The primary plant cell wall is composed of cellulose, hemicellulose, lignin and protein in a stable matrix. The concomitant depolymerization of lignin by laccase and of hemicelluloses by xylanase can improve lignocellulose degradation in the production of second generation biofuels. A thermophilic variant of xylanase A (XynAG3) and the thermostable laccase (CotA), both from Bacillus subtilis, were produced in co-transformed Pichia pastoris strain GS115. Mobility changes in SDS-PAGE after Endo H digestion indicat63 that both enzymes were glycosylated. The maximum catalytic activity of the XynAG3(Pp) and the CotA(Pp) was observed at 58 degrees C and 75 degrees C, respectively, and both enzymes presented high activity at pH 5.0. The half-life at 60 degrees C of XynAG3(Pp) and CotA(Pp) was 150 min and 540 min, respectively. The relative levels of CotA(Pp) and XynAG3(Pp) in culture broths were altered by the concentration of methanol used for induction, and CotA(Pp):XynAG3(Pp) ratios of 1:1.5 and 1:2 were evaluated against milled sugar-cane bagasse. The highest activity was observed at a 1:2 ratio of CotA(Pp):XynAG3(Pp), and was 44% higher as compared to the sum of the activities of the individual enzymes in the same assay conditions. These results demonstrate the synergistic action between an endoxylanase and a laccase against the natural lignocellulosic substrate. (C) 2014 Elsevier Ltd. All rights reserved.