Resume

A β-glucosidase from Aspergillus japonicus was produced by submerged fermentation using sugar cane bagasse in nature as carbon source, at 30 ºC, for 72 hours. The dialyzed crude extract containing the active β-glucosidase was applied to chromatographic columns, in three successive steps (DEAE-fractogel, MANAE- agarose and octyl-sepharose). The enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 114 kDa. The β-glucosidase activity assay was determined using p-Nitrophenyl-β-D-glucopyranoside (β-pNPG) as substrate, in a reaction time of ten minutes. The purified enzyme was immobilized by ionic interaction on MANAE agarose and DEAE-celulose. Soluble β-glucosidase presented a half-life of 20 min, at 60ºC, while the MANAE-agarose and DEAE-celulose derivatives presented a half-life of 25 and 48 hours respectively. The optima pH for soluble β-glucosidase, MANAE-agarose and DEAE-cellulose derivatives was 4.5. The optima temperature for both derivatives and soluble enzyme was 65ºC. The amino acid sequence determined by mass spectrometry demonstrated a similar structure for the β-glucosidase of Aspergillus niger and A. kawachii. The Km values for soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives using β-pNPG as substrate were 0.4, 1.6 and 0.96 mg/mL, respectively. The Vmax values were 24, 25.8 and 13.6 U/mg protein for the soluble enzyme, MANAE-agarose and DEAE-cellulose derivatives, respectively. Because of these characteristics, the MANAE-agarose and DEAE-cellulose derivatives can be successfully applied in processes which involve high temperatures.