Miguel A. Galvagno

Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices

Abstract The effects of vacuum-drying and freeze- drying on the cell viability of a commercial bakers yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10–20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the Tg values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 °C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems.

Variations in the levels of cyclic adenosine 3′:5′-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3t:́5′-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Abstract Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn 2+ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K m (1 μ m ) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn 2+ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.

cAMP levels and in situ measurement of cAMP related enzymes during yeast-to-hyphae transition in Candida albicans

Intracellular levels of cAMP and specific activities of adenylate cyclase, cAMP phosphodiesterase and cAMP-dependent protein kinase were measured during filamentation in the dimorphic fungus Candida albicans. Enzymatic assays were performed in permeabilized cells under conditions prevented endogenous proteolysis. The variations observed in cAMP levels were mainly accounted for by variations in the specific activities of adenylate cyclase and cAMP phosphodiesterase at different stages during germ tube formation. cAMP-dependent protein kinase, measured with kemptide as exogenous substrate, was developmental regulated. Some properties of the enzymatic activities from cell-free extracts are described.

Relationship between cyclic adenosine 3′:5′-monophosphate and germination inCandida albicans

Abstract Germination of yeast-like cells of Candida albicans is preceded by a significant decrease in intracellular levels of cyclic AMP during the early stage of germ-tube induction. These levels increased thereafter as germ-tube formation proceeded. The intracellular concentration of cyclic AMP was measured with a cyclic AMP radioimmunoassay and with a competitive assay method using a cyclic AMP-binding protein. Under inducing conditions, germtube formation was inhibited by the addition of cyclic AMP or compounds that are known to elevate the intracellular cyclic nucleotide concentration.

Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts.

Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars. The resulting glass transition temperatures were found to decrease with increasing moisture content. The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in bakers yeast. The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO2) of dry yeast was studied. First‐order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed. Significant losses of fermentative activity were observed in vitrified yeast samples. The dependence of rate constants with temperature was found to follow Arrhenius behavior. The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss.

Control of Mucor rouxii adenosine 3′:5′-monophosphate phosphodiesterase by phosphorylation-dephosphorylation and proteolysis☆

Partially purified cAMP phosphodiesterase from Mucor rouxii can be reversibly activated from 1.5- to 3-fold by treatment with MgATP, cAMP, and cAMP-dependent protein kinase, without change in its sedimentation behavior. Deactivation of activated enzyme can be observed in crude extracts under conditions which promote dephosphorylation; deactivation is prevented by 20 mm phosphate. cAMP phosphodiesterase can also be irreversibly activated by treatment with trypsin. The extent of activation by proteolysis is similar to that obtained by phosphorylation, but is accompanied by a decrease in the sedimentation coefficient of the enzyme. Activation by phosphorylation and proteolysis are not additive, suggesting that both mechanisms involve the same region of the enzyme molecule.

In situ measurement of cAMP related enzymes in the dimorphic fungus Mucor rouxii.

Abstract Yeast cells of the dimorphic fungus Mucor rouxii have been permeabilized by treatment with toluene:ethanol. The permeabilization allowed the in situ measurement of pyruvate kinase, cAMP phosphodiesterase and adenylate cyclase activities. Using a small peptide as substrate, cAMP dependent protein kinase activity could also be measured. Permeabilized cells showed higher cAMP phosphodiesterase and adenylate cyclase activities than cellular homogenates. The main catalytic properties of the enzymes were similar to that previously found in in vitro studies.

Activation of the cAMP cascade by steroidogenic hormones and glucagon in the pathogenic fungus Candida albicans

Incubation of Candida albicans yeast cells with human luteinizing hormone (hLH), human chorionic gonadotrophin (hCG) or glucagon produced a significant rise in cAMP total levels. The effect of these hormones in permeabilized cells of the fungus produced a 2-3 fold increase in the Mg2+, GTP-dependent adenylyl cyclase activity as well as full activation of the cAMP-dependent protein kinase (PKA) activity. These results indicate that the interaction of the mammalian hormones with the fungus triggered the cAMP activation cascade in a similar way to that found in higher eukaryotic organisms.

cAMP levels and in situ measurement of adenylate cyclase and cAMP phosphodiesterase activities during yeast-to-hyphae transition in the dimorphic fungus Mucor rouxii

Intracellular levels of cAMP and specific activities of adenylate cyclase and cAMP phosphodiesterase were measured during yeast-to-hyphae transition in the dimorphic fungus M. rouxii. Enzymatic activities were measured in permeabilized cells under conditions preventing protein dephosphorylation and proteolysis. A two-fold decrease in intracellular cAMP levels occurred shortly after exposure of the yeast to air quite before morphological changes became evident. Morphogenesis to hyphae after exposure to air was inhibited by the addition of 10 mM dibutyryl cAMP to the culture medium, and the yeast morphology was maintained for at least 24 hours. The decrease in cAMP levels that occurs shortly after exposure of yeast culture to air was mainly accounted for by variations in the state of activation of cAMP phosphodiesterase while the specific activity of adenylate cyclase did not vary significantly during yeast-to-hyphae transition.

Cyclic adenosine 3′, 5′-monophosphate phosphodiesterase from Mucorrouxii: Regulation of enzyme activity by phosphorylation and dephosphorylation

Abstract Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg 2+ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ 32 P]ATP resulted in a significant amount of [ 32 P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg 2+ and was accompanied by the release of [ 32 P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.