Production and characterization of a thermostable extracellular β-d-fructofuranosidase produced by Aspergillus ochraceus with agroindustrial residues as carbon sources

The filamentous fungus Aspergillus ochraceus produced high levels of a thermostable extracellular β-d-fructofuranosidase (EC 3.2.1.26) when cultured for 96 h, at 40 °C, in Khanna medium supplemented with sugar cane bagasse as carbon source. The enzyme was purified 7.1-fold, with a recovery of 24%, by two chromatographic steps in DEAE-cellulose and Sephacryl S-200. The purified enzyme was homogeneous according to electrophoretic criteria. β-d-Fructofuranosidase was a homodimeric glycoprotein with 41% carbohydrate content and apparent molecular mass of 135 kDa, estimated by gel filtration in Sephacryl S-200, or 79 kDa by SDS-PAGE. Optima of pH and temperature were 4.5 and 60 °C, respectively. The enzyme showed a t50 of 60 min at 60 °C. The enzyme activity was stimulated by Mn2+ (57%), Mg2+ (50%), Na+ (35%) and Ba2+ (20%), and inhibited by Cu2+ and Hg2+. Glucose at 40 mM stimulated the A. ochraceus extracellular β-fructofuranosidase in about 2.68-fold. The enzyme hydrolyzed raffinose, sucrose and inulin, exhibiting Km of 7.37, 13.4 and 2.66 mM, and Vmax of 22.39, 42.13 and 3.14 U mg−1 protein, respectively. Transfructosylation reactions were not detected, since glucose and fructose were the only products from sucrose hydrolysis.