Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies

Abstract

Candida antarctica lipase B immobilization by covalent attachment on chitosan and on chitosan–alginate complex previously activated by different strategies was studied. Hydroxyl and amine groups of support were activated using glycidol and glutaraldehyde. Ethylenediamine (EDA) was also used in the activation process. FT-IR analysis confirmed the reaction of these activating agents with the supports. Several activation–immobilization strategies were performed and the best derivatives showed activities of 422.44 ± 50.4 and 378.30 ± 34.70 U/g-support for chitosan and chitosan–alginate complex, respectively, slightly less in comparison to the commercial immobilized lipase Novozym 435 (529.78 ± 11.7 U/g-support). Best results of thermal stability (incubation at 60 °C) and operational stability (repeated cycles of synthesis of butyl oleate) were obtained for enzyme immobilized on chitosan–alginate, activated with 2% glutaraldehyde. This derivative was 33 times more thermally stable than the soluble enzyme, and it did not lose its initial activity after 8 cycles of a 12-h synthesis of butyl oleate. Chitosan, activated with 72% glycidol, EDA and 5% glutaraldehyde, showed less operational (loss of 16.7% of its initial activity) and thermal stabilities (only 12.5 times more thermally stable than soluble enzyme). Conversion of 100% was obtained in a 12-h reaction of butyl oleate synthesis, using the best derivatives (lipase immobilized on chitosan–Gly72%–EDA–Glu5% and on chitosan–alginate–Glu2%).