Study of the interaction of the oxidoreductase laccase of Pleurotus ostreatus with gold nanoparticles and phenolic compounds through empirical and in silico techniques

Abstract

The evaluation of the interaction of gold nanoparticles and the phenolic compound chlorogenic acid with laccase isoforms produced by the white root fungus P. ostreatus was performed. Key interactions were evaluated both through in silico techniques and laboratory measurements. In the computational study, the stability of chlorogenic acid in the laccase active site in the presence of a gold nanoparticle was analyzed using molecular dockings and molecular dynamics. Parametrization of the non-standard 4 copper center active site of the enzyme was performed. Distances between copper ions and residues were close enough to those reported in the literature for laccases and multicopper oxidases. Chlorogenic acid was most stable near the three nuclear copper center, which is surprising because it is different from the expected kinetic configuration. Stable configurations near the mononuclear copper center were rarely found and the ligand showed a preference for histidines 143 and 145 in the enzyme active site. Presence of gold nanoparticle near the trinuclear center access of the catalytic pocket retained the ligand for longer inside the active site. Stabilization of chlorogenic acid in the laccase catalytic pocket can be related to higher activity during the oxidation processes, gold nanoparticle interference in ligand leaving active site could inhibit such oxidation as seen in the experimental work. The in silico study led to conclude that the laccase isoforms 2 and 10 share homology, 3D configuration and are 99% identical prior the translation processes. For all experiments, an enzyme extract was used where the presence of the laccase isoforms 2 and 10 was ensured. Furthermore, the experimental work was aimed to determine the influence of gold nanoparticles on chlorogenic acid oxidation and vice versa, that is, the influence of chlorogenic acid on the synthesis of gold nanoparticles. Using a central composite experimental design with temperature, amount of chlorogenic acid, and enzymatic activity as factors, it was found that the best reaction conditions for gold nanoparticle synthesis were 75,2°C and enzyme activity of 113 UL-1 in the presence of chlorogenic acid. Also, below 50°C and 80 UL-1 chlorogenic acid might inhibit nanoparticle synthesis. On the other hand, low chlorogenic acid oxidation rates were found when gold nanoparticles were present. Nanoparticle characterization was done though SEM and EDX analysis. Spherical 20 nm to 48 nm nanoparticles that agglomerate in 200 nm structures were found. Characterization suggests that a bionanoconjugate of laccase and gold nanoparticles may have formed. Key words: gold nanoparticle synthesis, laccase, active site, interaction, oxidation, stability.