Elimination of bisphenol a and triclosan using the enzymatic system of autochthonous colombian forest fungi.

Bisphenol A (BPA) and triclosan (TCS) are known or suspected potential endocrine disrupting chemicals (EDCs) which may pose a risk to human health and have an environmental impact. Enzyme preparations containing mainly laccases, obtained from Ganoderma stipitatum and Lentinus swartzii, two autochthonous Colombian forest white rot fungi (WRF), previously identified as high enzyme producers, were used to remove BPA and TCS from aqueous solutions. A Box-Behnken factorial design showed that pH, temperature, and duration of treatment were significant model terms for the elimination of BPA and TCS. Our results demonstrated that these EDCs were extensively removed from 5 mg L(-1) solutions after a contact time of 6 hours. Ninety-four percent of TCS and 97.8% of BPA were removed with the enzyme solution from G. stipitatum; 83.2% of TCS and 88.2% of BPA were removed with the L. swartzii enzyme solution. After a 6-hour treatment with enzymes from G. stipitatum and L. swartzii, up to 90% of the estrogenic activity of BPA was lost, as shown by the yeast estrogen screen assay. 2,2-Azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) was used as a mediator (laccase/mediator system) and significantly improved the laccase catalyzed elimination of BPA and TCS. The elimination of BPA in the absence of a mediator resulted in production of oligomers of molecular weights of 454, 680, and 906 amu as determined by mass spectra analysis. The elimination of TCS in the same conditions produced dimers, trimers, and tetramers of molecular weights of 574, 859, and 1146 amu. Ecotoxicological studies using Daphnia pulex to determine lethal concentration (LC50) showed an important reduction of the toxicity of BPA and TCS solutions after enzymatic treatments. Use of laccases emerges thus as a key alternative in the development of innovative wastewater treatment technologies. Moreover, the exploitation of local biodiversity appears as a potentially promising approach for identifying new efficient strains for biotechnological applications.

Effect of culturing processes and copper addition on laccase production by the white-rot fungus Fomes fomentarius MUCL 35117.

AIM: To produce high laccase activities from the white-rot fungus Fomes fomentarius. METHODS AND RESULTS: Different culturing methods, viz, cell immobilization on stainless steel sponges and plastic material and solid-state fermentation (SSF) using wheat bran as substrate were used for laccase production by the white-rot fungus F. fomentarius. The SSF study expresses the highest laccase activities, nearly to 6400 U l(-1) after 13 days of laboratory flasks cultivation. When the wheat bran medium was supplemented with 2 mmol l(-1) copper sulfate, laccase activity increased by threefold in comparison to control cultures, reaching 27 864 U l(-1). With the medium thus optimized, further experiments were performed in a 3 l fixed-bed bioreactor (working volume 1.5 l) leading to a laccase activity of about 6230 U l(-1) on day 13. CONCLUSIONS: The results obtained clearly showed the superiority of wheat bran for laccase production over stainless steel sponges and plastic material. Supplementing the wheat bran solid medium with 2 mmol l(-1) copper sulfate allowed obtaining high activities at flask scale. The system was scaled to fixed-bed laboratory reactor. SIGNIFICANCE AND IMPACT OF THE STUDY: The high enzyme production along with the low-cost of the substrate, showed the suitability of the system F. fomentarius- SSF for industrial purposes.

Olive oil mill wastewater purification by combination of coagulation- flocculation and biological treatments.

In order to define an efficient pre-treatment of Olive Oil Mill Wastewater (OOMW) to overcome major obstacles to biological treatment, various organic and mineral coagulants have been tested. In particular, the application of quicklime until a pH around 12 - 12.4 was reached, allowed the reduction of almost 37% of the initial COD, and approximately 88% and 71% of the colour and phenolic content of the waste. Hence, further biological treatments with an adapted aerobic consortium (AC) and a white rot fungus (WRF) strain were improved. The WRF Coriolopsis polyzona was more efficient than AC to reduce colour and polyphenols when the waste was prior diluted or pre-treated; however, it was less effective in COD removal. The combined treatment: lime - AC of OOMW having initial COD of 102 g l(-1) led to the elimination of about 77, 91 and 63%, of the COD, phenols and colour, respectively. Interestingly, the opposite combination AC - lime permitted better COD, phenols and colour reduction to respectively, 21, 11 and 11% of the initial values. This latter condition is technically recommended since only one step separation was needed and no pH correction was necessary before undergoing aerobic treatment. Moreover, the process would produce a sludge potentially rich in organic matter, and consequently, useful as an agricultural amendment or/and as an additive in animal nutrition.

In situ localization of manganese peroxidase production in mycelial pellets of Phanerochaete chrysosporium.

The ultrastructure of Phanerochaete chrysosporium hyphae from pellets in submerged liquid cultures was investigated in order to learn more about the interrelation between fungal architecture and manganese peroxidase (MnP) production. At day 2 of cultivation, some subapical regions of hyphae in the outer and middle zones of the pellet initiated differentiation into intercalary thick-walled chlamydospore-like cells of about 10 micro m diameter. At the periphery of the cytoplasm of these cells, a large number of mitochondria and Golgi-like vesicles were observed. The sites of MnP production were localized at different stages of cultivation by an immunolabelling procedure. The immunomarker of MnP was mainly concentrated in the chlamydospore-like cells and principally distributed in Golgi-like vesicles located at the periphery of the cytoplasm. The apices of hyphae in the outer layer of the pellets were apparently minor sites of MnP production. Maximal MnP release into the culture supernatant coincided with apparent autolysis of the chlamydospore-like cells. Production of extracellular autolytic chitinase and protease coincided with the disappearance of these structures from the pellets. The chlamydospore-like cells observed in the mycelial pellets of P. chrysosporium could be metabolically active entities operating as an enzyme reservoir, delivering their content into the surrounding medium possibly by an enzyme-mediated autolytic process.

Glutathione metabolism of Acremonium chrysogenum in relation to cephalosporin C production: is gamma-glutamyltransferase in the center?

Methionine increased the intracellular glutathione (reduced) (GSH) pool and the specific gamma-glutamyltransferase (gamma-GT) activity in the cephalosporin C (CPC) producer Acremonium chrysogenum. The accelerated turnover of GSH might be indicative of the existence of a functioning gamma-glutamate cycle, and might supply the CPC biosynthetic machinery with L-cysteine. The gamma-GT was not subject to nitrogen metabolic repression but was more active in cells exposed to different oxidative-stress-generating agents. Exogenous cysteine hindered both the uptake of methionine and the induction of gamma-GT, and was not beneficial for CPC production. There was no causal relationship between the redox status of the cells and the observed cell morphology.

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. METHODS AND RESULTS: Contaminated soil from a sawmill site was mixed with farm animal manure and composted in a 0.5 m3 silo under fully aerobic conditions. The disappearance and fate of PCP was monitored by gas chromatography (GC-ECD) and extensive mineralization confirmed in experiments with 14C-radiolabelled PCP. The disappearance of PCP was rapid and virtually complete within 6 days, prior to the onset of thermophilic conditions. Dechlorination of the PCP was found to be both reductive and sequential. CONCLUSIONS: PCP removal from contaminated soil by aerobic composting with animal manure is efficient and proceeds via reductive dechlorination to virtually complete mineralization. This contrasts with other chlorophenol composting regimes in which mineralization is achieved but dechlorination intermediates do not accumulate to detectable levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study demonstrate that anaerobic reductive dechlorination can proceed in an aerobic composting environment and contribute to efficient pentachlorophenol removal. Farmyard manure composts may represent a rapid, low-cost, low-technology option for treatment of chlorophenol-contaminated soils.

gamma-Glutamyl transpeptidase in the yeast Saccharomyces cerevisiae and its role in the vacuolar transport and metabolism of glutathione.

In the yeast Saccharomyces cerevisiae, the enzyme gamma-glutamyl transpeptidase (gamma-GT; EC 2.3.2.2) is a glycoprotein that is bound to the vacuolar membrane. The kinetic parameters of GSH transport into isolated vacuoles were measured using intact vacuoles isolated from the wild-type yeast strain Sigma 1278b, under conditions of gamma-GT synthesis (nitrogen starvation) and repression (growth in the presence of ammonium ions). Vacuoles devoid of gamma-GT displayed a K(m) (app) of 18+/-2 mM and a V(max) (app) of 48.5+/-5 nmol of GSH/min per mg of protein. Vacuoles containing gamma-GT displayed practically the same K(m), but a higher V(max) (app) (150+/-12 nmol of GSH/min per mg of protein). Vacuoles prepared from a disruptant lacking gamma-GT showed no increase in V(max) (app) with nitrogen starvation. From a comparison of the transport data obtained for vacuoles isolated from various reference and mutant strains, it appears that the yeast cadmium factor 1 (YCF1) transport system accounts for approx. 70% of the GSH transport capacity of the vacuoles, the remaining 30% being due to a vacuolar (H(+)) ATPase-coupled system. The V(max) (app)-increasing effect of gamma-GT concerns only the YCF1 system. gamma-GT in the vacuolar membrane activates the Ycf1p transporter, either directly or indirectly. Moreover, GSH accumulating in the vacuolar space may exert a feedback effect on its own entry. Excretion of glutamate from radiolabelled GSH in isolated vacuoles containing gamma-GT was also measured. It is proposed that gamma-GT and a L-Cys-Gly dipeptidase catalyse the complete hydrolysis of GSH stored in the central vacuole of the yeast cell, prior to release of its constitutive amino acids L-glutamate, L-cysteine and glycine into the cytoplasm. Yeast appears to be a useful model for studying gamma-GT physiology and GSH metabolism.

Nitrification and autotrophic nitrifying bacteria in a hydrocarbon-polluted soil.

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.

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 nitrogen-limited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, saccharose, cellulose, or cellobiose. A two- 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. Experiments are 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, textile strips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.

Thiram and dimethyldithiocarbamic acid interconversion in Saccharomyces cerevisiae: a possible metabolic pathway under the control of the glutathione redox cycle.

A rapid decrease of intracellular glutathione (GSH) was observed when exponentially growing cells of Saccharomyces cerevisiae were treated with sublethal concentrations of either dimethyldithiocarbamic acid or thiram [bis(dimethylthiocarbamoyl) disulfide]. The underlying mechanism of this effect possibly involves the intracellular oxidation of dimethyldithiocarbamate anions to thiram, which in turn oxidizes GSH. Overall, a linear relationship was found between thiram concentrations up to 21 microM and production of oxidized GSH (GSSG). Cytochrome c can serve as the final electron acceptor for dimethyldithiocarbamate reoxidation, and it was demonstrated in vitro that NADPH handles the final electron transfer from GSSG to the fungicide by glutathione reductase. These cycling reactions induce transient alterations in the intracellular redox state of several electron carriers and interfere with the respiration of the yeast. Thiram and dimethyldithiocarbamic acid also inactivate yeast glutathione reductase when the fungicide is present within the cells as the disulfide. Hence, whenever the GSH regeneration rate falls below its oxidation rate, the GSH:GSSG molar ratio drops from 45 to 1. Inhibition of glutathione reductase may be responsible for the saturation kinetics observed in rates of thiram elimination and uptake by the yeast. The data suggest also a leading role for the GSH redox cycle in the control of thiram and dimethyldithiocarbamic acid fungitoxicity. Possible pathways for the handling of thiram and dimethyldithiocarbamic acid by yeast are considered with respect to the physiological status, the GSH content, and the activity of glutathione reductase of the cells.

In vitro inactivation of yeast glutathione reductase by tetramethylthiuram disulphide.

Previous in vivo investigations have shown that glutathione reductase is one of the sites of action of the dithiocarbamate fungicide tetramethylthiuram disulphide (thiram) in the yeast Saccharomyces cerevisiae. The inactivation of glutathione reductase by thiram has now been demonstrated in vitro. This inactivation was time-dependent and occurred only with the enzyme in the reduced state and in the absence of glutathione. Since the turnover rate of the enzyme with thiram as a substrate was significantly higher than the rate of enzyme inactivation, it was suggested that more than one enzyme-inhibitor complex was involved in the reaction. Arguments supporting a covalent modification of glutathione reductase were further obtained by experiments carried out with [14C]thiram and gel filtration. A kinetic scheme for the inactivation is proposed and the relevance of the in vitro data to previous in vivo studies is discussed taking into consideration current concepts of glutathione reductase inactivation by affinity reagents.

Acid-precipitable polymeric lignin from hardwood lignocellulose: production by a Streptomyces sp.

A Streptomyces sp. isolate, from decayed wood shavings, solubilized lignocellulose (LC) and lignin of Pinus radiata, producing about 50 mg acid-precipitable polymeric lignin per g LC. The product was poor in protein and carbohydrates and contained mainly vanillin, guaicol, vanillic and ferulic acids. Hardwood LC is thus suitable for producing APPL as a phenolic chemical feedstock.

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.

Lignocarbohydrate solubilization from straw by actinomycetes.

Actinomycetes grown on wheat straw solubilized a lignocarbohydrate fraction which could be recovered by acid precipitation. Further characterization of this product (APPL) during growth of Streptomyces sp. strain EC1 revealed an increase in carboxylic acid and phenolic hydroxyl content, suggesting progressive modification. This was also observed in dioxane-extracted lignin fractions of degraded straw, and some similarity was further suggested by comparative infrared spectroscopy. However, the molecular weight profile of APPL was relatively constant during growth of Streptomyces sp. strain EC1 on straw, while analysis of the dioxane-extracted lignin fractions appeared to show fragmentation followed by repolymerization. Lignocarbohydrate solubilization could be monitored in all cultures by routine assay of APPL-associated protein, which accounted for up to 20% of the extracellular culture protein in some cases. Interestingly, this protein fraction was found to include active hydrolytic and oxidative enzymes involved in the degradation of lignocellulose, and specific enzyme activities were often increased in the acid-insoluble fractions of culture supernatants. This was particularly important for peroxidase and veratryl oxidase activities, which could be readily detected in the acid-precipitable lignocarbohydrate complex but were virtually undetectable in untreated culture supernatants.

[Characterization of gamma-glutamylamidase-glutaminase activity in Saccharomyces cerevisiae]. / Caractérisation d'une activité gamma-glutamylarylamidase-glutaminase chez Saccharomyces cerevisiae.

In a foregoing paper we have shown the presence in the yeast Saccharomyces cerevisiae of an enzyme catalyzing the hydrolysis of L-gamma-glutamyl-p-nitroanilide, but apparently distinct from gamma-glutamyltranspeptidase. The cellular level of this enzyme was not regulated by the nature of the nitrogen source supplied to the yeast cell. Purification was attempted, using ion exchange chromatography on DEAE Sephadex A 50, salt precipitations and successive chromatographies on DEAE Sephadex 6B and Sephadex G 100. The apparent molecular weight of the purified enzyme was 14,800 as determined by gel filtration. As shown by kinetic studies and thin layer chromatography, the enzyme preparation exhibited only hydrolytic activity against gamma-glutamylarylamide and L-glutamine with an optimal pH of about seven. Various gamma-glutamylaminoacids, amides, dipeptides and glutathione were inactive as substrates and no transferase activity was detected. The yeast gamma-glutamylarylamidase was activated by SH protective agents, dithiothreitol and reduced glutathione. Oxidized glutathione, ophtalmic acid and various gamma-glutamylaminoacids inhibited competitively the enzyme. The activity was also inhibited by L-gamma-glutamyl-o-(carboxy)phenylhydrazide and the couple serine-borate, both transition-state analogs of gamma-glutamyltranspeptidase. Diazooxonorleucine, reactive analog of glutamine, inactivated the enzyme. The physiological role of yeast gamma-glutamylarylamidase-glutaminase is still undefined but is most probably unrelated to the bulk assimilation of glutamine by yeast cells.

Identification and evolution of the cellulolytic microflora present during composting of cattle manure: on the role of Actinomycetes sp.

Population changes in cellulolytic microflora were studied during composting of cattle manure. Six bacterial and nine actinomycetes species were isolated. Among the isolates, two actinomycetes, Micromonospora chalcae and Pseudonocardia thermophila, were cellulolytic strains which were numerically dominant and might therefore play a notable role in the degradation of cellulose in cattle manure. Fungi were practically absent during the process, but two cellulose-degrading bacterial species, Sporocytophaga myxococcoides and Cytophaga hutchinsonii, appeared to coexist in equilibrium with actinomycetes during the maturation phase.

Growth and cellulase production of Micromonospora chalcae and Pseudonocardia thermophila.

Beta(1,4)glucosidases and carboxymethylcellulases were demonstrated in both strains when using carboxymethylcellulose as a carbon source for growth. Beta(1,4)Glucosidases appeared mainly as cell-bound activities, whereas carboxymethylcellulases were evenly distributed between the incubation fluids and the cellular fractions. In both microorganisms, glucose appeared to repress biosynthesis of the enzymes, and cellobiose and carboxymethylcellulose acted as inducers of the cellulase complex.

Glutathione metabolism in yeast Saccharomyces cerevisiae. Evidence that gamma-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.

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae.

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.