Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.     

Erzeugung eines zellwandlytischen Enzymkomplexes aus Arthrobacter GJM-I zur Herstellung von Protoplasten aus Candia spec. H

Growing conditions have been found out for the bacterium Arthrobacter GJM-I to produce a lytic enzyme system, which converts cells of the yeast Candida spec. H to protoplasts quickly and in a good yield. Estimating the activities of α-mannanase and β-glucanase we found out the optimal culture time to gain the lytic enzyme system from the culture filtrate. It was shown that radioactive labeling of the yeast cells makes it possible to estimate quantitatively the conversion to protoplasts and the simultaneous lysis. The obtained lytic enzyme system can substitute the snail cnzyme system which was used for cell conversion of Candida spec. H to protoplasts till now.     

Incorporation of mannoproteins into the walls of aculeacin A-treated yeast cells

Inhibition of the synthesis of alkali-insoluble glucan by aculeacin A in Saccharomyces cerevisiae cells caused a decrease in the incorporation of a high molecular weight heterogeneous mannoprotein material and of a 33000 mannoprotein into the wall network. This was concomitant with the excretion of the latter molecule into the growth medium. Regenerating yeast protoplasts liberated considerable amounts of the heterogeneous material to the medium independently of the presence of aculeacin. The protoplast walls did lack this component and contained only minor amounts of the 33000 molecule, which was also completely absent from walls of aculeacin-treated protoplasts. Considerable levels of the 33000 species were immunodetected in the supernatants from treated and untreated protoplasts. These results point to the existence of specific interactions between the glucan network of the yeast cell surface and some of the wall mannoproteins. On the other hand, the presence of a population of SDS-solubilizable mannoproteins in the wall was independent of glucan levels.Abbreviations SDS sodium dodecyl sulphate - YNB Yeast nitrogen base     

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.     

Chitin synthesis in Candida albicans: comparison of yeast and hyphal forms.

Chitin synthesis was studied in both yeast and hyphae of the dimorphic fungus Candida albicans. Incorporation of N-acetyl-d-[1-(3)H]glucosamine ([(3)H]GluNAc) into an acid-alkali-insoluble fraction was 10 times greater in hyphal-phase cells. A crude preparation of chitin synthetase was obtained from sonically treated protoplasts of both forms of Candida. Enzyme activity, which was determined by using [(14)C]UDP-GLuNAc as a substrate, was exclusively associated with the 80,000 x g pellet from sonically treated protoplasts of both forms. It was determined that enzyme activity (nanomoles of [(14)C]UDP-GluNAc incorporated per milligram of protein) was approximately 2 times greater in hyphae versus yeast cells. Enzyme activity in both yeast and hyphae increased six- to sevenfold when the enzyme preparations were preincubated with trypsin. A vacuolar fraction, obtained from yeast cells but not from hyphae, stimulated enzyme activity when incubated with either yeast or hyphal enzyme preparations. Membrane fractions from protoplasts coated with [(3)H]concanavalin A before disruption were isolated by Renografin density gradient centrifugation. Chitin synthetase activity was preferentially associated with the concanavalin A-labeled fraction, suggesting that the enzyme was located on the plasma membrane. In addition, enzyme activity in protoplasts treated with cold glutaraldehyde before disruption was significantly greater than in protoplasts that were sonically disrupted and then treated with cold glutaraldehyde, indicating that the enzyme resides on the inner side of the plasma membrane.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Studies on the Glucanase of Sclerotinia Libertiana

Preparations of glucan obtained from baker’s yeast and sclerotia of Sclerotinia Libertiana were found to be completely hydrolysed by enzymes of the Sclerotinia fungus. Some differences in the molecular structure of the glucans were found upon examination of the modes of degradation by the successive action of Rhizopus- and Sclerotinia enzyme preparations of which the former had only a partial hydrolytic effect.

The dissolution of glucan in intact cells of yeast, that could be estimated from the rate of autolysis of the cells, was proved to be insignificant on the action of glucanase alone in the Sclerotinia enzyme solution. The combined action of glucanase with lipolytic enzyme in the fungus enzyme solution are shown to promote the solubilization of intact yeasts and sclerotium cells.     

Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae.

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.     

Lysis of yeast cell walls. Lytic beta-(1 leads to 6)-glucanase from Bacillus circulans WL-12.

When grown in a mineral medium with yeast cell walls or yeast glucan as the sole carbon source, Bacillus circulans WL-12 produces wall-lytic enzymes in addition to non-lytic beta-(1 leads to 3) and beta-(1 leads to 6)-glucananases. The lytic enzymes were isolated from the culture liquid by adsorption on insoluble yeast glucan in batch operation. After digestion of the glucan, the mixture of enzymes was chromatographed on hydroxylapatite on which the lytic activity could be resolved into one lytic beta-(1 leads to 6)glucanase and two lytic beta-(1 leads to 3)-glucanase was further purified by chromatography over diethylamino-ehtyl-agarose and carboxymethyl cellulose. Its specific activity on pustulan was 6.2 units per mg of protein. The enzyme moved as a single protein with a molecular weight of 54000 during sodium dodecylsulphate electrophoresis in slab gels. Hydrolysis of pustulan went thorugh a series of oligosaccharides, leading to a mixture of gentiotriose, gentiobiose and glucose. The enzyme also produced small amounts of gentiobiose from laminarin and pachyman and on this basis its lytic activity on yeast cell walls,was attribut beta-(1 leads to 3)-linked oligosaccharides were not detected. The lytic beta-(1 leads to 6)-glucanase has an optimum pH of 6.0. Pustulan hydrolysis followed Michaelis-Menten kinetics. A Km of 0.29 mg pustulan per ml and a V of 9.1 micro-equivalents of glucose released/min per mg of enzyme were calculated. The enzyme has no metal ion requirement. The lytic beta-(1 leads to 6)-glucanase differs in essence from the non-lytic beta-(1 leads to 6)-glucanase of the same organism by its positive action on yeast cell walls and yeast glucan and its much lower specific activity on soluble pustulan.     

Preliminary evidence for a glucan acceptor in the yeast Candida albicans.

Two membrane preparation containing glucan synthase activity were obtained by lysis of regenerating sphaeroplasts (enzyme A) or mechanical breakage (enzyme B) of yeast (Candida albicans) cells. The reaction products of both enzymes (glucans A and B respectively) were characterized as linear beta-1,3-linked glucans on the basis of chemical and enzymic analysis. In addition, two pools of glucan could be distinguished in glucan A preparations on the basis of their susceptibility to an exoglucanase. In no case were the reaction products synthesized de novo; rather the radioactive chains were added to the non-reducing end of non-radioactive preformed glucan chains or to an acceptor of a different nature. At least some of the performed chains of glucan A, but not those of glucan B, showed a free reducing terminal. Glucan A preparations were endowed with endoglucanase activity, which, under appropriate conditions, released glucose, laminaribiose and laminaritriose. These sugars were also found in cell-wall autolysates. On the basis of the origin of both enzyme preparations it is suggested that glucan molecules are synthesized while they are bound to a non-glucan acceptor that is subsequently excised, presumably by cell-wall-associated glucanases.     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

A physical method for separating Saccharomyces cerevisiae cells according to their ploidy

A centrifugation technique, using genetically marked Saccharomyces cerevisiae strains, has been developed to separate Saccharomyces cerevisiae cells of different ploidy levels from exponential phase cultures. The method involves the conversion of yeast cells to protoplasts, the separation of the protoplasts on an osmotically stabilized Nycodenz gradient, and their regeneration. This type of selection may be of importance where selectable markers are not available.     

Protoplast isolation in the marine brown alga Dictyopteris prolifera (Dictyotales)

Protoplasts were isolated from thalli of Dictyopteris prolifera using a mixture of crude enzymes from vicera of live oysters (Crassostrea gigas) and the following commercial enzymes: an abalone enzyme, cellulase, polygalacturonase and hemicellulase. The enzyme mixtures produced up to 3.3 × 10⁷ cells per l g of tissue fresh weight. The conversion to protoplasts of the cells was about 100% using the oyster enzyme or the abalone enzyme alone. The optimum pH for protoplast isolation was 6.0 and 20 hours were required for conversion to protoplasts.     

The influence of Congo red on the cell wall and (1 → 3)-β-d-glucan microfibril biogenesis in Saccharomyces cerevisiae

Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1 » 3)--d-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1 » 3)--d-glucan elementary fibrils depicted by electron microscopy.The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.In memory of Dr. D. R. Kreger of the University of Groningen, The Netherlands, who died on 7 January 1992     

Purification and Properties of Lytic β-Glucanase from an Arthrobacter Bacterium

A glucanase was isolated from a culture fluid of an Arthrobacter bacterium. The purified enzyme preparations consisted of the glucanase components having the same enzymatic activity. The enzyme was stable in a broad pH range, but lost its activity rapidly at above 60°C. Optimum pH values were found to be 5.5~6.5.

The glucanase attacked the following glucan preparations and liberated a relatively small amount of reducing power: Saccharomyces cerevisiae glucan, Candida albicans glucan, Saccharomyces fragilis glucan, pachyman, curdlan and laminaran. The most prominent sugar spot on the chromatogram of the digest from yeast glucan was identified with laminan-pentaose, and the other faint spots with a series of laminaridextrins. The β-1,6 glucosidic bonds in yeast glucan were not hydrolyzed and concentrated in a soluble fraction which was found near the origin of the chromatogram.     

Vesicle penicillinase of Bacillus licheniformis: existence of periplasmic-releasing factor(s).

In earlier studies of the membrane-bound penicillinase of Bacillus licheniformis 749/C, the enzyme present in the vesicles that were released during protoplast formation and the enzyme retained in the plasma membrane of protoplasts appeared to differ (i) in their behavior on gel permeation chromatography in the presence or absence of deoxycholate and (ii) in their tendency to convert to the hydrophilic exoenzyme (Sargent and Lampen, 1970). We have now shown that these vesicle preparations contain a soluble, heat-sensitive enzyme(s) that is released along with the vesicles during protoplast formation. The enzyme will convert the vesicle penicillinase to a form that resembles exopenicillinase, and this conversion can be inhibited by deoxycholate under certain circumstances. Sedimentation of such vesicle preparations at 100,000 X g produces vesicles which contain penicillinase that behaves as the plasma membrane enzyme obtained from protoplasts. Exopenicillinases released by growing cells at pH 6.5 and by washed cells or protoplasts at pH 9.0 have the same NH2-terminal residues (lysine and some glutamic acid); in addition, the various release systems show a parallel sensitivity to inhibition by deoxycholate, quinacrine, chloroquine, and o-phenanthroline. The formation of exopenicillinase (by cleavage of the membrane-bound enzyme) may well be dependent on the action of the releasing enzyme.     

The use of colloid gold labelling in the detection of plasma membrane from symbiotic and non-symbiotic Glycine max root cells

Glycine max L. Merr. cv. Maple Arrow protoplasts were prepared from both tissue-cultured root cells and symbiotically-infected (fix⁺) nodule cells. Whilst both cell types showed glucan synthetase II (GS II; EC 2.4.1.29) activity, neither cell type, whole or gently disrupted, showed glucan synthetase I activity. After sucrose density gradient centrifugation one of the several GS II activity peaks co-sedimented with the single radioactive particulate peak from [¹²⁵I]-labelled protoplasts at 1.14 g ml⁻¹. This peak is presumed to be the plasma membrane peak because labelling of protoplasts with colloidal gold prior to disruption moved the ¹²⁵I peak and the corresponding glucan synthetase II activity into denser regions of the gradient, leaving endoplasmic reticulum-contaminating IDPase (EC 3.1.3.31) and other glucan synthetase II peaks unmoved. Results are discussed in relation to various strategies of plasma membrane isolation.     

Activity of yeast FLP recombinase in maize and rice protoplasts.

We have demonstrated that a yeast FLP/FRT site-specific recombination system functions in maize and rice protoplasts. FLP recombinase activity was monitored by reactivation of beta-glucuronidase (GUS) expression from vectors containing the gusA gene inactivated by insertion of two FRTs (FLP recombination targets) and a 1.31 kb DNA fragment. The stimulation of GUS activity in protoplasts cotransformed with vectors containing FRT inactivated gusA gene and a chimeric FLP gene depended on both the expression of the FLP recombinase and the presence and structure of the FRT sites. The FLP enzyme could mediate inter- and intramolecular recombination in plant protoplasts. These results provide evidence that a yeast recombination system can function efficiently in plant cells, and that its performance can be manipulated by structural modification of the FRT sites.     

Studies on the Glucanase of Sclerotinia libertiana

The digestion of yeast cells with the glucanase of Sclerotinia was found to be significantly increased by pretreating the cells with papaya lysozyme, as well as by pretreating with dilute sodium hydroxide solution. Defatted chlorella cells were digested to a certain extent with the glucanase alone. Pretreatment with lipolytic enzyme slightly stimulated the digestion of yeast cells by glucanase, but this effect was not found with the yeast cells treated by soaps or the defatted chlorella cells. Egg white lysozyme had no effect on digestion of yeast cells. The effect of papaya lysozyme seemed to have relation with the liberation of hexosamine compounds from the yeast-cell walls. It is suggested that, in normal cells, the glucan is present to form a further complex structure with certain other component, becoming insusceptible to the action of glucanase.     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.