Expressão de xilanases de Aspergillus nidulans em Pichia pastoris: purificação, caracterização e aplicação na hidrólise do bagaço de cana-de-açúcar

Abstract

Sugarcane bagasse is the most abudant agricultural waste in Brazil and has a high potential to be used in the production of second generation ethanol. However, the high complexity of lignocellulosic biomass requires different enzymes for efficient hydrolysis. In this study, two xylanases from Aspergillus nidulans, AN1818.2 and AN3613.2, cloned in Pichia pastoris were purified, biochemically characterized, kinetically characterized and applied in sugarcane bagasse hydrolysis. BMMY medium was used to induce xylanases gene expression. This medium contains methanol, which induces AOX1 promoter, involved in methanol metabolism. The enzymatic extract was submitted to affinity (His Trap HP) and gel filtration (Superdex 200) chromatographies. Xylanase AN 1818.2 is optimally active at 60 oC and in pH 7,5. AN3613.2 was optimally active at 50 oC and in pH 6,0. Both xylanases were higly thermostable at 50 oC and in pH range from 2,5 to 13 and less stable at 60 oC. HgCl2, SDS, CuSO4 and xylotriose inhibited enzymatic activities. Wheat arabinoxylan was the best substrate for AN1818.2 hydrolysis and AN3613.2 is better suitable for beechwood xylan hydrolysis. Km for AN1818.2 and AN3613.2 was 1,66 and 4,23 mg/mL, respectively, using beechwood xylan as substrate. Sugarcane bagasse hydrolysis with Multifect CL Genencor® was more efficient when xylanase AN1818.2 was added with xylanase AN3613.2. Sugarcane bagasse hydrolysis with Accellerase 1500 Genencor® was more efficient when only xylanase AN3613.2 was added.