Enzyme immobilization in silica-hardened organogels

In this study we describe a novel method for immobilizing enzymes in a solid nanocomposite matrix based on gelatin gels, which are subsequently hardened by in situ polymerization of tetraethoxysilane (TEOS). Chromobacterium viscosum lipase is taken as the example. This immobilization method possesses the advantages of enzyme entrapment in microemulsions, together with newly beneficial qualities, such as transparency, which permits direct spectroscopic investigation, and considerable mechanical stability in both aqueous and organic solvents, which results in the maintenance of enzymatic activity for several months. The first step is enzyme solubilization in AOT reverse micelles, followed by transformation of this solution into an organogel by the addition of gelatin. The enzyme-containing gel, is then hardened by the formation of silicate polymer. A glassy nanocomposite is obtained, which is optically transparent, so that the protein can be studied directly spectroscopically. Circular dichroic spectra of cytochrome-c are shown as an example. The nanocomposite material can be dried and ground, yielding a powder that is stable in both aqueous and organic solvents. After extensive washing with water, the enzyme-containing nanocomposite showed good activity in cyclohexane. The synthesis of water-insoluble fatty acid esters was carried out in this solvent with yields close to 90%. In this case, the enzyme preparations can be used over a period of several months without loss of activity or chemical yield.