Cell wall enzymatic lysis of the yeast form of Pullularia pullulans and wall regeneration by protoplasts.

During the process of degradation of the cell wall of the yeast form of Pullularia pullulans by the lytic system of micromonospora chalcea samples were withdrawn at different times and observed under phase contrast and electron microscope. The progressive lysis of the walls reveals a fibrillar component inside the apparently amorphous wall. Freeze etched preparations of cells during the formation and regeneration of protoplasts show that the cellular membrane is split and this method allows the smooth external face of the membrane and other internal face covered by particles to be seen. The fact that the smooth face of the membrane is only visible during the preparation or the regeneration of protoplasts and very rarely when intact cells are fractured, suggests a strong adherence between cell wall and this external layer of the membrane. During the regeneration which takes place as in most of the yeasts and moulds, a special study of the extension of the cell wall is made and a possible mechanism for this extension of the regenerated cell wall is proposed.     

Plasmalogens in the yeast Pullularia pullulans

Choline and ethanolamine phosphoglycerides have been found in plasmalogen form in P. pullilans. Plasmalogens had not been described in yeasts up to now.     

Cell wall regeneration by protoplasts of Chlamydomonas

Protoplasts from Chlamydomonas smithii prepared by the action of C. reinhardii gamete autolysine have been studied with respect to cell wall regeneration. Natural protoplasts within sporangia were also investigated for purposes of comparison. In both cases a new cell wall is completed within 2–3 h of the onset of regeneration. The first visible stages of wall regeneration are to be seen after 40–60 min as a fine fringe outside of the plasmalemma. The development of the typical central triplet follows within the next 1 h. Cell wall regeneration is reversibly inhibited by cycloheximide (10g ml^-1) and reversibly disturbed by concanavalin A (50 g ml^-1). Actinomycin D at concentration over 100g ml^-1 also inhibit but the inhibition is irreversible and peculiar membrane effects are observed. Chelators (ethylenediamine tetraacetic acid; ethyleneglycol-bis-aminoethyl ether) and 2-deoxyglucose slightly retard or have no effect on cell wall regeneration.Abbreviations EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis(aminoethyl ether) - N,N tetraacetic acid     

Utilization of n-Alkanes by Pullularia pullulans

Pullularia pullulans was tested for its ability to utilize a series of n-alkanes for growth. It utilized hydrocarbons containing higher C-numbers (13, 14, 16, and 18) to a greater degree than those containing lower numbers; in addition, an induction phenomenon was noted. Gas-liquid and thin-layer chromatography of ether extracts of the growth media revealed that oleic and palmitic acids were formed from tridecane, tetradecane, hexadecane, and octadecane.     

d-Xylose as inducer of the xylan-degrading enzyme system in the yeast Pullularia pullulans

Summary The specificity of induction of wooddegrading enzymes from Pullularia pullulans was investigated using series of mono-, di- and (14)--trisaccharides or glycanes. A strain of P. pullulans (1740), unable to grow on Avicel or carboxymethyl-cellulose (CMC), uses xylan and steamexploded wood as carbon sources. This strain, thus grown, was evaluated for various enzyme activities. d-Xylose was the nutritional inducer of -xylosidase and -xylanase. d-Glucuronic acid induced activity on CMC and -glucosidase activity was observed regardless of carbon source used. (14)--Xylobiose was not an inducer of -xylanase production, but high levels of this enzyme were obtained with either structural isomers (12) or (13)-. Since synthesis of this enzyme was stimulated by increasing xylose concentration yp to 40 g/l, it is suggested that xylose enters the cells by passive transport and is unable to induce a permease system.Affiliated to the Scientific, Technological and Medical University of Grenoble     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

Comparison of Extracellular Polysaccharide Produced by Pullularia pullulans with Polysaccharides in the Cells and Cell Wall

The purified swep hydrolase appeared homogeneous on SDS disc electrophoresis, and the molecular weights of the enzyme and the subunit were 280,000 and 70,000, respectively. The isoelectric point of this enzyme was pH 4.8.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

Optimization of enzymatic lysis of yeast

In enzymatic lysis of yeast for the recovery of intracellular proteins, the rupture of whole cells is caused by the action of a lytic system consisting primarily of protease and glucanase. A first-principles mechanism for the lytic reaction based on a two-layer model of the wall structure and a burst model for the disruption of cells is pre sented. The fed-batch model results in a dynamic optimization problem, with the enzymes, activities being the control variables. Orthogonal collocation is applied to discretize the state equations, and the resulting non linear program is solved using successive quadratic pro gramming to determine the enzyme and protease inhibitor add-in rates and pH control profiles that maximize the recovery of intracellular protein. Applying the proposed approach, optimal profiles were determined such that a significant increase of the production of proteins in a fed-batch reactor is realized. Also, the optimal control policies in a series of continuous-flow stirred tank reactors (CFSTRs) are determined.     

Isolation and identification of trehalase from Pullularia pullulans

Trehalase has been isolated from Pullularia pullulans. The enzyme, which is specific for trehalose, was purified approximately 800-fold. The optimal pH was found to be 4.0 and the Michaelis dissociation constant, K(m), was determined to be 3.2 x 10(-3)m.     

Uptake of Glucose-1-14C by Pullularia pullulans

Pullularia pullulans cells were grown in a medium containing yeast extract, malt extract, glucose, and nutrient salts, in addition to glucose-1-(14)C. The lipids extracted from the cells were fractionated by use of a single column packed with silicic acid. Of the total radioactive carbon added to the culture medium, the neutral lipid fractions contained 24.8%, whereas the phospholipid portions contained only 2.1%. The largest amount (16.5%) of (14)C among the neutral lipids was found in the fraction containing the free sterols. Among the phospholipids, the largest amount (1.1%) was found in phosphatidylserine and phosphatidylethanolamine. The second largest amount (9.6%) of the total (14)C used was found in trehalose, followed by carbon dioxide (7.3%).     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

The Degradation of Gallic Acid and its Alkyl Esters by Pullularia pullulans

By elective techniques an isolate of Pullularia pullulans was obtained which possessed the ability to utilize gallic acid (3,4,5-trihydroxybenzoic acid) and its alkyl esters together with other aromatic substrates as the sole carbon source for growth in a mineral salts medium. Quantitative measurements were made of the disappearance of the gallates from culture media and of their metabolism by washed suspensions by the Warburg technique. The pattern of oxygen uptake with various substrates by suspensions adapted to gallic acid was examined in the light of Stanier's simultaneous adaptation postulates in an attempt to identify intermediates of gallic acid metabolism. As a result, β-ketoadipic acid was suspected of being an intermediate. A keto compound was isolated as its 2,4-dinitrophenylhydrazone during gallic acid metabolism but it was not β-ketoadipic acid or levulinic acid.     

Yeast protoplasts immobilized in alginate: Cell wall regeneration and reversion to cells

Yeast protoplasts may regenerate the cell wall and revert to cells if immobilized in a 2%–5% Ca-alginate gel and cultured in an osmotically stabilized medium. The method of protoplast immobilization and subsequent isolation from the gel is described in detail. The reversion yield is dependent of the actual gel concentration, gel shape (beads vs. sheets) and of a medium molarity, and it may be up to 90%. The morphology of the cell wall regeneration and morphology of reversion to the cell forms correspond to protoplast development in gelatin or agar gels.