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

摘要

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 K_m and k_(cat) 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 ℃ 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.