Intracellular pH of baker’s yeast

The distribution of bromophenol blue between the cell and the medium was used to calculate the intracellular pH of yeast. In buffered media the intracellular pH exhibited a plateau at pH _i =5.8 for low external pH values and another at pH _i =7.6 for high external pH values. The production of H ions by the yeast during utilization of glucose is not accompanied by an alkalinization of the cell interior. The pH _i even decreases somewhat in the presence of glucose and K ions.

1. (1)
   Внутриклеточный pH дрожжей вычисляли на основании распределения бромфеноловой сини между клетками и средой.     
   
   

Transport of ethanol in baker’s yeast

Ethanol is transported into various strains of baker's yeast by simple diffusion (no effect of inhibitors and a linear concentration dependence of the initial rate of uptake and final distribution in cells). It distributes itself in 96.6 +/- 16.2% of intracellular water.     

NAD+ regeneration in a microreactor using permeabilized baker’s yeast cells

NAD(P)-dependent oxidoreductases represent a great interest in the field of biotechnology and biotransformation. Although they have many advantages, the biggest drawback and limitation of oxidoreductase usage is the price of the coenzymes. In order to solve this problem, many in situ methods for regeneration of coenzymes have been studied and developed. Unfortunately, although results indicate that those methods are suitable for regeneration procedure, most of the processes need additional optimization to make them more sustainable. As an alternative, microreactor technology could be used as a new technique for coenzyme regeneration processes due to many advantages.In this study regeneration of coenzyme NAD⁺ was carried out in a microreactor by acetaldehyde reduction to ethanol using enzyme alcohol dehydrogenase (ADH). Suspended and immobilized whole permeabilized baker’s yeast cells were used as the source of the ADH enzyme. A 65.3% conversion of NADH was achieved with suspended permeabilized baker’s yeast cells for a residence time of τ = 36 s and equimolar concentration of substrates (c_i,NADH = 5.5 mmol/dm³, c_{i,acetaldehyde} = 5.5 mmol/dm³). When working with immobilized cells, conversion achieved for the same residence time was 10 fold lower. When permeabilized baker’s yeast cells were used for coenzyme regeneration process was stabile for 6 days of continuous operation which makes this system a good alternative for coenzyme regeneration.     

Isolation of total RNA from baker’s yeast

A simple method of production of total RNA from baker’s yeast was developed. Total RNA was isolated from yeast (Saccharomyces cerevisiae) biomass using lysis with sodium dodecyl sulfate at 100°C for 40–60 min and subsequent precipitation of the target product with 3 M NaCl. The preparation obtained was characterized in detail: yield of total RNA from 1 kg of pressed yeast, 9.25 g; optical density at 260 nm of 1 mg of RNA dissolved in 1 ml of water, 20.2 U; content of the acid-soluble fraction, 2.02%; and protein content, 1.8%. Total tRNA was isolated from total RNA by fractional precipitation with ethanol followed by gel filtration.     

Effect of inhibitors on acid production by baker’s yeast

Glucose-induced acid extrusion, respiration and anaerobic fermentation in baker’s yeast was studied with the aid of sixteen inhibitors. Uranyl(2+) nitrate affected the acid extrusion more anaerobically than aerobically; the complexing of Mg²⁺ and Ca²⁺ by EDTA at the membrane had no effect. Inhibitors of glycolysis (iodoacetamide, N-ethylmaleimide, fluoride) suppressed acid production markedly, and so did the phosphorylation-blocking arsenate. Fluoroacetate, inhibiting the citric-acid cycle, had no effect. Inhibition by uncouplers depended on their pK_a values: 2,4,6-trinitrophenol (pK_a 0.4) < 2,4-dinitrophenol (4.1) < azide (4.7) < 3-chlorophenylhydrazonomalononitrile (6.0). Inhibition by trinitrophenol was only slightly increased by its acetylation. Cyanide and nonpermeant oligomycin showed practically no effect; inhibition by dicyclohexylcarbodiimide was delayed but potent. The concentration profiles of inhibition of acid production differed from those of respiration and fermentation. Thus, though the acid production is a metabolically dependent process, it does not reflect the intensity of metabolism, except partly in the first half of glycolysis.     

Extrusion of metabolites from baker’s yeast during glucose-induced acidification

Extrusion of metabolites (glycerol, lactic, malic, and succinic acid) during the medium acidification caused by resting baker’s yeast supplied with 200mm glucose was studied under aerobic and anaerobic conditions and in the absence and presence of 14mm KCl. The maximum levels of glycerol and of the sum of acids (about 13 and 8mm, respectively) were attained anaerobically; aerobiosis reduced the levels by 40–50 % and the presence of K⁺ ions by another 10–20 %. The time courses of glucose consumption and medium acidification were similar aerobically and anaerobically. The glucose consumption ourves exhibited a short plateau about 2 min after glucose addition, caused probably by a rapid osmotic equilibration of glucose across the cell mambrane. Metabolite extrusion indicates that at high glucose concentrations the alcohol dehydrogenase reaction is supplemented by other reactions aiding in the maintenance of a balanced NAD⁺/NADH ratio in the cells.     

Synthesis of hydroquinone-α-glucoside by α-glucosidasefrom baker’s yeast

Hydroquinone-α-glucoside was synthesised from hydroquinone and maltose as glucosyl donor by transglucosylation in a water system with α-glucosidase from baker’s yeast. Only one phenolic –OH group was α-anomer-selectively glucosylated. The optimum conditions for transglucosylation reaction were at 30 °C for 20 h with 50 mM hydroquinone and 1.5 M maltose in 100 mM sodium citrate/phosphate buffer at pH 5.5. The glucoside was obtained at 0.6 mg/ml with a 4.6% molar yield with respect to hydroquinone.     

Is the microwave irradiation a suitable method for measuring soil microbial biomass?

Soil microbial biomass (SMB), the living part of soil organic matter, is used to quantify the total biomass of microorganisms present in the soil. The importance of studies about SMB has emphasized the need to identify methods which can measure the size of SMB. Among the methods currently available, chloroform-fumigation extraction and incubation are the most commonly used for estimation of SMB. However, several studies have proposed the microwave (MW) irradiation as a quick, simple and safe alternate method. There are different opinions about suitability of this method for measuring SMB. There is a question to do “Is the microwave irradiation a suitable method for measuring soil microbial biomass?” Most of the published papers comparing MW and chloroform-fumigation showed strong relationship between both methods. Therefore, we consider MW a suitable method for measuring SMB; however, it is necessary to calibrate the MW methods in different soils with a range of properties, such as clay content, to find an appropriate conversion factor in order to generate correct values for SMB with MW method.     

Differences in glucose sensing and signaling for pexophagy between the baker’s yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris

The mechanism(s) of glucose sensing for inducing the autophagic peroxisome degradation (pexophagy) is not known. Recently, we have found that defects in the S. cerevisiae PKA-cAMP signaling pathway due to knockouts of GPR1 and/or GPA2 suppressed glucose-induced degradation of peroxisomal thiolase. Here we report that single defects of high (SNF3) and low (RGT2) affinity glucose sensors involved in glucose-dependent induction of hexose transporters have only a slight effect on glucose-induced degradation of peroxisomal thiolase, although simultaneous defects of both sensors, SNF3 and RGT2 (which are known to strongly affect glucose transport) strongly inhibit this process in S. cerevisiae. Most likely, glucose is sensed for pexophagy using the Gpr1 sensor involved in the PKA-cAMP signaling pathway. In the methylotrophic yeast P. pastoris, however, knock out of S. cerevisiae orthologs of GPR1 and GPA2 did not affect glucose-induced degradation of oleate-induced thiolase or the methanol-induced key peroxisomal protein, alcohol oxidase. This implies that glucose sensing for pexophagy is different in baker’s and methylotrophic yeasts.

Addendum to: Nazarko VY, Thevelein JM, Sibirny AA. G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae. Cell Biol Int 2007; doi:10.1016/j.cellbi.2007.11.001.     

Behaviour of dehydrated baker’s yeast during reduction reactions in a biphasic medium

The behaviour of dry bakers yeast (Saccharomyces cerevisiae type II, Sigma) used as biocatalyst without preliminary growth for the synthesis of 2-heptanol from 2-heptanone in a biphasic system is presented. Cells undergo intracellular trehalose consumption with a stoichiometric ethanol production during the first 15 h of the process. This metabolism is then replaced by acetate accumulation. These reactions are disconnected from the biocatalytic reaction and do not provide reduced cofactors. 2-Heptanone is metabolised by two pathways. The first leads to 2-heptanol (molar yield close to 55%, enantioselectivity higher than 99%, with a slight decrease at the end of the process) and the second corresponds to material incorporation into the biomass. This latter phenomenon is assumed to provide the biocatalyst with the reduced cofactors needed for the reduction process. Overall, the process yielded ca. 1.4 g/l 2-heptanol in 50 h reaction, which is close to that observed with fresh cells previously grown for 15 h.     

Physiological responses of pressed baker’s yeast cells pre-treated with citric, malic and succinic acids

Summary The dough-leavening power of baker’s yeast, Saccharomyces cerevisiae, is strongly influenced by conditions under which the pressed yeast is maintained prior to bread dough preparation. In this study, the influence of the yeast cell’s pre-treatment with organic acids (malic, succinic, and citric acids) was investigated at a wide range of pH values when the pressed yeast samples were exposed to 30 °C. Increased fermentative activity was observed immediately after pre-treatment of the cells with organic acids. When the pH of the pressed yeast containing added citric acid was raised from 3.5 to 7.5, increases in both fermentative and maltase activities were obtained. Improvements in viability and levels of total protein were also observed during storage in the presence of citric acid, notably at pH 7.5. Glycerol-3-phosphate dehydrogenase activity and levels of internal glycerol also increased in the presence of citrate. On the other hand, pressed yeast samples containing succinic acid at pH 7.5 showed decreased viability during storage despite the maintenance of high levels of fermentative activity, similar to pressed yeast containing malic acid at pH 4.5 and 7.5. Decreases in intracellular levels of trehalose were observed during storage in all cases. Overall, the results of this study revealed the potential benefits of adding organic acids to pressed yeast preparations for baking purposes.     

A new method of quantitative determination of contamination of baker’s yeast by wild yeasts or by dissociation forms of the production culture

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A method for the staining of intraosseous nerve fibers using Sihler’s staining technique

Abstract

Understanding nerve fiber distribution in the jaw bone is important when performing invasive surgical treatments. Both microscopic and macroscopic anatomical techniques have been developed to study innervation. Conventional methods of removing and staining these structures, however, often alter structure and lack reproducibility of the resulting specimens. We sought to optimize Sihler’s staining technique to stain intraosseous nerves in mandibles. Four cadaver specimens were used. The best staining of intraosseous nerve fibers was achieved by using the Plank-Rychlo solution. When the Styrene monomer was used, the resulting transparency was better than that obtained with glycerin under the same conditions. No significant differences were found between Sihler's staining procedure performed according to the conventional method and the procedure in which the second decalcification step was omitted. Our results demonstrate that applying Sihler’s staining technique to bones makes them transparent and allows observation of nerves while preserving the external shape of the bone and maintaining the position of intraosseous nerve fibers. Our findings suggest our Sihler staining method for intraosseous nerve fibers can provide an intermediate resolution between macroscopic and microscopic techniques.     

The estimation of alcohol dehydrogenase activity in aerobic and anaerobic “permeabilised” baker’s yeast cells

The in vivo and in vitro activity of alcohol dehydrogenase from baker's yeast maintained under aerobic and anaerobic conditions was measured. In vivo measurements were made in cells "permeabilised" with toluene. Michaelis constants (NAD+ as substrate) were found to be almost identical as those reported for purified preparations. In addition the Km of the enzyme from cells incubated under anaerobic conditions was virtually identical to that from cells from aerobic conditions. The activity of the enzyme was found to be greater (in both "permeabilised" cells and extracts) in cells maintained under nitrogen than air. Cells metabolizing glucose in N2 produced greater levels of ethanol than in air and the rate of NAD+ reduction was also found to be greater in N2 than in air. The results indicate that it was feasible to determine rates of this enzyme in vivo and that the difference in activity of alcohol dehydrogenase under N2 and air may conceivably account for differences in rates of glucose utilisation, ethanol production and NAD+ reduction in air and nitrogen.     

Transition rate kinetics from ethanol oxidation to glucose utilisation within a structured model of baker’s yeast

 The transition rate kinetics from ethanol oxidation to glucose utilisation, within a structured model of baker’s yeast, described previously, were experimentally identified. The shift in metabolism has been assessed through glucose pulses during batch growth on ethanol. The influence of glucose concentration (between 0.25 g l^-1 and 0.90 g l^-1) and initial biomass concentration (between 0.61 g l^-1 and 1.44 g l^-1) on the transition rate was determined. The transition rate can not be described by a first-order saturation-type kinetics with respect to glucose only. A corrective term, which takes into account biomass concentration should be included. Received: 28 April 1995/Received revision: 6 July 1995/Accepted: 22 August 1995     

Different affinities of the α- and β-anomers ofD-glucose,D-mannose andD-xylose for the glucose uptake system of baker’s yeast

A recording technique for measuring the sugar uptake by cell suspensions using a polarimeter is described. The method makes it possible to calculate the uptake rates of the α-and β-anomers. The constitutive monosaccharide transport system ofSaccharomyces cerevisiae andSaccharomyces fragilis exhibits a higher affinity for the α-anomers ofd-glucose,d-manose andd-xylose than for the corresponding β-anomers, this resulting in a preferential uptake of the α-anomers from a mixture. The α-anomer ofd-xylose is preferred both during influx and efflux. The membrane transport ofd-xylose inSaccharomyces cerevisiae is not associated with a change of the anomer configuration. The facilitated diffusion system appears to possess a regulatory role for the utilization ofd-glucose andd-mannose in both yeast species investigated.