Facile fabrication of lipase to amine functionalized gold nanoparticles to enhance stability and activity

Abstract

Among various techniques of immobilization, EDC/NHS cross linking is a simple and single step process for covalent coupling between enzymes and nanoparticles. Here we describe immobilization of lipase on amine functionalized gold nanoparticles (AuNPs-NH₂) to attain enhanced activity and stability. To achieve a suitable orientation, it is necessary to understand the contribution of different functional groups on the enzyme's surface. Therefore, the crystal structure of lipase was analyzed using a computational method (PyMOL) to find the exposed acidic amino acid residues that can be exploited for conjugation. Confirmation of conjugation (AuNP-NH₂-lipase) was determined by various techniques such as agarose gel electrophoresis, zeta measurement, FTIR-spectroscopy and TEM. Further, catalytic parameters (V_max, K_M,app, K_cat, and K_cat/K_M,app) have been studied to establish activity enhancement upon immobilization. The data also suggested that, AuNP-NH₂-lipase has desirable improved parameters such as temperature and storage stability. The thermodynamic parameters for the kinetics of deactivation (, and ) of the AuNP-NH₂-lipase and free lipase demonstrated better stability of the conjugate. CD and fluorescence spectroscopic studies revealed minor structural rearrangements in the enzyme upon conjugation. Thus the AuNP-NH₂-lipase conjugate represents a novel enzyme preparation with attributes of high activity and stability that could be an attractive choice in diverse applications ranging from catalysis to diagnostics.