Enhanced expression of an industry applicable CotA laccase from Bacillus subtilis in Pichia pastoris by non-repressing carbon sources together with pH adjustment: Recombinant enzyme characterization and dye decolorization

• The CotA laccase gene from Bacillus subtilis was expressed in Pichia pastoris.
• Sorbitol together with pH adjustment enhanced the rCotA production by 76-fold.
• The rCotA laccase demonstrated a remarkable stability under alkaline conditions.
• Recombinant laccase showed an excellent capacity for repetitive decolonization of indigo carmine.

In this study, protease-deficient Pichia pastoris strain SMD1168H was selected for heterologous expression of the CotA laccase from Bacillus subtilis. Four non-repressing carbon sources were individually applied to facilitate the recombinant laccase production. An up to 76-fold increase in laccase activity was achieved through sorbitol addition in combination with pH adjustment. Recombinant CotA (rCotA) demonstrated a remarkable stability under alkaline conditions, the enzyme retained 637% and 94.37% of its initial activity after 10-day incubation at pH 9.0 and 10.0, respectively. The rCotA laccase showed an outstanding thermostability and exhibited higher tolerance towards organic solvents than the spore laccase. The Km and kcat values of rCotA laccase for ABTS were of 146.4 ± 2.7 μM and 14.4 ± 0.1 s−1 and for SGZ of 12.7 ± 2.6 μM and 6.9 ± 0.6 s−1, respectively. A repeated-batch decolorization experiment was carried out at pH 10.0, 40 °C to evaluate the capacity of rCotA for repetitive decolorization of indigo carmine under alkaline conditions. During the first six repeated cycles, more than 95% decolorization was observed in 10 min and total color was removed within one hour. The decolorization efficiency stayed above 90% after twenty decolorization cycles.

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