Juan Martín Sánchez

Biosynthesis of triacylglycerols and volatiles in olives

For many reasons (history, myth, oil quality, etc.) olive is unique among the commercially important oil crops. The biochemistry of the olive tree is also singular. From the photosynthetic point of view olive is one of the few species capable of synthesising both polyols (mannitol) and oligosaccharides (raffinose and stachyose) as the final products of the photosynthetic CO2 fixation in the leaf cell. These carbohydrates, together with sucrose, can be exported from the leaves to the fruits to fulfil the metabolic requirements for oil synthesis. On the other hand, contrary to oilseeds, which are absolutely dependent on the leaves to supply photoassimilates for the synthesis of storage oil, developing olives contain active chloroplasts capable of fixing CO2. Thus, the olive contributes to its own carbon economy. In fact, detached olives have been demonstrated to be capable of fixing radiolabelled CO2 in the light and using the reduced photosynthetic products to form storage oil. Soluble fractions from olive pulp have been demonstrated to catalyse the synthesis of fatty acids from malonyl-CoA. The properties of this subcellular fraction indicate that fatty acids are formed by the same type of fatty acid synthase complex established for other plant systems. By the same token, glycerolipids, including storage triacylglycerols, are formed from glycerophosphate and acyl-CoAs according to the Kennedy pathway, as it has been demonstrated in particulate fractions from olive pulp and tissue culture. Also unique to virgin olive oil is its characteristic aroma. The most abundant volatile compounds in the aroma of olive oil are aldehydes and alcohols of six carbon atoms. Such compounds are formed from linoleic and α-linolenic acids through a sequence of enzymatic reactions known as the lipoxygenase pathway, which is stimulated when olives are crushed during the process of oil extraction. The properties of the different reactions involved in the metabolic pathway leading to the formation of such volatile compounds are described in this paper.

Differential presence of oleosins in oleogenic seed and mesocarp tissues in olive (Olea europaea) and avocado (Persea americana)

Abstract Olive drupes accumulate triacylglycerol (TAG) in the fleshy mesocarp of the fruit and also in the endosperm and embryo tissue of their seeds. Ultrastructural analysis has shown that, whereas the TAG in the seed tissues is stored in small, relatively regular oil bodies with diameters in the region of 0.5–2.0 μm, it is present in large, irregular oil bodies of 10–20 μm in the mesocarp. The fatty acid profiles of the TAG in oil bodies isolated from seed and mesocarp tissues are very similar, but oil bodies from these tissues differ dramatically in their protein content. Oil bodies from olive seed endosperm and embryo tissues contain about 10% (w/w) protein, whereas no significant protein was detected in oil bodies from mesocarp tissue of olive or avocado. Major polypeptides of 22 kDa and 50 kDa were purified from olive seed oil bodies and antibodies were raised against them. The 22-kDa oil-body protein was demonstrated to be an oleosin, based on its exclusive localisation on the surface of oil bodies of mature seed tissues, as shown by immunogold electron microscopy. It is concluded that oleosins, such as the 22-kDa polypeptide in olive, are present in the long-term storage oil bodies from the embryo and endosperm tissues of the seed and are absent from oil bodies of the mesocarp.

Hydroperoxide lyase from olive (Olea europaea) fruits

Abstract Hydroperoxide lyase catalyses the cleavage of hydroperoxides from polyunsaturated fatty acids to yield oxoacids and volatile aldehydes. Some of these aldehydes are constituents of the aroma of many fruits and vegetables, and are the major components of the aroma of virgin olive oil. The enzyme has been extracted by solubilization of membrane fractions prepared from the pulp tissues of developing olive fruits. Partial purification by ion-exchange and hydroxyapatite chromatography resulted in the resolution of two isoforms of the enzyme of similar properties: both showed optimal pH at 6.0 and were active with 13-fatty acid hydroperoxides only. The activity measured with 13- EZZ- hydroperoxy-linolenate was 2.5-fold higher than that measured with 13- EZ -hydroperoxylinoleate. Both enzymes showed high affinity for 13-hydroperoxides, with K m values in the micromolar range. The involvement of this enzyme activity in the formation of volatile aldehydes, present in the aroma of olive oil, is discussed.

Mini-transposons in microbial ecology and environmental biotechnology

Abstract Mini‐transposon is the generic name given to the members of a collection of genetic assets derived from transposons Tn10 and Tn5, in which the naturally occurring functional segments of DNA have been rearranged artificially to originate shorter mobile elements. In the most widespread design (that known as the pUT system), any heterologous DNA segment can be conveniently cloned within the boundaries of a mini‐Tn5 vector and finally inserted into the chromosome of target Gram‐negative bacteria after a few simple genetic manipulations. The large variety of antibiotic, non‐antibiotic and excisable selection markers available has been combined at ease with DNA fragments encoding one or more phenotypes of interest for ecological or biotechnological applications. These include the tagging of specific strains in a community with selectable and/or optical marker genes, the production of stable gene fusions for monitoring transcriptional regulation in single cells, the metabolic engineering of strains destined for bioremediation, the non‐disruptive monitoring of gene transfer and the assembly of gene containment and strain containment circuits for genetically manipulated microorganisms.

Lipid requirements of the plasma membrane ATPases from oat roots and yeast

Abstract The lipid specificity of the plasma membrane ATPases from oat roots and yeast has been investigated by reconstituting delipidated enzyme with phospholipid vesicles and with micelles of lysophospholipids and other detergents. The plant ATPase is activated by Triton X-100 and by all phospholipid and lysophospholipid species, exhibiting only a slight preference for zwitterionic polar heads (phosphorylcholine and phosphorylethanolamine). No unsaturation is required on the hydrophobic chain. On the other hand, the yeast ATPase requires a negatively charged polar head (with preference for phosphorylglycerol and phosphorylinositol) and an unsaturated hydrophobic chain.

Alcohol dehydrogenases from olive (Olea europaea) fruit

Alcohol dehydrogenase activity was detected in extracts from the pericarp tissues of developing olive fruits using hexanal as the substrate. Total activity in the crude extract was 20-fold higher with NADPH than with NADH. Three discrete enzymes were resolved by means of a purification protocol involving ammonium sulfate fractionation followed by ion-exchange and affinity chromatography. One of the enzymes was NAD-dependent and displayed a high K(m) for hexanal (K(m) = 2.1 mM). Two NADP-dependent alcohol dehydrogenases were resolved, one showing a high K(m) for hexanal (K(m) = 1.9 mM) and the second with a lower K(m) for the same substrate (K(m) = 0.04 mM). The three enzymes have been partially purified and their kinetic parameters and specificities for various aldehydes determined. The involvement of these enzymes in the biogenesis of six carbon alcohols constituent of the aroma of olive oil is discussed.

Lipoxygenase pathway in olive callus cultures (Olea europaea)

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.

Biogenesis of Olive Oil Aroma

Contrary to other vegetable oils, virgin olive oil is prepared from fresh olives by means of physical procedures carried out under mild conditions (chapter 2), thus resulting in a fruit juice highly priced for its delicate flavour. As described in chapter 12, the aroma of virgin olive oil is formed by a complex mixture of volatile compounds, including aldehydes, alcohols, ketones, hydrocarbons, and esters, which can be analyzed and quantified by gas chromatography—mass spectrometry (GC-MS) (Morales et al. 1995; Olias et al. 1980). Interestingly, among these compounds, C6 aldehydes (hexanal, 3(Z)-hexenal and 2(E)-hexenal), alcohols (hexanol, 3(Z)-hexenol and 2(E)-hexenol), and their acetyl esters (hexylacetate and 3(Z)-hexenyl acetate), constitute 60–80% of total volatile compounds in all of the different oils (from various Spanish and Italian cultivars) analyzed so far (Morales et al. 1995; Olias et al. 1980; Ranalli & De Mattia 1997), with 2(E)-hexenal being the most prominent component. These C6 volatile compounds, which are found in the aroma of many other vegetable products, are responsible for the so-called green notes characteristic of green leaves. It has been established for other plant species that all of those volatile compounds are formed from polyunsaturated fatty acids through a cascade of biochemical reactions collectively known as the lipoxygenase pathway. This biochemical pathway, which is induced in higher plants upon disruption of tissues, involves a series of enzymes that oxidize (lipoxygenase) and cleave (hydroperoxide lyase) polyunsaturated fatty acids to yield aldehydes, which are subsequently reduced to alcohols (alcohol dehydrogenases) and esterified to produce esters (alcohol acyltransferase).

Glycerolipid synthesis by microsomal fractions from Olea europaea fruits and tissue cultures

Abstract Glycerolipid synthesis in olive, Olea europaea, was investigated using both olive fruits and tissue cultures. Microsomal fractions were isolated from fruits and tissue cultures and incubation conditions with [14C]glycerol 3-phosphate were optimised. Characteristics of these incubations, including the incorporation of radioactivity into intermediates of the triacylglycerol synthetic pathway, were compared. The effect of temperature on triacylglycerol synthesis via the Kennedy pathway was also examined. Increasing the temperature of the incubations from 20 to 30°, but not from 30 to 40°, increased the rates of triacyglycerol synthesis for microsomes from both fruits and tissue cultures. In the case of olive callus cultures, microsomes prepared from callus tissue cultured at 35° displayed greater levels of triacylglycerol synthesis than microsomes prepared from 25° maintained cultures. Possible reasons for these differences are discussed.

Incorporation of free fatty acids into acylthioesters and lipids of developing sunflower seeds

Abstract The synthesis of lipids from radioactive fatty acids in developing sunflower seeds has been examined. Lauric, palmitic, stearic and oleic acids were us