Lysis of Yeast Cells Showing Low Susceptibility to Zymolyase

Lysis of commercial baker’s yeast cells was examined using Zymolyase. The lysis was stimulated by the addition of sodium sulfite or potassium chloride or both. The effect of potassium chloride was less than that of sodium sulfite, but the two compounds acted synergistically. The cells were effectively lysed by Zymolyase in the presence of 0.1 M sodium sulfite and 0.8 M potassium chloride. The extent of lysis was similar to that of brewery yeast cells obtained from a brewhouse. Cells pretreated with sodium sulfite did not show much of an increase in susceptibility to Zymolyase, but were effectively lysed by the enzyme in the presence of potassium chloride. Potassium chloride stimulated lysis only in the presence of Zymolyase. Yeast cells treated with cupric ions in the presence of sodium sulfite became highly susceptible to Zymolyase, suggesting irreversible destruction of the sodium sulfite-sensitive and potassium chloride-sensitive structure of the cell wall.

Cells of Saccharomyces cerevisiae prepared under various culture conditions were completely lysed by Zymolyase in the presence of sodium sulfite or potassium chloride or both.     

An efficient technique for the isolation of yeast spores and the preparation of spheroplast lysates active in protein synthesis

A procedure for isolation of yeast spores and preparation of yeast spheroplasts with the use of the bacterial lytic enzyme, Zymolyase, is described. The high lytic activity of Zymolyase, allows isolation of the yeast spores in a rapid and simple manner. The resulting spores are not contaminated with vegetative cells and retain their full activity in germination. Moreover, the enzyme appears to be very efficient in preparation of yeast lysates, actively synthesizing proteins. The use of Zymolyase for other purposes is suggested.     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

A Protease that Participates in Yeast Cell Wall Lysis during Zymolyase Digestion

Zymolyase B decreased the turbidity of a yeast cell wall suspension by about 50% and caused release of peptide-mannan from the cell walls. However cell walls treated with the enzyme still maintained the cell shape. The effect of the enzyme on the cell walls was inhibited by yeast mannan and completely counteracted by treatment of the enzyme with DFP. The activity was not affected by pH, but was considerably reduced by incubation of the enzyme at 55°C for 15 min, a treatment that did not affect the proteolytic activity. Heat-treatment decreased the molecular weight of the enzyme from 29,000 to 22,500 and its sensitivity to yeast mannan. Yeast mannan caused noncompetitive inhibition of the proteolytic activity of the native enzyme and competitive inhibition of that of the heat-treated enzyme. Modification of tryptophan residues of Zymolyase B resulted in decreased sensitivity to yeast mannan and a decrease in the activity of the enzyme on yeast cell walls as well as heat-treatment. On the basis of these results, it is hypothesized that Zymolyase B binds to the cell wall mannans and changes their conformation, making the attached proteins susceptible to proteolysis, and then releases peptide-mannan from the cell walls.     

Shochu brewing characteristics and properties of a trichothecin-resistant shochu yeast mutant

An antibiotic-resistant strain of Saccharomyces cerevisiae was isolated from shochu yeast. Three mutants were used for shochu brewing and gave higher ethanol productivities than the parent. The mutants were resistant to cycloheximide, cerulenin, trichothecin and other organic compounds such as lauric acid. In the presence of 20% (v/v) ethanol, the viability of the mutants was 87–96%, but that of the parent was 77%. Zymolyase treatment for 3 h, decreased the viability of the parent by 44% but that of the mutants only by 11–32%. Thus the higher ethanol productivity of these mutants is related to their high ethanol tolerance and resistance to various organic compounds.     

Effect of anH-2 mutation on cytotoxic T cell recognition. Specific antigen can determine the pattern ofH-2 restriction

Hz1 (H-2 ^bm1 ) mice, an H-2 mutant strain derived from C57BL/6(H-2 ^b ), were either injected with vaccinia virus or had their spleen cells sensitized in vitro with syngeneic TNP-modified cells. The cytotoxic cells generated were tested for their activity against target cells that were either infected with vaccinia virus, TNP-modified, or both vaccinia infected and TNP-modified.Hz1 anti-TNP cytotoxic cells specifically lysed syngeneic target cells that were trinitrophenylated but not infected with vaccinia virus, while anti-vaccinia cells specifically lysed vaccinia infected target cells but not TNP-cells. Hz1 (H-2K ^bm1 D ^b ) anti-TNP effector cells killed B10.A(5R)-TNP (H-2K ^b D ^d ) targets, indicating that there is cross-reactivity between TNP-H-2K^b and TNP-H-2K^bm1. On the other hand, there is no cross-reactivity between vaccinia-H-2K^b and H-2K^bm1, since Hz1 anti-vaccinia effector cells did not kill vaccinia infected B10.A(5R) targets.Since Hz1 anti-TNP effector cells lysed B10.A(5R) target cells that were first infected with vaccinia virus and then derivatized with TNP, virus does not mask cross-reactive determinants shared by TNP-H-2K^b and H-2K^bm1. Additional experiments showed that Hz1 anti-TNP effector cells lysed TNP-modified and vaccinia infected B10.A(5R) target cells irrespective of the virus concentration used for infection or the time of addition of virus. Further, there are no detectable quantitative differences between C57BL/6 and Hz1 anti-TNP effector cells in their ability to kill TNP-5R targets.The cytotoxic effect of Hz1 anti-TNP effector cells on B10.A(5R)-TNP targets could not be blocked with TNP derivatized inhibitor cells that carry theH-2D ^d region allele. Thus, the ability of anti-TNP H-2K^b effector cells to cross-react with H-2K^bm1 cannot be explained by a cross-reaction between H-2K^bm1 and H-2D^d.Abbreviations used in this paper TNP trinitrophenol - PFU plaque forming unit - Con A Concanavalin A - BSS balanced-salt-solution - FCS fetal calf serum - TNBS trinitrobenzene sulfonic acid - PBS phosphate-buffered-saline     

The Synergistic Effects among β-,3-Glucanases from Oerskovia sp. CK on Lysis of Viable Yeast Cells

Oerskovia sp. CK produced three types of β-1,3-glucanases designated as F-L, F-0 and F-2. F-L showed high lytic activity to viable yeast cells and weak activity to yeast glucan. F-0 and F-2 had little or no lytic activity and strong β-1,3-gIucanase activity.

F-0 or F-2 showed high lytic activities to yeast cells pretreated with small amounts of F-L which did not lysed the cells. Lytic activity of F-0 or F-2 also increased when cells were treated with alkaline pH or with both reducing agents and pH.

From these results, it is supposed that the ineffectiveness of F-0 or F-2 on the lysis of yeast cells might be attributed to a spatial inaccessibility of enzymes to the yeast glucan layer. However, the treatment of F-L, alkaline pH and reducing agents would bring about a modification of cells to give F-0 or F-2 access to the wall glucan and consequently the lysis of cells would occur.     

An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae

Protoplasts from various strains of red-pigmented yeasts were generated at high frequency using improved procedures. The use of sulphur-containing amino acids and 2-deoxyglucose in growth media led to impaired cell wall synthesis and rendered cells very susceptible to treatment with mercapto-ethanol and various lytic enzymes. Use of individual lytic enzymes separately resulted in relatively low frequencies of protoplasts from most of the red yeasts examined, whilst use of -glucoronidase, Novozyme and Zymolyase in series markedly increased stable protoplast formation. The latter effects were shown to be strain specific. The ability to generate large numbers of red yeast protoplasts prompted the attempt to examine intergeneric fusion between auxotrophs of a strain of Saccharomyces cerevisiae and Rhodotorula rubra. Putative hybrids were selected as variously-pigmented prototrophic colonies growing on minimal medium and stabilised by subculturing on the latter medium. Unusual cream, orange and yellow hybrid colonies were generated, composed of cells of varying morphologies (chains, multibudded). The majority of stable hybrids contained one nucleus, although several heterokaryons were also observed. Some hybrids possessed the phenotypes of both parents: fusant wcat41 grew as rapidly as the S. cerevisiae parent but also contained an inducible phenylalanine ammonia-lyase (PAL) which appeared to be more active than that of the Rhodotorula parent.     

Settlement inhibition of fouling invertebrate larvae by metabolites of the marine bacterium halomonas marina within a polyurethane coating

The marine bacterium, Halomonas marina (ATCC 27129), was shown to inhibit settlement and development of the sessile invertebrates Balanus amphitrite and Bugula neritina. Different bacterial treatments were employed to investigate this interaction. Filmed bacteria and liquid suspensions of whole cells, lysed cells and culture filtrate all reduced settlement of B. amphitrite. Polyurethane coatings containing whole cells were partially inhibitory while lysed cells caused complete inhibition of B. amphitrite larval settlement. In contrast, culture filtrate in a polyurethane matrix stimulated settlement of B. amphitrite larvae. Whole cells, culture filtrate, and lysed cells embedded in a polyurethane coating also controlled B. neritina settlement and maturation.     

Mechanism of Fermentation Conversion from Polyalcohol Fermentation to Ethanol Fermentation by Pichia miso

A possible mechanism of fermentation conversion is described from polyalcohol fermentation to ethanol fermentation by Pichia miso. Little alcohol dehydrogenase activity was found in polyalcohol-producing cells, whereas higher enzyme activity was induced by ethanol-producing cells. The fermentation conversion may be caused by the different levels of alcohol dehydrogenase activity between polyalcohol- and ethanol-producing cells. It was also shown that yeast growth was inhibited and that yeast cells were lysed by ethanol (at 6g/100ml) that accumulated in 24 hr.     

Enzymatic Release of Invertase from Cell Ghosts of Candida utilis

1. The liberation of invertase (β-fructofuranosidase, EC 3.2.1.26) from Candida utilis at autolysis of the cells was found to begin after the autolysis was almost completed. The autolysis residue at this stage consisted mainly of cell walls (ghosts). A suspension of washed cell ghosts released invertase on further incubation and this liberation was stimulated by the addition of reducing agents such as mercaptoethanol, or proteolytic enzymes such as papain, as has been known in the release of the invertase of Saccharomyces cerevisiae.

2. The invertase activity of the cell ghosts was not lost when the suspension was heated at 60°C. However, the invertase of the heated cell ghosts was not liberated even if the above stimulative agents were added.

3. Several commercial enzymes were shown to stimulate the liberation of invertase from the heated cell ghosts and “Zymolyase,? one of the effective enzymes, was fractionated. One fraction isolated from the preparation showed a striking effect on the liberation of invertase but this fraction did not show lytic activity on brewer’s yeast cells.     

A comparison of the lytic action ofCytophaga johnsonii on a eubacterium and a yeast

The non-fruiting myxobacteriumC. johnsonii will not attack living cells ofAerobacter aerogenes orSaccharomyces cerevisiae. Autoclaved cells of both organisms are however lysed but in different ways. WithA. aerogenes the cell contents are dissolved leaving an outer membrane which is not further attacked. With the yeast the wall is lysed and a structure resembling a spheroplast remains. Glucanase is produced by the myxobacterium when grown on autoclaved whole yeast. No glucanase is produced when the organism is grown on autoclavedA. aerogenes.     

A method for fractionation of cloned protein in recombinantSaccharomyces cerevisiae

In a spheroplasting method which allows the fractionation and quantification of cloned invertase activity in recombinantSaccharomyces cerevisiae cells, the yeast cell is selectively degraded with the enzyme Zymolyase for 60 minutes at 45°C to separate periplasmic proteins from cytoplasmic proteins. Most of the glucose-6-phosphate dehydrogenase (a cytoplasmic marker protein) was found in the cytoplasmic fraction.     

Yeast entrapment in Duckweeds: Immobilization performance and biomass localization within the support particles

Cell structured support material (CSM) prepared from Wolffia arrhiza fronds were loaded with Saccharomyces cereevisiae by the propagation of yeast cells introduced in the aerenchyma. The loading process could be strongly accelerated by preventing the growth of freely suspended yeasts in the fermenter. The pathway of the inoculation through the stomata could be visualized by transparent light microscopy of amylase and protease treated CSM. Slices of the loaded CSM clearly showed that the framework of the inner chlorenchyma cells remains intact during the propagation of the included yeast cells. Most of the yeast cells were found to be localized as a dense suspension within unbroken cells. During long-term treatment of the loaded CSM at high convection, about one third of the yeasts was released into water with a constant rate greater than 0.34 h^?1. The residual amount of yeast cells remained in a stable compartment from which further loss did not occur for the period tested (20–48 h). It is assumed that the stable compartment is identical with the interior of chlorenchyma cells. The results are discussed in relationship to the possible use of the immobilized biomass in fermentation.     

Release of virus-like particles by osmotic shock from a mutant strain of yeast deficient in cell integrity

Summary A yeast system based on a S. cerevisiae mutant strain deficient in cell integrity, was used to achieve the release of yeast expressed virus-like particles (VLPs) by simple osmotic shock. Yeast cells, grown in the presence of 1M sorbitol, lysed and released the intracellular content upon being transferred to non-osmotically stabilized medium. The release of VLPs was followed by Western blotting determination of the p1 protein.     

Evidence for the occurrence of autolytic enzymes in Methanobacterium wolfei

Cultures of the pseudomurein-containing archaebacterium Methanobacterium wolfei regularly lysed a short while after the energy source H₂ was exhausted, or when H₂ in growing cultures was replaced by N₂. During lysis of cells, the DNA was released into the culture medium.No intact cell wall sacculi of lysed cells could be detected, but a soluble fragment of the pseudomurein was isolated and characterized.The lysate of Methanobacterium wolfei was used to lyse other species of the genus Methanobacterium. Since no phages were detected, autolytic enzymes probably are responsible for cell lysis.     

Production of 21% (v/v) ethanol by fermentation of very high gravity (VHG) wheat mashes

Summary Very high gravity wheat mashes containing 300 g or more sugares per liter were prepared by enzymatic hydrolysis of starch and fermented with a commercial preparation of active dry yeast. The active dry yeast used in this study was a blend of several strains ofSaccharomyces cerevisiae. The fermentation was carried out at 20°C at different pitching rates (inoculation levels) with and without the addition of yeast extract as nutrient supplement. At a pitching rate of 76 million cells per g of mash an ethanol yield of 20.4% (v/v) was obtained. To achieve this yeast extract must be added to the wheat mash as nutrient supplement. When the pitching rate was raised to 750 million cells per g of mash, the ethanol yield increased to 21.5% (v/v) and no nutrient supplement was required. The efficiency of conversion of sugar to ethanol was 97.6% at the highest pitching rate. This declined slightly with decreasing pitching rate. A high proportion of yeast cells lost viability at high pitching rates. It is suggested that nutrients released from yeast cells that lost viability and lysed, contributed to the high yield of ethanol in the absence of any added nutrients.     

Thermosensitivity of the <Emphasis Type="Italic">Saccharomyces cerevisiae gpp1gpp2</Emphasis> double deletion strain can be reduced by overexpression of genes involved in cell wall maintenance

A Saccharomyces cerevisiae strain in which the GPP1 and GPP2 genes, both encoding glycerol-3-phosphate phosphatase isoforms, are deleted, displays both osmo- and thermosensitive (ts) phenotypes. We isolated genes involved in cell wall maintenance as multicopy suppressors of the gpp1gpp2 ts phenotype. We found that the gpp1gpp2 strain is hypersensitive to cell wall stress such as treatment with β-1,3-glucanase containing cocktail Zymolyase and chitin-binding dye Calcofluor-white (CFW). Sensitivity to Zymolyase was rescued by overexpression of SSD1, while CFW sensitivity was rescued by SSD1, FLO8 and WSC3—genes isolated as multicopy suppressors of the gpp1gpp2 ts phenotype. Some of the isolated suppressor genes (SSD1, FLO8) also rescued the lytic phenotype of slt2 deletion strain. Additionally, the sensitivity to CFW was reduced when the cells were supplied with glycerol. Both growth on glycerol-based medium and overexpression of SSD1, FLO8 or WSC3 had additive suppressing effect on CFW sensitivity of the gpp1gpp2 mutant strain. We also confirmed that the internal glycerol level changed in cells exposed to cell wall perturbation.     

Enzymatic production of β-D-glucose-1-phosphate from trehalose

β-D -Glucose-1-phosphate (βGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of βGlc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, βGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline βGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.     

Lysis of yeast cells by Oenococcus oeni enzymes

Oenococcus oeni exhibited extracellular β (1→3) glucanase activity. This activity increased when cells were cultivated with glycosidic cell-wall macromolecules. In addition, the culture supernatant of the organism effectively lysed viable or dead cells of Saccharomyces cerevisiae. This lytic activity appeared in the early stationary phase of bacterial growth. Yeast cells at the end of the log phase of growth were the most sensitive. The optimum temperature for lysis of viable yeast cells was 40°C, which is very different from the temperatures observed in enological conditions (15–20°C). Moreover, the rate of the lytic activity was significantly lower in comparison with yeast cell wall-degrading activities previously measured in various other microorganisms. Therefore, yeast cell death that is sometimes observed during the alcoholic fermentation could hardly be attributed to the lytic activity of O. oeni. Journal of Industrial Microbiology & Biotechnology (2000) 25, 193–197. Received 27 December 1999/ Accepted in revised form 14 July 2000