Evaluation of the Effect of Chemical or Enzymatic Synthesis Methods on Biodegradability of Polyesters

International audienceThis work compares the biodegradability of polyesters produced by an esterification reaction between glycerol and oleic di-acid (D 18:1) issued from green chemical pathways, via either classical thermo-chemical methods, or an enzymatic method using the immobilized lipase of Candida antartica B (Novozym 435). An elastomeric polymer synthesized by enzymatic catalysis is more biodegradable than an elastomeric thermo-chemical polyester synthesized by a standard chemical procedure. This difference lies in percentage of the dendritic motifs, in values of the degree of substitution, and certainly in cross-links inducing an hyper-branched structure less accessible to the lipolytic enzymes in a waste treatment plant. However, when the elastomeric polymer synthesized by enzymatic catalysis is processed at high temperature as required for certain industrial applications, it presents an identical rate of biodegradation than the chemical polyester. The advantages of the thermo-chemical methods are greater speed and lower cost. Enzymatic synthesis appears be suited to producing polyesters, devoid of metallic catalysts, which must be used without processing at high temperature to keep a high biodegradability

Effects of pollutants on laccase activities of Marasmius quercophilus, a white-rot fungus isolated from a Mediterranean schlerophyllous litter

International audienceMarasmius quercophilus is a white-rot fungus involved in carbon recycling in Mediterranean ecosystems because of its laccase production. Here we described the effect of metal ions and halide salts, on laccase activity in order to point out the action of such environmental pollutants on this enzyme of major importance. Furthermore we tested organic solvent effects on laccase reaction since reaction mixture including solvent can be used in the transformation of xenobiotics. In the case of metal ions, we found that chloride ions were responsible for inhibition while CuSO 4 and MnSO 4 enhanced laccase activity. When halides were tested, we showed the following degree of inhibition: F À > Cl À > Br À. Furthermore we found that I À was oxidized by laccase with I 2 as the product of the reaction. With ABTS, 50% of the laccase activity remains for solvent concentration ranging from 40% to 60% depending on the solvent used while with syringaldazine solvent concentration ranged from 50% to 70%. The organic solvent effects observed were probably a result of enzyme denaturation and of both enhancement of oxidised product solubilisation and of substrate solubilisation (for syringaldazine). These results show that lac-case from M. quercophilus is not rapidly inhibited by certain environmental pollutants which sustains its role in carbon turnover under pertubation. However the strong effect of chloride ion on laccase activity should be further investigated with in situ studies since this could drastically influence carbon recycling in litters from Mediterranean littoral locations

A new substrate to measure laccase activities in complex environments: Application to litters

International audienceMeasuring laccase activities in natural environments requires improvement since this oxidation usually produces radical compounds which can interfere with other molecules from these complex media, leading to misinterpretation of the results. Here the syringaldazine skeleton was used to synthesise a new substrate whose quinone is soluble in water and chemically stable. Hydrophilic moieties were added to syringaldazine at the 3 and 5 positions on both aromatic rings to enhance its solubility in water. Syringaldazine analogue 6c was selected. Laccases from two white-rot fungus strains were first tested and a strong affinity for this substrate was observed. Moreover the laccase activity found using a litter extract was higher with 6c which can be explained by the strong stability of its quinone in water. (C) 2010 Elsevier Ltd. All rights reserved

The importance of water availability in the reaction equilibrium of hydrolases in forest litters from a Mediterranean area: a study on lipases

International audienceWater is one of the main variables affecting the carbon cycle (carbon storage or assimilation) in soils or litters from the Mediterranean area, though in most studies it has been considered via soil moisture measurements only. Here, we investigated the effect of water availability as characterized by water activity, a w , on the equilibrium of enzyme reactions (hydrolysis/synthesis) for litters from three species characteristic of the Mediterranean area (Quercus pubescens L, Q. ilex L. and Pinus halepensis L.). Lipases were used as models and an organic phase was used as the reaction medium to adjust water amount. We found that the activities of hydrolysis/transesterification increased with a w , showing that both can occur at the same a w in litter. Furthermore, these lipase activities in litter decreased for a w close to 1, which has been described with purified lipases in organic media, indicating potential enzyme aggregation. Variations in a w with moisture were studied with sorption isotherms, which were found to be similar (isotherm type 2) for all the studied litters. Water activity is a crucial indicator for soils under hydric stress at small water contents, which should be considered for describing more precisely enzyme functioning and giving valuable information about carbon dynamics in soils or litters

Alkylphenol oxidation with a laccase from a white-rot fungus: Effects of culture induction and of ABTS used as a mediator

International audienceWe investigated the potential of the laccase from the white-rot fungus Marasmius quercophilus to transform certain alkylphenols (p-nonylphenol, p-octylphenol and p-t-octylphenol). We tested the reactivity of this enzyme under different conditions: in liquid cultures and using the partially purified laccase with and without 2,2 0-azino-bis-3-ehtylbenzothiazoline-6-sulfonicacid (ABTS) as a mediator. The percentage of p-t-octylphenol disappearance in liquid cultures was 69.0 ± 1.5% and 81 ± 5% after a 8-d or 15-d incubation , respectively, with p-nonylphenol, these percentages were 62 ± 4% and 91 ± 6% and with p-octyl-phenol 37 ± 3% and 65 ± 1% after a 15-d and a 21-d incubations, respectively. Induced pre-cultures were also used to inoculate the liquid cultures to enhance p-octylphenol transformation: the percentages of disappearance were 91.0 ± 0.5% and 97 ± 1% after a 8-d and a 15-d incubation, respectively. Mass spec-trometry analysis showed that the products of oxidation of p-octylphenol were dimers with a mass of 411 m/z. Furthermore, we identified a purple compound (m/z 476) formed when ABTS was added to the reaction medium with the purified laccase. This result confirms that, in complex environments such as soils or litters where many molecules can interact with the enzyme substrate or the product of oxidation , laccase activities and those of other phenoloxidases should not be measured with ABTS

Purification of a laccase from Marasmius quercophilus induced with ferulic acid: reactivity towards natural and xenobiotic aromatic compounds

International audienceIn this study, we describe the biochemical features and the ability of a new laccase isoform from a Marasmius quercophilus strain collected on evergreen oak litter to transform various aromatic compounds. This laccase, induced with ferulic acid, exhibits interesting enzymatic properties, such as great thermal stability at 30 and 40 • C (24 h), no inhibition with EDTA, Cystein or SDS. These biochemical features are quite different from those observed with the laccase of another M. quercophilus strain collected in a different area of the South of France. Thus, this study shows the degree of variation in the properties of this enzymatic system within the same species. Furthermore we demonstrate that several natural aromatic compounds were transform leading to polymerization. The transformation of chlorophenols such as 2-chlorophenol, 2,4-dichlorophenol and 2,4,6-trichlorophenol is also observed without adding mediators (2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) or 1-hydroxybenzotriazole) to the reaction mixture. Thus, the mediators used do not seem to extend the substrate range for this laccase under these experimental conditions

Identification de nouvelles molécules extraites du liège de Quercus suber L

International audienceVarious methods of suberin extraction have been used in order to identify monomers of this complex polymer. Pre-extraction of waxes has allowed us to identify for the first time 3-friedelanol as a terpen from cork. Moreover, the wax chemical composition found here varied from previous results since cerin was not identified while friedelin and betulin were. Three fractions were obtained: a polymeric, a monomeric and a low molecular weight fraction, the last of which has never before been described. 2,6-heptanediol was found to be the main compound of this fraction. Furthermore, depolymerisation at room temperature gives the same yields as those obtained at reflux, defining an easier and cheaper methodology.Différentes méthodes d'extraction de la subérine ont été utilisées afin d'identifier les constituants de ce polymère. Le 3-friedelanol a été identifié pour la première fois comme un terpène du liège. De plus, la composition chimique des cires trouvée ici diffère de résultats précédents. La dépolymérisation de la subérine a conduit à trois fractions : une polymérique, une monomérique et une comprenant des composés de faibles poids moléculaires, n'ayant jamais été décrite précédemment avec comme composé majoritaire le 2,6-heptanediol. La dépolymérisation à température ambiante a donné les mêmes taux d'extraction qu'à reflux, favorisant l'identification des molécules et définissant une méthodologie plus facile à mettre en œuvre et moins chèr

Infra-red spectroscopy reveals chemical interactions driving water availability for enzyme activities in litters of typical Mediterranean tree species

International audienceIn Mediterranean ecosystems, water is one of the main drivers of the microbial activities that support organic matter turnover in soils or litters. In addition to drought stress, coastal areas are subject to os-motic stress linked to sea spray exposure. Here we explored i) how water availability, characterized by water activity a w , is impacted by adding NaCI to litter, ii) the chemical interactions between water, NaCl and the litter matrix and iii) whether microbial activities (using lipase as a model) are affected under these conditions. Litters of two vegetal species typical of the Mediterranean area (Quercus pubescens and Pinus halepensis) were subjected to FTIR-ATR (Fourier-Transformed Infra-Red e Attenuated Total Reflectance) spectroscopy for chemical characterization. Q. pubescens and P. halepensis litters were characterized by cutin and aromatics respectively. Sorption isotherms were identical for both species litters; when NaCl was added, a shift in isotherm shape was observed at a w ranging from 0.75 to 1. FTIR also discriminated samples with and without added NaCl and revealed that cellulose is probably the polymer in interactions with ions. Very interestingly, no differences were found between lipase hydro-lytic activities with and without added NaCl: salt addition had no effect on these activities