Charles Jaspers

Glutathione as an endogenous sulphur source in the yeast Saccharomyces cerevisiae

Glutathione-deficient mutants (gshA) of the yeast Saccharomyces cerevisiae, impaired in the first step of glutathione (GSH) biosynthesis were studied with respect to the regulation of enzymes involved in GSH catabolism and cysteine biosynthesis. Striking differences were observed in the content of the sulphur amino acids when gshA mutants were compared to wild-type strains growing on the same minimal medium. Furthermore, all mutants examined showed a derepression of gamma-glutamyltranspeptidase (gamm-GT), the enzyme initiating GSH degradation. However, gamma-cystathionase and cysteine synthase were unaffected by the GSH deficiency as long as the nutrient sulphate source was not exhausted. The results suggest that the mutants are probably not impaired in the sulphate assimilation pathway, but that the gamma-glutamyl cycle could play a leading role in the regulation of the sulphur fluxes. Studies of enzyme regulation showed that the derepression of gamma-GT observed in the gshA strains was most probably due to an alteration of the thiol status. The effectors governing the biosynthesis of cysteine synthase and gamma-cystathionase seemed different from those playing a role in gamma-GT regulation and it was only under conditions of total sulphate deprivation that all these enzymes were derepressed. As a consequence the endogenous pool of GSH was used in the synthesis of cysteine. GSH might, therefore, fulfil the role of a storage compound.

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants.

The white-rot fungus Cerrena unicolor C-139 produced 450 000 U l(-1) of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K(m) value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.

Pathways of glutathione degradation in the yeast Saccharomyces cerevisiae

Abstract The degradation of glutathione (GSH) in the yeast Saccharomyces cerevisiae appears to be mediated only by γ-glutamyltranspeptidase and cysteinylglycine dipeptidase. Other enzymes of the γ-glutamyl cycle, γ-glutamyl cyclotransferase and 5-oxo- l -prolinase, are not present in the yeast. In vivo transpeptidation was shown in the presence of a high intracellular level of γ-glutamyltranspeptidase, but only when the de-repressing nitrogen source was a suitable acceptor of the transferase reaction. In contrast, when the de-repressing source was not an acceptor of the transferase reaction (e.g. urea), only glutamate was detected. Intracellular GSH is virtually inert when the level of γ-glutamyltranspeptidase is low. Possible roles for in vivo transpeptidation are discussed.

Evidence for a role of manganese peroxidase in the decolorization of Kraft pulp bleach plant effluent by Phanerochaete chrysosporium: Effects of initial culture conditions on enzyme production

Abstract A role for Mn-peroxidase (MnP) produced by the white rot fungus Phanerochaete chrysosporium growing on Kraft pulp bleach plant was demonstrated. This enzyme, but not lignin peroxidase (LIP), was detected when growing the fungus under pellet form in the effluent. Enzyme production depended on the density of conidial inoculation, a factor, in turn, determining the shape of pellets. Decolorization and MnP production were obtained only when the fungus grew under the form of a fluffy pelleted material. In vitro experiments with purified MnP and LIP, have shown that only MnP has a decolorizing activity, but this was limited to about 25%. Full ‘in vivo’ decolorization, which attain more than 80%, may therefore depend on the production of other enzyme components by the fungus.

Production of laccase by immobilized cells of Agaricus sp.: induction effect of xylan and lignin derivatives.

Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogenlimited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, sacch arose, cellulose, or cellobiose. Atwo-to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experimentsare consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textilestrips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.

Glutathione metabolism in yeast Saccharomyces cerevisiae. Evidence that γ-glutamyltranspeptidase is a vacuolar enzyme

In a first experiment we have shown that S. cerevisiae beta-glutamyltranspeptidase is associated with a particulate fraction obtained by differential centrifugation. We have subsequently shown that this enzyme activity followed accurately the distribution of vacuolar markers. Liberation of vacuoles was carried out by mechanical disruption of spheroplast under isotonic conditions and the vacuoles were purified by centrifugation of Ficoll gradients. Yeast beta-glutamyltranspeptidase could be implicated in the exchanges of amino acids between the cytoplasm and the vacuolar sap.

Successive rapid reductive dehalogenation and mineralization of pentachlorophenol by the indigenous microflora of farmyard manure compost

Aims: To determine whether composting with animal manure can be used to effectively remediate soil from a pentachlorophenol (PCP)‐contaminated site, and to establish the fate of the degraded xenobiotic.

Adsorption effects in the decolorization of a Kraft bleach plant effluent by Phanerochaete chrysosporium

SummaryPhanerochaete chrysosporium was proposed for the decolorization and degradation of chlorinated compounds (AOX) present in the E1 stage effluent of the Kraft process. We observed that pellets of the fungus inoculated in the E1 effluent, not supplemented with nutrients, strongly absorb colour and AOX. The data were in agreement with a model describing a multilayer adsorption.

Molecular and kinetic properties of purified γ-glutamyl transpeptidase from yeast (Saccharomyces cerevisiae)

Abstract The enzyme γ-glutamyl transpeptidase was purified from the yeast Saccharomyces cerevisiae by a procedure involving protamine sulfate treatment, chromatography on DEAE-Sephadex A 50, salt fractionation, successive chromatography on Sephadex G 150 and lentil lectin sepharose 6B. The procedure achieves 25 % yield and 4200-fold purification. The final preparation is a glycoprotein ( M , 90 000) containing 31.4 % carbohydrates and composed of two non-identical subunits ( M , 64 000 and 23 000). The specificity patterns of the yeast enzyme are rather similar to those of mammalian and higher plant transpeptidases. The enzyme mechanism might be of the double displacement (ping-pong) type.

Production of a high level of laccase by submerged fermentation at 120-L scale of Cerrena unicolor C-139 grown on wheat bran

Submerged fermentation in a stirred bioreactor of the white rot fungus Cerrena unicolor C-139 was done at a 120-L scale in the presence of wheat bran as a cheap lignocellulosic substrate for fungus growth and laccase production. Enzyme monitoring showed that laccase production started after 2 days of cultivation, attaining a maximum activity of 416.4 U·mL(-1) at day 12 of fermentation. After treatment of culture liquid by successive micro- and ultrafiltration (5kDa), a liquid concentrate containing 22203176 units of laccase was obtained. Obtaining large amount of laccase is essential for various industrial applications, including detoxification of industrial effluents, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated area, stabilization of beverages, production of cosmetics, manufacture of anti-cancer drugs, and nanobiotechnology. The cultivation method and the fungal strain used here provided a substantial amount of enzyme produced at a price lower than 0.01 € cent/unit enzyme.