Yeast Candida Utilis Research Articles

Homeostatic regulation of cellular energy metabolism: experimental characterization in vivo and fit to a model

Measurements in isolated liver cells, cultured kidney cells, protozoa (Tetrahymena pyriformis), and yeast (Candida utilis) indicate that homeostatic regulation of cellular energy metabolism is of common origin. In every case near equilibrium is maintained between the transfer of reducing equivalents from the intramitochondrial NAD couple to cytochrome c and the phosphorylation of cytosolic ADP to ATP. Under conditions of constant energy demand, changes in the intracellular phosphate concentration cause an adjustment in the [ATP]/[ADP] to maintain a constant [ATP]/[ADP][Pi] and constant respiratory rate. The regulation of mitochondrial respiration occurs as part of the reactions by which reduced cytochrome c is oxidized by molecular oxygen. At similar values for the [ATP]/[ADP][Pi] the respiratory rate increases with increasing reduction of cytochrome c. A model for mitochondrial respiratory control is found to give a good fit to the data in all of the different types of cells tested.

Continuous single cell protein production fromCellulomonas sp. andCandida utilis grown in mixture on barley straw

Continuous fermentations with mixed cultures of the cellulolytic bacteriaCellulomonas sp. and the yeastCandida utilis were examined. Fermentations were carried out in an aerated 5-l fermenter with different preparations of wet disintegrated barley straw as the cellulose source (3.6–4.2%). The straw was pretreated with NaOH (3.2–8.5 kg NaOH/100 kg dry straw) under high pressure and temperature in a feedstuff pellet press. The quantity of dry cell mass produced and the breakdown of the straw were measured. Crude protein and ash content in cell dry matter and residual fiber were determined. The experiments showed thatCellulomonas sp. andCandida utilis may be grown together in a continuous culture (dilution rate D=0.12–0.14 h−1) for at least 3 days without washing out one of the organisms. Highest productivity was 1.39 g cell dry matter/l/h when using straw pretreated with 5.7% NaOH. The dry cell product contained 58–66% crude protein and up to 51% of the organic fiber dry matter was solubilized. The yield constants were 0.32–0.61 g cell dry matter per g solubilized organic fibers.

Regulation of cellular metabolism by intracellular phosphate

1. Incubation of liver cell suspensions with 10 mM glycerol or 10 mM fructose caused a decrease in intracellular [P i] and a fall in the [ ATP] [ ADP] while the respiratory rate, the redox state of the intramitochondrial NAD couple, and the [ ATP] [ ADP] · [ P i ] remained essentially unaltered. 2. The concentration of intracellular phosphate in yeast Candida utilis was found to be dependent on phosphate concentration in the growth medium: it was 25–30 μmol/g wet weight in cells grown in the presence of 50 mM P i, 10–12 μmol/g wet weight in cells grown in 5 mM P i, and 3–4 μmol/g wet weight in cells grown with 1 mM P i. 3. The [ ATP] [ ADP] ratios were found to be highest in yeast cells grown in the presence of 50 mM P i (⩾ 30) and lowest in cells grown with 1 mM P i (8–9). The increase in [ ATP] [ ADP] was due to an increase in [ATP]. 4. The endogenous levels of glucose 6-phosphate in washed and aerated cells were lowest in cells grown in the presence of 50 mM P i and highest in those grown in the presence of 1 mM P i. A suggestion is made that the high intracellular [P i] stimulates phosphorolytic breakdown of glycogen and depletes the cellular stores of this substrate. 5. It is postulated that the cellular respiratory rate and the NADH generation rate are regulated by [ ATP] [ ADP] · [ P i ] and not by the “energy charge” as defined by Atkinson (Biochemistry 7, 4030–4034, 1968). 6. The concentration of intracellular [P i] is implicated as an important contributor to the regulatory mechanisms of cellular metabolism.

The number of Fe atoms in the iron-sulphur centers of the respiratory chain

1. From the 57Fe hyperfine interaction in EPR spectra of reduced submitochondrial particles from the yeast Candida utilis, grown with 57Fe, it is concluded that all Fe-S centers in these particles detectable in spectra at 35–80 K are [2Fe-2S] 2−(2−;3−) centers. These are the centers 1 of NADH and succinate dehydrogenase, the Rieske Fe-S center and possibly center 2 of succinate dehydrogenase. 2. The signals of the reduced particles detectable only at temperatures below 20 K are [4Fe-4S] 2−(2−;3−) clusters. These are the centers 2, 3 and 4 of NADH dehydrogenase. 3. The EPR spectra of the [2Fe-2S] 3− centers of Complex I and II, but not that of Complex III, display a great inequality of the Fe nuclei in the effective hyperfine interaction in the x− y direction.

Heterogeneity of Size and Distribution of Intramembrane Particles in the Plasma Membrane of Yeast

The matrix of biological membranes consists of a lipid bilayer into which proteins or protein aggregates are intercalated. Freeze-fracture techni- ques permit these proteins, perhaps in association with lipids, to be visualized in the hydrophobic regions of the membrane. Thus, numerous intramembrane particles (IMP) have been found on the fracture faces of membranes from a wide variety of cells (1-3). A recognized property of IMP is their tendency to form aggregates in response to changes in experi- mental conditions (4,5), perhaps as a result of translational diffusion through the viscous plane of the membrane. The purpose of this communica- tion is to describe the distribution and size of IMP in the plasma membrane of yeast (Candida utilis).Yeast cells (ATCC 8205) were grown in synthetic medium (6), and then harvested after 16 hours of culture, and washed twice in distilled water. Cell pellets were suspended in growth medium supplemented with 30% glycerol and incubated for 30 minutes at 0°C, centrifuged, and prepared for freeze-fracture, as described earlier (2,3).

Effect of crotonaldehyde on the metabolism of Candida utilis during the production of single cell protein from ethanol.

The effect of 20 low-boiling compounds on the yeast Candida utilis 49 was assessed by a screening test on agar medium. The highest toxicity was exhibited by crotonaldehyde, allyl alcohol and acrolein. Oxidation and assimilation experiments in a slightly aerobic environment showed that an increase in the level of crotonaldehyde in the medium in the range of 10--20 mg 1-1 brings about a lowering of intensity of metabolic processes in Candida utilis, suppression of ethanol utilization and aerobic oxidation rate, a drop in biomass yield, prolongation of cultivation time, etc. The inhibitory effect of crotonaldehyde depends strongly on the manner of its dosage into the medium (single or continuous) and other cultivation conditions (intensity of medium aeration, physiological state of the culture, etc). Crotonaldehyde is lost from the medium partially by volatilization and partially due to chemical and biochemical transformation.

Purification of an exo-1,3-beta-glucanase from Candida utilis.

An exo-1,3-beta-glucanase (EC 3.2.1.-) has been purified from the culture fluid of the yeast Candida utilis, and its biochemical properties have been studied. The amino acid analysis revealed a high content of acidic amino acids. The purified enzyme had 20% carbohydrate and a net negative charge showing higher affinity for laminarin than for p-nitrophenyl-beta-D-glucopyranoside and yeast cell-wall 1,3-beta-glucans. In addition, the enzyme hydrolyzed the substrates starting from the nonreducing ends, releasing glucose as the exclusive hydrolysis product. The enzyme activity was strongly inhibited by lactones and also by some heavy-metal ions.

A soluble acyl-coenzyme a oxidase from the yeast Candida utilis

trans-2-Enoyl-CoA and two unidentified polar compounds were synthesized from the corresponding long-chain acyl-CoA by a particle-free supernatant fraction obtained from Candida utilis. The enzyme was unreactive toward free fatty acids but desaturated all long-chain acyl CoAs tested (14:0, 16:0, 18:1, 18:2). Molecular oxygen was the only required cofactor. Phenazine methosulfate and 2,6-dichloroindophenol did not replace the requirement for oxygen. The activity was inhibited specifically by NADPH and stimulated by linoleic acid or linolenic acid. The enzyme was not active in log phase cultures, but was detected only in stationary phase cells. Introduction of the trans-2-double bond was confirmed by gas-liquid chromatography, thin-layer chromatography, mass spectrometry, catalytic hydrogenation, oxidative cleavage, and chemical reactivity of the product toward nucleophilic addition.

Assimilation spectrum of the yeast Candida utilis 49 used for producing fodder yeast from synthetic ethanol.

Oxidizing and assimilating ability of the yeast Candida utilis 49 was tested with 21 different low-boiling organic compounds which come as components of raw synthetic ethanol. The highest yields of yeast dry weight were obtained with ethanol (72.0%), propanol (48.2%), ethyl acetate (43.4%) and acetic acid (34.2%). To a minor extent, the yeast was capable of utilizing also 2-propanol, butanol and 2-butanol; it oxidized most of the compounds tested.

The hydrothermal degradation of cellulosic matter to sugars and their fermentative conversion to protein

For the hydrothermal degradation of cellulosic matter, an apparatus was developed in which water is used as extraction medium. Samples, 0.15 g each, of pure cellulose (filter paper), natural straw, and 14C-labeled straw were treated at temperatures of between 200° and 275°C. Of the inserted cellulose, 65.7% was recovered at the optimum temperature as sugars and hydroxymethylfurfural. It was possible to degrade the straw selectively: at lower temperatures, the hemicellulose part of the plant matter was converted to xylose and arabinose; and then at higher temperatures, the cellulose was converted to glucose and cellobiose. At the same time, a certain amount of the sugars was transformed to furfural compounds. The growth behavior of the yeast Candida utilis (strain Weissenbach) was analyzed, using cellobiose, xylose, and glucose (standard) as carbon sources. The growth curves applying cellobiose were nearly identical to those of glucose. Xylose showed lower productivity than the hexoses. The main products of the hydrothermal degradation can, therefore, be used favorably as nutritive substances for this proteinproducing yeast.

Method of Purifying β-(1-3)-Glucanase from Candida utilis

A method has been developed to obtain a rapid purification of the beta-(1-3)-glucanase present in culture fluids of the yeast Candida utilis.

Characterization of nitrate reductase from the yeastCandida utilis

The assimilatory nitrate reductase [NAD (P) H: nitrate oxidoreductase, EC 1.6.6.2] of the nitrate-utilizing yeastCandida utilis was prepared in soluble form from cells grown aerobically with nitrate as the nitrogen source, and some of its properties studied. The enzyme is cytoplasmic in intracellular distribution and its activity is NAD (P) H-dependent. The enzyme utilized both NADH and NADPH as electron donors but not reduced viologen dyes, flavins and other reductants. Flavin compounds, at higher concentrations, decreased the enzyme activity, while at very low concentrations, FAD (10−5 M) showed little stimulation. The optimal activity of the enzyme was obtained when the reaction was carried out for 10 min at 37° C at pH 7·0 with 20 mM KNO3, 0·1 mM NAD (P) H, 0.01 mM FAD and 140 µg enzyme protein. Complete loss of enzyme activity was observed on exposure to 50° C for 3 min. pHMB, cyanide, azide and EDTA, in that order, inhibited the enzyme activity effectively. The inhibition caused by pHMB was largely reversed by DTT.

Antigenic independence of some microbial urate oxidases

Antisera were prepared to urate oxidase derived from three microbial species, a yeast (Candida utilis), a mold (Aspergillus flavus), and a bacterium (Bacillus fastidious). The antisera inhibited enyme activity to a limited extent. Cross-reaction studies with preparations of the enzyme from these and other species indicated that the microbial enzyme exhibits a high degree of antigenic independence. This appeared to be particularly true of the bacteria studied.

Mitochondrial structure studied by high voltage electron microscopy of thick sections of Candida utilis.

Mitochondrial structure in yeast cells under various physiological conditions has been studied by high voltage electron microscopy of sections that are 0-5 to 2-0 mum thick. Such thick sections of the yeast Candida utilis had a small number of long, branched tubular mitochondria per cell. The mitochondria extended into cell buds and unseparated daughter cells. It was apparent from parallel studies with thin sections that most of the rounded mitochondrial profiles viewed in thin sections should not be interpreted as being numerous small individual mitochondria. Attempts to study thick sections of the yeasts Saccharomyces cerevisiae and Schizossaccharomyces pombe were frustrated by poor contrast.

Flow Behavior of Concentrated Suspensions of Yeast Grown on Sauerkraut Waste

The flow behavior of suspensions of yeast (Candida utilis) grown on sauerkraut waste was studied as a function of concentration at 25.8 C. Suspensions containing 5.8, 21.7, 22.6, 23.6, 24.7 and 27.0% dry solids behaved as Newtonian fluids. The viscosity of these suspensions increased exponentially with the concentration of dry solids. A 29.2% suspension showed time dependent shear-thinning behavior. The non-Newtonian characteristics of the suspension could be described by both the Power law and the Casson flow models.

Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells : Subunit structure and molecular weight determination

Cytochrome c oxidase was purified from mitochondria of Candida utilis yeast cells. The purification procedure involved the hypotonic incubation of mitochondria followed by washes with increasing concentrations of KCl. The membrane fragments derived from this procedure were subjected to ammonium sulfate fractionation in the presence of 2% cholate. The purified active enzyme contained 8.5–9.2 nmol heme a per mg protein and was free of other types of hemoproteins. Upon Sephadex G200 gel filtration in the presence of cholate, an apparent molecular weight of 200,000 was estimated. A single band was observed for the active enzyme upon DEAE-cellulose chromatography, sucrose density gradient centrifugation, and Sephadex G200 gel filtration. Electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate resolved the enzyme into six polypeptide bands with apparent molecular weights of 49,000, 32,000, 28,000, 20,000, 13,500, and 8,000, respectively. The six components were also resolved by gel filtration on Sephadex G200, equilibrated with 0.1% sodium dodecyl sulfate, giving apparent molecular weights of 46,000, 35,000, 23,000, 19,000, 12,500, and 7,800.

Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells : Function of copper in the assembly of active cytochrome c oxidase

1. 1. Cytochrome c oxidase from Candida utilis yeast cells grown in copper deficient and copper sufficient media has been studied by labeling the apoprotein with radioactive leucine ( 33H- and 14C-labeled leucine). The six subunits of cytochrome c oxidase were found to be present in copper deficient cells. 2. 2. The kinetics of reconstitution of cytochrome c oxidase when copper deficient cells are grown in copper supplemented medium suggest that an active process is involved in the transfer and/or integration of copper into cytochrome c oxidase. 3. 3. When copper deficient cells are grown in copper supplemented media containing cycloheximide so that no cytoplasmic protein synthesis occurs, cytochrome c oxidase reconstitution is totally inhibited. 4. 4. When copper deficient cells are grown in copper supplemented media containing either chloramphenicol which blocks mitochondrial translation, or ethidium bromide which inhibits mitochondrial transcription, cytochrome c oxidase reconstitution is 20–25% of the control value. 5. 5. The data show that: (a) the apoprotein of cytochrome c oxidase is synthesized in copper deficient cells; (b) the insertion of copper is not necessary for the integration of the apoprotein into the membrane; (c) the transfer of copper and/or its integration into cytochrome c oxidase necessitates a cytoplasmic protein which is sensitive to cycloheximide. Thus copper plays a key role in the assembly of active cytochrome c oxidase.

Influence of Aeration Rate on Yeast Production in Sauerkraut Brine

The optimal rate of of aeration for production of food yeast (Candida utilis) in sauerkraut brine was found to be approximately 24mM O2/1/h.

Uptake of some cations by Candida utilis.

The affinity of a number of bivalent and tervalent cations for the yeastCandida utilis was shown to decrease from Zn2+ to Ca2+. Ferric ions inhibited the uptake of Zn2+ but not vice versa. Inhibition of Zn2+ uptake in a synthetic medium with Fe3+ does not decrease the content of crude protein in cells.

Amino acid pools and metabolism during the cell division cycle of arginine-grown Candida utilis.

Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed.