Studio cinetico della laccasi da Trametes versicolor

Laccases (EC 1.10.3.2; LC) are multi-copper oxidases (MCOs) widely studied for their potentialbiotechnological applications. Laccase catalyses the oxidation of a broad substrate range which includesdiphenols, aromatic amines, polyphenols, and methoxy substituted phenols, using O₂ as terminal electronacceptor, with water as the sole by-product. This makes laccase a promising green and sustainablebiocatalyst. These enzymes, particularly the one derived from Trametes versicolor, exhibit remarkablecatalytic properties, making them valuable in various industrial processes, including pharmaceutical, foodand chemical synthesis as well as bioremediation, textile dye degradation, and lignin modification. T.versicolor is a basidiomycete fungus belonging to the white rot fungi (WRF) family, known to be amongthe largest natural producers of ligninolytic enzymes.This study aims to provide a clear determination of the reaction kinetics of laccase from T. versicolor andto investigate how environmental factors such as temperature and pH influence its activity. Understandingthese effects is crucial for identifying optimal conditions for potential industrial applications. This studyfocuses on the kinetic characterization and thermal stability of T. versicolor laccase. A detailed kineticstudy was conducted using the chromophore ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)) as a model substrate. The oxidation reaction was monitored via UV-Vis spectrophotometry, allowingprecise determination of kinetic parameters under different experimental conditions. The effect of pH wasinvestigated within the range of 3 to 6,5 using McIlvaine buffer to ensure stable and controlled conditions.Meanwhile, the influence of temperature was examined between 15°C and 35°C using a water bath and athermoelectric cell holder. The study also explored the enzyme’s thermal stability through heatdenaturation assays at various temperatures ranging from 20°C to 80°C.The reaction followed typical Michaelis-Menten kinetics, with kinetic constants Vmax and km determinedusing the Lineweaver-Burk linearization method. The results revealed that while the enzyme exhibitedoptimal activity at 35°C and pH 3, prolonged exposure to these optimal conditions led to progressiveinactivation. The thermal stability study demonstrated that elevated temperatures compromise theprotein’s structural integrity, leading to a loss of catalytic activity. Kinetic data were analysed to estimatethe activation energy (Ea) of the reaction, which was found to be 24.65 kJ/mol. The enzyme's catalyticefficiency was evaluated in terms of the turnover number (kcat), providing insight into its performanceacross different temperature conditions and corroborating the observed kinetic trends.This study may contribute to a deeper understanding of laccase kinetics and thermal behaviour, providingcritical data for optimizing enzymatic processes in biotechnological and environmental applications.The results confirm that time, pH, and temperature are key factors influencing enzyme activity andhighlight the importance of identifying appropriate temperature and pH conditions to achieve high catalyticactivity. Future research should focus on optimizing the process and evaluating the possibility ofimmobilising the enzyme on suitable supports to enhance its stability and allow its reuse in large-scaleapplications.