Sunday, April 29, 2007

Immobilization of the highly active Yarrowia lipolytica lipase – a comparison of physical adsorption and covalent attachment techniques

Aline G. Cunha1, Gloria Fernández-Lorente2, Mireille A. W. Alloue3, Juliana V. Bevilaqua4, Jacqueline Destain3, Lucia M. C. Paiva1, Denise M. G. Freire1, Roberto Fernández-Lafuente2, and Jose M. Guisán2. (1) Departamento de Bioquimica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil, (2) Department of Biocatalysis, Institute of Catalysis, CSIC, Madrid, Spain, (3) Bio-industries, Faculté Universitaire des Sciences Agronomiques de Gembloux, Passage des Déportés, 2, Gembloux, 5030, Belgium, (4) Cenpes, Petrobras, Rio de Janeiro, Brazil

Microbial lipases have been increasingly employed in a broad range of applications. Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, enzyme-free products, predictable decay rates and improved economics.
This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase by physical adsorption and covalent attachment immobilization techniques. The enzyme was physically adsorbed on octyl-sepharose and octadecyl-Sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE-sepharose support by ionic adsorption. BrCN-sepharose was used as support for the covalent attachment immobilization method. The highest activity retention yields were 60% and 61%, using octadecyl-sepabeads and MANAE-sepharose as supports, respectively.
Thermal deactivation kinetics study at a temperature range from 35 to 45 oC and pH varying from 5.0 to 9.0 was performed. It revealed that the hydrophobic adsorption on octadecyl-Sepabeads produced an appreciable stabilization of the biocatalyst and changed thermal deactivation profile. Enzyme half life time (t1/2 ) values at pH 7.0 and 45°C showed that lipase immobilized via hydrophobic adsorption was almost 10-fold more stable than free lipase. On the other hand, the Y. lipolytica lipase immobilized on octyl-sepharose and MANAE-sepharose supports presented low stability, similarly to the free enzyme. The interfacial activation on octadecyl-Sepabeads increased the stability of Y. lipolytica lipase under different conditions of pH and temperature. Immobilization onto that support led to improved stabilization what could be due to the enhanced lipase stability when adsorbed at open form.