Fate of active form of proteins in selected environmental matrices : investigation with green fluorescent protein, β-glucosidase and amyloid fibrils in wastewater sludges, biofilms and soils

Thèse de doctorat en sciences agronomiques et ingénierie biologique -- UCL, 200

Monitoring the Active Conformation of Green Fluorescent Protein (GFP) and β-Glucosidase Adsorbed on Soil Particles

In order to determine the effect of various soil components on the activity of proteins, we monitored the fluorescence and the enzymatic activity of, respectively, green fluorescent protein (GFP) and β-glucosidase adsorbed on fine soil particles. We also monitored the activity of these proteins in the presence of components that are representative of soil colloids: a montmorillonite clay, goethite and organic matter extracted from soil. Upon adsorption on clay and goethite, GFP lost its fluorescence properties while β-glucosidase suffered only a partial loss of its catalytic activity. Extractable organic matter had an inactivating role on GFP while it did not cause inactivation of β-glucosidase. When GFP and β-glucosidase adsorbed on particles from natural soil samples, their behaviour was consistent with the behaviour observed for these proteins in the presence of the separate components, suggesting that the macroscopic activity of proteins adsorbed on soil particles corresponds to an average of the activities of proteins adsorbed on a mixture of surfaces. The monitoring of the proteins on soil particles with different organic matter contents has also shown that organic matter can have different effects (protecting or inactivating) on different proteins

Fate of amyloid fibrils introduced in wastewater sludge.

Laboratory data on the behaviour of the pathogenic form of the prion protein (PrP(Sc)) in environmental matrices such as sewage sludge is scarce. Direct experiments with this misfolded protein require strict safety measures, pathogen class-3 facilities and costly reagents. However, preliminary data can be generated by non-pathogenic model systems that involve lower costs and simpler manipulation. We chose amyloid-like fibrils formed from the well-studied protein lysozyme as a model because, in the case of an accidental contamination of sewage sludge, PrP(Sc) would most likely be in the form of amyloid fibrils. All amyloid fibrils have similar structural features and tend to bind to thioflavin-T, thereby enhancing the fluorescence yield of the dye. We used this fluorescence enhancement to monitor amyloid fibrils introduced into activated sludge. We observed that, in the presence of sludge flocs, the concentration of amyloid fibrils that are detectable through the enhancement of thioflavin-T fluorescence decreased as a function of time, most likely due to hydrolysis of the fibrils by sludge proteases. Some of the fluorescence loss seems also due to the binding of sludge exopolymers to amyloid fibrils

Factors affecting the fate of active proteins introduced in wastewater sludges: investigation with green fluorescent protein.

Green fluorescent protein (GFP) was used as a reporter protein to investigate the interactions and fate of the active conformation of proteins in wastewater sludge. GFP was chosen because its fluorescence is dependent on the integrity of its native conformation. We identified factors that cause the loss of GFP fluorescence when this protein is introduced in aerobic or anaerobic sludge. In both systems, soluble polymers present in the liquid fraction caused an initial loss of fluorescence, but stabilized the remaining fluorescent GFP molecules. In aerobic sludge, interaction of GFP with the sludge solids initially caused an additional loss of fluorescence but the remaining fluorescence kept fairly constant over the following hours. In anaerobic sludge, on the contrary, interaction of GFP with the sludge solids caused a temperature dependent loss of fluorescence, probably related to the presence of precipitated ferrous sulfide. No direct relationship was found between sludge protease activity and loss of GFP fluorescence, suggesting that proteases are not the primary factor controlling the fate of the active form of proteins in wastewater sludges

Microbial biomass and cellulase activity in soils under five different cocoa production systems in Alto Beni, Bolivia

Cocoa is one of the most important crops of the ‘‘Alto Beni’’ region in Bolivia. This crop is produced in different systems, among them monoculture and agroforestry. In order to determine the effect of the production system on microbiological soil characteristics, we measured microbial biomass carbon, microbial biomass nitrogen and cellulase activity and we determined the microbial quotient in soil under five different cocoa production systems (conventional monoculture, organic monoculture, conventional agroforestry, organic agroforestry and successional agroforestry) and in fallow plots. The measurements were carried out in dry and rainy season. Soil from fallow plots and soil under agroforestry had higher microbial biomass than soils under monocultures, probably due to the effect of fresh organic matter input on microbial biomass. No significant difference for microbial biomass in soil from plots subjected to organic management and soil from plots subjected to conventional management was observed, possibly because of the short time elapsed from the initial establishment of the plots. In dry season, the microbial quotient showed a significantly higher value in soils under conventional agroforestry than in soils under organic monoculture, suggesting that besides the input of fresh organic matter, mineral fertilization may play a role on the fraction of available carbon. Cellulase activity was not affected by any of the factors tested, indicating that, under our assay conditions, it was not a good indicator of changes in soil

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The fate of active proteins in environmental matrices such as wastewater sludge, soil, and organic amendments is not well understood . In the present paper, we report the use of green fluorescent protein (GFP) as a probe protein to investigate the behaviour of proteins in wastewater sludge. We developed a procedure to quantitatively detect the active form of this protein in such a matrix. The procedure is based on the fluorimetric analysis of GFP in the separated liquid and solid fractions of sludge. We then tested the suitability of the approach by monitoring GFP added to aerobic and anaerobic sludge. Under aerobic conditions at 20 degrees C, most GFP immediately associated with the sludge solid fraction. About 20% of the fluorescence due to solid fraction associated GFP was still present after 72 h, which suggests a relative persistence of proteins in this system. Under anaerobic conditions at 35 degrees C, fluorescence signal due to GFP was reduced by 90% after only 6 h