No abstract

While the Pycnoporus cinnabarinus laccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression in Saccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the ␣-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved ␣-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase in k cat . While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted by Aspergillus niger (ϳ23 mg/ liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

The capabilities of p-coumaric acid (PCA), ferulic acid (FA), and sinapic acid (SA) as laccase mediators are compared in oxidation of industrial dyes and polycyclic aromatic hydrocarbons (PAH). SA behaved as highly efficient mediator in decolorization of dyes, including the recalcitrant Reactive Black 5. This mediating capacity was related to the specificity constant of the enzyme oxidizing this p-hydroxycinnamic acid, which was 16 times higher than for the typical substrate 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS). The kinetics of ABTS oxidation by laccase in the presence of p-hydroxycinnamic acids suggested that the stable phenoxyl radical of a SA transformation product acts as laccase mediator. On the other hand, FA and, especially PCA, easily mediated benzo[a]pyrene oxidation, the latter also promoting the oxidation of the more recalcitrant phenanthrene. Phenanthrene transformation by laccase-PCA was enhanced by Tween 80. This fact, together with the detection of TBARS (thiobarbituric acid-reactive-substances) from unsaturated fatty acids, revealed that laccase can also initiate lipid peroxidation reactions in the presence of p-hydroxycinnamic acids enabling oxidation of the most recalcitrant PAH.