Active bionanoconjugates of laccase and gold nanoparticles: kinetic and structural studies

Thesis for the master degree in Structural and Functional Biochemistry

The work presented here had the objective of using gold nanoparticles (AuNPs) functionalized with mercaptoundecanoic acid (MUA), or the peptide CALNN, to develop bionanoconjugates (BNCs) with the enzyme Rhus vernicifera (Rv) laccase. Laccases are multi copper oxidases able to catalyze the oxidation of a variety of phenolic compounds with the reduction of molecular oxygen to water, and conjugating this enzyme with AuNPs could potentially contribute to enhance its catalytic activity. Spectroscopic, electrophoretic, kinetic, and computer modelling studies were carried out in order to characterize the enzyme structurally and investigate its stability and activity when conjugated with AuNPs. The studies performed revealed that Rv laccase is a monomer with 5.65 ± 0.83 nm in diameter and presents an optimal activity at pH 7.5, when syringaldazine was used as substrate. Laccase was successfully adsorbed to AuNP-CALNN but not to AuNP-MUA. BNCs with laccase and AuNP-CALNN remained active in the experimental conditions tested at pH from 6 – 8.5. Laccase followed a Michaelis-Menten kinetic model using syringaldazine as a substrate, in the pH range 6 – 8.5. Parallel studies with free laccase were conducted to evaluate the influence of AuNP-CALNN on laccase activity. These kinetic studies were performed by following substrate consumption and product formation. The kinetic results with less error were obtained when the reaction of product formation was followed. Therefore, the results for product formation were considered to be more reliable than the results obtained for substrate consumption. The results for product formation revealed that the catalytic efficiency of BNCs was more than 50% higher at pH 7, almost 100% higher at pH 7.5 and 30% higher at pH 8 in comparison with free laccase. At pH 6 there was a decrease of approximately 38% in the catalytic efficiency. The catalytic efficiency was similar in BNCs and free laccase at pH 6.5 and 8.5. In the pH range 6.5 to 8.5, AuNP-CALNN proved beneficial by either maintaining or increasing the catalytic activity of laccase. Using AuNP-CALNN to immobilize laccase might allow decreasing industrial costs, recycling the enzyme and improving the outcome of processes usually achieved with free laccase, such as delignification, wine clarification, textile dye bleaching or waste water detoxification.