0000000000064409
AUTHOR
Jean-marc Belin
Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n- alkane-assimilating yeast Yarrowia lipolytica
ABSTRACT We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n -alkane-assimilating yeast Yarrowia lipolytica , encoded by the POX1 through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to …
Involvement of acyl coenzyme A oxidase isozymes in biotransformation of methyl ricinoleate into gamma-decalactone by Yarrowia lipolytica.
ABSTRACT We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes ( POX1 through POX5 ) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δ pox3 , which produced 220 mg of γ-decalactone per liter after 24 h. The Δ pox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotra…
Optimization of Yarrowia lipolytica’s β-oxidation pathway for γ-decalactone production
The yeast Yarrowia lipolytica growing on methyl ricinoleate produces various lactones, γ-decalactone, the worthy aroma compound, 3-hydroxy-γ-decalactone without sensorial properties and two decenolides of various interest. Unfortunately, these three latter lactones are produced at high levels by this yeast, decreasing yields and complicating the extraction of γ-decalactone. In this study, the production of γ-decalactone was increased through a genetic engineering of the strain and the accumulation of the three other lactones was lowered. Theses results show that it is possible to improve the mastering of the complex β-oxidation pathway (the metabolic pathway involved in these bioconversions…
Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach.
International audience; beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with ce…
Purification of hydroperoxide lyase from green bell pepper (Capsicum annuum L.) fruits for the generation of C6-aldehydes in vitro.
The aim of this work was to compare the efficiency of different extracts of hydroperoxide lyase from green bell peppers in producing aldehydes: a crude extract, a chloroplastic fraction, and a purified enzyme were investigated. From a crude extract, the HPO lyase was purified by ion-exchange chromatography with a 22.3-fold increase in purification factor. Analysis by SDS-PAGE electrophoresis under denaturating conditions showed only one protein with a molecular weight of 55 kDa, whereas size-exclusion chromatography indicated a molecular weight of 170 kDa. A maximum of 7500 mg of aldehydes per g of protein was obtained with the purified enzyme within 20 min of bioconversion compared to 392 …
Bacterial Bioconversion of Primary Aliphatic and Aromatic Alcohols into Acids: Effects of Molecular Structure and Physico-chemical Conditions
The biotransformation of four alcohol substrates (butanol, 2-methylbutanol, 3-methylbutanol and 2-phenylethanol) into their acids was studied using a strain of Acetobacter aceti. Bioconversion yields depended on the molecular structure of the alcohol. Biotransformation of high concentrations of alcohols was possible until the precursor reached an inhibiting concentration (3.8 g dm -3 for butanol and 3-methylbutanol, 4.2 g dm -3 for 2-methylbutanol). In contrast, biotransformation of 2-phenylethanol decreased when alcohol concentration was higher than 0.3 g dm -3 . Dissolved oxygen concentrations and pH conditions of the medium were important factors in improving bioconversion. Transformatio…
Apple pomace: An enzyme system for producing aroma compounds from polyunsaturated fatty acids
This paper reports on the possibility of obtaining C-6 (hexanal) and C-10 (2,4-dedadienal) volatile aldehydes by degradation of linoleic acid (C18∶2 Δ 9–12) under the action of the intrinsic enzyme systems found in apple pomace. More aroma compounds are produced by micronization of the pomace and by adding SO2 (60 ppm) and vitamin C (500 ppm), thereby synergistically counteracting oxidation of phenolic compounds, which is a limiting factor in bioconversion.
Mechanisms underlying the toxicity of lactone aroma compounds towards the producing yeast cells
M. A G U E D O , L. B E N E Y , Y. W A C H EA N D J. - M. B E L I N. 2003. Aims: To study the fundamental mechanisms of toxicity of the fruity aroma compound c-decalactone, that lead to alterations in cell viability during its biotechnological production by yeast cells; Yarrowia lipolytica that is able to produce high amounts of this metabolite was used here as a model. Methods and Results: Lactone concentrations above 150 mg l )1 inhibited cell growth, depolarized the living cells and increased membrane fluidity. Infrared spectroscopic measurements revealed that the introduction of the lactone into model phospholipid bilayers, decreased the phase transition temperature. Moreover, the H + -…
Unsaturated fatty acid bioconversion by apple pomace enzyme system. Factors influencing the production of aroma compounds
Productions of volatile compounds (hexanal and 2,4-decadienal) obtained from polyunsaturated fatty acids by action of specific apple pomace enzyme system were quantitatively improved by increasing substrate and enzyme concentrations in the reaction medium. The importance of an exogenous supply of oxygen during bioconversion was also shown. Some physico-chemical factors involved in the pomace enzyme system expression were screened. A temperature of 25°C was favourable to the bioconversion. The control of alkaline or acidic conditions in the reaction medium may orientate the reaction preferentially to the production of one or other aldehyde.
Production of 6-pentyl-?-pyrone byTrichoderma harzianum from 18?n fatty acid methyl esters
Biosynthesis of 6-pentyl-α-pyrone byTrichoderma harzianum in two different media was evaluated. Best yields were found in nitrogen deficient medium (C/N=60). Limited growth seems to favour the production of this lactone. When fungal cells, precultured in low nitrogen medium, were incubated on methyl ricinoleate (10 g/l, C/N=60) an increase in 6-pentyl-α-pyrone production was observed in comparison with the media containing methyl oleate or methyl linoleate.
Cell surface properties affect colonisation of raw milk by lactic acid bacteria at the microstructure level
Cell localisation in the matrix microstructure of fermented food is important for microbial activity and ripening. To evaluate the importance of physicochemical factors in these phenomena, the physicochemical behaviour of four strains of lactic acid bacteria were investigated after inoculation of raw milk or milk fractions. To discriminate between physicochemical and metabolic properties, media were (i) rapidly acidified to avoid cell division or (ii) bacteria were allowed to ferment milk. To evaluate the impact of milk components, cells were dispersed in protein or fatty globule fractions. From two Lactococcus lactis subsp. lactis biovar. diacetylactis strains selected from their different…
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…
Increased electron donor and electron acceptor characters enhance the adhesion between oil droplets and cells of Yarrowia lipolytica as evaluated by a new cytometric assay.
International audience; The adhesion of methyl ricinoleate droplets to cells of the yeast Yarrowia lipolytica was investigated. A new cytometric method, relying on the double staining of fatty globules with Nile Red and of cells with Calcofluor, enabled us to quantify methyl ricinoleate droplet adhesion to cells precultured on a hydrophilic or on a hydrophobic carbon source. In this last case, droplet adsorption was enhanced and a MATS (microbial adhesion to solvents) test revealed that this increase was due to Lewis acid-base interactions and not to an increase in the hydrophobic properties of the cell surface. These preliminary results demonstrate that the developed cytometric method is p…
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…
Interactions between bacterial surfaces and milk proteins, impact on food emulsions stability
Bacteria possess physicochemical surface properties such as hydrophobicity, Lewis acid/base and charge which are involved in physicochemical interactions between cells and interfaces. Moreover, food matrices are complex and heterogeneous media, with a microstructure depending on interactions between the components in media (van der Waals, electrostatic or structural forces, etc.). Despite the presence of bacteria in fermented products, few works have investigated how bacteria interact with other food components. The objective of the present study was to determine the effects of the surface properties of lactic acid bacteria on the stability of model food emulsions. The bacteria were added t…
Production of 3-hydroxy-γ-decalactone, the precursor of two decenolides with flavouring properties, by the yeast Yarrowia lipolytica
3-Hydroxy-γ-decalactone is the precursor of dec-2 and dec-3-en-4-olides which are valuable aroma compounds not yet produced. To promote the accumulation of this lactone, the yeast Yarrowia lipolytica was placed in different environmental conditions aiming at altering β-oxidation fluxes. The concentration of substrate, pH, aeration and dissolved oxygen level were modified. We observed an important accumulation at low aeration (0.40 molar yields) and, to a lesser extent, at lower pH (0.15). As oxygen played a key-role, we evaluated its effect at fixed dissolved oxygen and at the pH which was the most favourable to the biotransformation (pH 4.5). At 5% and 30% dissolved oxygen, yields reached …
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…
Quantitative separation of Trichoderma lipid classes on a bonded phase column.
Bond Elut aminopropyl columns were used to purify the different lipid classes of Trichoderma harzianum and Trichoderma viride. This methodology permitted good separation of the fungal lipid classes in less time than traditional techniques. The incorporation of [1 (14)C]linoleic acid into neutral lipids, free fatty acids and phospholipids was quantified for both strains. The fatty acid profile of the different lipid classes of these fungal strains was determined as a function of culture time.
Production of volatile compounds in model milk and cheese media by eight strains of Geotrichum candidum Link
SummaryEight strains of Geotrichum candidum isolated from cheese were compared for aroma production. Twenty-four components, including alcohols, methyl ketones, fatty acids and esters, were identified by gas–liquid chromatography and gas chromatography–mass spectrometry of the volatile compounds produced by G. candidum cultures. Strains could be differentiated by their ability to produce dimethyldisulphide, phenol, phenylethanol and methyl ketones.
Medium-size droplets of methyl ricinoleate are reduced by cell-surface activity in the gamma-decalactone production by Yarrowia lipolytica.
International audience; Size of methyl ricinoleate droplets during biotransformation into gamma-decalactone by Yarrowia lipolytica was measured in both homogenized and non-homogenized media. In non-homogenized but shaken medium, droplets had an average volume surface diameter d32 of 2.5 microm whereas it was 0.7 microm in homogenized and shaken medium. But as soon as yeast cells were inoculated, both diameters became similar at about 0.7 microm and did not vary significantly until the end of the culture. The growth of Y. lipolytica in both media was very similar except for the lag phase which was lowered in homogenized medium conditions.
Intracellular pH-dependent efflux of the fluorescent probe pyranine in the yeast Yarrowia lipolytica.
International audience; 8-Hydroxypyrene-1,3,6-trisulfonic acid (pyranine) can be used as a vital intracellular pH (pH(i)) indicator. In the yeast Yarrowia lipolytica, a partial efflux of the probe was detected by using the pH-independent wavelength of 415 nm. A simplified correction of the fluorescent signals was applied, enabling to show for this species a good near-neutral pH(i) maintenance capacity in a pH 3.9 medium. Octanoic acid, which is known to have toxic effects on yeast, decreased the pH(i) and increased the 260-nm-absorbing compounds leakage. However, this acid inhibited the fluorescent probe efflux linearly with its concentration suggesting a pH(i)-dependent efflux of pyranine …
Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes
Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Fatty acid hydroperoxide lyase (HPO lyase) is a cytochrome P450 acting on fatty acid hydroperoxides in many organisms. The expression of green bell pepper HPO lyase in the yeast Yarrowia lipolytica is described for the first time. HPO lyase activity from yeast extract and whole yeast cells is measured and aldehydes production from yeast extract and whole yeast cells is compared. 1200 U/L reaction medium were obtained after 96 h of culture on olive oil rich me…
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…
Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species
The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U- 14 C]linoleic acid or [5- 14 C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [ 14 C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [ 14 C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-…
Cloning and characterization of the peroxisomal acyl CoA oxidaseACO3 gene from the alkane-utilizing yeastYarrowia lipolytica
The ACO3 gene, which encodes one of the acyl-CoA oxidase isoenzymes, was isolated from the alkane-utilizing yeast Yarrowia lipolytica as a 10 kb genomic fragment. It was sequenced and found to encode a 701-amino acid protein very similar to other ACOs, 67.5% identical to Y. lipolytica Aco1p and about 40% identical to S. cerevisiae Pox1p. Haploid strains with a disrupted allele were able to grow on fatty acids. The levels of acyl-CoA oxidase activity in the ACO3 deleted strain, in an ACO1 deleted strain and in the wild-type strain, suggested that ACO3 encodes a short chain acyl-CoA oxidase isoenzyme. This narrow substrate spectrum was confirmed by expression of Aco3p in E. coli.
Production of volatile compounds in liquid cultures by six strains of coryneform bacteria
The aromatic profiles of four strains of Brevibacterium linens, one strain of Brevibacterium sp. and one strain of Microbacterium sp. were determined with some pure cultures of these microorganisms in standard trypcase soy liquid medium, which enabled four of these six strains to produce flavour compounds of ripened cheese. Thirty-two flavour compounds were identified by gas chromatography and mass spectrometry. The identified flavour compounds included the following: fatty acids, alcohols, methylketones, sulphur compounds, aromatic compounds and a pyrazine. Some important differences were found among the six strains studied. The four strains of B. linens had similar flavour profiles. Their…
Modification génétique de la levure Yarrowia lipolytica pour maîtriser la β-oxydation
Les biotechnologies font regulierement appel a la β-oxydation pour produire des composes a haute valeur ajoutee ou depolluer des effluents industriels. Cette voie metabolique est difficile a maitriser car elle implique un nombre reduit d'enzymes pour une voie tres longue. En particulier, la production d'intermediaires reactionnels est delicate car les enzymes de la voie de reconsommation sont souvent les memes que celles impliquees dans la production. Ainsi, la production de γ-decalactone a partir de ricinoleate de methyle chez Y. lipolytica implique la β-oxydation (Endrizzi et al., 1996). Cette levure est un bon modele pour etudier le metabolisme des acides gras car elle possede cinq acyl-…
Interaction of an odorant lactone with model phospholipid bilayers and its strong fluidizing action in yeast membrane
International audience; Some odorant lactones are naturally present in fruits or in fermented products; they can also be used as food additives and can be produced by microorganisms at the industrial scale by biotechnological processes. Gamma-decalactone was previously shown to have antimicrobial properties. We determined by infrared spectroscopy measurements that this compound rapidly diffused into model phospholipid bilayers (within 2 min), modifying the general physical state of a dimyristoyl-L-alpha-phosphatidylcholine (DMPC) film. In vivo, the lactone strongly increased membrane fluidity in the model yeast Yarrowia lipolytica, as evaluated by fluorescence anisotropy measurements. This …
Peroxisomal beta-oxidation activities and gamma-decalactone production by the yeast Yarrowia lipolytica.
International audience; gamma-Decalactone is a peachy aroma compound resulting from the peroxisomal beta-oxidation of ricinoleic acid by yeasts. The expression levels of acyl-CoA oxidase (gene deletion) and 3-ketoacyl-CoA thiolase activities (gene amplification on replicative plasmids) were modified in the yeast Yarrowia lipolytica. The effects of these modifications on beta-oxidation were measured. Overexpression of thiolase activity did not have any effect on the overall beta-oxidation activity. The disruption of one of the acyl-CoA oxidase genes resulted in an enhanced activity. The enhancement led to an increase of overall beta-oxidation activity but reduced the gamma-decalactone produc…
Comparison of Volatile Flavor Compounds Produced by Ten Strains of Penicillium camemberti Thom
Abstract Volatile compounds produced by Penicillium camemberti Thom in a milk culture medium were identified using gas chromatography coupled with mass spectrometry. Volatile compounds were mainly methyl ketones and their corresponding secondary alcohols, fatty acids, and the alcohols 3-methylbutanol, 2-methylpropanol, 3-octanol, and 1-octen-3-ol. Comparison of 10 strains of P. camemberti Thom resulted in their being grouped into 6 aromatic strain groups, This grouping seems to be useful for the selection of strains for cheese making.
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…
An air-lift biofilm reactor for the production of γ-decalactones by Yarrowia lipolytica
Decalactones are interesting flavouring compounds that can be produced from ricinoleic acid. In this study, the production of lactones in biofilms using Yarrowia lipolytica is investigated. The hydrophobia of cells increased for increased aeration rates resulting in higher adhesion when the reactor wall was hydrophobic (plastic). To increase adhesion, sheets of methyl-polymethacrylate (PMMA) were added in the reactor and the production of lactones increased with the surface of plastic added, reaching 850 mg/L of 3-hydroxy-γ-decalactone for 60 cm2. In an Airlift bioreactor made of PMMA, biofilms were present at the top of the reactor for increased aeration. In the meantime, a metabolic shift…
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…
Lipid accumulation inTrichodermaspecies
Two filamentous fungi, Trichoderma harzianum and Trichoderma viride, were compared for their ability to synthesize lipids on different carbon and nitrogen sources. Three culture media were selected for each strain after preliminary screening. All the test media were nitrogen-deficient (C/N = 60) so as to stimulate lipid accumulation. For both microorganisms the glucose-ammonium sulphate medium was the most conducive to lipid production: a lipid accumulation of 17% (w/w) of biomass dry weight was obtained for T. harzianum and of 32% (w/w) of biomass dry weight for T. viride. In sucrose-sodium nitrate medium T. harzianum was able to accumulate almost 25% (w/w) of its biomass in lipid form. Ho…
Retention of aroma compounds by lactic acid bacteria in model food media
The interactions between aroma compounds and other particles in foods, particularly with macromolecules, have been greatly studied in order to better understand the binding of flavors in food matrices. Bacteria possess many macromolecules on their cellular surface that provide them surface properties which are involved in the physicochemical interactions between cells and interfaces. However, the interactions between bacteria and aroma compounds have not received so much attention despite the presence of bacteria in many fermented products. In order to study the retention of aroma compounds by bacteria, we have investigated the retention of esters by lactic acid bacteria with static headspa…
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.
Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action
Abstract Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of…
A simple protocol for extraction and detection of pyrazines produced by fungi in solid state fermentation
A protocol for the extraction and detection of pyrazines produced by fungi in Solid State Fermentation that gave good recovery of these compounds was devised together with specific techniques for detection by Gas Chromatography (GC). The protocol involved solid-liquid and liquid-liquid extractions, concentration of the extracts and GC analysis. Extraction yields were measured from a model system in which synthetic pyrazines were added. Extracts were analysed with a gas chromatograph equipped with a thermo-ionic detector to allow specific pyrazine detection.
Improved co-oxidation of β-carotene to β-ionone using xanthine oxidase-generated reactive oxygen species in a multiphasic system
beta-Ionone, an aroma compound exhibiting flower notes, can be obtained from beta-carotene in a cooxidation system utilizing xanthine oxidase-generated reactive oxygen species (ROS). ROS have to be controlled as, although they can give rise to beta-ionone, they may also degrade it. In this work, the biotransformation of beta-carotene into beta-ionone was investigated in systems containing variable proportions of decane to extract beta-ionone before degradation. The use of 50% or 90% decane resulted in increased production yields. Tween 80, which was added to further improve the production, slightly decreased the reactivity of the medium and the extraction of beta-carotene, but increased the…