Affinity partitioning with polymer-bound Cibacron blue F3G-A for rapid,large-scale purification of phosphofructokinase from baker's yeast

Phosphofructokinase has been isolated in homogenous form from baker's yeast. The first two steps, fractional precipitation with polyethylene glycol and affinity partitioning in aqueous biphasic systems containing Cibacron blue F3G-A-polyethylene glycol, gave a 58-fold purification within 3 h. In these steps the amount of contaminating proteases was reduced by 2 orders of magnitude. After concentration using DEAE-cellulose followed by gel chromatography, homogeneous enzyme was obtained. The advantages of affinity partitioning for large-scale preparations are discussed.     

Occurrence of proteinase a isoinhibitors in wild type yeast strains and commercial baker's yeast

The occurrence of the proteinase A inhibitors 2 and 3 was investigated in wild type strains of $\text{Saccharomyces}\text{cerevisiae}$ and $\text{Saccharomyces}\text{carlsbergensis}$ as well as in several strains of commercial baker's yeast. Haploid and diploid strains of $\text{Saccharomyces}\text{cerevisiae}$ contain only proteinase A inhibitor 3 whereas in $\text{Saccharomyces}\text{carlsbergensis}$ only proteinase A inhibitor 2 is found. Strains of commercial baker's yeast contain either proteinase A inhibitor 3 or both inhibitors in a constant ratio of 1:3. Single cell cultures isolated from a strain of commercial baker's yeast also contain a mixture of the two inhibitors. Therefore, baker's yeast is not a mixture of two different cell types but the genome for both inhibitors is present in each single cell. In general, the results indicate that the occurrence of the two proteinase A inhibitors is determined genetically and, therefore, they may be called “isoinhibitors”.     

Experiences with a 20 litre industrial bead mill for the disruption of microorganisms

Suspensions of several yeast strains and bacterial species were disrupted in a continuously operating industrial agitator mill of 22.7 litre internal working volume. The influence of agitator speed, flow rate, concentration of microorganisms in the slurry, packing density of glass beads and bead diameter on the disruption process was studied using baker's yeast (Saccharomyces cerevisiae). Cell disintegration was followed by assaying the appearance of protein and the activities of d-glucose-6-phosphate dehydrogenase [d-glucose-6-phosphate:NADP⁺ oxidoreductase, EC 1.1.1.49] and α-d-glucosidase [α-d-glucoside glucohydrolase, EC 3.2.1.20] in the soluble fraction. The best operating conditions for the disintegration of baker's yeast with respect to activity yield appeared to be at a rotational speed of 1100 rev/min, a flow rate of 100 litre h^?1 and a cell concentration of 40% (w/v). The location of the desired enzyme in the cell is of importance for the choice of bead diameter and packing density of the glass beads. Temperature increase and power consumption during disintegration are also strongly influenced by the bead loading in the mill. With optimized parameters, 200 kg baker's yeast can be processed per hour with a degree of disintegration >85%. The disruption process in the mill was found to be very effective for several yeast species tested, e.g. Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Candida boidinii. The usefulness of the Netzsch LME 20-mill for the disruption of bacteria species was demonstrated with Escherichia coli, Brevibacterium ammoniagenes, Bacillus sphaericus and Lactobacillus confusus. As expected, the mill capacity for bacterial disruption was significantly smaller than for the yeast. Between 10 and 20 kg per h bacteria may be processed, depending on the organism.     

Synthesis of 4-carboxy-2-polyprenylphenols by a particulate fraction of baker's yeast

The preparation ofa cell-free homogenate and 10000 g particulate fraction with polyprenylpyrophosphate-p-hydroxybenzoate polyprenyltransferase activity from 0 to 7-day-old blocks of compressed baker's yeast is described. The synthesis of 4-carboxy-2-triprenylphenol from p-hydroxybenzoate and FPP by the particulate fraction has been studied in some detail. In particular it has been shown that the transferase catalysing the reaction is activated by Mg²⁺, has a pH optima of 7 and is inhibited by phosphate buffer. Intracellular distribution studies have established that in freshly grown cells of Saccharomyces carlsbergensis the greater part of the polyprenyl transferase activity is present in the mitochondria.     

Biological significance of methylation of cytochromes from ascomycetes and plants

The K_m and V_max values characterizing the reaction of baker's yeast iso-I-cytochrome c, whether tri-methylated or not at lysine residue 72, with crude preparations of cytochrome c peroxidase, cytochrome c oxidase and succinate cytochrome c oxidoreductase from Saccharomyces cerevisiae are similar. These results, as well as the redox potential values, the auto-oxidability parameters and the circular dichroism spectra, strongly suggest that the biological methylation of yeast cytochrome c does not alter its functional properties. The functional characteristics of baker's yeast iso-I-cytochrome c are similar to those of horse heart cytochrome c and yeast iso-2-cytochrome c.     

Determination and characterization of long-chain fatty acyl-CoA thioesters from yeast and mammalian liver

The long-chain fatty acyl-CoA content of various biological materials, i.e., baker's yeast and mammalian liver, has been determined under standard and several other metabolic conditions, using optimized methods for cell disruption, separating acid-soluble and acid-insoluble CoA from each other, and assaying. After studying the optimization of the extraction of long-chain acyl-CoA compounds and the purification of the extracts, acyl-CoA fractions from several biological sources have been isolated and characterized on behalf of their fatty acid residues by gas-liquid chromatography of the methyl ester derivatives.     

Nature of the uptake of d-galactose, d-glucose and α-methyl-d-glucoside by Saccharomyces cerevisiae

Both glucose-grown baker's yeast after induction and galactose-grown yeast appear to take up d-galactose by a system not requiring phosphorylation and only up to a diffusion equilibrium, as shown by pulse labelling, sampling at very short intervals and chromatographic analysis of extracts. Part of the sugar taken up is transformed into trehalose which is present in substantially greater amounts in cells than the transported sugar itself. The effect of 2,4-dinitrophenol and of iodoacetamide, as well as the nature of the efflux of sugars from preloaded cells, support the results. d-Glucose and α-methylglucoside are also taken up without phosphorylation.     

Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Enzymes of pentose biosynthesis. The quaternary structure and reacting form of transketolase from baker's yeast

Transketolase from baker's yeast is a dimeric enzyme with a molecular weight of 158,000 ± 4000. Sedimentation velocity and sedimentation equilibrium experiments indicate that the enzyme dissociates at low concentrations (less than 0.1 mg/ml) in the absence of the coenzyme, thiamine pyrophosphate. However, no such dissociation was detected in the presence of coenzyme. Reacting enzyme sedimentation velocity measurements showed that the reacting species of the enzyme is a dimer with an s_20,w of 7.7 S.     

Glucose/molasses effects on enzyme activities and fermentative activity of fully aerobic continuous cultures of baker’s yeast

Supplementing a molasses medium with glucose was expected to have deleterious effects on the quality of industrially grown baker’s yeast. This was investigated in the laboratory using beet molasses and glucose in fully aerobic continuous cultures of baker’s yeast.     

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.     

Purification of some glycoside hydrolases by affinity chromatography

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1→3)-linked β-d-glucosyl residues, some of which are attached by (1→6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1→3)-β-d-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an α-d-mannosidase from jack-bean meal.     

The intrinsic zinc atoms of yeast alcohol dehydrogenase

The intrinsic Zn content of yeast alcohol dehydrogenase (YADH) has been determined by three highly sensitive analytical techniques. The enzyme prepared from baker's yeast has a specific activity of 430–460 U/mg and contains 4 intrinsically bound Zn atoms per tetrameric enzyme of molecular weight 150,000. The enzyme is homogeneous by disc gel electrophoresis and analytical ultracentrifugation and remains stable and fully active on prolonged storage. YADH samples from commercial sources, while of high activity, can initially contain more than 4 g-atom of Zn/mole, but dialysis against EDTA removes these adventitious Zn atoms which do not bear a consistent relationship to enzymatic activity, in accord with earlier investigations. Apparently, they are bound to the enzyme in a manner different from that of the catalytically essential Zn atoms and likely represent contamination. The 4 intrinsic Zn atoms exchange fully with ⁶⁵Zn(II) resulting in [(YADH)⁶⁵Zn₄] which exhibits the same specific activity and stability as the native enzyme.     

The binding of thiamine pyrophosphate with transketolase in equilibrium conditions

The binding between thiamine pyrophosphate and transketolase, purified from baker's yeast, in equilibrium conditions has been studied. In the presence of Ca²⁺, the enzyme molecule has been shown to possess two binding sites for the coenzyme, whose dissociation constants are 3.2 × 10^?8 and 2.5 × 10^?7M; besides, there are site(s) where the binding of the coenzyme is less firm. In the presence of Mg²⁺, a positive cooperative interaction between the binding sites of thiamine pyrophosphate has been observed. Regardless of the cation used, the major part of the catalytic activity of the transketolase molecule manifests itself in the binding of one molecule of the coenzyme.     

Baker’s yeast mediated asymmetric reduction of cinnamaldehyde derivatives

The enantioselective reduction of cinnamaldehyde derivatives is an attractive strategy to prepare various optically active multifunctional molecules that can be used as chiral building blocks for the synthesis of some HIV-protease inhibitors. The asymmetric reduction with pH adjusted to 5.5 of α-substituted-cinnamaldehydes (Br, N₃) mediated by baker’s yeast (Saccharomyces cerevisiae) yielded α-substituted-3-phenyl-1-propanol in excellent enantiomeric excesses and yields.     

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.     

Interaction of myasthenic serum globulin with the acetylcholine receptor.

A serum factor from patients with myasthenia gravis which inhibited the binding of 125I-labeled alpha-bungarotoxin to acetylcholine receptor extracted with Triton X-100 from rat muscle has been studied in detail. The inhibitory activity was localized to the IgG fraction based upon the fractionations by sodium sulfate precipitation and DEAE chromatography as well as reaction with anti-IgG globulin. The myasthenic globulin inhibited toxin binding to receptors extracted from degenerated muscle but did not inhibit toxin binding to normal junctional receptors. At saturation levels of myasthenic globulin, the number of denervated acetylcholine receptors available for toxin binding was reduced approx. 50 percent. The myastehnic globulin was found to bind to denervated acetylcholine receptors but not to normal acetylcholine receptors by a radioimmunoassay technique in which myasthenic globulin incubated with 125I-labeled alpha bungarotoxin-receptor complexes was precipitated by anti-IgG serum. The globulin binding was saturable over the same range as inhibition of toxin binding. The data suggest that the myasthenic IgC binds to a site on the receptor complex juxtaposed to the acetylcholine receptor site. The myasthenic globulin appears to be a useful probe for investigation differences between acetylcholine receptors extracted from normal and denervated muscle and for investigating the pathogenesis of myasthenia gravis.     

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.     

Cellulase immobilized magnetic nanoparticles for green energy production from Allamanda schottii L: Sustainability research in waste recycling

This study presents ethanol''s fabrication by fermenting the golden trumpet flower (Allamanda schottii L) with the yeast strain Saccharomyces cerevisiae. The changes in different parameters during fermentation were studied and optimized while producing the ethanol and the end product was subjected to emission test study by blending petrol and ethanol. The Allamanda floral substrate contains 65% polysaccharides. The strain S. cerevisiae was obtained in the form of baker’s yeast from a domestic shop. For 100 ml of slurry, the highest bioethanol yield recorded was about 18.75 ml via optimization of different culture conditions, including a 1:8 ratio for slurry preparation, maintained under 35 ⁰C, 5.5 pH, 72 h. old inoculum with a quantity of 3.75 g 100 ml⁻¹, fermented for120 h. The highest yield of bioethanol was acquired under the addition of urea. This technique & design is capable of industrial-scale fabrication of bioethanol by using A. schottii floral substrates. This research was conducted to fabricate ethanol by fermentation (A. schottii L) floral substrate with S. cerevisiae. The optimum physiochemical parameters required to obtain the highest yield of bioethanol from A. schottii flower by fermentation was studied. The immobilization strategy with a cheap agricultural substrate and magnetic nanoparticles were also studied. The engine performance and emission studies were done with different blends of petrol and bio-ethanol.