Lysed Yeast Cells Research Articles

Characterization of an antifungal β-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial β-1,3-glucanases for fungal cell wall β-glucan degradation.

Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli. The sequence shows that GlxGluA belongs to glycoside hydrolase family 17 (GH17). To investigate how GlxGluA acts to degrade fungal cell wall β-glucan, it was compared with β-1,3-glucanase with different substrate specificities. We obtained recombinant β-1,3-glucanase (designated as CcGluA), which belongs to GH64, from the bacterium Cellulosimicrobium cellulans. GlxGluA inhibited the growth of the filamentous fungus Trichoderma viride but was unable to lyse the yeast Saccharomyces cerevisiae. In contrast, CcGluA lysed yeast cells but had a negligible inhibitory effect on the growth of filamentous fungi. GlxGluA degraded the cell wall of T. viride better than CcGluA, whereas CcGluA degraded the cell wall of S. cerevisiae more efficiently than GlxGluA. These results suggest that the target substrates in fungal cell walls differ between GlxGluA (GH17 class I β-1,3-glucanase) and CcGluA (GH64 β-1,3-glucanase).

Enzymatic hydrolysis of spent Saccharomyces cerevisiae derived from sago bioethanol fermentation

Spent Saccharomyces cerevisiae is a by-product of bioethanol fermentation. The spent yeast is abundant in valuable components which can be used for many applications. One of the ways to prepare yeast extract is through enzymatic hydrolysis which is by rupturing the yeast cell walls using exogenous enzymes under certain conditions that promote the leakage of intracellular compounds. To date, enzymatic hydrolysis of spent S. cerevisiae derived from the production of sago bioethanol is yet to be thoroughly explored. In the present study, we examine the feasibility of enzymatic hydrolysis of spent S. cerevisiae generated from sago bioethanol fermentation. The effect of two enzymes namely alcalase and cellulase and their concentrations (0.1-0.5% (v/v) on the release of protein and carbohydrate in the hydrolysate was also investigated. Additionally, the surface morphology of the hydrolysed yeast cells was observed using a Scanning electron microscope (SEM). Our results showed that the optimal concentration of alcalase and cellulase for enzymatic hydrolysis of spent S. cerevisiae was 0.4% (v/v) and 0.5% (v/v) respectively. In addition, cellulase was found to be more superior than alcalase with respect to the protein content in the hydrolysate. The enzymatic hydrolysis of spent yeast by alcalase and cellulase yielded improvements of 1.1 to 1.8-fold and 3.5 to 5.6-fold of protein and total carbohydrate concentration respectively in comparison to that achieved via autolysis. It was evident from the SEM analysis that there was a notable change in the surface morphology of the lysed yeast cells indicating the lysis of the yeast cells throughout the enzymatic hydrolysis. In summary, the current work provides useful insights into the strategies of valorising spent S. cerevisiae generated from sago bioethanol production. This will further help the development of value-added products from the waste, hence promoting a sustainable economy besides reducing the environmental impacts associated with the disposal of spent S. cerevisiae.

Mining the nanotube-forming Bacillus amyloliquefaciens MR14M3 genome for determining anti-Candida auris and anti-Candida albicans potential by pathogenicity and comparative genomics analysis

There is a global health concern associated with the emergence of the multidrug-resistant (MDR) fungus Candida auris, which has significant mortality rates. Finding innovative and distinctive anti-Candida compounds is essential for treating infections caused by MDR C. auris. A bacterial strain with anti-Candida activity was isolated and identified using 16 S rRNA gene sequencing. The whole genome was sequenced to identify biosynthesis-related gene clusters. The pathogenicity and cytotoxicity of the isolate were analyzed in Candida and HFF-1 cell lines, respectively. This study set out to show that whole-genome sequencing, cytotoxicity testing, and pathogenicity analysis combined with genome mining and comparative genomics can successfully identify biosynthesis-related gene clusters in native bacterial isolates that encode antifungal natural compounds active against Candida albicans and C. auris. The native isolate MR14M3 has the ability to inhibit C. auris (zone of inhibition 25 mm) and C. albicans (zone of inhibition 25 mm). The 16 S rRNA gene sequence of MR14M3 aligned with Bacillus amyloliquefaciens with similarity (100%). Bacillus amyloliquefaciens MR14M3 establishes bridges of intercellular nanotubes (L 258.56 ± 35.83 nm; W 25.32 ± 6.09 nm) connecting neighboring cells. Candida cell size was reduced significantly, and crushed phenotypes were observed upon treatment with the defused metabolites of B. amyloliquefaciens MR14M3. Furthermore, the pathogenicity of B. amyloliquefaciens MR14M3 on Candida cells was observed through cell membrane disruption and lysed yeast cells. The whole-genome alignment of the MR14M3 genome (3981,643 bp) using 100 genes confirmed its affiliation with Bacillus amyloliquefaciens. Genome mining analysis revealed that MR14M3-coded secondary metabolites are involved in the biosynthesis of polyketides (PKs) and nonribosomal peptide synthases (NRPSs), including 11 biosynthesis-related gene clusters with one hundred percent similarity. Highly conserved biosynthesis-related gene clusters with anti-C. albicans and anti-C. auris potentials and cytotoxic-free activity of B. amyloliquefaciens MR14M3 proposes the utilization of Bacillus amyloliquefaciens MR14M3 as a biofactory for an anti-Candida auris and anti-C. albicans compound synthesizer.

Autolysis of spent baker’s yeast generated from sago bioethanol fermentation: a preliminary study

Spent Baker’s yeast (Saccharomyces cerevisiae) is obtained as a by-product of ethanol fermentation industry and is mainly discarded as waste. The spent yeast can be potentially converted to yeast extract through autolysis process in which the yeast cells are lysed under certain conditions to release valuable and bioactive substances. To date, there are still limited studies on the autolysis of spent S. cerevisiae generated following the production of sago bioethanol. This research aims to investigate the feasibility of autolysis of spent Baker’s yeast generated from sago bioethanol fermentation by focusing on two important parameters namely initial pH and incubation time. The spent Baker’s yeast was autolysed at different initial pH values (3, 5 and 7) and at different incubation times (24, 48, 72 and 96 h). The protein and carbohydrate concentrations of the resulting lysates were analysed using Lowry assay and phenol-sulphuric acid assay respectively. Additionally, the surface morphology of the autolysed yeast cells was observed using Scanning electron microscope. Our results showed that the autolysis conducted at the initial pH of 3 gave the highest concentration of protein and total carbohydrate released from the lysate which were 2.5-fold and 2-fold over that in the control samples respectively. Meanwhile, the optimal incubation time for the autolysis of spent Baker’s yeast for both maximum concentrations of protein and carbohydrate released was found to be 72 h whilst prolonging beyond that did not result in any significant change of both the protein and total carbohydrate concentration. The morphological analysis of the lysed yeast cells clearly showed a significance change of the surface morphology indicative of cell lysis throughout the incubation period. In conclusion, the present work gives significant insights into the feasibility of autolysis of spent Baker’s yeast generated from sago bioethanol fermentation. This will in turn serves as useful fundamental information for developing potential side process streams within an integrated sago biorefinery in the future.

Development of nanocomposite biodegradable film containing iron nanoparticles biosynthesised by Saccharomyces cerevisiae

SummaryThe use of nanoparticles in biodegradable films has been shown to improve their characteristics in food packaging. The study was designed to investigate the effect of iron oxide nanoparticles (IONPs) and lysed yeast cells residue (YSR) on the properties of whey protein concentrate (WPC) films. IONPs (0, 0.5, 1.0 and 1.5 mm) biosynthesised by Saccharomyces cerevisiae and YSR were used to prepare WPC films. The addition of IONPs and YSR increased the tensile strength (TS) of the films by approximately 2.6 MPa compared with the control films (P < 0.05). Percent elongation and water vapour permeability of the films decreased significantly (P < 0.05) by the contribution of IONPs. All the films did not inhibit the growth of tested microorganisms. The incorporation of YSR in the film also did not make a significant change in any properties of the film (P > 0.05). The results indicated the usefulness of IONPs incorporation into WPC films to enhance mechanical but not antimicrobial properties.

Lysing Yeast Cells with Glass Beads for Immunoprecipitation.

Yeast cells display cell walls that must first be broken before the addition of detergents for lysis. This method describes the use of glass beads in combination with a mechanical bead beater to disrupt cell walls of both Saccharomyces cerevisiae or Schizosaccharomyces pombe directly in a nonionic detergent Lysis buffer containing 0.1% Nonidet P-40. Alternatively, this protocol can be applied for the lysis of yeast cells in Lysis buffer without detergent; upon completion of the bead beating, Triton X-100 is added to complete lysis. Yeast cells are cultured and collected while in log phase before being washed once and mixed together with glass beads in a tube. The applied shaking process facilitates disruption of the cell walls, upon which separation of yeast and glass beads is accomplished by forcing lysed cells through a hole created in the bottom of the tube during the centrifugation process. An alternative bead-beating protocol details the use of Lysis Buffer 2, which does not contain detergents and calls for the addition of Triton X-100 after cell lysis in the presence of glass beads. Use of Lysis Buffer 2 without detergent may avoid bubble and foam formation during the bead-beating process that could potentially denature proteins.

Lysing Yeast Cells for Immunoprecipitation Using a Coffee Grinder

This protocol describes the freezing of yeast in liquid nitrogen (LN2) to form small "beans" that can be ground using a simple propeller-blade coffee grinder. The method is ideally suited for lysate preparations from larger yeast cultures ranging from 50 mL to 5 L and displays the advantage that samples remain cold during the preparative steps. Cells are cultured and collected by centrifugation while in log phase, and the resultant cell pellets are mixed with deionized distilled water and dropped into LN2 to form small frozen beans. Before the freezing process, it is imperative to keep all cell pellets at 4°C on ice. The frozen yeast beans are ground by using a simple kitchen coffee grinder, and the yeast powder is collected for immediate lysis or storage at -80°C for subsequent use. Protective clothing and safety glasses should be worn at all times when working with liquid nitrogen. Plasticware may shatter upon repeated cooling in liquid nitrogen, and appropriate care should be taken.

Effect of glutathione on wheat dough properties and bread quality

Reduced glutathione (GSH), released from lysed yeast cells, is well-known for weakening dough structure. However, its influence on bread texture and staling has not yet been completely elucidated. Herein, this study aims at assessing the effects of GSH on dough properties and bread quality, especially bread staling, using Rheofermentometer analysis, texture profile analysis (TPA), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and low-field nuclear magnetic resonance (LF-NMR). The results revealed that GSH substantially decreased the dough height and gas-retention capacity during fermentation. The weakened dough structure was attributed to GSH-induced disulfide bond cleavage in gluten proteins and the depolymerization of glutenin polymers. The addition of 0.005%–0.01% GSH resulted in acceptable bread quality. However, bread supplemented with 0.015%–0.03% of GSH exhibited a coarser and more open crumb microstructure, as well as high level of hardness and low resilience during aging. GSH promoted crumb water loss and drove the water shift from the immobilized to the bound state. The DSC and XRD analyses further confirmed that GSH promoted starch retrogradation and recrystallization. These results suggest that apart from the gluten structure, GSH also altered gluten−starch interactions and water redistribution, ultimately decreasing bread quality and accelerating bread staling.

Destabilization of wheat dough: Interrelation between CO 2 and glutathione

Abstract Minor variations in the metabolic profile of yeast strongly affect wheat dough structure, the resulting bread volume and its crumb texture. However, it is complex to analyze the impact of single yeast metabolites on the development of dough during processing. In this study, a chemical-leavening system based on glucono-delta-lactone and NaHCO3 was applied to analyze aeration and gas retention in presence of glutathione independent from the yeast metabolism. The addition of glutathione-amplified gas release during kneading and reduced the time to reach the maximum gas formation rate. The results indicated that the inflation of dough is not limited by the amount of CO2 nor its release kinetics; however, during baking further aeration is restricted. The oven rise was impaired by high initial CO2 volumes, which is enhanced by the presence of glutathione. Industrial relevance This investigation addressed the effect of yeast equivalent glutathione on the properties of aerated dough and the resulting bread properties. Through the application of a chemical-leavening system the yeast metabolism can be avoided. GSH from lysed yeast cells had a weakening effect, which confirms observations of structure-loss after freeze–thaw processes of dough. Moreover, the use of chemical-leavening agents reduces bread production costs, since the fermentation process can be neglected.

A study on significant microbial interaction leading to decolorization and degradation of textile dye Rubine 3GP

The present study evaluates an obligatory interaction between the yeast Saccharomyces cerevisiae NCIM 3312 and the bacterium Pseudomonas sp. strain BCH3 for the biodegradation of the dye Rubin 3GP (R3GP). No significant degradation of R3GP was observed either by Saccharomyces cerevisiae NCIM 3312 or by Pseudomonas sp. strain BCH3, when both the cultures were tested individually under their respective optimum medium conditions. However, when both of them were allowed to intermingle with each other, R3GP was found to be degraded within 72 h, with a steady increase in β -1,3-glucanase, chitinase and protease activity in the culture supernatant; indicating the possible role of Pseudomonas sp. strain BCH3 in cell wall lysis of S. cerevisiae NCIM 3312. The present study elucidates a rare microbial interaction where the bacterium Pseudomonas sp. strain BCH3 utilizes lysed yeast cells as the sole source of nutrients for its own growth and subsequently performs decolorization and degradation of R3GP. Enzymatic status showed involvement of various oxidoreductive enzymes like lignin peroxidase, laccase, DCIP reductase and azo reductase, indicating their role in decolorization and degradation of R3GP. Degradation was confirmed using HPLC, FTIR analysis and the biochemical pathway of degradation was elucidated by using GC-MS analysis.

Structural analysis of compounds with actions similar to local anesthetics and antipsychotic phenothiazines in yeast

Local anesthetics and antipsychotic phenothiazines cause a rapid shutdown of both actin polarization and translation initiation in yeast cells, like some environmental stresses. These compounds all have an amphiphilic structure, surfactant activity and the ability to lyse yeast cells. To elucidate the structures responsible for the shutdown activity and cell lysis, we investigated a variety of amphiphiles. In the hydrophobic region, the straight alkyl structure was sufficient for the shutdown of actin polarization and translational initiation. In the hydrophilic region of the straight alkyl compounds, cationic trimethyl ammonium (TMA) and non-ionic hydroxyl structure (alcohols) shut down both reactions, while an anionic structure, sulphate, with a long alkyl chain (≥C6) shut down actin polarization only. On the compounds that shut down both reactions, including the clinical drugs, TMA compounds and alcohols, the potencies of shutdown and lysis exponentially increased with increasing the number of carbons in the hydrophobic region, whereas safety was affected by the structures of both hydrophilic and hydrophobic regions. These results indicate that the yeast system can easily evaluate clinical drugs, and provide a structural basis for designing compounds to shut down intracellular reactions.

Preparation of Extracts from Yeast

Because yeast is exceptionally well suited to genetic analysis, both classical and molecular, it is an attractive system for expressing recombinant animal proteins for purification purposes. Methods available for lysing yeast cells include autolysis, pressure cells (e.g., French press), abrasives (glass bead vortexing), and enzymatic lysis (e.g., zymolase). One of the simplest methods, discussed in this protocol, involves the abrasive action of well-agitated glass beads. This is a very effective method for both low volumes (e.g., <1 mL using a microcentrifuge tube) and many liters using a specialized DynoMill apparatus. Cell breakage is typically >95%, as assessed by phase-contrast microscopy.

Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR

The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood.

Effects of pH and Temperature on Recombinant Manganese Peroxidase Production and Stability

The enzyme manganese peroxidase (MnP) is produced by numerous white-rot fungi to overcome biomass recalcitrance caused by lignin. MnP acts directly on lignin and increases access of the woody structure to synergistic wood-degrading enzymes such as cellulases and xylanases. Recombinant MnP (rMnP) can be produced in the yeast Pichia pastoris alphaMnP1-1 in fed-batch fermentations. The effects of pH and temperature on recombinant manganese peroxidase (rMnP) production by P. pastoris alphaMnP1-1 were investigated in shake flask and fed-batch fermentations. The optimum pH and temperature for a standardized fed-batch fermentation process for rMnP production in P. pastoris alphaMnP1-1 were determined to be pH 6 and 30 degrees C, respectively. P. pastoris alphaMnP1-1 constitutively expresses the manganese peroxidase (mnp1) complementary DNA from Phanerochaete chrysosporium, and the rMnP has similar kinetic characteristics and pH activity and stability ranges as the wild-type MnP (wtMnP). Cultivation of P. chrysosporium mycelia in stationary flasks for production of heme peroxidases is commonly conducted at low pH (pH 4.2). However, shake flask and fed-batch fermentation experiments with P. pastoris alphaMnP1-1 demonstrated that rMnP production is highest at pH 6, with rMnP concentrations in the medium declining rapidly at pH less than 5.5, although cell growth rates were similar from pH 4-7. Investigations of the cause of low rMnP production at low pH were consistent with the hypothesis that intracellular proteases are released from dead and lysed yeast cells during the fermentation that are active against rMnP at pH less than 5.5.

Immunoprecipitation: Lysing Yeast Cells Using Glass Beads

INTRODUCTIONFor yeast, the preferred method of lysis is mechanical shearing by vortexing the cells in the presence of glass beads as described in this protocol. This is a powerful lysis method but imparts extensive energy into the lysis procedure and results in considerable denaturation of the samples. For this reason, yeast immunoprecipitations are not as powerful a technique as similar studies in mammalian cells and should not be relied upon for determination of protein properties that require authentic protein structure. The lysate prepared in this procedure is ready for preclearing.

Study of the Lytic Activities of Actinomycetes Isolated from Different Soils in Georgia

The lytic activities of 310 cultures from the Collection of Actinomycetes of the Institute of Biochemistry and Biotechnology, National Academy of Sciences of Georgia, were studied; 18% of these strains appeared capable of lysing yeast cell wall. The active producer of the enzyme was selected. This culture was isolated from chestnut soil in Gardabani raion (Central Georgia). Its cultural–morphological, biochemical, and antagonistic properties allowed the culture to be ascribed to the species Geodermatophilus obseurusLuedemann, 1968. The maximal lytic activity under submerged cultivation conditions, exceeding the activity of Actinomyces griseinusby twofold, was observed during the logarithmic growth phase.

Genetically controlled cell lysis in the yeastSaccharomyces cerevisiae

The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast cells in order to release their intracellular contents. To develop such a method, a tightly regulated promoter, pMET3, has been used to control three genes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of these regulation cassettes, pMET3-SRB1/PSA1 and pMET3-PKC1, have been integrated at the chromosomal loci of the respective genes in order to overcome problems of plasmid instability. Although repression of PDE2 did not cause cell lysis, cells depleted of Srb1p/Psa1p gradually lost their viability and integrity, releasing about 10% of total protein into the medium. Repression of PKC1 led to extensive cell lysis, accompanied by the release of 45% of cellular protein into the medium. A double mutant, carrying both pMET3-SRB1/PSA1 and pMET3-PKC1 cassettes in place of SRB1/PSA1 and PKC1, was constructed and found to permit the efficient release of both homologous and heterologous proteins. &copy 1999 John Wiley & Sons, Inc.,

The Tail of a Yeast Class V Myosin, Myo2p, Functions as a Localization Domain

Myo2p is a yeast class V myosin that functions in membrane trafficking. To investigate the function of the carboxyl-terminal-tail domain of Myo2p, we have overexpressed this domain behind the regulatable GAL1 promoter (MYO2DN). Overexpression of the tail domain of Myo2p results in a dominant-negative phenotype that is phenotypically similar to a temperature-sensitive allele of myo2, myo2-66. The tail domain of Myo2p is sufficient for localization at low- expression levels and causes mislocalization of the endogenous Myo2p from sites of polarized cell growth. Subcellular fractionation of polarized, mechanically lysed yeast cells reveals that Myo2p is present predominantly in a 100,000 x g pellet. The Myo2p in this pellet is not solubilized by Mg++-ATP or Triton X-100, but is solubilized by high salt. Tail overexpression does not disrupt this fractionation pattern, nor do mutations in sec4, sec3, sec9, cdc42, or myo2. These results show that overexpression of the tail domain of Myo2p does not compete with the endogenous Myo2p for assembly into a pelletable structure, but does compete with the endogenous Myo2p for a factor that is necessary for localization to the bud tip.

Localization of a protein a‐tagged kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes

We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.

Characterization of Rarobacter faecitabidus Protease I, a Yeast-Lytic Serine Protease Having Mannose-Binding Activity

Rarobacter faecitabidus protease I, a yeast-lytic serine protease, was characterized in order to elucidate the mechanism of lysis of yeast cells by this enzyme. The N-terminal amino acid sequence of the enzyme was found to be homologous to those of Lysobacter enzymogenes alpha-lytic protease and Streptomyces griseus proteases A and B around the catalytic His residue, showing that it is a mammalian type serine protease. In a study of its substrate specificity, it preferentially hydrolyzed the ester of alanine among amino acid p-nitrophenylesters. It also efficiently hydrolyzed succinyl Ala-Pro-Ala p-nitroanilide, the specific synthetic substrate for pancreatic elastase. With oxidized insulin B-chain, it hydrolyzed almost exclusively the peptide bond between valine 18 and cysteic acid 19 in the early step of the reaction, and thereafter it partially hydrolyzed Val12-Glu13, Ala14-Leu15, and Leu15-Tyr16. These results indicate that Rarobacter protease I is elastase-like in its substrate specificity, preferentially hydrolyzing the peptide bond of aliphatic amino acids. Its affinity for yeast cells was also investigated, and while Rarobacter protease I was adsorbed by yeast cells, pancreatic elastase was not. This difference was thought to account for the failure of pancreatic elastase to lyse yeast cells, even though its specificity is similar to that of the yeast-lytic enzyme. Rarobacter protease I was adsorbed by a mannose-agarose column and specifically eluted from the column with a buffer containing D-mannose or D-glucose. These monosaccharides also inhibited its yeast-lytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)