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 mu M and 14.4 +/- 0.1 s(-1) and for SGZ of 12.7 +/- 2.6 mu M and 6.9 +/- 0.6 s(-1), respectively. A repeated-batch decolorization experiment was carried out at pH 10.0, 40 degrees 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. (C) 2014 Elsevier Ltd. All rights reserved.