0000000000606082
AUTHOR
Mirna P. Santiago-gómez
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…
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…
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…
Characterization of Purified Green Bell Pepper Hydroperoxide Lyase Expressed by Yarrowia Lipolytica: Radicals Detection during Catalysis
International audience; The optimization of the expression of recombinant 6-His-tagged HPO lyase in Yarrowia lipolytica is described: 1800U/L of culture was detected at 24h of culture on a medium containing olive oil as the sole carbon source. The enzyme was purified by IMAC and showed an optimal pH at 5.5, an optimal temperature at 20^\circC and a Km value of 9μM with 13-HPOD substrate. The participation of radicals during the catalysis of purified bell pepper fruit hydroperoxide lyase has been observed by electron paramagnetic resonance spectroscopy and the yet unidentified radical species might be an alkyl or alkoxyl radical linked to the enzyme.