Stabilization of Candida rugosa lipase on nanosized zirconia-based materials

We synthesized and characterized three novel materials on the basis of zirconia. Despite their three-to six fold higher specific surface area, nanoZrO2-CeO2 (150 m2/g) and nanoZrO2-B (296 m2/g) they proved to be less effective supports for a lipase from Candida rugosa than nanoZrO2-A. For the last, we achieved protein loading of 23 mg/g, distributed in a monolayer, which means that 61% of the carrier surface was occupied by the protein. The immobilized on nanoZrO2-A lipase (nanoZrO2-A-CRL) exhibited enhanced thermal and solvent stability in comparison to the native enzyme. NanoZrO2-A-CRL has a half-life at 55 °C of 73 min, while for the native enzyme it was only 5.3 min. The immobilized enzyme preserved 20% of its activity in six consecutive cycles of the reaction hydrolysis of tributyrin. The immobilization influenced the enantioselectivity of CRL. Using nanoZrO2-A-CRL under optimal reaction condition in the reaction of esterification of lauric acid with (±)-menthol, we achieved menthol conversion of 43% (i.e. 82% of (−) methyl laurate), enantiomeric excess of 97% and enantiomeric ratio of 144. The conformational analysis proved that upon immobilization no serious change in the secondary structure of the lipase from Candida rugosa had occurred.