Obtenção e caracterização de uma lacase amarela recombinante de Leucoagaricus gongylophorus

Abstract

Laccases are enzymes that have a wide range of applications including their use in bioremediation. These enzymes have been described as multi copper oxidases that reduce oxygen to water while oxidizing compounds. Some laccases not present absorbance at 600 nm and blue color which are common in these enzymes and, for this reason, they have been denominated yellow laccases. An important feature of yellow laccases is the ability to degrade non-phenolic substrates in the absence of chemical mediators. However, the origin and structural factors that differentiate the blue and yellow laccases are not well understood. The objectives of this work were to express, purify and characterize a recombinant yellow laccase (LacRLg) from a cDNA library of Leucoagaricus gongylophorus, the symbiont fungus of leaf cutting ants. From the clone of the fungus cDNA library, the laccase ORF was cloned into the pPICZαA expression vector, resulting in the clone pPICZαA-Lac which was transformed into three strains of the yeast Pichia pastoris, X-33, KM71H and GS115. This system proved to be efficient in expressing the enzyme in soluble and active form, and a colony of the X-33 lineage was more effective in the expression of the enzyme. LacRLg was expressed in the extracellular medium and purified on a molecular exclusion column, followed by an ion exchange column. The recombinant enzyme has an apparent molecular mass of approximately 66 kDa as assessed by SDS-PAGE. LacRLg does not absorb light at 600 nm and therefore does not show blue coloration and can be classified as a yellow laccase. The enzyme presented catalytic activity against syringaldizine, considered a specific substrate for laccases. The enzymatic extract was used in to evaluate the potential of degradation of polycyclic aromatic hydrocarbons (HPA's) in the absence of mediators. HPA's are organic pollutants of persistence and their degradation plays a role in maintaining the environment. The degradability of HPA's by LacRLg was evaluated against the anthracene, phenanthrene and pyrene in a LC system to UV-Vis detector and SPD M10A. LacRLg was able to degrade, in 24 hours, 95% of anthracene, 87% of phenanthrene and 88% of pyrene. These results suggest that this enzyme, due to its ability to degrade HPA's in the absence of chemical mediators, has potential use in bioremediation processes.