Two-step modification of silica nanoparticles for covalent lipase immobilization

Abstract

Lipases are enzymes very well known for their laboratory and industrial application. Various immobilization supports and techniques were examined in order to improve lipase stability and activity for industrial application. Lipase can be immobilized by adsorption, entrapment or by covalent binding [1]. Different supports are considered for enzyme immobilization, organic or inorganic, natural or synthetic, but there is no unique solution. Ideal support should posses enough active groups to interact with enzyme, but to be inert to reaction media; it should be mechanically stable, renewable for many cycles and inexpensive. Lately, nanoparticles of silica are used for enzyme immobilization because of its extremely high surface area and controllable pore size. Nanoparticles of silica are characterized by surface to volume ratio that is significantly higher than commonly used supports. Some of the authors have also presented that nanoparticles of silica have stabilization effect for the immobilized enzyme molecules[2]. Also in some cases immobilization on nanoparticles also provides temperature stability of immobilized enzyme[3]. In this study, nanoparticles of silica were modified in two-step process. The goal was to introduce new reactive groups on silica surface, and make silica surface more suitable for immobilization of lipase. In first step, nanoparticles of silica were treated with (3- aminopropyl)-trimethoxysilane (APTMS), and then in step two, silica particles were treated with cyanuric chloride (CTC) (temperature and molar ratio silica/CTC were variated). This way nanoparticle of silica became rich in chloride groups, which enabled covalent immobilization of lipase. FTIR analysis was performed after each modification step, and confirmed presence of new active groups. Better results were obtained when second step of modification was performed at 0 °C and high molar ratio silica/CTC. Lipase from Candida rugosa was immobilized on modified nanoparticles of silica. Amount of proteins bound was in range between 55 and 78%, but activity retention after immobilization process was approximately 30%. Immobilized enzyme was used in reaction of aroma ester synthesis, and reached conversion rate of 30% within 8 h.