Production and manipulation of blue copper oxidases for technological applications

Simeng Zhou (1) , Pierre Rousselot-Pailley (1) , Lu Ren (1) , Yolande Charmasson (1) , Elise Courvoisier Dezord (1) , Viviane Robert (1) , Thierry Tron (1) , Yasmina Mekmouche (1) , Elise Courvoisier Dezord (1)

1 ISM2 - Institut des Sciences Moléculaires de Marseille

Fungal laccases are robust multicopper oxidoreductases. Perfectly amenable to synthetic evolution, the fungal laccase scaffold is a potential generic for the production of tailored biocatalysts, which, in principle, can be secreted at substantial levels in industrially relevant organisms. In this chapter, the strategy we have developed for the rapid production of hundreds of milligram of laccase variants is detailed. It is based on the use of two heterologous expression hosts: the yeast Saccharomyces cerevisiae for a rapid upstream screening and the fungus Aspergillus niger for downstream production. Methods for screening active and nonactive laccase variants, convenient setups for enzyme production in both organisms as well as a methodology for efficient purification of large amounts of recombinant enzymes are given. The general procedure for developing new materials for artificial catalysis is also described

Mots clés: Laccase heterologous expression; Saccharomyces cerevisiae; Homeologous recombination; Directed mutagenesis; Hybrid catalyst; Surface modification; Aspergillus niger

Domaines: Chimie organique; Catalyse; Biotechnologies; Biochimie [q-bio.BM]

Simeng Zhou, Pierre Rousselot-Pailley, Lu Ren, Yolande Charmasson, Elise Courvoisier Dezord, et al.. Production and manipulation of blue copper oxidases for technological applications. Methods in Enzymology, 2018, 613, pp.17-61. ⟨10.1016/bs.mie.2018.10.015⟩. ⟨hal-02396850⟩