M embrane deterioration is an early and characteristic feature of plant senescence engendering increased permeability, loss of ionic gradients, and decreased function of key membrane proteins such as ion pumps (1). One of the clearest manifestations of this is the onset of membrane leakiness measurable as increased conductivity of diffusates from intact tissue. This is detectable in carnation petals, for example, well before petal inrolling, the first morphological manifestation of senescence in this tissue, and also before the climacteric-like rise in ethylene production (2). The decline in membrane structural integrity at the onset of senescence appears to be largely attributable to accelerated metabolism of membrane lipids and ensuing change in the molecular organization of the bilayer. Indeed, loss of membrane phospholipid is one of the best documented indices of membrane lipid metabolism during senescence and has been demonstrated for senescing flower petals, leaves, cotyledons and ripening fruit (3, 4).The selective depletion of phospholipid fatty acids from the membranes of senescing tissues results in an increase in the sterol:fatty acid ratio in the bilayer and a consequent decrease in bulk lipid fluidity. This has been demonstrated by fluorescence depolarization and electron spin resonance for microsomal membranes from senescing cotyledons, flowers, leaves, and ripening fruit (5-8) and for plasmalemma of ripening fruit and senescing flowers (5). The decrease in lipid fluidity is engendered by an enrichment of free sterols relative to fatty acids in the bilayer as fatty acids are cleaved from the membrane lipids and selectively removed, reflecting the fact that free sterols are known to restrict the mobility of phospholipid fatty acids (9). As well, in some senescing tissues, the decrease in bulk lipid fluidity appears to be caused in part by a selective depletion of polyunsaturated fatty acids from membranes and an ensuing increase in the saturated-to-unsaturated fatty acid ratio (6). There are also reports that the large changes in bulk membrane lipid fluidity accompanying senescence may alter the conformation of membrane proteins, rendering them prone to proteolysis (10, 11).Recent data suggest that free fatty acids arising from the metabolism of membrane lipids may be removed from the bilayer by blebbing of lipid particles highly enriched in free fatty acids from the membrane surface into the cytosol (12-14). These lipid particles appear to be structurally analogous to oil bodies. Indeed, there is growing evidence that the free fatty acids released from senescing membranes are metabolized by glyoxylate cycle enzymes also induced at the onset of senescence (15). However, it is also clear that free fatty acids accumulate in senescing membranes and induce lipid-phase separations. The resulting mixture of liquid-crystalline and gel phase lipid domains in the bilayer contributes to the leakiness of senescing membranes because of packing imperfections at the phase boundaries (16).Deesterification of membr...

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In recent years there has been an increase in the interest in the production of compounds by isolation from natural sources or through processes that can be deemed “natural”. This is of particular interest in the food and beverage industry for flavors and aromas. Hexanal, organoleptically known to possess “green character”, is of considerable commercial interest. The objective of this study was to determine if the enzyme template known to be responsible for the synthesis of hexanal from linoleic acid (18:2) in tomato fruits could be harnessed using a hollow‐fiber reactor. A hollow‐fiber reactor system was set up and consisted of a XAMPLER ultrafiltration module coupled to a reservoir. The enzyme template was extracted from ripe tomato fruits and processed through an ultrafiltration unit (NMWC of 100 kDa) to produce a retentate enriched in soluble and membrane‐associated lipoxygenase (LOX) and hydroperoxide lyase (HPL). This extract was recirculated through the lumen of the hollow‐fiber ultrafiltration unit with the addition of substrate in the form of linoleic acid, with buffer addition to the reaction flask to maintain a constant retentate volume. Product formation was measured in the permeate using solid phase microextraction (SPME) developed for this system. At exogenous substrate concentrations of 16 mM and a transmembrane pressure of 70 kPa, hexanal production rates are in the order of 5.1 μg/min. Addition of Triton X‐100 resulted in membrane fouling and reduced flux. The reactor system has been run for periods of up to 1 week and has been shown to be stable over this period. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 372–377, 2000.

Hexanal is a key organoleptic element of green-note that is found in both fragrances and flavors. We report a novel process for the production of hexanal using immobilized enzyme templates extracted from different plant sources in combination with hollow-fiber ultrafiltration for in situ separation. Enzyme templates, known to be responsible for the synthesis of hexanal from linoleic acid (18:2), were isolated from naturally enriched tissues including carnation petals, strawberry and tomato leaves. These templates were immobilized in an alginate matrix and used as a biocatalyst in a packed-bed bioreactor. Continuous product recovery was achieved using a hollow-fiber ultrafiltration unit. The effects of pH, reaction temperature, and substrate and enzyme concentrations were studied and their effects on hexanal generation identified and optimized. Utilizing optimized conditions, hexanal production 112-fold higher than endogenous steady-state levels in a corresponding amount of plant tissue could be achieved over a 30-minute period. Based on the reactor studies, product inhibition also appears to be an important factor for bioreactor-based hexanal production.