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W A Scheffers

Publications and source records attributed to W A Scheffers.

At least 37 records · Page 2Linked to original sources
PubMed1989

Localization and kinetics of pyruvate-metabolizing enzymes in relation to aerobic alcoholic fermentation in Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621.

The role of pyruvate metabolism in the triggering of aerobic, alcoholic fermentation in Saccharomyces cerevisiae has been studied. Since Candida utilis does not exhibit a Crabtree effect. this yeast was used as a reference organism. The localization, activity and kinetic properties of pyruvate carboxylase (EC 6.4.1.1), the pyruvate dehydrogenase complex and pyruvate decarboxylase (EC 4.1.1.1) in cells of glucose-limited chemostat cultures of the two yeasts were compared. In contrast to the general situation in fungi, plants and animals, pyruvate carboxylase was found to be a cytosolic enzyme in both yeasts. This implies that for anabolic processes, transport of C4-dicarboxylic acids into the mitochondria is required. Isolated mitochondria from both yeasts exhibited the same kinetics with respect to oxidation of malate. Also, the affinity of isolated mitochondria for pyruvate oxidation and the in situ activity of the pyruvate dehydrogenase complex was similar in both types of mitochondria. The activity of the cytosolic enzyme pyruvate decarboxylase in S. cerevisiae from glucose-limited chemostat cultures was 8-fold that in C. utilis. The enzyme was purified from both organisms, and its kinetic properties were determined. Pyruvate decarboxylase of both yeasts was competitively inhibited by inorganic phosphate. The enzyme of S. cerevisiae was more sensitive to this inhibitor than the enzyme of C. utilis. The in vivo role of phosphate inhibition of pyruvate decarboxylase upon transition of cells from glucose limitation to glucose excess and the associated triggering of alcoholic fermentation was investigated with 31P-NMR. In both yeasts this transition resulted in a rapid drop of the cytosolic inorganic phosphate concentration. It is concluded that the relief from phosphate inhibition does stimulate alcoholic fermentation, but it is not a prerequisite for pyruvate decarboxylase to become active in vivo. Rather, a high glycolytic flux and a high level of this enzyme are decisive for the occurrence of alcoholic fermentation after transfer of cells from glucose limitation to glucose excess.

Candida↗
PubMed1989

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation.

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.

Candida↗
PubMed1989

Glucose transport in crabtree-positive and crabtree-negative yeasts.

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.

Biological Transport↗
PubMed1989

Competition for glucose between the yeasts Saccharomyces cerevisiae and Candida utilis.

The competition between the yeasts Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 for glucose was studied in sugar-limited chemostat cultures. Under aerobic conditions, C. utilis always successfully completed against S. cerevisiae. Only under anaerobic conditions did S. cerevisiae become the dominant species. The rationale behind these observations probably is that under aerobic glucose-limited conditions, high-affinity glucose/proton symporters are present in C. utilis, whereas in S. cerevisiae, glucose transport occurs via facilitated diffusion with low-affinity carriers. Our results explain the frequent occurrence of infections by Crabtree-negative yeasts during bakers' yeast production.

Aerobiosis↗
PubMed1989

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetate-CoA Ligase↗
PubMed1988

Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess.

When chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621, grown under glucose limitation, were pulsed with excess glucose, both organisms initially exhibited similar rates of glucose and oxygen consumption. However, striking differences were apparent between the two yeasts with respect to the production of cell mass in the culture and metabolite excretion. Upon transition from glucose limitation to glucose excess, S. cerevisiae produced much ethanol but the growth rate remained close to that under glucose limitation. C. utilis, on the other hand, produced little ethanol and immediately started to accumulate cell mass at a high rate. This high production rate of cell mass was probably due to synthesis of reserve material and not caused by a high rate of protein synthesis. Upon a glucose pulse both yeasts excreted pyruvate. In contrast to C. utilis, S. cerevisiae also excreted various tricarboxylic acid cycle intermediates, both under steady-state conditions and after exposure to glucose excess. These results and those of theoretical calculations on ATP flows support the hypothesis that the ethanol production as a consequence of pyruvate accumulation in S. cerevisiae, occurring upon transition from glucose limitation to glucose excess, is caused by a limited capacity of assimilatory pathways.

Adenosine Triphosphate↗
PubMed1988

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556.

From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45 degrees C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight at D = 0.1 h to 2 U mg of cell dry weight at D = 0.8 h. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.

Journal Article↗
PubMed1988

Hydrogen peroxide metabolism in yeasts.

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H(2)O(2) generated in the oxidation of methanol by alcohol oxidase. Not only H(2)O(2) generated intracellularly but also H(2)O(2) added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H(2)O(2) consumption during growth in chemostat cultures on mixtures of glucose and H(2)O(2), it appeared that the mutant was capable of decomposing H(2)O(2) at a rate as high as 8 mmol . g of cells . h. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H(2)O(2). When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H(2)O(2) at a high rate when cells were grown on mixtures of glucose and H(2)O(2). In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H(2)O(2), the level of catalase remained low, but CCP increased with increasing rates of H(2)O(2) utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H(2)O(2) detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.

Journal Article↗
PubMed1987

Mechanism of stimulation of endogenous fermentation in yeast by carbonyl cyanide m-chlorophenylhydrazone.

Addition of the uncoupler and protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to starved yeast cells starts endogenous alcoholic fermentation lasting about 20 min. Hexose 6-phosphates, fructose 2,6-bisphosphate, and pyruvate accumulate in less than 2 min after addition of CCCP from almost zero concentration to concentrations which correspond to 1/5-1/10 of the steady-state concentrations during fermentation of glucose. CCCP immediately causes a decrease of the intracellular cytosolic pH from 6.9 to 6.4. This change activates adenylate cyclase (Purwin, C., Nicolay, K., Scheffers, W.A., and Holzer, H. (1986) J. Biol. Chem. 261, 8744-8749) and leads to the previously observed transient increase of cyclic AMP. It is shown here that the following enzymes known from in vitro experiments to be activated by cyclic AMP-dependent phosphorylation are activated in the CCCP-treated starved yeast cells in vivo: glycogen phosphorylase, trehalase (pH 7), 6-phosphofructo-2-kinase. The activation of 6-phosphofructo-2-kinase leads to an accumulation of fructose 2,6-bisphosphate, which is known from in vitro experiments to activate 6-phosphofructo-1-kinase and to inhibit fructose-1,6-bisphosphatase. All effects observed in the intact yeast cells fit with the idea that the CCCP-initiated activation of adenylate cyclase leads to a sequence of events which by protein phosphorylation and allosteric effects initiates endogenous alcoholic fermentation.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1986

Mechanism of control of adenylate cyclase activity in yeast by fermentable sugars and carbonyl cyanide m-chlorophenylhydrazone.

The phosphorylation of fructose-1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP-dependent protein kinase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Possible mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby lead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that neither sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoacridine fluorescence after addition of fermentable sugars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cyanide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-1,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately 2-fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent Km for ATP with no change in Vmax. These studies suggest that activation of adenylate cyclase by decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose-1,6-bisphosphatase.

Adenylyl Cyclases↗
PubMed1986

A comparative radiorespirometric study of glucose metabolism in yeasts.

A comparative radiorespirometric study of glucose metabolism in glucose-limited chemostat cultures of Saccharomyces cerevisiae, Candida utilis and Rhodosporidium toruloides was performed in an attempt to estimate the contribution of the hexose monophosphate (HMP) pathway to glucose metabolism. Radioactively labelled glucose was administered directly to the cultures in a constant substrate feed, without disturbance of the steady state. The 14CO2 yields from [1-14C]- and [6-14C]-glucose demonstrated that the HMP pathway activities for the three yeasts were very similar. Furthermore, a quantitative analysis of results indicated that the HMP pathway activities were close to the theoretical minimum needed to cover the NADPH requirement for biomass formation.

Candida↗
PubMed1986

Alcoholic fermentation by 'non-fermentative' yeasts.

All type strains of 'non-fermentative' yeasts, available in the culture collection of the Centraalbureau voor Schimmelcultures, were reinvestigated for their capacity to ferment glucose in the classical Durham tube test. Although visible gas production was absent, nearly all strains produced significant amounts of ethanol under the test conditions. Under conditions of oxygen-limited growth, even strong alcoholic fermentation may occur in a number of yeasts hitherto considered as non-fermentative. Thus, shake-flask cultures of Hansenula nonfermentans and Candida silvae fermented more than half of the available sugar to ethanol. It is concluded that the taxonomic test for fermentation capacity, which relies on detection of gas formation in Durham tubes, is not reliable for a physiological classification of yeasts as fermentative and non-fermentative species.

Candida↗
PubMed1986

Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts.

Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.

Ascomycota↗
PubMed1985

Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis.

Xylose reductase from the xylose-fermenting yeast Pichia stipitis was purified to electrophoretic and spectral homogeneity via ion-exchange, affinity and high-performance gel chromatography. The enzyme was active with various aldose substrates, such as DL-glyceraldehyde, L-arabinose, D-xylose, D-ribose, D-galactose and D-glucose. Hence the xylose reductase of Pichia stipitis is an aldose reductase (EC 1.1.1.21). Unlike all aldose reductases characterized so far, the enzyme from this yeast was active with both NADPH and NADH as coenzyme. The activity with NADH was approx. 70% of that with NADPH for the various aldose substrates. NADP+ was a potent inhibitor of both the NADPH- and NADH-linked xylose reduction, whereas NAD+ showed strong inhibition only with the NADH-linked reaction. These results are discussed in the context of the possible use of Pichia stipitis and similar yeasts for the anaerobic conversion of xylose into ethanol.

Aldehyde Reductase↗
PubMed1984

Microbial contamination of a vitamin A formulation, prepared in local pharmacies, and its preservation against yeasts and moulds.

The microbial contamination of 44 samples of a vitamin A preparation in sucrose syrup was investigated. The contaminants were almost exclusively yeasts and moulds. Microbiological and physicochemical studies showed that sorbic acid was the preservative of choice for this formulation. The results are discussed with respect to the preservation of non-sterile pharmaceuticals.

Drug Contamination↗
PubMed1984

Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect).

Aerobic growth of the yeast Brettanomyces intermedius CBS 1943 in batch culture on a medium containing glucose and yeast extract proceeded via a characteristic pattern. In the first phase of growth glucose was fermented to nearly equal amounts of ethanol and acetic acid. After glucose depletion, growth continued while the ethanol produced in the first phase was almost quantitatively converted to acetic acid. Finally, after a long lag phase, growth resumed with concomitant consumption of acetic acid. When the culture was made anaerobic during the first phase, growth, glucose consumption and metabolite production stopped immediately. This Custers effect (inhibition of alcoholic fermentation as a result of anaerobic conditions) was transient. After 7-8 h the culture was adapted to anaerobiosis, and growth and ethanol production resumed. The lag phase could be shortened at will by the introduction of hydrogen acceptors, such as oxygen or acetoin, into the culture. Glycerol production was not observed during any phase of growth. These results support the hypothesis that the Custers effect in this yeast is due to a disturbance of the redox balance, resulting from the tendency of the organism to produce acetic acid, and its inability to restore the balance by production of glycerol.

Aerobiosis↗
PubMed1984

Alcoholic Fermentation of d-Xylose by Yeasts.

Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.

Journal Article↗