Evaluation of new biorecognition elements for environmental monitoring

To date, environmental monitoring is mainly focused on traditional chemical techniques, or on the assessment of specific biomarkers. However, these analyses are affected by several limitations: mainly, they are expensive, spot-sampling and time-consuming. In order to overcome these drawbacks, new biological monitoring methods, such as biosensors and biological early warning system (BEWS) are under development. These kinds of devices, built around whole cells, enzymes and antibodies, are well-suited to cooperatively and continuously monitor the environmental conditions. The key-factor of this very promising approach is the biological sensing element. Whole cell systems and enzymes are well suited for environmental monitoring: they are able to determine the bioavailable and toxic concentration of xenobiotics, especially if the source and nature of the compound cannot be predicted. Microorganisms usually detect a broad spectrum of chemicals, and represent a good opportunity for low cost, long shelf-life, and wide range of conditions in which they can be applied. Besides, enzymes are effective when a particular kind of pollutant would be detected because is possible to fine tune their metabolic behaviour by means of protein engineering. In this work, three biological sensing elements, related to three different index of toxicity were evaluated, in order to develop new biosensors for environmental monitoring: a broad toxicity index associated to the decrease of light emission (EC50 or half effective concentration) of a bioluminescent bacterium, Vibrio fischeri, a metal toxicity connected to the metal-regulated production of a siderophore (pyoverdine) by the soil and water microorganism Pseudomonas fluorescens, and finally an index of toxicity given by PAHs, was related to the metabolization of these compounds by laccase of Trametes versicolor. One of the first step during the assessment of a new biological sensing element is the study of the effect of physical-chemical parameters. The tested physical-chemical parameters (temperature, pH, inoculum percentage (v/v) and carbon source) influenced both microbial sensible elements (V. fischeri and P. fluorescens), therefore, these sensible elements can be used in a whole-cell biosensor for in-situ application, even if the response is affected by the environmental variables. Furthermore, the light emission of V. fischeri was highly variable, although a more stable bioluminescence was obtained by means of a glucose fed-batch: this is one step towards the direct application of this system, usually tailored for laboratory assays, to estimate the broad acute toxicity directly in situ in a portable device. Regarding the interaction between P. fluorescens and Fe3+, Cu2+, and Zn2+, the minimum inhibitory concentration (MIC) and the pyoverdine critical concentration (PCC) obtained values were compared to those indicated in the WHO Guidelines for drinking water quality and in European directive 98/83/EC: MICs of Fe3+, Cu2+ and Zn2+ are always above the threshold specified, whilst PCCs are very near to the recommended thresholds for iron and copper. The PCC was not determined for zinc in the tested range of concentration and conditions. These results highlighted that this sensible element should be further investigated for the development of a biosensor able to monitor metals in the environment. The last and most promising sensing element assessed in this work was the lccβ laccase of T. versicolor. A combination of computational docking (SwissDock) and molecular biology techniques was used to generate rationally engineered laccases with increased ability to process large and persistent PAHs. These mutated isoforms were produced by heterologous expression in P. pastoris, successfully purified, and characterized by means of biochemical assays. The activity of the enzymes was initially tested and characterized with phenolic and non phenolic substrates at different pH (3.0-8.0): the best mutated enzyme F162A/L164A (M1) showed an increased specific activity (UI/mg) in comparison with the wild type, in every tested condition. This result was in agreement with those obtained by computational docking simulations (estimated free binding energy), validating the rational design approach. Moreover, decolourization assays of large aromatic dyes, used as model compounds, have shown that the mutated enzymes are reactive towards molecules with chemical structure resembling that of aromatic organic pollutants. By means of example, enzyme mutants with a larger binding pocket (e.g. M1) showed higher activity against triphenylmethane dyes (e.g. Methyl Green), especially without a mediator of the reaction (HBT), and high stability under a variety of temperature conditions (4, 22 °C, room temperature). Therefore, the best enzyme should be integrated on an appropriate transducer (e.g. electrode), and coupled to a wireless platform generating a BEWS for environmental monitoring.