UVB light influence on the laccase enzyme catalytic activity in reverse micelles and in homogeneous aqueous medium.

Laccase is a versatile enzyme widely used for the oxidation of environmental contaminants and exhibits great potential in many others applications; however, it undergoes photo-degradation when irradiated with UVB light. The photo-stability of this biomolecule can be improved by immobilization in different encapsulation media and reverse micelles have been employed with this purpose. The laccase activity using syringaldazine as substrate has been studied in the absence and in the presence of reverse micelles of 0.15 M of sodium 1,4-bis (2-ethylhexyl) sulfosuccinate (AOT) in isooctane at W0 ([H2O]/[AOT]) = 30, before and after irradiation of the enzyme with UVB light. The kinetic parameters, i.e., Michaelis-Menten constant (KM), catalytic constant (kCAT), and catalytic efficiency (kCAT/KM), were determined by spectroscopic measurements in the micellar system and in homogeneous aqueous medium. The distribution of the substrate in two pseudo-phases (micelle and organic solvent) was taking into account in the kinetic parameters' determinations. The results obtained indicate that the nano-aggregate system confers a solubilization media in the water core of the micelle, both for the enzyme and the substrate, in which the catalytic function of the enzyme is preserved. On the other hand, in homogeneous aqueous medium kCAT/KM value, it is reduced by ~50% after UVB irradiation of the enzyme, while in micellar medium, less than 10% of the activity was affected. This mean that the enzyme achieves a considerably photo-protection when it is irradiated with UVB light in reverse micelles as compared with the homogeneous aqueous medium. This phenomenon can be mainly due to the confinement of the biomolecule inside the micelle. Physical properties of the nano-environment could affect photochemical reactions.

Effect of UVB solar irradiation on Laccase enzyme: evaluation of the photooxidation process and its impact over the enzymatic activity for pollutants bioremediation.

The multi-copper Laccase enzyme corresponds to one of the most investigated oxidoreductases for potential uses in xenobiotic bioremediation. In this work, we have investigated the photo-degradation process of Laccase from Trametesversicolor induced by UVB light and the influence on its activity over selected substrates. Laccase undergoes photo-degradation when irradiated with UVB light, and the process depends on the presence of oxygen in the medium. With the kinetic data obtained from stationary and time resolved measurements, a photo-degradation mechanism of auto-sensitization was proposed for the enzyme. Laccase generates singlet oxygen, by UVB light absorption, and this reactive oxygen species can trigger the photo-oxidation of susceptible amino acids residues present in the protein structure. The catalytic activity of Laccase was evaluated before and after UVB photolysis over hydroxy-aromatic compounds and substituted phenols which represent potential pollutants. The dye bromothymol blue, the antibiotic rifampicin and the model compound syringaldazine, were selected as substrates. The values of the kinetic parameters determined in our experiments indicate that the photo-oxidative process of Laccase has a very negative impact on its overall catalytic function. Despite this, we have not found evidence of structural damage by SDS-PAGE and circular dichroism experiments, which indicate that the enzyme retained its secondary structure. We believe that, given the importance of Laccase in environmental bioremediation, the information found about the stability of this kind of biomolecule exposed to UV solar irradiation may be relevant in the technological design and/or optimization of decontamination strategies.