Degradation of Phospholipids and Liquefaction of Pressed Baker’s Yeast by Acetic Acid Vapour

When pressed baker’s yeast (Saccharomyces cerevisiae) was exposed to the vapour of acetic acid, autolysis of yeast cells was induced in 3 or 4 hr. In order to elucidate the mechanism of the autolysis caused by the AcOH-treatment, we investigated variations in the lipid content of yeast cells during the treatment. The degradation of phospholipids and the accumulation of free fatty acids occurred within 3 hr. Formic acid exerted a similar effect on the pressed yeast. The effect of propionic acid was not seen in 3hr but was after 18 hr. When the homogenate of fresh yeast cells was incubated in the acidic region below pH 4.5 for 1 hr, phospholipids were hydrolyzed and free fatty acids were accumulated. Such deacylation of phospholipids was observed even at pH 6 on incubation for 12hr, but not observed at pH 7 or above pH 9. At pH 8, although phospholipids were somewhat degraded, free fatty acids almost never accumulated but diacylglycerol did accumulate.

Therefore, yeast cells have inherently phospholipid-acylhydrolases and, on AcOH-treatment, such enzymes may degrade membrane phospholipids to induce the autolysis of pressed yeast.     

Regulation of Acid Phosphatase Synthesis in Baker’s Yeast Protoplast by Phosphate Compounds

The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure.     

Purification and Properties of a Thermo-labile Antigen from Baker’s Yeast Cells

A thermo-labile antigen (TLA) on the yeast cell surface was isolated from a yeast cell autolyzate and purified to a homogeneous state by chromatography on an immunoadsorbent affinity column. The molecular weight of TLA was about 1.45 x 10⁵ on SDS-polyacrylamide gel electrophoresis and about 1.5 x l0⁵ on gel chromatography on Sephadex G-200. The TLA contained 74.5% protein and 25.5% sugar. It was characterized by high contents of glycine, glutamic acid, serine and aspartic acid. Half-cystine, methionine, histidine and arginine were not found. The sugar moiety was composed of galactose, mannose, N-acetylglucosamine and fucose. The antigenic determinant of TLA was distinct from that of cell wall mannan in the Ouchterlony immunodiffusion test. No precipitin line against anti-TLA serum was observed, when TLA was heated at 90°C for 10 min. Oxidation with periodate had little effect on antigenicity, but digestion with Pronase or treatment with protein denaturants resulted in loss of the antigenicity. These results suggest that the protein moiety plays an important role as the antigenic determinant of TLA. Moreover, the antiserum specific to TLA agglutinated fresh yeast cells, and the distribution of TLA was apparent on the yeast cell surface by immunofluorescence staining. These findings suggest that TLA molecules were exposed on the outer surface of the yeast cell wall.     

Degradation of Cellular Phospholipids and Softening of Pressed Baker’s Yeast

Variations in lipid components of washings and homogenate of pressed baker’s yeast were investigated during the storage of pressed baker’s yeast at 30°C. Washings represents the substances which had leaked out from cells. Homogenate represents those contained in whole cells. Lipids in yeast washings increased toward softening, the phospholipids in yeast homogenate decreased continuously during storage. Two stages, an earlier period of storage (Stage I) and a later period of storage (Stage II) were observed in the degradation of phospholipids. Free fatty acid which was the main degradation product of phospholipid accumulated in Stage II, particularly at softening. The order in phospholipid degradation was PC>PE>PI + PS (PI>PS). Moreover, when washings of stored yeast at softening were assayed using ¹⁴C-acyl PC, the release of ¹⁴C-acyl fatty acid was observed.

These results suggest that phospholipids were degraded by some phospholipid-deacylating enzymes toward softening. From the results of lipid analysis, we inferred that the responsible enzymes were phospholipases.     

Effect of Specific Growth Rate on Fermentative Capacity of Baker’s Yeast

The specific growth rate is a key control parameter in the industrial production of baker’s yeast. Nevertheless, quantitative data describing its effect on fermentative capacity are not available from the literature. In this study, the effect of the specific growth rate on the physiology and fermentative capacity of an industrial Saccharomyces cerevisiae strain in aerobic, glucose-limited chemostat cultures was investigated. At specific growth rates (dilution rates, D) below 0.28 h⁻¹, glucose metabolism was fully respiratory. Above this dilution rate, respirofermentative metabolism set in, with ethanol production rates of up to 14 mmol of ethanol · g of biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. A substantial fermentative capacity (assayed offline as ethanol production rate under anaerobic conditions) was found in cultures in which no ethanol was detectable (D < 0.28 h⁻¹). This fermentative capacity increased with increasing dilution rates, from 10.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.025 h⁻¹ to 20.5 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.28 h⁻¹. At even higher dilution rates, the fermentative capacity showed only a small further increase, up to 22.0 mmol of ethanol · g of dry yeast biomass⁻¹ · h⁻¹ at D = 0.40 h⁻¹. The activities of all glycolytic enzymes, pyruvate decarboxylase, and alcohol dehydrogenase were determined in cell extracts. Only the in vitro activities of pyruvate decarboxylase and phosphofructokinase showed a clear positive correlation with fermentative capacity. These enzymes are interesting targets for overexpression in attempts to improve the fermentative capacity of aerobic cultures grown at low specific growth rates.The quality of commercial baker’s yeast (Saccharomyces cerevisiae) is determined by many parameters, including storage stability, osmotolerance, freeze-thaw resistance, rehydration resistance of dried yeast, and color. In view of the primary role of baker’s yeast in dough, fermentative capacity (i.e., the specific rate of carbon dioxide production by yeast upon its introduction into dough) is a particularly important parameter (2).In S. cerevisiae, high sugar concentrations and high specific growth rates trigger alcoholic fermentation, even under fully aerobic conditions (6, 18). Alcoholic fermentation during the industrial production of baker’s yeast is highly undesirable, as it reduces the biomass yield on the carbohydrate feedstock. Industrial baker’s yeast production is therefore performed in aerobic, sugar-limited fed-batch cultures. The conditions in such cultures differ drastically from those in the dough environment, which is anaerobic and with sugars at least initially present in excess (23).Optimization of biomass productivity requires that the specific growth rate and biomass yield in the fed-batch process be as high as possible. In the early stage of the process, the maximum feasible growth rate is dictated by the threshold specific growth rate at which respirofermentative metabolism sets in. In later stages, the specific growth rate is decreased to avoid problems with the limited oxygen transfer and/or cooling capacity of industrial bioreactors (10, 27). The actual growth rate profile during fed-batch cultivation is controlled primarily by the feed rate profile of the carbohydrate feedstock (4, 22). Generally, an initial exponential feed phase is followed by phases with constant and declining feed rates, respectively (8).From a theoretical point of view, the objective of suppressing alcoholic fermentation during the production phase may interfere with the aim of obtaining a high fermentative capacity in the final product. Process optimization has so far been based on strain selection and on empirical optimization of environmental conditions during fed-batch cultivation (e.g., pH, temperature, aeration rate, and feed profiles of sugar, nitrogen, and phosphorus [5, 10, 23]). For rational optimization of the specific growth rate profile, knowledge of the relation between specific growth rate and fermentative capacity is of primary importance. Nevertheless, quantitative data on this subject cannot be found in the literature.The chemostat cultivation system allows manipulation of the specific growth rate (which is equal to the dilution rate) while keeping other important growth conditions constant. Similar to industrial fed-batch cultivation, sugar-limited chemostat cultivation allows fully respiratory growth of S. cerevisiae on sugars (21, 37, 39). This is not possible in batch cultures, which by definition require high sugar concentrations, which lead to alcoholic fermentation, even during aerobic growth (6, 18, 37). Thus, as an experimental system, batch cultures bear little resemblance to the aerobic baker’s yeast production process. Indeed, we have recently shown that differences in fermentative capacity between a laboratory strain of S. cerevisiae and an industrial strain became apparent only in glucose-limited chemostat cultures but not in batch cultures (30).The aim of the present study was to assess the effect of specific growth rate on fermentative capacity in an industrial baker’s yeast strain grown in aerobic, sugar-limited chemostat cultures. Furthermore, the effect of specific growth rate on in vitro activities of key glycolytic and fermentative enzymes was investigated in an attempt to identify correlations between fermentative capacity and enzyme levels.     

Accumulation of Nicotinamide Adenine Dinucleotide in Baker’s Yeast by Secondary Culture

A study was made to determine a method for the production of NAD using baker’s yeast, and a suitable secondary culture condition for the accumulation of NAD was established. From the study the following results were obtained: Nicotinamide, nicotinic acid and adenine were effective on the accumulation of NAD. However, ribose or tryptophan — one of the precursor of NAD — was not effective. NaF, KCN or NaN₃ — metabolic inhibitors — inhibited the accumulation of NAD. Baker’s yeast obtained from commercial source was cultured secondarily in the medium containing 0.3% adenine, 0.6% nicotinamide in 0.2 M K₂HPO₄ (50% fresh yeast was added), pH 4.5. Under this optimal condition, NAD content reached about 12 mg/g dry cells (corresponding to 2.0 mg/ml medium), and it corresponded to about 20 times that of the initial content.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Separation and Characterization of Two Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidases from Baker’s Yeast

We have fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against β-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the β-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.     

Influence of Incubation Conditions on Hydrolysis Efficiency and Iodine Enrichment in Baker’s Yeast

The influence of incubation conditions, enzyme type, hydrolysis time, and potassium iodide concentration on hydrolysis and iodine enrichment were studied in supernatant and pellets of Saccharomyces cervisiae hydrolysates. The type of enzyme used and incubation time significantly influence hydrolysis efficiency and protein concentration in supernatant and pellet. The highest protein hydrolysis efficiency was obtained by 24-h incubation with papain. Significantly lower values were observed for pepsin and autolysis. The potassium iodide concentration influences the iodine content of supernatant and pellet, but not hydrolysis. Iodide enrichment of supernatant and pellet depends on the concentration of iodide using during incubation. High concentration of iodide and long incubation times were the conditions for optimal iodide enrichment and high-protein hydrolysates. The optimal hydrolysis efficiency and iodine enrichment were obtained during 24-h incubation with papain in a 4.5-mM potassium iodide medium. The efficiency reached 98.22% with iodine concentrations of 2,664.91 and 9,200.67 μg/g iodine in pellet and supernatant, respectively.     

The Presence of Apopyruvate Carboxylase and Synthesis of Holopyruvate Carboxylase in Biotin Requiring Baker’s Yeast

Pectin transeliminase from the culture medium of Aspergillus sojae was purified 940-fold by ammonium sulfate fractionation, column chromatography on CM-Cellulose, DEAE-Sephadex and SE-Sephadex and gel filtration. The purified enzyme was free from polygalacturonase, hemicellulase, cellulase and protease, and was almost homogeneous on disc electrophoresis. Using gel filtration a molecular weight of about 32,000 was estimated for the enzyme. The pH optimum of the enzyme for pectin (68% esterified) was 5.5, but was 7.0 for polymethyl polygalacturonate methyl glycoside (98% esterified). The addition of divalent cation to pectin stimulated the activity and shifted the pH optimum to neutral side. This tendency by divalent cation, however, was not observed when polymethyl polygalacturonate methyl glycoside was used as substrate. The enzyme is stable between pH 4 to 7. Heating the enzyme solution at 70°C for 10 min caused complete loss of activity. A marked inhibition of activity was produced by oxidizing reagents such as iodine and N-bromosuccinimide. In contrast, no effect emerged from the action of chelating agents, reducing reagents and those reagents which convert SH-groups in protein into mercaptides. The limits of degradation of pectin and polymethyl polygalacturonate methyl glycoside by the enzyme were 26.3 and 39.8%, respectively.     

Incorporation of 14C-Labelled Glycerol and γ-Aminobutyric Acid into Vitamin B6 in a Cell-suspension of Achromobacter cycloclastes

Incorporation of ¹⁴C from various ¹⁴C-labelled compounds into vitamin B₆ (abbreviate as B₆) in a cell-suspension of B₆-producing bacteria, Achromobacter cycloclastes A.M.S. 6021, was studied by using a newly designed purification procedure of the radioactive B₆. The procedure consisted of Sephadex G–25 gel filtration, Dowex 50W–X8 column chromatography, Amberlite CG–50 column adsorption, powdered cellulose partition column chromatography, crystallization and sublimatography. It was observed that the labels both from 1,3- and from 2-labelled glycerol were clearly incorporated into B₆ and the label of ¹⁴C-labelled γ-aminobutyric acid was also incorporated. The incorporation of ¹⁴C from ¹⁴C-labelled glycerol or γ-aminobutyric acid was affected by the addition of cold γ-aminobutyric acid or glycerol. The implication of these compounds as precursors of B₆ was discussed.     

Thiol-disulfide Transhydrogenase from Baker’s Yeast and a New Method for the Direct Assay of an Enzyme-catalyzed Thiol-disulfide Interchange Activity

This paper presents a study on the enzyme reduction of the disulfide bond and the following results have been found.

In enzyme preparation, antioxidants showed a stability effect and EDTA appeared to have both enzyme stabilization and solubilization. On the distribution of the enzyme activity in subcellular fractions, the water soluble fraction appeared to contain the major released enzyme activity. The enzyme was inhibited with several metals. Hg²⁺ and transition metals were the most toxic. The substrate specificity of this enzyme was wide for the low molecular substrates, but the protein disulfide reducing activity was not detected in this preparation. It was assumed that the thiol-disulfide transhydrogenase was coupled with glutathione reductase and the disulfide substrates were reduced by the system involving the two enzymes. A new method for the direct recording of an enzyme-catalyzed thiol-disulfide interchange using diphenyl disulfide and p,p-dinitro diphenyl disulfide was devised.     

Differences in the Mode of Growth Inhibition of Baker’s Yeast Brought about by Arsenate and Methanearsonate

The bite force of three surimi gels with molars was measured in the mouth using a multiple-point sheet sensor. A peak force appeared at the breaking point of each sample, and then the force increased again, accompanied by a decrease in the opening between the upeer and lower teeth. Low values in the peak force, pressure, and time at the first peak, the time at which the maximum contact area was engaged, impulse, and slope of bite curve were observed in samples with low breaking force and low breaking deformation found by the mechanical measurement of gel strength, and with less toughness in the sensory assessment. The duration of the bite force, the second peak time, and active bite pressure at the second peak did not change with a change in the surimi texture. The active pressure at the breaking point of each gel was affected by gel strength, while that at the second peak was independent of the gel strength.     

Purification and Properties of an Asymmetric Reduction Enzyme of 2-Methyl-3-oxobutyrate in Baker’s Yeast

The benzyl 2-methyl-3-hydroxybutyrate dehydrogenase was purified from the cells of baker’s yeast by streptomycin treatment, Sephadex G-50 gel filtration, SP-Sephadex C-50 chromatography, and Toyopearl HW-60F gel filtration. The purified enzyme preparation was homogeneous and the molecular weight was about 31,000 to 32,000. The enzyme was NADPH-dependent and its maximum activity was at pH 7.0 and 45°C. It was stable between pH 6 and 9. The Km values at pH 7.0 were 0.42 mM for benzyl 2-methyl-3-oxobutyrate (1) and 4.2 mM for α-methyl β-hydroxy ester [syn-(2) and anti-(3)]. This enzyme reduced only benzyl 2-methyl-3-oxobutyrate (1) but had no effect on other synthetic substrates.

The reduced products [syn-(2) and anti(3)] produced by the purified enzyme were identified by 400 MHz NMR.     

Preparation of Diphosphopyridine Nucleotide from Bakers’ Yeast

A method for the preparatoin of diphosphopyridine nucleotide (DPN) from bakers’ yeast is described. This method consists of partial purification of crude extract of yeast by charcoal chromatography according to Pontis and coworkers, ion-exchange chromatography on Dowex-l acetate, and precipitation of DPN as the free acid with ethanol. 0.71~.1.1 g of DPN with a purity of 85~90% was obtained from 5 kg of fresh bakers’ yeast by this method.     

A Comparison of the Beneficial Effects of Live and Heat-Inactivated Baker’s Yeast on Nile Tilapia: Suggestions on the Role and Function of the Secretory Metabolites Released from the Yeast

Yeast is frequently used as a probiotic in aquaculture with the potential to substitute for antibiotics. In this study, the involvement and extent to which the viability of yeast cells and thus the secretory metabolites released from the yeast contribute to effects of baker’s yeast was investigated in Nile tilapia. No yeast, live yeast or heat-inactivated baker’s yeast were added to basal diets high in fishmeal and low in soybean (diet A) or low in fishmeal and high in soybean (diet B), which were fed to fish for 8 weeks. Growth, feed utilization, gut microvilli morphology, and expressions of hsp70 and inflammation-related cytokines in the intestine and head kidney were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Gut alkaline phosphatase (AKP) activity was measured after challenging the fish with Aeromonas hydrophila. Results showed that live yeast significantly improved FBW and WG (P < 0.05), and tended to improve FCR (P = 0.06) of fish compared to the control (no yeast). No significant differences were observed between inactivated yeast and control. Live yeast improved gut microvilli length (P < 0.001) and density (P < 0.05) while inactivated yeast did not. The hsp70 expression level in both the intestine and head kidney of fish was significantly reduced by live yeast (P < 0.05) but not inactivated yeast. Live yeast but not inactivated yeast reduced intestinal expression of tnfα (P < 0.05), tgfβ (P < 0.05 under diet A) and il1β (P = 0.08). Intestinal Lactococcus spp. numbers were enriched by both live and inactivated yeast. Lastly, both live and inactivated yeast reduced the gut AKP activity compared to the control (P < 0.001), indicating protection of the host against infection by A. hydrophila. In conclusion, secretory metabolites did not play major roles in the growth promotion and disease protection effects of yeast. Nevertheless, secretory metabolites were the major contributing factor towards improved gut microvilli morphology, relieved stress status, and reduced intestinal inflammation of Nile tilapia fed diets supplemented with baker’s yeast.