Comparative studies of flocculation and deflocculation of Saccharomyces uvarum and Kluyveromyces bulgaricus

Summary Flocculation of Kluyveromyces bulgaricus and Saccharomyces uvarum occurred when these yeasts were grown in a peptone glucose medium enriched with calcium ions. K. bulgaricus and S. uvarum flocculated at the beginning and at the end, respectively, of the exponential growth phase. After growth, both yeasts were washed with an EDTA solution, flocculated again in an acetate buffer, and optimum flocculation was obtained at pH 4.5 in the presence of 3.75 mM Ca⁺⁺. K. bulgaricus flocculation was irreversibly suppressed by incubation at 80° C for 6 min. S. uvarum needed an incubation at 100° C for 20 min to be irreversibly deflocculated. For both yeasts, flocculation stability depended on the presence of sugars. Mannose, mannose 6P and oligosaccharides bearing a mannose in a terminal non-reducing position reversed flocculation of S. uvarum, while galactose, galactose 6P and oligosaccharides bearing a galactose in a terminal nonreducing position reversed flocculation of K. bulgaricus. It is suggested that sugars specifically reverse flocculation because cell-to-cell aggregation of these yeasts is a lectin-carbohydrate-linked mechanism; not any sugar is capable of deflocculating any yeast, but the mechanism is specific.     

Effect of amphotericin B on the sterol composition of Kluyveromyces bulgaricus and Kluyveromyces lactis

The degree of sensitivity of the yeasts Kluyveromyces bulgaricus and K. lactis to amphotericin B is linked to a difference in the sterol composition of their membranes. No direct proportionality was found between sensitivity and the quantity of sterols present. At sublethal doses, amphotericin B perturbed sterol synthesis, resulting in ergosterol precursor accumulation. An ergosterol pathway is proposed for Kluyveromyces.     

Hydrolysis of lactose solutions and wheys by whole cells of Kluyveromyces bulgaricus

Summary Some factors affecting the hydrolysis of lactose solution and whey by whole cells of Kluyveromyces bulgaricus were studied. The K_m values were 59 mM and 32 mM for whole cells and cell-free extracts respectively. Optimum hydrolysis activity was observed at 48°C. At this temperature, 80% hydrolysis was obtained in lactose solution and whey after 3.5 and 9 min respectively by yeast cells (15 mg/ml) with an activity of 1.13 U/mg. Protein concentration in whey did not have an inhibitory effect. In whey permeate, cells were reused eight times with a hydrolysis degree of more than 80% but in lactose phosphate buffer, the hydrolysing capacity was lost quickly.     

Comparative studies onChlorella cell walls: Induction of protoplast formation

Among 12 strains ofChlorella ellipsoidea, C. vulgaris, andC. saccharophila tested, 4 strains (1,C. ellpsoidea; 2,C. vulgaris; 1,C. saccharophila) formed osmotically labile protoplasts after treatment with mixtures of polysaccharide degrading enzymes. The relationship between enzymatical digestibility and structure or composition ofChlorella cell walls were studied by electron microscopy and staining techniques with some specific dyes. The cell wall structures of the 12Chlorella strains were grouped into three types: (1) with a trilaminar outer layer, (2) with a thin outer monolayer, and (3) without an outer layer. Protoplasts were formed only from the strains with a cell wall of Type 2. In the strains with a cell wall of Type 1, the outer layer protected the inner major microfibrillar layer against enzymatic digestion. The cell wall of Type 3 was totally resistant to the enzymes; the chemical composition of the cell wall would be somewhat different from that of other types.     

Development of a new coulter counter system: Measurement of the volume,internal conductivity,and dielectric breakdown voltage of a single guard cell protoplast ofVicia faba

Summary Two intracellular microelectrodes were used to study electrotonic interaction between cultured embryonic (16- to 20-day-old) chick myocardial cells reaggregated into small spheresin vitro. Under different culture conditions, reaggregates with two types of functional membrane properties were produced: (i) highly differentiated reaggregates, and (ii) reverted reaggregates. In the highly differentiated state, the cells had high stable resting potentials and produced rapidly-rising tetrodotoxin (TTX)-sensitive action potentials in response to electric field stimulation. In the reverted state, the cells exhibited slowrising spontaneous action potentials having prominent pacemaker potentials and TTX-insensitive upstrokes. These states resemble electrophysiological properties of the highly differentiated (18 daysin ovo) and less fully differentiated (3 daysin ovo) intact embryonic chick heart, respectively. Both types of reaggregates had similar ultrastructural appearance, with many elongated cells and intercalated disc-like structures; gap-like junctions were not seen. The highly differentiated cells had input resistances of about 5 M, and exhibited only little electrotonic interaction in response to intracellular current injection either when the cells were at rest or during the action potential plateau. Intracellular stimulation produced propagating action potentials which triggered contraction of the entire reaggregate. Large hyperpolarizing current pulses applied during the action potential plateau caused premature repolarization which also propagated to the other impaled cell. In the reverted reaggregates, electrotonic interaction was weak or absent in about 52% of the impaled cell pairs, moderate in 30%, and strong in 18% (encountered only at interelectrode distances of less than 100 m). The difference in degree of electrotonic interaction may be due to the state of differentiation with respect to the membrane electrical properties.     

Influence of carbon source and surface hydrophobicity on the aggregation of the yeast Kluyveromyces bulgaricus

Aggregation of the yeast Kluyveromyces bulgaricus is mediated by the galactose-specific lectin KbCWL1. This lectin contains hydrophobic amino acids and its activity is calcium dependent. A specific fluorescent probe, 1-anilinonaphthalene-8-sulfonic acid in the free acid form (ANS; Sigma Chemical Co., St. Louis, Missouri), was used to study the hydrophobic areas on the cellular surface of K. bulgaricus. Changes in surface hydrophobicity during the growth and aggregation of yeast cells were studied. Surface hydrophobicity increased during growth and depended on the amount of yeast cells in the culture medium. During growth, the size of the hydrophobic areas on the cell surface was measured using ANS and was found to increase with the percentage of flocculating yeasts. Our results strongly suggest that the hydrophobic areas of the cell walls of yeast cells are involved in the aggregation of K. bulgaricus.     

The use of a coulter counter to study the alcoholic fermentation in enological conditions

Summary A Coulter counter was used to rapidly determine the exact stage of alcoholic fermentation of wine musts on a growth and mortality curve. By this technique it was also possible to check the yeast population involved in this fermentation and detect assimilable nitrogen deficiencies of musts.     

Factors affecting protoplast formation and regeneration by four species of Streptomyces

Four species of Streptomyces, Streptomyces canescens, S. limosus, S. griseus and S. griseolus , were used to study the effects of glycerine and gelatin on the formation and regeneration of protoplasts. For each species efficient protoplast formation with high protoplast concentrations and low levels of non-protoplast units was obtained with mycelia grown in medium without glycerine. The low regeneration frequencies of protoplasts of S. canescens and S. limosus on R2 medium were increased substantially by the addition of 1% gelatin. The use of single colonies, rather than spores, to establish mycelial cultures was found routinely to produce good protoplast preparations.     

Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.     

Hydrolysis of whey by whole cells of Kluyveromyces bulgaricus immobilized in calcium alginate gels and in hen egg white

Summary Whey hydrolysis was compared in column reactors containing whole yeast cells immobilized in Ca-alginate or in hen egg white in relation to cell -galactosidase activity, flow rates, temperature and time. With cells of 1.3 U/mg dry weight (ONPG method) immobilized in Ca-alginate, 80% hydrolysis was obtained at 4° and 20° C with, respectively 0.50 and 1.65 bed volume/H; the values were 0.2 and 0.74 with cells entrapped in hen egg white. When the flow rate was expressed as ml/H/g wet yeast, no significant difference was observed between both matrices and 80% hydrolysis was reached with a flow rate 1.7 and 5 according to the temperature. The best performance was achieved by the yeast egg white reactor. At 4°C, hydrolysis decreased by 10% after 13 days; by 20% after 17 days. The presence of lactose transport inhibitors in whey did not significantly influence lactose hydrolysis.M. Decleire et al.: Hydrolysis of whey by immobilized whole cells of Kluyveromyces bulgaricus     

Rapid determination of kinetic constants by competitive spectrophotometry

The use of competitive spectrophotometry to measure kinetic constants for enzyme-catalyzed reactions is described. The equation for the progress curve characterizing the kinetic behavior of an enzyme acting simultaneously on two alternative substrates is derived. By the addition of a competition term to the integrated Michaelis-Menten equation, the kinetic constants of an alternative substrate can be evaluated by measuring the competition with a substrate of known kinetic constants in a single experiment. Studies are presented involving the enzymes leucine aminopeptidase (LAP) and carboxypeptidase A (CPA). The results obtained with LAP and CPA showed that the kinetic constants determined using competitive spectrophotometry were in agreement with values cited in the literature or with values determined by single substrate enzyme kinetics.     

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.     

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source. This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30. Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity. The optimal hydrolysis temperature for sucrose, raffinose and inulin was 55°C and the optimal pH for sucrose was 4.75. The apparent K _m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively. Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides. The results obtained suggest the hypothesis that enzyme production was constitutive. Journal of Industrial Microbiology & Biotechnology (2000) 25, 63–69. Received 17 November 1999/ Accepted in revised form 30 May 2000     

保加利亚乳杆菌原生质体的制备与回复研究

目的:通过对保加利亚乳杆菌的原生质体的制备和回复的方法学探讨,为乳杆菌的基因操作和其相关研究提供技术思路和实验条件.方法:用酶浓度分别为1 μg/ml、4 μg/ml、10μg/ml Mutanolysin(变溶菌素)对保加利亚乳杆菌进行处理,脱去细胞壁以探讨其原生质体形成与时间和酶浓度的关系;并选用较为适宜的酶浓度制备其原生质体,在自制的双层再生培养基上观察其原生质体在普通培养、CO2培养、厌氧培养时的回复生长情况.结果:保加利亚乳杆菌对Mutanolysin较敏感,酶浓度为1 μg/ml时只需40min大部分菌体形成原生质体.经1μg/ml Mutanolysin处理后制得的保加利亚乳杆菌原生质体倾入自制的双层再生培养基中,置于CO2和厌氧环境条件下培养能很好的回复生长.结论:本文的研究为乳杆菌的基因工程方面的研究提供了相关的技术条件和实验基础.     

Kluyveromyces bulgaricus yeast lectins. Isolation of two galactose-specific lectin forms from the yeast cell wall

Incubation of galactose treated Kluyveromyces bulgaricus yeast cells in EDTA/phosphate-buffered saline led to an extract possessing hemagglutinating and yeast flocculating properties. Purification of this extract by affinity chromatography and gel filtration gave two lectin forms, Kb-CWL I and Kb-CWL II, with an apparent molecular mass of 38,000 and 150,000 Da, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Kb-CWL I and Kb-CWL II were dimeric and octameric of a subunit of 18,900 Da. At high concentration, purified Kb-CWL I associated to give Kb-CWL II. This association seemed to be independent on pH. The two lectin forms were glycoproteins, the peptide counterpart was very rich in Lys, Glu, and Gly, and the carbohydrate part represented 1% of the whole molecule and was composed of Glc, Man, and Ara. The two lectin forms (KB-CWL I and Kb-CWL II) agglutinated human red blood cells and flocculated EDTA-treated K. bulgaricus yeast cells. The activity of both lectin forms required Ca2+ ions, while Sr2+ showed some competitive inhibition. Optimal activity was obtained within a pH range of 4-6.5 for both forms. Temperatures of 80-90 degrees C for 20 min, or proteolytic treatment reduced irreversibly the activity of Kb-CWL I and Kb-CWL II. The role of the cell wall phosphopeptidomannan as a ligand and a potential physiological receptor of these lectin forms was demonstrated.