Abstract

Debaryomyces hansenii is a halotolerant yeast capable of growing at substantially higher salt concentrations than Saccharomyces cerevisiae, by accumulating glycerol as a compatible solute under osmotic stress conditions [1, 2]. In an adaptation of yeast cells to an hyperosmotic medium, the plasma membrane plays an important role due to its action as a diffusion barrier; its selective permeability to different ions and solutes allows the cells to restore and maintain the osmotic equilibrium between the cell interior and the environment. However, it is not yet clear how and to which extent the presence of a high salt concentration in the external medium affects other transport processes across the plasma membrane. This contribution reports on the kinetic characterization of the monosaccharide uptake in S. cerevisiae UCD 522 (industrial wine yeast), in the halotolerant yeast D. hansenii NRRL Y-7393 and in one of the hybrids, HM92, previously obtained by protoplast fusion between the above two strains. The hybrid strain is also halotolerant [3]. Yeast cultures were maintained on complete YEPD solid medium (yeast extract 1%, peptone 2 %, glucose 2 % and agar 1.5 %). For uptake assays, the cells were grown in a liquid medium, YEPD or YEPD + 2 mol/L NaC1, The culture flasks were inoculated with a preculture to give a cell density of 5 x 106 cells per mL. The cultures were incubated at 30 ~ at 3.3 Hz. The cells were harvested in the early stationary phase, washed twice with distilled water and resuspended to a 5 % aqueous suspension. The cell suspension was aerated on a magnetic stirrer for 4 h at room temperature (cell starvation). Following aeration, the cells were washed once again and resuspended as before. After mixing with 100 mmol/L KH2PO4 buffer pH 6.5, the suspension was shaken in a water bath at 30 ~ and the experiment was started by adding the appropriate monosaccharide (D-xylose or D-glucose). In experiments with cells grown in 2 mol/L NaC1, the salt stress conditions were maintained during the washing and the aeration of the cells, Samples were taken at intervals. The uptake of D-xylose was measured by the filtration method [4] using the orcinol reagent [5]. The uptake of D-glucose was measured enzymically in the supernatant after centrifugation using hexokinase and glucose-6-phosphate dehydrogenase in the presence of ATP and NAD +. Kinetic parameters, K T and Vmax, were determined from Eadie-Hofstee plots. D-Xylose was chosen as a transport substrate for the kinetic studies since it shares the uptake system with D-glucose and is very slowly metabolized. Contrary to S. cerevisiae, in which glucose and xylose are taken up by facilitated diffusion, the uptake of monosaccharides in D. hansenii and also in the hybrid HM92 was dependent on metabolic energy and led to intracellular accumulation of the pentose. Moreover, the hybrid accumulated xylose about two times higher than its parent strain D. hansenii (Fig. 1). Adding the uncoupler 2,4-dinitrophenol (DNP) to preloaded cells of both D. hansenii parent strain and the hybrid induced a complete outflow of DNP BO

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