Recombinant expression of a laccase from Coriolopsis gallica in Pichia pastoris using a modified α-factor preproleader

- The gene from laccase of Coriolopsis gallica UAMH8260 was isolated and sequenced.
- The protein was expressed in Pichia pastoris, using a modified α-factor preproleader reported to enhance laccase secretion.
- The recombinant enzyme showed similar kinetic constants to those of the wild type enzyme.
- The expression system is amenable for protein engineering of this versatile enzyme.

In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhanced secretion and pAOX1, a methanol-responsive promoter. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in this heterologous system. A volumetric activity of 250 U/L was achieved after 12-day culture in Fernbach flasks. The protein was recovered from the supernatant and purified, obtaining a preparation with 90% electrophoretic purity. The catalytic constants of the recombinant enzyme are almost identical to the fungal enzyme, thus rendering this system a useful tool for protein engineering of laccase from C. gallica.