Purification and partial characterization of a flocculin from brewer's yeast.

Analysis of a shear supernatant from flocculent, "fimbriated" Saccharomyces cerevisiae brewer's yeast cells revealed the presence of a protein involved in flocculation of the yeast cells and therefore designated a flocculin. The molecular mass of the flocculin was estimated to be over 300 kDa, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel permeation chromatography of the flocculin yielded an aggregate with an apparent molecular weight of > 2,000. The flocculin was found to be protease sensitive, and the sequence of its 16 N-terminal amino acids revealed at least 69% identity with the predicted N terminus of the putative protein encoded by the flocculation gene FLO1. The flocculin was isolated from flocculent S. cerevisiae cells, whereas only a low amount of flocculin, if any, could be isolated from nonflocculent cells. The flocculin was found to stimulate the flocculation ability of flocculent yeast cells without displaying lectinlike activity (that is, the ability to agglutinate yeast cells).     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Chemical modifications of the cell-surface components of Lactobacillus fermentum FTPT 1405 and their effect on the flocculation of Saccharomyces cerevisiae

The effect of treatment of Lactobacillus fermentum with several protein- and carbohydrate-modifying reagents on the bacterium's ability to flocculate Saccharomyces cerevisiae was investigated. The proteinaceous nature of the cell-surface components of L. fermentum which are responsible for floc formation was confirmed by inactivation of floc formation following photo-irradiation, with Methylene Blue or Rose Bengal as sensitizer, or acylation with acetic anhydride, maleic anhydride or acetylimidazole, and by the reaction of the components with nitrous acid, I₂ and performic acid.The phenolic hydroxyl group of tyrosine and the indole group of tryptophan appear essential for flocculation. Proteinaceous components of the yeast cell surface and carbohydrate components on the bacterial cell surface were not required for flocculation but carbohydrate residues on the yeast surface were essential.     

Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer''s Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer's yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.     

Glucose tolerance factor potentiation of insulin action in adipocytes from rats raised on a torula yeast diet cannot be attributed to a deficiency of chromium or glucose tolerance factor activity in the diet

The nature of the dietary component responsible for adipocytes having the ability to respond to Glucose Tolerance Factor (GTF) was investigated. Rats were raised on either a control diet or one of three diets differing only in the protein source (torula yeast, brewer's yeast, or casein). Only in adipocytes from rats fed the torula yeast diet did a GTF fraction prepared from brewer's yeast potentiate the action of suboptimal concentrations of insulin in the incorporation of label fromd-[1-¹⁴C]-glucose andd-[U-¹⁴C]-glucose into CO₂ and fatty acids. It was concluded that this potentiation was not the result of a deficiency of GTF activity in torula yeast, because a GTF fraction prepared from torula yeast had similar insulin potentiating activity. Differences in response among diets were not owing to differences in levels of amino acids or owing to concentrations of 22 (Al, As, B, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mo, Na, Ni, P, Pb S, Se, Si, Sn, Sr, Zn) of the 23 trace elements investigated. The level of Mn, was low in all diets, but particularly low in the torula yeast diet. Mn deficiencies have previously been implicated in perturbations of glucose metabolism, so that it is possible that this deficiency may be responsible for the effects attributed to the torula yeast diet.     

The flocculation of wine yeasts: biochemical and morphological characteristics in Kloeckera apiculata

The floc-forming ability of flocculent strains of Kloeckera apiculata, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Three different flocculation phenotypes were discriminated by protease digestion, whereas the inhibition of flocculation by sugars distinguished two definite patterns: one mechanism of flocculation involved a galactose-specific protein and the other a broad-specificity lectin. SEM and TEM observation of the cell surface of two different Kloeckera strains revealed fine fibrils and a diffuse structure at the point of contact in one strain, and thick masses of mucus on the cell wall of the other strain.     

Applications of yeast flocculation in biotechnological processes

A review on the main aspects associated with yeast flocculation and its application in biotechnological processes is presented. This subject is addressed following three main aspects—the basics of yeast flocculation, the development of “new” flocculating yeast strains and bioreactor development. In what concerns the basics of yeast flocculation, the state of the art on the most relevant aspects of mechanism, physiology and genetics of yeast flocculation is reported. The construction of flocculating yeast strains includes not only the recombinant constitutive flocculent brewer's yeast, but also recombinant flocculent yeast for lactose metabolisation and ethanol production. Furthermore, recent work on the heterologous β-galactosidase production using a recombinant flocculentSaccharomyces cerevisiae is considered. As bioreactors using flocculating yeast cells have particular properties, mainly associated with a high solid phase hold-up, a section dedicated to its operation is presented. Aspects such as bioreactor productivity and culture stability as well as bioreactor hydrodynamics and mass transfer properties of flocculating cell cultures are considered. Finally, the paper concludes describing some of the applications of high cell density flocculation bioreactors and discussing potential new uses of these systems.     

Inducing flocculation of non-floc-forming <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

The present article reviews several approaches for inducing flocculation of Escherichia coli cells. The common industrially used bacterium E. coli does not naturally have floc-forming ability. However, there are several approaches to induce flocculation of E. coli cells. One is induction by flocculants—polyvalent inorganic salts, synthetic polymeric flocculants, or bio-based polymeric materials, including polysaccharide derivatives. Another method is the induction of spontaneous flocculation by changing the phenotypes of E. coli cells; several studies have shown that physical treatment or gene modification can endow E. coli cells with floc-forming ability. Coculturing E. coli with other microbes is another approach to induce E. coli flocculation. These approaches have particular advantages and disadvantages, and remain open to clarification of the flocculation mechanisms and improvement of the induction processes. In this review, several approaches to the induction of E. coli flocculation are summarized and discussed. This review will be a useful guide for the future development of methods for the flocculation of non-floc-forming microorganisms.     

The flocculation of wine yeasts: biochemical and morphological characteristics inZygosaccharomyces — flocculation inZygosaccharomyces

The floc-forming ability of flocculent strains ofZygosaccharomyces bailii andZ fermentati, isolated from musts, was tested for susceptibility to proteinase and sugar treatments.Z. fermentati was found highly resistant to the proteolytic enzymes tested, whereasZ. baili was only trypsin-resistant.The inhibition of flocculation by sugars distinguished two types: inZ. fermentati flocculation was completely inhibited by mannose, inZ. bailli by various sugars.By SEM observation, the cell surface ofZygosaccharomyces revealed the presence of a column structure, resulting from fusion of vesicles present on the cell surface.     

富铬酵母的理化性质和氨基酸分析

用２００～３００ｎｍ的波长范围对富铬酵母及普通酵母的溶液进行紫外扫描,发现在λ_(２６０ｎｍ)处有一特征紫外吸收峰,富铬酵母细胞的铬含量与其吸收峰的光密度呈线性关系;在不同的温度和ｐＨ值条件下,通过紫外吸收峰的测定,富铬酵母溶液在酸性条件下稳定,在碱性条件下不稳定。溶液的λ_(２６０ｎｍ)紫外吸收峰随着温度的上升而升高。从氨基酸含量分析结果看,富铬酵母的谷氨酸、甘氨酸、组氨酸、丙氨酸和赖氨酸含量高于普通酵母,但其他氨基酸含量比普通酵母低。     

Removal of heavy metals using a brewer's yeast strain of Saccharomyces cerevisiae: the flocculation as a separation process

In this work, a brewer's yeast strain was used to remove heavy metals from a synthetic effluent. The solid-liquid separation process was carried out using the flocculation ability of the strain. The yeast strain was able to sediment in the presence of Cu2+, Ni2+, Zn2+, Cd2+ and Cr3+, which evidences that the flocculation can be used as a cheap and natural separation process for an enlarged range of industrial effluents. For a biomass concentration higher than 0.5 g/l, more than 95% of the cells were settled after 5 min; this fact shows that the auto-aggregation of yeast biomass is a rapid and efficient separation process. Cells inactivated at 45 degrees C maintain the sedimentation characteristics, while cells inactivated at 80 degrees C lose partially (40%) the flocculation. The passage of metal-loaded effluent through a series of sequential batches allowed, after the second batch, the reduction of the Ni2+ concentration in solution for values below the legal limit of discharge of wastewater in natural waters (2mg/l); this procedure corresponds to a removal of 91%. A subsequent batch had a marginal effect on Ni2+ removal (96%). Together, the results obtained suggest that the use of brewing flocculent biomass looks a promising alternative in the bioremediation of metal-loaded industrial effluents since the removal of the heavy metals and cell separation are simultaneously achieved.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure.

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Modification by glucose of the flocculent phenotype of a <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> wine strain

We have evaluated the induction of the flocculent phenotype of Kloeckera apiculata by glucose mc1 and propose a pathway involved in carbohydrate flocculation induction. Pulses of glucose were given to cells growing in glucose-poor medium (2 g l(-1)) and the flocculation percentage was measured. To elucidate the mechanism involved in flocculation induction, cycloheximide was injected into the cultures 120 min before the glucose pulse. 2,4-Dinitrophenol or cAMP was added to the media instead, or simultaneously with glucose, while a protein kinase A (PKA) inhibitor was added 30 min before the glucose pulse. With 20 and 50 g l(-1) glucose pulse, the yeast flocculation percentage arises to 55 and 65%, respectively. The quantity of proteins and the reflocculating capacity of a lectinic protein extract from the yeast cell wall increase as the concentration of glucose pulse was higher. Cycloheximide prevented the glucose-induced flocculation, while cAMP or 2,4-dinitrophenol increased it 4- and 5-fold, respectively. PKA inhibitor completely prevented the glucose induction flocculation. The flocculent phenotype of K. apiculata mc1 was induced by glucose and the mechanism seems to imply de novo protein (lectin) synthesis via the PKA transduction pathway. This work contributes to the elucidation of the mechanism involved in flocculation induction by glucose of a non-Saccharomyces wine yeast, K. apiculata, which has not been reported. The induction of flocculation by glucose could be a biotechnological tool for the early removal of the indigenous microorganisms from the grape must before the inoculation of a selected starter strain to conduct the alcohol fermentation.     

Flocculation of cell walls of brewer's yeast and effects of metal ions,protein-denaturants and enzyme treatments

Effects of metal ions, protein-denaturants and enzyme treatments on flocculation of cell walls of Beer Yeast IFO 2018 were investigated. Cell walls from flocculent cells grown in a complete medium were able to form flocs as were whole cells, but cell walls from non-flocculent cells, such as “Mg²⁺-deficient” cells, “early-phase” cells and “low-pH” cells, were not. The cell walls dispersed in distilled water reflocculated in solutions containing Ca²⁺ or other metal ions. Of the alkali metal ions tested, only Na⁺ inhibited flocculation of flocculent cell walls at a concentration more than 0.1 M. Ca²⁺ or Sn⁴⁺ was absolutely required for flocculation of cell walls in the physiological saline (NaCl, 150 mM), but the effect of Sn⁴⁺ seems rather non-specific, because it promoted flocculation of non-flocculent cell walls as well. Sr²⁺ and Ba²⁺ were antagonistic to Ca²⁺ and inhibited flocculation. Flocculation of cell walls was also depressed by high concentrations of protein-denaturants, e.g. urea and guanidine·HCl. Treatment with proteolytic enzymes deprived cell walls of floc-forming ability. Effect of metal ions, protein-denaturants and treatment with enzymes on the flocculation of intact cells was investigated as control. Since flocculating properties of cell walls were very similar to those of intact cells, flocculation must be an inherent property of cell walls.     

Isolation OP DNA Prom Yeast by Chromatography on Hydroxyapatite

A simple procedure is described for isolation of purified non degraded total DNA from yeast cells. The procedure involves conversion of the cells into sphero-plasts by enzymatic treatment, lysis of the sphero-plasts in 8 M urea - 0.24 M sodium phosphate buffer -0.01 M EDTA (ethylendiamintetraacetic acid, sodium salt) - 1% SDS (sodium dodecyl sulphate), deproteiniza-tion of the lysate with chloroform-phenol and separation of the DNA from proteins, RNA and other contaminants by hydroxyapatite chromatography. The yield is about 90% of the DNA in the starting material (sphero-plasts).     

Effect of higher alcohols on the activity of biosynthetic L-threonine dehydratase from Saccharomyces carlsbergensis brewer's yeast]

The effects of low (1 . 10(-4) M) and high (1 . 10(-3) M) concentrations of n-propanol, isobutanol and isoamylols on the kinetic behaviour of "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlsbergensis 776 were studied. It was concluded that these alcohols control the activity of the first enzyme of the L-threonine biosynthetic pathway.     

Endothelial heparan sulphate: Compositional analysis and comparison of chains from different proteoglycan populations

From cultures of human umbilical vein endothelial cells incubated with³H-glucosamine or³⁵S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG heparan sulphate proteoglycan - DSPG dermatan sulphate proteoglycan - GlcNAc(6S) N-acetylglucosamine 6-sulphate - GlcNAc6R glucosamine with either-OH or-OSO₃ at C-6 - GlcNR glucosamine with either-SO₃ or-COCH₃ as N-substituent - GlcNSO₃ N-sulphated glucosamine - GlcNSO₃(3S) N-sulphated glucosamine 3-sulphate - GlcA d-glucuronic acid - IdoA l-iduronic acid - IdoA(2S) iduronic acid 2-sulphate - HexA hexuronic acid - DHexA hexuronic acid with a 4,5-double bond - Xyl xylose - SAX strong anion exchange - d.p. degree of polymerization (a disaccharide has d.p.=1 etc) - AUFS absorbance units full scale The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6].     

Isolation,purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun.60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ?-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A₁ and A₂ peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.     

Effect of hydrolytic enzymes and protein-modifying reagents on gonadotropin receptors in bovine corpus luteum cell membranes.

Preincubation of membranes with various concentrations of pronase, trypsin, lipase, phospholipase A from Vipera russelli and from Crotalus durissus terrificus, phospholipase C from Bacillus cereus and from Clostridium welchii, acetic anhydride, 2,4-dinitrofluorobenzene and tetranitromethane resulted in a dose-dependent inhibition of 125I-labeled human choriogonadotropin binding. At the submaximal concentrations of enzymes and at both submaximal and maximal concentrations of protein-modifying reagents, the losses were always greater with 125I-labeled human choriogonadotropin than with 125I-labeled human lutropin. The inhibition of binding was a consequence of changes in the membranes rather than changes in the hormone caused by the agents being carried over to the final incubation. Inhibition of binding was non-competitive and irreversible. In untreated membranes, the 125I-labeled human choriogonadotropin binding was homogeneous (Kd = 1.7.10(-10) M; N = 60 fmol/mg protein). Treatment of membranes with various enzymes and protein-modifying reagents except tetranitromethane resulted in heterogeneous binding. The number of available high affinity receptors was greatly reduced in every case. However, the affinity of these sites were either unchanged (trypsin, lipase, phospholipase A from V. russelli, dinitrofluorobenzene and the tetranitromethane) or decreased (pronase and acetic anhydride). The newly appeared second receptor site had a Kd which varied from 3.2.10(-10) to 7.1.10(-9) M depending on the agent used, and the receptor numbers were low in all cases except acetic anhydride. Receptor occupancy conferred the receptors with marked protection against various hydrolytic enzymes, dinitrofluorobenzene and tetranitromethane. These data suggest that inhibition of binding by the above agents was primarily a consequence of changes in the receptor molecules themselves.