Tuesday, May 3, 2011
β-glucosidase is an enzyme of great biotechnological potential. For example, this enzyme is one of the enzymatic ingredients of cellulolytic enzyme complexes that are used for degradation of cellulose in order to produce ethanol from lignocellulosic residues. Furthermore, β-glucosidases have numerous applications in food and pharmaceutical industries (formation of aroma compounds in wines and juices, removal of bitter compounds of fruits, deglycosylation of soy isoflavones, etc). An industrial process would be more economical by using immobilized systems that allow the reuse of the enzyme and improve the enzyme stability against different inactivation agents. In this work, we immobilized commercial β-glucosidase on the derivatized supports: MANAE-agarose, glyoxyl-agarose, glutaraldehyde-chitosan and cationic polyacrylic resin. The immobilization reactions were performed at 25oC and at different pH values (4.8, 7.0 or 9.0, depending on the support and immobilization protocol). It was necessary modify the surface of the native enzyme by chemical amination of their acidic groups (reaction between ethylenediamine and carboxyl groups of Asp and Glu residues) in order to allow the immobilization on glyoxyl-agarose and glutaraldehyde-chitosan. With this strategy was possible to prepare derivatives ca. 18 times more stable than the soluble enzyme. However, the recovered activity was low due to enzyme inactivation at alkaline immobilization conditions. Although adsorption on solid supports produce derivatives less stable due to weak interaction enzyme-support, the immobilization on cationic polyacrylic resin yield derivatives very actives (72% of immobilization yield and 40% of recovered activity). In this work, glucose production profiles using soluble and immobilized enzymes were also obtained.