A modified method based on arsenomolybdate complex to quantify cellulase activities : application to litters

In this study a simplified methodology adapted from that of Somogyi-Nelson is described in order to quantify cellulase activities in natural environments such as litters. We recommend (i) reducing drastically Na2SO4 amounts (from 90 to 4g) to improve solubility and (ii) reading absorbance at 870 nm since the highest values for the reduced arsenomolybdate complex were obtained for this wavelength

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

A modified method based on p-nitrophenol assay to quantify hydrolysis activities of lipases in litters

Lipases are glycerol ester hydrolases (EC 3.1.1.3) produced by a wide range of microorganisms. They catalyse the hydrolysis of different esters depending on the water content of the reaction medium. Here, we developed a simple methodology to quantify lipase hydrolysis activities using two different litters: a litter of Quercus pubescens (QP) and a litter of both Q. pubescens and Q. ilex. Different p-nitrophenyl esters were used to test hydrolysis in a reaction medium with an organic solvent (heptane). We showed that these activities depended on the amount of litter, the incubation time and the substrate concentration and that they increased with temperature. Furthermore, the lipases from the studied litters were still active after 2 h at 70 degrees C. These activities showed common properties of lipases: the highest activities were obtained with a medium-acyl chain substrate, p-nitrophenyl laurate. Moreover abiotic hydrolysis with short-chain acyl substrates was observable. The following parameters are recommended to quantify hydrolysis activities of lipases in litters: 10 mM of p-nitrophenyl laurate in 2 ml of heptane, 1 g of litter, 2 ml of water incubated at 30 degrees C for 2 h

Effects of anthracene on microbial activities and organic matter decomposition in a Pinus halepensis litter from a Mediterranean coastal area

Due to increasing atmospheric pollution, it has become highly important to investigate how anthropic chronic contaminations may affect ecosystem functioning. To explore the effect of polycyclic aromatic hydrocarbons (PAHs) on indigenous microbial activities, anthracene was used as a model PAH in a mesocosm experiment with Pinus halepensis litter from the Massif of Marseilleveyre (Marseille, France). The effects of anthracene on microbial activities were followed after 1- and 3- month incubations by: Catabolic Level Physiological Profile (CLPP) using ECO and FF plates and four enzyme activities (cellulase. beta-glucosidase, acid phosphatase and lipase), Moreover the chemical variations in organic matter were evaluated by solid-state C-13 NMR and C/N ratio. These experiments revealed an increase in cellulase, beta-glucosidase and phosphatase activities and a decrease in lipase activities after a 3-month incubation in the presence of anthracene. Principal Component Analysis (PCA) from CLPP showed that bacterial catabolic diversity is more influenced than that of fungal communities by anthracene. Correlation between both chemical and biological indicators revealed that the increase in lignocellulolytic enzymes (cellulase, laccase and beta-glucosidase) was significantly correlated to the decrease in phenolic compounds. In addition, aromaticity ratio also decreased in the presence of anthracene suggesting that transformation of the recalcitrant part of organic matter was enhanced. Our results highlight the difference in sensitivity of bacterial and fungal communities to PAHs, the later especially active while exposed to high concentrations of pollutant. This suggests that microbial communities inhabiting P. halepensis litters in Mediterranean coastal areas may resist to chronic pollution involving PAH

Identification of various laccases induced by anthracene and contribution to its degradation in a Mediterranean coastal pine litter

Mediterranean coastal ecosystems are known to be highly subject to natural and anthropic environmental stress. In this study, we examine the effects of anthracene as a common pollutant on the total microbial communities from a Pinus halepensis litter of a typical Mediterranean coastal site (Les Calanques, Marseille). The main objective was to identify the microbial factors leading the resilience of this ecosystem. Two questions were addressed: (i) how lignin-degrading enzymes (Laccase, Lignin-peroxidase and Mn-peroxidase) are affected by the presence of this molecule, (ii) whether the indigenous consortia are involved in its degradation in mesocosms under favorable incubation conditions (25 degrees C, 60% WHC) and after different time intervals (1 and 3 month(s)). We found a strong increase in laccase production in the presence of anthracene after 3 months, together with anthracene degradation (28% +/- 5). Moreover 9,10-anthraquinone is detected as the product of anthracene oxidation after 3 months. However neither lignin-peroxidase activity nor Mn-peroxidase activity is detected. Laccase proteins directly extracted from litter were sequenced via Nano-LC-MS/MS and reveal twelve different peptide sequences induced by the presence of anthracene in the mesocoms. Our study confirms the major detoxification role of this enzymatic system and highlights the high degradation potential of fungal species inhabiting P. halepensis litter, a factor in the resilience of Mediterranean ecosystems

Does anthracene affect microbial activities and organic matter decomposition ? A comparative study in Pinus halepensis litters from Mediterranean coastal and inland areas

The widespread concern about pollution caused by Polycyclic Aromatic Hydrocarbons (PAHs) raises the question of how they affect soil microbial communities which are potentially involved in the transformation of these pollutants. Using microcosms, we describe the effect of anthracene, a model PAH, on microbial communities inhabiting a Pinus halepensis litter from both coastal (COS) and inland (INL) Mediterranean sites. The microcosms were incubated over 3 months (25 degrees C, 60% WHC) and the effects of anthracene on microbial activities of both litters were monitored. Different enzyme activities (laccase, cellulase, beta-glucosidase and acid phosphatase) and microbial respiration were measured and variations in litter chemical composition over incubation were determined using C-13 Nuclear Magnetic Resonance (NMR) from both sites. Our results show that lignocellulolytic enzymes increased markedly after a 3-month incubation in COS microcosms, especially in the presence of anthracene, whereas INL microcosms were not similarly affected. These results show that anthracene not only has no toxic effect on the microbial activities tested but actually enhances the lignocellulolytic activities of the fungal communities from coastal litters, demonstrating the detoxification potential and resistance of stressed Mediterranean coastal ecosystems

A halotolerant laccase from Chaetomium strain isolated from desert soil and its ability for dye decolourization

13 p.-6 fig.-3 tab.A novel fungal laccase produced by the ascomycete Chaetomium sp. isolated from arid soil was purified and characterized and its ability to remove dyes was determined. Extracellular laccase was purified 15-fold from the crude culture to homogeneity with an overall yield of 50% using ultrafiltration and anion-exchange chromatography. The purified enzyme was found to be a monomeric protein with a molecular mass of 68 kDa, estimated by SDS-PAGE, and with an isoelectric point of 5.5. The optimal temperature and pH value for laccase activity toward 2,6-DMP were 60 °C and 3.0, respectively. It was stable at temperatures below 50 °C and at alkaline conditions. Kinetic study showed that this laccase showed higher affinity on ABTS than on 2,6-DMP. Its activity was enhanced by the presence of several metal ions such as Mg2+, Ca2+ and Zn2+, while it was strongly inhibited by Fe2+, Ag+ and Hg2+. The novel laccase also showed high, remarkable sodium chloride tolerance. Its ability to decolorize different dyes, with or without HBT (1-hydroxy-benzotriazole), as redox mediator, suggests that this protein may be useful for different industrial applications and/or bioremediation processes.Peer reviewe