Stimulation of Zero- trans Rates of Lactose and Maltose Uptake into Yeasts by Preincubation with Hexose To Increase the Adenylate Energy Charge

Abstract

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three-to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular ␤-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [ 14 C]lactose uptake reactions were started. A short (1-to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5-to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 mol ⅐ g dry yeast

Adaptive evolution of the lager brewing yeastSaccharomyces pastorianusfor improved growth under hyperosmotic conditions and its influence on fermentation performance

“…(). Intracellular trehalose was extracted with boiling water and assayed enzymatically as described (Guimarães et al ., ). Intracellular glycerol was extracted with boiling water and assayed enzymatically with glycerol kit (R‐Biopharm AG, Germany).…”

Proton-solute coupling mechanism of the maltose transporter from Saccharomyces cerevisiae

“…Cells were resuspended in assay buffer and kept on ice until used within 4 hours Most transport assays were performed at 30 °C using cells at OD 600 of 4, 8 or 16, except when the kinetic parameters of transport (K m and V max ) were determined and OD 600 of 20 to 27.5 were used. Cells were incubated at 30 °C for 5 min to increase the adenylate energy charge 48 , after which [U- 14 C]maltose (600 mCi/mmol; American Radiolabeled Chemicals, Inc.) was added to approximately 48100 Bq/mL to start the uptake reaction; the maltose concentrations varied from 0.25 mM to 50 mM. At given time intervals, 50 μL samples were added to 2 mL ice-cold KPi or KCP and rapidly filtered on cellulose-nitrate filters with 0.45 μm pores (GE-Healthcare, Little Chalfont, UK) pre-soaked in KPi or KCP plus 1 mM of maltose to block non-specific adsorption of 14 C-maltose.…”

Metabolic engineering ofSaccharomyces cerevisiaefor lactose/whey fermentation

“…We have determined recently a K m of 1.0-1.8 mM for K. lactis CBS2359, and our results of lactose uptake by two recombinant S. cerevisiae strains expressing the K. lactis LAC12 gene are also consistent with such K m values. 18 The transport of lactose in K. lactis is an active process, requiring an energy-generating system, which permits the intracellular accumulation of lactose against a concentration gradient. 16,17 The transport is inhibited by the proton ionophore 2,4-dinitrophenol, 16,17 and therefore it has been suggested that the transporter operates, at least in part, by a proton symport mechanism.…”

“…15 The β-galactosidase (lactase) is encoded by the LAC4 gene 23 and is described to be intracellular. 18,[24][25][26] This enzyme has a K m for lactose of 12-17 mM and its pH optimum is around 7. 25 β-galactosidase hydrolyzes lactose into glucose and galactose.…”