Biocatalyst Engineering

Evolution of laccases to improved biocatalysts through strategies of directed evolution techniques is being addressed in our lab. For that purpose, efficient and reliable expression systems and high-throughput screenings have been established (Brissos et al. 2009). Reproducible high-throughput screenings for the oxidation of standard oxidizing substrates and for the decolourisation of high redox potential dyes have been developed and optimized. Our present aim is to select variants with improved stability towards organic solvents. Indeed, CotA-laccase presents a rather low stability towards methanol, DMSO and acetonitrile, solvents required to solubilize natural mediators and lignin related substrates.

PpAzoR from P. putida shows a broad substrate acceptance but a low kinetic thermostability. Five rounds of mutagenesis/recombination followed by high-throughput screening (≈ 10,000 clones) yielded the hit 1B6 showing 300-fold higher half life at 50°C than that of homodimeric PpAzoR azoreductase from Pseudomonas putida MET94 (Brissos et al. Submitted). The characterization using fluorescence, calorimetry and light scattering shows that 1B6 superior kinetic stability is due to increased resistance of the unfolded monomers to aggregation. 1B6 shows a dimeric folded state less stable than the wild type (slightly lower melting and optimal temperature) but in contrast it hardly suffers irreversible denaturation. Directed evolution approaches have been widely taken to improve stability of the native (functional) state of proteins and rarely to impart non-aggregation behaviour. The aggregation resistance of 1B6 is mainly due to mutations that disturb hydrophobic patches and introduce surface net charges. We have also examined 2A1 and 2A1-Y179H variants with increased thermodynamic stability (10 to 20°C higher melting and optimal temperatures than wild type) and show that mutations that lead to improved structural robustness are involved in strengthening solvent-exposed loops or inter-dimer interactions of the folded state.


MET | Microbial & Enzyme Technology 2013