.5 mg ml −1 , respectively. The V max value for the enzymic coupled reaction lipoxygenase–hydroperoxide lyase was 6 μg of 1-octen-3-ol ml −1 min −1 , with a K m value of 0.3×10 −3 M. The highest enzymic activity was observed in the presence of pure oxygen (22.9 μg of 1-octen-3-ol ml −1 ), with a bioconversion yield of 36%. The production of 380 μg 1-octen-3-ol per g of mushroom homogenate was demonstrated using…
Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica
International audience; Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Comb…