Mimicking the darwinist algorithm of natural selection, through several rounds of random mutagenesis and/or DNA recombination coupled to massive high-throughput screenings, directed molecular evolution allows to tailor enzymes more robust, stable, efficient and in general with many improved features. In this evolutionary scenario, semi-rational analysis - where researches are taking advantage from protein structural information to create and explore libraries constructed by saturation mutagenesis- constitutes also a powerful methodology. Laccases from white-rot fungi are remarkable biocatalysts due to their broad substrate specificity, with potential applications in bioremediation, lignocellulose processing, organic synthesis and more. Most of these transformations must be carried out at high concentrations of organic cosolvents where laccase undergoes unfolding, therefore loosing its catalytic performance. We have evolved the thermostable laccase from the Ascomycete Myceliophthora thermophila to tolerate high concentrations of cosolvents. The genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae, variant R2, was capable to resist a wide array of miscible cosolvents of biotechnological significance at concentrations as high as 50% (v/v). Intrinsic electrochemical laccase features such as the redox potential at the T1 and T2/T3 sites and the geometry and electronic structure of the catalytic coppers were altered during the course of the in vitro evolution experiment. Some mutations located at the surface of protein contributed to the reinforcement of the protein architecture at different key-denaturation regions by establishing new hydrogen bonds and salt bridges. Moreover, the mutations introduced at the C-terminal extension affected the protein folding at the post-translational maturation steps. Additionally, we have developed a new methodology named in vivo overlap extension (IVOE) which is based on the high homologous recombination frequency of Saccharomyces cerevisiae. This methodology provides a simple manner to build combinatorial saturation mutagenesis libraries avoiding extra PCR reactions, by-products formation and in vitro ligation steps. Several positions that seem to be related with the redox potential at the T1 site were targeted for combinatorial saturation mutagenesis. After exploring over 170000 clones, the best variant revealed a direct relationship between the highly conserved tripeptide 509VSG511, located in the vicinity of the T1 site, and the C-terminal plug. The Km O2 value of the mutant was increased 1.5-fold and the electron transfer pathway between the reducing substrate and the T1 copper ion was altered, thus improving catalytic efficiencies about 3- and 8-fold towards nonphenolic and phenolic substrates, respectively. Although the copper geometry at the T1 site was perturbed upon mutation, paradoxically the laccase redox potential was not significantly altered. Taking together, the present study suggests that 509VSG511 tripeptide may play a hithertounrecognized role in regulating the traffic of O2 to the T2/T3 copper cluster, in combination with the C-terminal plug, which is dependent on the binding of the reducing substrate at the copper T1.

204 páginas, 64 figuras y 29 tablas

Peer reviewed